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United Nations Scientific Committee
on the Effects of Atomic Radiation

1977 report to the General Assembly, with annexes,:··J..'::J ti:"'" ii
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UNITED NATIONSNew York, 1977

NOTE
The report of the Committee without its annexes appears as Official Records of the General
Assembly, Thirty-second Session, Supplement No. 40 (A/32/40).
In the text of each annex, Arabic numbers in parentheses are references listed at the end.
The designations employed and the presentation of material in this publication do not imply theexpression of any opinion whatsoever on the part of the Secretariat of the United Nations concerning
the legal status of any country, territory, city or area, or of its authorities, or concerning thedelimitation of its frontiers or boundaries.

UNITED NATIONS PUBLICATION
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Produced by UNIDO, Vienna

ANNEXH
Genetic effects of radiation

CONTENTSParagraphs

Paragraphs

INTRODUCTION . . . • . . . . . . . . . . . . . . .

I. RELEVANT HUMAN DATA •....•.••
A.

B.

C.
D.
E.

F.
G.
H.

L

J.

The prevalence of naturally occurring
hereditary defects and diseases in humanpopulations • . . • • . • • . • . . • • • .
1. The results of the British Columbia
Survey and comparison with otherresults . . . . . . . . . . . . • . . .
2.
Summary and conclusions . . . . •Numerical and structural chromosome
abnormalities in new-born infants
1.
Spontaneous rates of incidence ••2. Mutation rates . . . . . • . • • . .
Chromosomal anomalies in spontaneousabortions .••. , •••....••••.
Parental x irradiation and spontaneousabortions . . • . • . • • . . . . • . • • _Parental x irradiation and Down's
syndrome . • . . . . . • • • • . • . . . .
Mortality in the children of the A-bomb
survivors in Hiroshima and Nagasaki • .
Cytogenetic survey of the offspring of
the A-bomb survivors in Hiroshima andNagasaki • . . . • . . . . . • . • . • • • .
Spontaneous non-disjunction in thehuman male . • . . . . . • . • • • . • • .
Mitotic non-disjunction in lymphocytesof young adults . . . • • . . . . • . • . .
Summary and conclusions . . • . • . • .

IL EFFECTS IN EXPERIMENTAL MAMMALS
A. Dominant lethals . • • . • . . . . . . • .
1.
Introduction . . . . . . • . . . . .
2.
Dominant lethals in male mice •.
3.
Dominant lethals in female mice4.
Species differences • . • . • . . . .
5.
Summary and conclusions •..•.
B. Translocations . . . . . . • . . . . . . .
1.
Dose-effect relationships in mouse
sperrnatogonia . . • . . • • . . . .
(a) X and gamma irradiation(b) Neutron irradiati1.,n and RBE
estimates

Dose rate • . • . . . • . • . • . • .
Dose fractionation •....••••(a) Short intervals . . . . . • . .
(b) Medium intervals . . • • . . •( c) Long intervals •..••.•.

106-110111-126
112-113114-121122-126
127-128

98-10598-102

Interaction with chemicals
Comparison between cytologicallyobserved and genetically recovered
frequencies of reciprocal transloca tions •••••...•••.•..
6. Summary and conclusions .•.••
7. Other male germ-cell stages . . . 8. Female mice •.••••.....•
9. Other species . . • . . • • • . • . •(a) Post-meiotic cells •.••1 • • •(b) Sperrnatogonia •.•.•••.
(c) Fractionation effects
10. Types of transloca tions and theireffects on fertility and viability •.
(a) The mouse .•.•.•••.•(b) Other species . . . . . • • • .
( c) Embryonic mortality in theprogenyof translocation
heterozygotes ••••...••
11. Summary • • • • . • • . . . . . • •Inversions ••••••••...••••••
Tandem duplication . • . . . . • . • • .
Loss or addition of chromosomes
1. Sex-chromosome losses ••••..
(a) Spontaneous rates .••.••(b) Induction in male germ cells(c) Induction in female germ
cells . • . . . . . • • . . . . .
2.
Non-disjunction . . . . • . • . . .
(a) Mouse • . . . . . . . • • • . .
(b) Hybrids between the house
mouse and tobacco mouse(c) A method to measure nondisjunction in the laboratory
mouse using Robertsoniantranslocations . . . . . . . . ,103-105

3.

2.

1

3.

2-58

2-20

4.
5.

2-15
16-20
21-26
21-22
23-26
27-31
32
33-37
38-39
40-41
42-45
46-48
49-58
59-469
59-88
59
60-64
65-68
69-81
82-88
89-218

C.
D.
E.

(d)

Microtus oeconomus .... .

Summary....•.......•.

129-132
133-147
148-154155-170
171-192
173-175
176-187
188-192
193-208
193-199200-204205-208
209-220221-225226
227-260
227-231227
228-229230-231232-254
233-244245-249

250-252
253-254
255-260425

Paragraphs

F.

G.

H.

I.

J.

426

Point mutations • • . • . • • • . . . . . .
1.
Specific-locus mutations in malemice . • . . . • • . • • • • • . . . .
2.
Dose-rate effects . . • . • . . . • •3.
Specific-locus mutations in female
mice •••.••. • · · · · · · · • ·{a) Dose-fractionation effects .•{b) Dose-rate effects .•.•.••
4.
Mutation processes at low andhigh radiation doses and dose
rates: criticism of a current model
5.
Nature of specific-locus mutations
6.
Autosomal recessive lethals •••.
7.
Dominant mutations . . . • . . • .
8.
Summary and conclusions ..•••
Effects of incorporated radioisotopes
1.
Plutonium-239 . . . • • • . • . . .
{a) Distribution of 2 3 9 Pu in themouse testis •....•..•.•{b) Induction of post-implan·tation loss through 2 3 9 Pu
treatment of male mice{c) Cytogenetic effects of 2 3 9 Pu
in male mice • . • • . . . . •2.
Tritium (3 H) .•••••••...•{a) Induction of dominant lethals
in mice • . . . . . . • . . . .
{b) Induction of specific-locus
mutations in male mice{c) Induction of chromosome
aberrations
inhuman
lymphocytes by tritiatedwater (HTO) •••.•.•.•
3.
Summary and conclusions ••...
The radiation response of sperma·
togonial cells and its relevance to the
int:rp~etation of genetic effects ofradiation • • • • • . . . • . • . . . . . . .
1.
Effects of dose fractionation ••.
2.
Effects of dose rate •••••••.
3.
Stem-cell killing and sterile period{a) Irradiation of adult male mice{b) Irradiation of young mice .•( c) Other species • • . • . • • . •4.
A model for stem cell heterogeneity in the adult and immaturemouse testis . . • . • • • . . . . . •5.
Summary and conclusions •.••.
Timing of ·oocyte development in theadult mouse ....•.•.••••.•••The induction of chromosome aberrations in somatic cells, the arm number
hypothesis, and its relevance to theevaluation of hazards from the induction of reciprocal transloca tions
1.
Introduction: the arm number
hypothesis . . • • • • . . • . . . . .
2.
Relationships between the frequen·cies of dicentrics in lymphocytes,
reciprocal translocations in spermatocytes, and heritable translocations in F I progeny • . • . . . .
3.
Further work on the- arm number
hypothesis • . . . . . . . • . . . • .
(a) The relationship between
dicentrics andreciprocaltranslocations in lymphocytes{b) Induction of dicentrics inlymphocytes of different
mammalian species • . . . . .
(c) Induction of dicentrics inmouse and human fibroblasts(d) Induction of reciprocal translocations in spermatogonia ofdifferent mammalian species(e) Relationship between dicentrics (lymphocytes) and translocations (spermatogonia) ..

Paragraphs

261-355
261-275276-285286-298286-289290-298299-310311-318319-329330-344345-355356-386357-371357-358359-365366-371372-377372-373374

375-377378-386

387-448398-399
400-409410-427410-420421-424425-427428-435
436-448449

450-469
450-451452-453454-465455
456-460
461-462

The relationship between thefrequency of translocati::ms
observed in spermatocytes
and in the F I progeny ..••
Problems, perspectives, summaryand conclusions . . • . . . . : ..•

{I)

4.

UL NATURE
OF
RADIATION-INDUCEDDAMAGE AND THE PHENOMENON OF
REPAIR
1.
Introduction . . • • . • . • . . . •2.
Progress in the enzymology ofrepair after uv irradiation
3.
Ionizing radiation: nature of thelesion produced and its repair ..•4. DNA repair in mouse germ cells
5. DNA repair in Drosophila . . • • .
6. Relationships between the radiation-induced lesion in DNA, its
repair, and the production of
mutations and chromosome aberra·
tions • . • . . . . . • . . . . . . . .
7. Conventional(non-molecularapproach) repair studies in Dros<r
phila

8.

•.•••••••••••••••

Summary and conclusions ••.•.

IV. INTERSPEClFIC COMPARISON OF MlJ"TATION RATE ESTIMATES • . . . • . • . . . .
I.
Introduction • . . • • . . • . . . .
2.
General considerations •.•...•
3.
Examination of the data used byAbrahamson et al. . • • • . . . . .
4.
Other relevant data •••.••..•
5.
Summary and conclusions . . . . .
Y. SOMATIC CELL GENETICS . . . • . . • . .
A. Introduction . • . . . . . . . . . . . . •B. Hypoxanthine-guanine ph~horibosyltransferase (HG-PRT) • . . • . • . . . .
1. Gene localization • • . . . . . . . .
2.
Screening methods and origin of
HG-PRT deficiency . • . . . . . .
3.
Factors affecting the recovery of
spontaneous and induced mutants
4.
Dose-effect relationships
C Thymidine kinase (TI() • . . • . . . . .
D. Ouabain resistance . . . . • . . • • . . .
E. Fructose utilization • . . . . . . . . . •F. Back mutations . . . . . . . • . • . . . .
G. Gene transfer through purified meta·
phase chromosomes . . . • • . . . . . .
H. Summary and conclusions . . • . . • . .
YI. EVALUATION OF GENETIC RADIATIONHAZARDS IN MAN . . . • . . • . . • . . . •A. A comparison of the main conclusionsof the UNSCEAR and BEIR reports
B. Current status . . . . • • . • . . . . • • .
I.
Introduction . . . . • • . . . . . .
2.
Direct method . . . . . . • • . . •(a) Recessive mutations . . . . .
(b) Dominant mutations . . . . •(c) The relationship between
rates of induction of recessive
and dominant mutations andthe effects of these mutationsin the progeny . . • . . . . .
(d) Reciprocal translocations ••{e) Other structural aberrations(!} Sex-chromosome losses . . .
3.
Doubling dose method . • . • . . .
4.
Summary and conclusions . . . • .

VII. SUGGESTIONS FOR FUTURE RESEARCH
463
Tabler464................... - .....

References . . . . . . . . . . . . . . . . . . . . . . .

465
466-469

470-523470
471-478479-486487-492
493-494495-501502-506
507-523524-527524-527. 528-530
531-535536-547548-552553-594553
554-569554-555556-557558-565566-569570-576577
578
579-580
581-584585-594595-659595-605606-659606
607-636607-613614-615616-626627-633634
635-636637-644645-659
660

Pages
514
540

Introduction
1.
The 1972 report of the Committee (589)
presented a comprehensive review of the genetic effectsof ionizing radiation. The present report will be devoted
to (a) an updating of the 1972 report, especially thoseparts that require significant revision in the light of
information that has accumulated during the last few
years, with particular attention being paid to thoseresults that bear on the problem of the evaluation ofgenetic radiation hazards in man, and (b) a comparisonof the main conclusions reached by the Committee in1972 with those of the Advisory Committee on the
Biological Effects of Ionizing Radiation (BEIR) of the
National Academy of Sciences of the United States ofAmerica (34).

I. RELEVANT HUMAN DATA
A. THE PREVALENCE OF NATURALLY OCCURRING HEREDITARY DEFECTS AND DISEASESIN HUMAN POPULATIONS
1. The results of the British Columbia Survey
and comparison with other results

2.
Trimble and Doughty (576) have published theresults of a major study whose primary objective was todeteqnine the frequency of live-born individuals affectedby hereditary or partially hereditary defects and diseases
in a large, geographically defined population, namelythat in the Canadian province of British Columbia, witha. current population in excess of two million people.
Smee these results have been obtained and interpretedusing infonnation on the inheritance of specific traits
that has been gained during the last fifteen years or so,and since knowledge of the amount of hereditarydiseases in human populations is crucial to an evaluationof genetic radiation hazards in man using the
doubling-dose method, they will be discussed insome detail in the following paragraphs.
3.
The data used for this study were derived from therecords of the British Columbia Registry for Handicapped Children and Adults and pertain to a period of
21 years (1952-1972). These include cases actuallyregistered with the Registry as well as those ascertained
through the province-wide Surveillance System ofCongenital Anomalies, both systems having multiple
sources of ascertainment. Of the total case-load of
48 212 individuals recorded with the Registry by theend of 1972, only about 44 per cent (21 290) were bornin the province during the period 1952-1972. In
addition: 9313 children also born in the province during
the penod 1964-1972 were ascertained through theCongenital Anomalies Surveillance .System. The data
relate to _this ~otal of 30 603 affected children among
756 304 live births. Adequate precautions were taken to
avoid duplicate entries from multiple sources ofascertainment. The details of the different kinds ofdiseases or disorders included in the analysis are given intables 1 to 8. Diseases of unknown aetiology andenvironmentally caused disorders (representing a total of4949 cases out of 30 603) are not listed in these tables.

4.
An examination of the frequencies of children
with one or another kind of genetic or partially genetic
disease in the successive annual birth cohorts showed
that, in general, these remained relatively constant up to
the year 1963 (2.5-3.8 per cent), increased from 1964 to1968 (5.1-6.0 per cent) but decreased in recent years (to2.6 per cent in 1972 for example). The higher figuresfrom 1964 onwards are due to individuals ascertained
through the Surveillance System. which contains
information only for children born since 1964. The~ecline in the more recent cohorts is only apparent,
since there is a tendency for the affected children born
4t later years not to be registered until they reach school
age. To arrive at the best estimates of the frequencies ofsingle gene, chromosomal and multifactorial diseases, theauthors used the data for the period 1964-1966; forcongenital malformations. they used those from
1967-1969. The reason was that, for these birth years,
there appeared to be nearly complete registration of
conditions that manifest themselves during childhood oryou":. The total thus estimated is about 6 per
100 live-born for all registered conditions, regardless ofthe particular aetiology of a condition (0.18 per cent,single gene diseases; 0.16 per cent, chromosomal
anomalies; 3.58 per cent, congenital anomalies; 1.58 per
cent, other multifactorial diseases; and 0.60 per cent,diseases of unknown aetiology).
5.
To allow for biases due to delay in disease onset,
migration and incompleteness of ascertainment, suitablecorrection factors were used to get "adjusted estimates''from "minimal estimates" given above and both theseare presented in table 9 together with the earlier ones of
Stevenson (566) for Northern Ireland, a division of theUnited Kingdom of Great Britain and Northern Irelandwhich were revised subsequently by UNSCEAR(586, 587). Examination of table 9 will show that there
are several differences between the estimated incidence
rates in British Columbia and in Northern Ireland: (a)the total frequency of serious diseases or handicaps
believed to be genetic is about 9 per cent in British
Columbia as compared with about 6 per cent inNorthern Ireland; (b) the incidence of diseases due tosingle dominant genes is lower by a factor of more than
10 in the present study; (c) the frequencies ofsingle-gene autosomal recessives and of chromosomal
anomalies are also lower (by a factor of 2) in British
Columbia; and (d) the incidence of diseases that fallunder the categories of congenital malformations andother multifactorial diseases is higher by a factor of
more than 2 in the present work (9 .0 per cent comparedwith 4.0 per cent). 1
6.
Nearly all (97 per cent) of the chromosomal
anomalies recorded in the British Columbia Survey are
due to Down's syndrome, whereas in the UNSCEAR
estimates, other chromosomal anomalies were also
included (see footnote c to table 9). The frequency ofcases with Down's syndrome in British Columbia isroughly the same as that reported in the UNSCEAR
reports. The low estimate of the total frequency ofregistered chromosomal anomalies (0.20 per cent versus
0.42 per cent) indicates that some of the individuals
with other chromosomal disorders such as K.linefelter's
1 The 9.0 per cent is from column 4 (4.28 + 4.73 = 9.01),
and the 4.0 per cent is from column 2 (2.50 + l .SO = 4.00)

427

or Turner's syndrome were not brought to the attentionof the Registry, presumably because they were notregarded as handicapped during childhood or had theaetiology of their disorders incorrectly classified as notdue to chromosomal aberrations. Many cases of
sex-chromosomal abnonnalities and structural anomaliesof the autosomes would have been classified ascongenital malformations or as diseases of unknown
aetiology because the correct cause of the defect would
not be readily recognized in the absence of routine
cytogenetic analyses. While the precise extent ofunderestimation of these cases is difficult to determine,
the authors sunnise that accounting for these individuals
could increase the estimate of the total frequency ofchromosomal anomalies by as much as threefold (i.e.,from 0.2 per cent to 0.6 per cent). The recent summary
estimates on the incidence of chromosomal anomalies innew-born babies (table 11) in fact give a total frequencyof 0.6 per cent.

of late onset such as Huntington's chorea". In the British
Columbia survey, only 2 cases of Huntington's chorea
were found, giving an incidence of 2.6 per million.
However, ad hoe surveys such as that of Shokeir (534)indicate a prevalence in Saskatchevan and Manitoba of
8.7 per 100 OOO which (since patients have the overt
disease for only about 15 years) corresponds to a birthfrequency of heterozygotes of about 1 in 2000.

7.
The major discrepancies between the present data
and the earlier ones based on the Northern Ireland
survey relate to the very low incidence of diseases
presumed to be caused by regular dominants (0.08 percent compared with 0.95 per cent) and in the rather highincidenGe of con.ditions that fall under the categories ofcongenital anomalies and multifactorial diseases (9.0 per
cent compared with 4.0 per cent). The discrepancy for
regular dominants is in part due to a considerable
underestimate of the incidence of dominant disorders
with onset in adult life in the British Columbia survey,
and in part, to the inclusion in the Northern Ireland
survey of conditions which would not now be regarded
as having a simple dominant mode of inheritance. This
discrepancy is of major concern since regularly inherited
dominant (and X-linked) conditions are expressed in theimmediate descendants of exposed individuals and are
expected to increase in direct proportion to an increasein mutation rate. They are therefore an importantcomponent of the genetic hazard from radiation
exposure.

11. Ad hoe surveys of the prevalence of individual
dominant conditions by observers skilled in the diagnosisof particular disorders and using all available means ofascertainment of cases, such as those quoted by Vogel
and Rathenberg (599) in estimating human mutation
rates and listed in table 10, with the addition of more
recently recognized dominant disorders such as monogenie hypercholesterolaemia (82a) suggest that the totalbirth frequency of such disorders may well be 1 percent. The total figure of 1 per cent dominants used in
the 1972 report may therefore be retained. though theindividual conditions contributing to it have beenreappraised.

8.
The 12 most frequent disorders in Stevenson's listof dominant diseases account for about 75 per cent of
all his cases of dominants. A review of the inheritance of
these 12 conditions 2 has shown that at least 7 of themwould not now be regarded as being inherited as simple
dominant disorders (360). This has been illustrated by
Newcombe (360) for hydrocephaly, which accounts for
one fifth of all the cases in Stevenson's dominantcategory. This condition is now known to beheterogeneous and the family patterns observed exclude
simple dominant (or even simple recessive) inheritance inany substantial proportion of cases.
9.
The British Columbia survey relates largely todiseases with onset in childhood and is based on afollow-up of individuals from birth to ages 1-21 years,
and Sutton (555) has expressed the view that the studyof Trimble and Doughty "clearly underestimates diseases
1 Cataracts (senile and pre-senile), hydrocephaly (internal
obst;11c~ve), alopecia areata, nystagmus (familial idiopathic),
cysuc disease of lungs, choroidal sclerosis, multiple exostoses,?euro~bromatosis, colobomata, ataxia (dominant hereditary
including Friedrich's), porphyria (dominant detectable) andcataracts (congenital).

428

10. Similar underestimates are likely to exist in the
British Columbia data for conditions such as neurofibromatosis, the adult type of polycystic disease of thekidney, multiple polyposis of the colon and monogenic
hypercholesterolaemia. The same limitations apply to
Edwards' (145) low estimates of the incidence ofdominant disease. Sutton (555) is of the opinion that "itwould seem wise to use the figure of 1 per centdominant disorders until additional reliable data indicate
this not to be correct".

12. X-linked and autosomal recessive conditions areusually more severe and already expressed in childhoodand therefore, the estimates of their incidence are morestraightforward. Edwards' (145) estimates of 0.05 percent for X-linked and 0.25 per cent for autosomal
recessive conditions would appear to agree well withestimates from ad hoe prevalence studies (82a).
However, some of the more common autosomal
recessive disorders are probably maintained by heterozygous advantage and a figure of 0.1 per cent is a more
realistic estimate for the incidence that is maintained bymutation.
13. Turning now to the incidence of diseases that falls
under the category of congenital anomalies, multifactorial and irregularly inherited diseases, 3 as was
3 In contrast to simple dominant diseases where affected
carriers transmit the disorder to half of their offspring on the
average, the dominants of incomplete penetrance constitutemutational{y-maintained conditions whose inheritance is not yet
fully understood and which at present, may be incorrectlyclassified as disorders with a multifactorial aetiology. Incomplete
penetrance may be due to environmental factors or due togenetic ones and in the latter case, the category of dominants ofincomplete penetrance merges \\ith the multifactorial category.
It is difficult at present to estimate the birth frequency of these
diseases due to dominant genes of incomplete penetrance or togive unequivocal examples. They may include some cases ofcommon congenital malformations and other common disordersof late onset. However, the great majority of cases of common
congenital malformations and constitutional and degenerative
diseases are thought to be truly multifactorial in origin and arecharacterized by high frequency, a non-dominant pattern ofinheritance but a familial pattern of incidence in twins; sibs,
cousins and so forth that is indicative of multilocal transmission.

mentioned earlier, it is higher by a factor of 2 in British
Colombia relative to Northern Ireland (9.0 per ce11t
compared with 4.0 per cent). These diseases constitute avery substantial proportion of human disorders, butthere are great uncertainties concerning the exact
mechanisms involved in their aetiology. It is not reallypossible to make any realistic quantitative apportionment of their causation between genetic and environmental factors, both of which play varying roles. The 9
per cent of Trimble and Doughty may include an
unknown proportion of incorrectly classified chromosomal anomalies, monogenic disorders and diseases oflargely environmental aetiology. It is therefore likely tobe somewhat of an overestimate. On the other hand,underestimations are likely to exist for constitutional
disorders of adult onset, for example, schizophrenia ordeath in the fifth decade from coronary heart disease.
14. Insofar as this major group of disorders is
genetically determined, the family pattern they show
suggests that the genetic component is usuallymultilocal, i.e., depending on genetic variation in at least
several gene loci (82b, 82c). Their high frequency, e.g., 1
in 250 to 1 in 2000 in different human populations forspina bifida cystica, 1 in 1OOO for the commonest
congenital heart defect (ventricular septal defect), and 1
in 100 for schizophrenia, indicates that selective
mechanisms are responsible for their maintenance andthat they are not mutation-dependent. Newcombe (361)
has recently stressed this point.
15. On the other hand, it is known that there are
relatively rare monogenic disorders already recognizable
among this group of disorders, for example thedominant conditions cleft lip and mucus pits of thelower lip (among the large group of multifactoriallydetermined facial cleft malformations). These are readily
assigned to the dominant category. It is likely, however,that there are further unrecognized rare regulardominant and irregularly dominant conditions in thisgroup which would be mutatio11ally maintained. The sizeof. this mutationally-maintained fraction is unknown.
BEIR (34) conservatively assumed that this component 4
is not likely to be more than 50 per cent or less than 5
per cent. The upper limit of 50 per cent is perhaps
implausibly high and a more realistic figure would bearound 10 per cent. In this report, for purposes ofhazard evaluation,. we shall use the figure of 9 per cent
for_ the incidence of the class of congenital, multifactonal and irregularly inherited diseases (based on the
British Columbia data) and assume that the mutational
component 4 is 5 per cent (best average estimate) (82).

2. Summary and conclusions
16. The rates of spontaneous incidence of different
kinds of genetic disease in man available from the survey
carried out in British Columbia represent a significant
addition to our knowledge on the load of genetic
ailments carried by the human population.
• The mutational component of a disease is the proportionof its incidence that is directly proportional to the mutationrate.

17. In this survey, the. total frequency of these diseases
has been estimated to be 9.44 per cent and can be
broken down into 0.12 per cent autosomal dominantand sex-linked diseases, 0.11 per cent recessive and 0.20
per cent chromosomal ones, 4.28 per cent congenital
malformations and 4.73 per cent other multifactorialdiseases.
18. Discrepancies exist between these rates and thoseobtained earlier by Stevenson (which, with somerevisions, are the ones that have thus far been used inhazard evaluations), particularly with respect to theincidence of dominant diseases and those included under
the category of congenital malformations. multifactorial
and irregularly inherited diseases. For the former, thepresent estimate is lower by about an order ofmagnitude (0.12 per cent compared with 1.0 per cent)and for the latter, higher by a factor of 2 (4.0 per centcompared with 9.0 per cent). In addition, the rate ofincidence of chromosomal abnormalities in British
Columbia is also lower by a factor of 3 relative to recent
results of new-born surveys (0.20 per cent comparedwith 0.60 per cent).
19. The Committee reappraised the above figures,
taking into account, among other things, the resultsfrom different ad hoe surveys for specific dominantconditions, those from new-born surveys for chromosomal anomalies and the uncertainties involved in the
aetiology of diseases that fall under the category ofcongenital malformations, multifactorial diseases andirregularly inherited conditions. The following figuresthat were arrived at will be used in the context of hazard
evaluations: 1.0 per cent dominant and X-linked
diseases, 0.1 per cent recessive diseases, 0.4 per centchromosomal diseases and 9 .0 per . cent congenital
malformations, multifactorial and irregularly inheritedconditions.
20. The magnitude of the mutational component for
the last mentioned category of diseases is thought to be
not more than 10 per cent (upper limit) in contrast to
the upper limit figure of 50 per cent used by BEIR; forthe purpose of calculations, the Committee will use afigure of 5 per cent as the best average estimate.

B. NUMERICAL AND STRUCTURAL CHROMOSOME ABNORMALITIES IN NEW-BORN INFANTS
1. Spontaneous rates of incidence

21. In its 1972 report, the Committee presented theresults of surveys carried out in different parts of theworld on the chromosomal constitution of live-born
infants; the data available at that time showed that outof 21 996 babies, 114 (0.52 per cent) had an abnormal
chromosome constitution. Additional data obtainedsince then in different laboratories (summarized by
Jacobs et al. (250), Hamerton et al. (212), and Nielsen
and Sillesen (363) and including the more recent workof Lin et al. (289) bring the total number of babies
examined to 55 679, with 336 of them (0.60 per cent)having abnormal karyotypes. All these karyotypes(except those in the work of Lin et al.) were examined429

with conventional techniques. The kinds and frequenciesof the different abnormalities recorded in these surveys
are given in table 11.
22. The data summarized in table 1 I permit thefollowing conclusions: (a) the total frequency varies
from 0.47 per· cent to 0.83 per cent in the different
surveys; the overall rates of incidence {based on pooled
results) are 0.22 per cent sex-chromosome anomalies,
0.14 per cent autosomal trisomies, 0.19 per cent euploid
structural rearrangements, and 0.05 per cent aneuploid
structural rearrangements (including supernumerarychromosomes); 5 (b) the frequencies of 47)(YY males
range from O in Moscow to 0.03-0.05 per cent in Boston,
Winnipeg and Arhus, 0.13-0.14 per cent in Edinburghand New Haven, to a high of 0.37 per cent in Ontario;( c) variations in the incidence rates of 4 7,XXV males isless (ranging from 0.07 per cent in Boston to 0.18 percent in New Haven), and in general the rates arecomparable; (d) the overall incidence of sex-chromosome anomalies in males is 0.26 per cent (93/34 872)
and in females, 0.14 per cent (29/20 807): (e) the
frequency of 45)( females is very low, being only 0.01
per cent (2/20 807); nearly all the other babies listed
under "Other" (column 10) were mosaics, suggestingthat the vast majority of XO conceptions do not survive
to term and those which do are indeed mosaics; (f) the
frequency of autosomal trisornies (trisomy-21; listed
under +G) is 0.12 per cent (compare this frequency withthat recorded in the British Columbia survey, 0.19 per
cent, table 9); (g) the incidence rates of euploid
structural rearrangements are reasonably consistent forsix of the surveys (Edinburgh, Winnipeg, Hamilton,
Boston, New Haven and Moscow: 0.14-0.21 per cent),
while in Arhus, the frequency is high (0.30 per cent) andin Ontario, low (0.05 per cent); the results also
demonstrate that the frequency of Robertsoniantranslocations (D/D and D/G types) and that of detectedbalanced reciprocal translocations are roughly the same;and (h) aneuploid structural rearrangements are
relatively uncommon, only 30 being observed in thewhole sample.

2. Mutation rates
23. Jacobs et al. (250) used the data pertaining to
43 558 babies to estimate the "rates of mutation" forthe different kinds of chromosomal abnormalities. For
numerical errors of chromosomes which result in
live-born children, this was 14.0 10-4 per gamete per
generation (mosaic individuals and those with 46)( inv(Y) were excluded). The estimate based on 55 679 babies (table 11) is nearly the same, being 14.2 10-4 per
gamete per generation (sex-chromosome anomalies:
7.5 10-4 per gamete per generation; autosomal anomalies: 6.7 10-4 per gamete per generation; mosaics and Y
chromosome inversions likewise excluded).
24. For structural abnormalities of the autosomes. theestimates of Jacobs et al. were based on the incidence in
5 A person was considered to have a supernumerarychromosome if he had 4 7 chromosomes 46 of which wereapparently normal, the additional chromos~me being equal to or
smaller than a member of Group G and clearly different inmorphology from a G-group chromosome (250).

430

the total sample of 43 558 and an analysis of the
proportion which was non-familial (and thus new
mutants) in a sample of 56 babies found to. carrynon-mosaic structural rearrangements: they found that
10 out of 50 euploid babies (20 per cent) and 2 out of 6
aneuploid ones (33 per cent) were new mutants.
Multiplying the incidence rate of0.19 per cent (euploid)by 0.2 and dividing by 2 (to correct for the possibility
that the mutation could have arisen in either sex), theauthors estimated that the rate for all euploid structural
rearrangements (Robertsonian translocations, reciprocaltranslocations and inversions) and 1.9 10-4 per gamete
per generation: for reciprocal translocations alone, thefigure was 1.33 10-4 per gamete per generation{36/43 558 multiplied by 0.33 (proportion of new
mutants: 8/24) and divided by 2). For aneuploid
structural abnormalities (translocations, inversions anddeletions), the rate was 0.30 10-4 per gamete per
generation (8/43 558 multiplied by 0.33 and divided
by2).
25. Under the assumption that the proportions of new
mutants found by Jacobs et al. are applicable to thepresent total sample of 55 679 babies, the following
rates can be estimated using calculation procedures asabove (para. 24): all euploid structural rearrangements, 0.0019 X 0.2 X 0.5 = 1.9 10-4 per gamete per
generation;
reciprocaltranslocations
alone,
0.00086 X 0.33 X 0.5 = 1.4 10-4
per gamete per
generation;
aneuploid
structuralabnormalities,
0.00027 X 0.33 X 0.5 = 0.45 10-4 per gamete per generation. The .above estimates are roughly similar to thosemade by Jacobs (249) from less extensive neo-natal data
and to those of Jacobs, Frackiewicz and Law (249) who
combined both neo-natal and post-natal data.
26. Jacobs et al. (247) have pointed out that the
mutation rates for autosomal rearrangements must be
underestimates of the true rates for at least two reasons:(a) only a fraction of all chromosome rearrangements in
man is detectable in somatic cells by conventional
staining techniques. Of the structural aberrations
considered here, only Robertsonian translocations allow
a detection rate approaching 100 per cent; and (b) many
aberrations may be selected against before they give rise
to live births; such selection is presumably almost
completely restricted to unbalanced rearrangements. On
the basis of unpublished results of Buckton 6 with
irradiated human lymphocytes, Jacobs et al. (247)concluded that approximately 75 per cent of symmetrical exchanges (reciprocal translocations) may escapedetection. However, human cytogeneticists who haveused the new banding techniques (the latter, among
other things, permit a. precise identification ofchromosomes or chromosome parts involved in translocations) are of the opinion that the efficiency of the new
banding techniques with respect to the detection ofreciprocal translocations is only slightly higher (by 10-20
per cent) than the conventional staining techniques; thismeans that the rate of 1.4 10-4 per gamete per
generation estimated for reciprocal translocations is not
likely to be strikingly lower than the true rate. Since
there remains the possibility that. even with the banding
techniques, small equal symmetrical exchanges may• The paper has recently been published (66).

escape detection, it is perhaps wise at present tomultiply the rate mentioned above by 2 to get an
estimate of the probable true rate, i.e., we shall assume
that the spontaneous rate of origin of reciprocaltranslocations in man is 2.8 10- 4 per gamete per
generation.

C. CHROMOSOMAL ANOMALIES INSPONTANEOUS ABORTIONS

27. The incidence of chromosomal anomalies inspontaneous abortions in man has been extensively
reviewed by Carr (78, 79) and Boue and Boue ( 46) andhas been considered to some extent in the Committee's
earlier reports (587, 589). It is well established thatchromosomal anomalies are frequent among spontaneous abortions with an incidence rate that is muchhigher than in live-born infants. The total frequency ofthese anomalies in the major abortion studies varies from
8 per cent in that of Stenchever et al. (565) to 64 percent in that of Szulman (559): the wide discrepancies aremost probably due to the operation of a variety offactors, such as geographical variation, maternal age,prevalence of unsuspected induced abortions in the
population, variations in hospital admission practices
and especially the procedure and scope (selected versus
unselected) of the collection of abortuses. Boue andBoue ( 46) (and several others) have found that thephenotype and gestational age of the concept us are both
related to the incidence of chromosomal anomalies: intheir large-scale study, they recorded one or anotherkind of chromosome abnormality in 66 per cent of theabortuses arrested in their development before 8 weeks
of gestation, this falling to 23 per cent of zygotes with
8-12 weeks of development. (In 1097 out of 1205
observations, the developmental arrest occurred before
the eighth week and these represent about 90 per cent of
all abortions; developmental arrests before the third
week are rare, since in these cases the pregnancy might
be unrecognized and such specimens are seldom received
for examination.)
28. Most of the chromosomal anomalies which havebeen described among abortuses are numerical such asmonosomy-X, autosomal trisomy, triploidy and tetraploidy. Boue and Boue (45, 46) recently summarized theresults of their continuing investigations and comparedthem with those obtained by some other workers whose
sample sizes were adequate enough to permit some
general inferences. These are given in table 12 inaddition to the data of Kajii et al. (253) and of Creasy etal. (127) not covered in the above paper. Since the dataof Boue and Boue are the most extensive, they can beused to establish a rank order of incidence of thedifferent abnormalities to inquire the extent to whichthese results agree with those of others and to gain an
insight into the kinds of aberrations that are more or lessfrequent in abortuses in contrast to live-borns. As will beevident from an inspection of table 13, the agreement
between these results is good. with trisomy constituting
the most predominant class of abnormalities (-50 percent), followed by monosomy-X, triploidy, tetraploidy
and others.

29. In trisomic abortuses, the extra chromosome isalmost always an autosome (in contrast to monosomy inwhich the missing chromosome is a sex chromosome).
Trisomies of all autosomal chromosome pairs are found,but their relative incidences are different, trisomy for C,
D, E, and G being more, and those for A, B and F less,frequent (table 14). All the three possible kinds oftriploidy (69,XXX, 69,XXY and 69,XYY) are seen
although again their relative frequencies appear different(XXY > XXX > XYY) (46). The small number of
triploid abortuses with the 69,XYY constitution does
not necessarily demonstrate the rarity of occurrence of
this class, since a similar result can be obtained if thesekaryotypes lead to very early developmental arrest. The
virtual absence of autosomal monosomy and the rather
low frequency of some kinds of trisomy (A, B and F)
might likewise be due to their very early developmental
arrest. This view is supported by the experimental workof Gropp (203) and of Ford and Evans {169), who
studied the time of action of the lethal effects of zygotic
aneuploidy in F 1 hybrids between the tobacco mouseand the laboratory mouse. It was found that autosomal
zygotes could be observed only before implantation: thetrisomic embryos however, died during the post-implantation period with a considerable reduction in numbers
between the earlier gestation period (8-11 days) and thelater period (12-15 days) and none was observed atbirth.
30. An analysis of the relationship of maternal age andthe different types of chromosomal anomalies (table 15)
shows that the mean maternal age is higher for lethal
trisomies relative to other abnormalities as well as toabortuses with normal karyotype. Close inspection oftable 15 will reveal that it is primarily for the trisomies
involving acrocentric chromosomes (D and G groups)that the influence of maternal age is marked; in lethal
G-trisomies, the curve of maternal age distribution (45)shows bimodality, with 60 per cent of the observationsin the age-dependent group, a pattern very similar tothat noted by Penrose and Smith ( 406) for children with
Down's syndrome. Thls suggests that whether a zygote
with a trisomy G will lead to a spontaneous abortion ora delivery at term may not depend on the age of themother.
31. It is instructive to compare the results fromnewborn surveys with those from the abortion studies( tables 11, 13 and 14). It will be evident thatmonosomy-X is one of the most frequently encounteredclass of abnormalities in spontaneous abortions whereas
it is very rare in new-barns. About 40 per cent of all
spontaneous abortions with chromosome anomalies {andabout 80 per cent of all trisomy) are due to trisomy for
D, E and G group chromosomes; in the new-born
population, trisomy for these chromosomes accounts for0.14 per cent of all new-born children (or about 23 percent of the abnormalities recorded in these). Structuralanomalies of chromosomes and mosaics constitute about
5 per cent of all abnormalities scored in the abortus
material, whereas in new-horns, about 40 per cent of allabnormalities scored are structural and involve theautosomes (both euploid and aneuploid structuralaberrations), 0.24 per cent of the children having
structural abnormalities.

431

D. PARENTAL X IRRADIATION AND
SPONTANEOUS ABORTIONS
32. Alberman et al. (5) made a comparison of thehistories of medical irradiation received by parents of
spontaneous abortuses of abnormal karyotypes withcorresponding histories in cases of abortions of normal
karyotype and with those in parents of live births. The
overall finding was that the mothers of chromosomalJy
abnormal foetuses had received the largest mean gonadal
dose of irradiation for medical reasons compared withthe other spontaneous abortions and the live-birthcontrols. As in their Down's syndrome study (6) theradiation doses tended to be rather small and the effectsof radiation appearing to be cumulative and, seemingly,
often maternal age-dependent. Irradiation appeared to
be an especially important factor in the case of triploidabortuses: the mean gonadal dose of mothers of triploids(about 0.74 rad) exceeded that received by mothers of
those with autosomal trisomy of one chromosome
including trisomy of the G group (0.30 rad) or of the
45,X abortuses (0.18 rad). The authors stress the pointthat most of the chromosomally abnormal foetuses are
not viable and are lost early in pregnancy.

E. PARENTALX IRRADIATION AND
DOWN'S SYNDROME
33. Even though several retrospective and prospective
studies have been conducted to find out whether
parental irradiation may increase the risk of producing
Down's syndrome (trisomy for chromosome 21) in theprogeny, the answer to the question remains equivocal.
For instance, the retrospective studies of Uchida andCurtis (581), Sigler et al. (537) and Cohen and
Lilienfield (115) have been interpreted as showing an
association between maternal irradiation and Down's
syndroIIJe. The investigation of Alberman et al. (6)showed that the mothers of those with Down's
syndrome had had more total x-ray examin~tions, bothin number and in dose, before the conceptions (relative
to those "control mothers") of children with a variety ofother serious congenital handicaps which are notthought to be in any way associated with parental
irradiation; the overall difference did not reach asignificant level. However, when the mean doses for thedifferent maternal age groups and conception histories
were compared (i.e., radiation doses received less than
6 years before. 6-10 years before and more than I O years
before the conceptions). (a) the variation with age wasmore marked in the mothers of the Down ·s syndromegroup ( than in the control) and (b) the effect was
greatest in the subgroup where x-ray exposures wereadministered more than I Oyears before the conceptions.
34. Jn contrast, the retrospective studies of otherinvestigators (83. 305. 324) failed to provide any
evidence for an association between maternal irradiation
and Down's syndrome in the progeny, but these reports
were based on small numbers of cases.
35. The prospective studies of Schull and Neel (563)
of cbildren born to mothers exposed to the A-bombradiation at Hiroshima likewise showed no association.
but the doses involved were of a different order of432

magnitude from those used for medical purposes. The
study of Stevenson et al. (561) also failed to support the
hypothesis that x-ray diagnostic procedures before
conception increase the frequency of Down's syndromein children subsequently born. However, the prospective
study of Uchida et al. (583) indicated that a significantly
greater number of trisomic children were born aftermaternal radiation exposure.
36. In a recent paper, Kochupillai et al. (260), claimed
that the incidence of Down's syndrome in the
population living in an area of high backgroundradioactivity in coastal Kerala in southern India wassignificantly higher relative to a control population andto the rates recorded in similar surveys by otherinvestigators elsewhere in the world. The exposure riskin the study population was estimated as 1.5-3 rad peryear (control, 0.1 rad per year) and the frequency of
Down's syndrome observed was 12 in 12 918, giving a
rate of 1 in 1076 (or- 0.93 per 1000). No Down's
syndrome cases were detected in the control (5938
individuals screened). In terms of maternal age, thedistribution of Down's syndrome cases in the study
population was as follows: 1 in 862 {age range 20-29), 1
in 81 (age range 30-39) and 1 in 266 (age range 4049).

37.

Sundaram (548) re-examined the data presented
by Kochupillai et al., and concluded that the author's
claim could not be substantiated for the following main
reasons: (a) only about 11 per cent of the females
included in their study receive radiation doses in therange 1.1-2 rad per year, and 2.8 per cent receive
exposures higher than 2 rad per year. This type of
dosimetric profile is due to the non-homogeneous
distribution of monazite-containing beach sands; (b) theage structure of the Kerala study population is quite
different from those of others with which the data arecompared, i.e., while in most surveys, only about 4 percent of all births is contributed by females in the agegroup 40 years and older, in the Kera la survey, thisfigure is around 20 per cent and this age group is known
to have the highest risk for children born with Down's
syndrome; (c) when the data are re-calculated toestimate the incidence at birth and at the time of study( taking into account the family size, the number offemales in each age group and the infant mortality rate),
one arrives at 9-10 cases for the population sample
studied, and the observed 12 cases therefore do notshow any significant difference; (d) the observation of
Kochupillai et al. that the risk in the maternal age group40-49 is only one third of that in the age group 30-39 ispuzzling and at variance with what is so far known about
the relationship between maternal age and the incidenceof Down's syndrome.

F.

MORT ALI TY IN THE CHILDREN OF TIIE
A-BOMB SURVIVORS IN HIROSHIMA AND
NAGASAKI

38. Neel et al. (359) and Kato (254) have publishedthe results of a continuing study of mortality rates
among children born to suf\1vors of the atomic
bombings of Hiroshima and Nagasaki which updated
those presented in an earlier report (255). Although theactual numbers of deaths since the previous review wassmall, the present report was prompted more by the factthat significant revision of the dose estimates had
become available.

39. The mortality experience pertains to (a) 18 946
children born live to parents both of whom were
proximally exposed (i.e., either one or both parents lessthan 2000 rn from the hypocentre and receiving jointly
an estimated dose equivalent of 117 rem); 7 (b)16 516 children born to distally exposed parents (i.e.,neither parent less than 2000 m, but either or both lessthan 2500 m. from the hypocentre and receiving
essentially no radiation) and (c) 17 263 children born toparents not in Hiroshima or Nagasaki at the time of thebombing. The average interval between birth andverification of death or survival was 17 years. Analysis of
the data has shown that no significant effects of parental
exposure on childhood mortality can be demonstrated. 8
However, the data permit an estimate of the lower limitof doubling dose 9 (for the type of damage 8 resulting indeath during the first 17 years of life among live-born
children conceived 0-13 years after parental exposure),
and this is equal to a gamma-ray dose of 46 rad forfathers and 125 rad for mothers. 1 0 Neel et al. suggest
that, on the basis of mouse data, the gametic doublingdose for chronic low-level radiation would be expected
to be 3-4 times the value of 46 rad for males (i.e., atleast 138 rad) and over 1OOO rad for females.

G. CYTOGENETIC SURVEY OF THE OFFSPRING
OF THE A-BOMB SURVIVORS IN HIROSHIMAAND NAGASAKI
40. A continuing survey of the offspring of thesurvivors of Hiroshima and Nagasaki bombings forstructural and numerical anomalies of the chromosomes
is in progress. The latest report published by Awa (15)shows that among 2885 children of A-bomb survivors 1 {(1386 males and 1499 females), a total of 18 individuals(0.62 per cent) with chromosome abnormalities havebeen found (3 XXY, 3 XYY, 2 XXX and one X/XXX'The unit used in the paper of Neel et al. is the rem andwas obtained by summing the dose equivalents of the gamma
and neutron components of the irradiation and assuming thatthe quality factor for neutrons is 5.
'It is assumed that the childhood mortality as measured
here is due to different kinds of genetic damage (point
mutations, small deletions. unbalanced translocation andnon-disjunction).
9 The 97 .5% lower confidence limit. The data exclude a
doubling dose equivalent lower than 46 rem for males and
125 rem for females.
1 0 The doubling dose is calculated from the relationshipd .. xy/z, where d Is the doubling dose, x is the mortality due tospontaneous mutation in the preceding generation (included
both gene and chromosome mutations), y is the contribution ofa given sex to the mortality, assumed to be 0.5 for each sex, andz = the maximum slope of the dose-effect curve (calculated in a
stepwise regression analysis) which can not be excluded bypresent data at the 5% confidence level. The numerical values
used in the above equation are, for males; x = 0.005, y = O.S andz =0.000054, and for females, x = 0.005, y =0.5 andz = 0.00002.
1 1 Subjects in the exposed group were those wh~separents (either one or both) were exposed to a dose of morethan 1 rad; the parents of the controls were either not exposedor received less than 1 rad.

mosaic, together 0.31 per cent; 9 structural abnormalities, including 3 t(Dq Dq), 1 t(Dq Gq) and 5 balanced
autosomal translocations, together 0.31 per cent). In thematched control 11 of 1090 subjects (510 males and580 females), 3 individuals with chromosomal anomalies
have been found ( 1 XXY and 2 balanced reciprocaltranslocations). Awa has pointed out that although theprevalence of sex-chromosome aneuploidy in thechildren of exposed parents is higher than in the controls(0.31 per cent compared with 0.09 per cent), thedifference is not significant.

41.

The above incidence rates can be compared withthose obtained in surveys of new-born infants(paras. 21-22 and table 11):
Hiroshima andNagasaki data
controls
I 090 subjects

AnomalyNum·herChildren of ex- Surveys of newposed parents born infants
2 885 subjects 55 679 subjects

PerNum- PerNumcentage ber
centage her

Sexchromosome
Numerical
autosome Structuralautosome 2

0.18

9

All

0.28

18

3

0.09

9

0.31

Percentage

122

0.22

76

0.14

0.31

137

0.24

0.62

336

0.60

It can be seen that the frequencies in new-born surveys
are similar to those in the children of exposed parents
from Hiroshima and Nagasaki and that there are no
significant differences. The control frequencies in the
Hiroshima and Nagasaki data however are lower than in
the other groups, but the differences are not significant.

H. SPONTANEOUS NON-DISJUNCTION

IN THE HUMAN MALE
42. One of the important recent developments in
mammalian cytogenetics has been the introduction ofdifferential staining techniques that have permitted theidentification of specific chromosomes in interphasenuclei. Thus, quinacrine staining of interphase cells has
shown that the human Y chromosome can be visualized
as an intensely fluorescent dot (404) and that it can beseen throughout the male germ-cell series (403) includi~ thespermatozoa (26). New techniques have now beendeveloped for differential staining of chromosomes 1
and 9 (37, 189, 401, 402). It has become clear that the
specific staining reactions are confined to regions ofconstitutive heterochromatin composed of highly
repetitive nucleotide sequences of DNA with varying
base ratios dependent upon the chromosome concerned(251). The general category of differential staining

involved here has been designated as C-banding.
43.

These technical developments have been founduseful to identify and measure the frequency ofaneuploid spermatozoa (aneuploid with respect tochromosomes Y, 1 and 9) in ejaculates. Initial estimatesfor Y chromosome, i.e., for those spermatozoa containing two Y bodies (and not one) were 1.3 per cent433

(37) and 2 per cent (403). A later study (401) confirmed
these frequencies for the Y chromosome and alsosuggested a similar frequency for chromosome 9. It
should be pointed out here that the above frequencies
refer only to those of hyperhaploid spermatozoa; if thereciprocal class of hypohaploids occurs at the same rates,
the calculated figures have to be doubled. The
implication of these findings is that, if non-disjunctionof other chromosomes occurs at about the same rates,
the proportion of aneuploid spermatozoa will beconsiderable and will lead to the production of many
trisomic embryos, most of which will be spontaneously
aborted.

47. Peripheral
blood
from
28 subjects
aged19-29 years was used for lymphocyte cultures; 7 ofthese were fathers and 7 were mothers of children withtrisomy-21; the remaining 14 were age- and sex-matched
controls with no known history of Down's syndrome
among close relatives. After irradiation ( 50 R of
13 7 Cs gamma rays at 29 R/min), the irradiation andcontrol samples were appropriately processed forchromosome studies. (After 69 h in culture, colcemid
was added; harvesting was at 72 h. The latter time period
was used to recover cells containing non-disjunctional
products in preference to chromosome breakage
products.)

44. The above data of Pearson and Bobrow (403),
Bobrow et al. (37) and Pawlowitzki and Pearson (401)have been criticized on a number of points (141) as
follows: (a) single Y chromosomes are known to have abifid structure in a proportion of interphase cells andthat extra dots in spermatozoa could be explained bysingle Y chromosomes being bifid; (b) the extra dots
might represent adventitious spots either throughstaining artefacts or by areas on other chromosomes
staining up with the same reaction; (c) such a highfrequency of YY spermatozoa is not reflected in thenumber of XYY individuals found either in live-born orabortion series, and, since there is no evidence ofaneuploid gametic selection in mice, there is likely to be
little or no selection in man which could account for thedifferences found in the frequency of YY spermatozoa
and XYY individuals.

48. The results were: (a) the total frequency ofhypermodal cells in the irradiated sample was 0.1 percent (29/28 OOO), which was higher than that in thecontrols (0.025 per cent); (b) in the unirradiatedlymphocytes exposed to irradiated serum, there was also
an increase in the frequency of hypermodal cells(28/28 OOO); (c) more hyperdiploid cells were found in
the irradiated samples from control males than fromfathers of progeny with trisomy-21; 2 of the control
males had a total of 12 hyperdiploid cells, suggesting an
increased susceptibility to non-disjunction; (d) of thetotal of 57 hyperdiploid cells, 1 was tetrasomic forchromosome 21, 1 had double trisomy and l, triple
trisomy. The extra chromosome could be accurately
identified in 38 cases; there appeared to be a
non-random non-disjunction of chromosomes, this being
higher for the G-group chromosomes (10 cases) and for
the X (C-group, 11 cases). All identifiable G-groupchromosomes were No. 21. These results therefore
suggest that the X and chromosome 21 may beparticularly susceptible to somatic non-disjunction.

45. In a subsequent paper, Pearson et al. ( 405)
obtained results that confirmed their earlier ones andwere compatible with their expectations (rates of"non-disjunction" for chromosomes 1, 9 and Y being
respectively 3.5, 5 and 2 per cent). but have not beenable to rule out the criticisms mentioned above. It isperhaps worth remembering that the specific staining
reactions used in these studies are confined to regions ofconstitutive heterochromatin and that chromosomes 1,
9, Y (and 16) have well defined and prominent blocks ofthese at the centromeric region (except in the Y where it islocalized in the distal portion of the long arm) (14). To ·what extent heterochromatin per se may play a role innon-disjunction and whether one can generalize from thebehaviour of those chromosomes with large blocks ofheterochromatin to others which do not have these, arequestions that have to be satisfactorily answered before
the results discussed above can be used in the con text of
estimating total non-disjunction frequencies (for all
chromosomes).

I. MITOTIC NON-DISJUNCTION IN LYMPHOCYTESOF YOUNG ADULTS
46. Uchida et al. (584) conducted experiments to see
whether low-dose irradiation of human lymphocytes
would lead to non-disjunction of chromosomes. It was
realized that a positive fmding in a study of this typedoes not necessarily imply that these results can beextrapolated to meiotic segregational errors; rather, it
was thought that such a finding may indicate theusefulness of somatic cell systems as models for the
study of meiotic non-disjunction.
434

J. SUMMARY AND CONCLUSIONS
49. Since the publication of the 1972 report,
additional data have become available on the incidenceof chromosomal anomalies in new-born babies. All theseresults considered together show that the total incidence
is 0.60 per cent of which roughly one third (0.22 percent) attributed to sex-chromosome anomalies, one
quarter (0.14 per cent) to autosomal numerical anomalies, one third (0.19 per cent) to autosomal euploid
structural anomalies and one twelfth (0.05 per cent) to
autosomal aneuploid structural anomalies.
50. The total frequency of chromosome anomalies
recorded in the children of the A-bomb survivors ofHiroshima and Nagasaki (0.62 per cent) is very similar tothat found in.new-born surveys (0.60 per cent) although,in the control group for the former, this figure is only
0.28 per cent. The differences however, are notsignificant.
51. On the basis of the total data from new-born
surveys and the family history of some of theabnormalities studied, spontaneous mutation rates can be
estimated. These are 14.2 10-4 per gamete per
generation for all numerical errors of chromosomes
which result in Jive-born children, 7.5 10-4 forsex-chromosome anomalies alone and 6.7 10-4 forautosomal numerical anomalies. The corresponding
figures for all euploid structural rearrangements of themes is 1.9 10-4 and for balanced autosomal
aut~so ca1 translocations alone, 1.40 10-4. Consideringr~c1 0 t that the efficiency with which the latter type oft be a~·on can be scored in somatic cells may be lower
3 err~~O per cent, it is thought that the true rate may be~ 10 -4 per ga11:ete per ge~eration, for those gametes
which give rise to live-born children.

58. Data have become available which show that
non-disjunction can be induced by irradiation of human
lymphocytes and that the X chromosome andchromosome 21 may be particularly susceptible tosomatic non-disjunction.

~,.,
chromosome anomalies are frequent among:,_. ntaneous abortions, with an incidence rate that issp~ch higher than in live-born infants and most of the~ rmer are numerical anomalies. It has been found thatb:th the phenotype and gestational age of the conceptus
are related to the incidence of chromosome anomalies inspontaneous abortions. In over 90 per cent ofobservations, the developmental arrest occurred before
the eighth week of gestation.

II. EFFECTS IN EXPERIMENT AL
MAMMALSr

53. In spontaneous abortions, trisomy for one or
another autosome is the predominant type accounting
for about 50 per cent of all the abnormalities, followedby monosomy-X, triploidy, tetraplody and others. In
contrast, in new-borns, autosomal trisomies (especiallyof D, E and G) accounts for only one quarter of allabnormalities. A second difference lies in the highfrequency of monosomy-X in spontaneous abortions(about 20 per cent of all anomalies) and its rather low
frequency (0.6 per cent of all anomalies) in new-boms.
'Thirdly, structural anomalies of chromosomes andmosaics account for about 5 per cent of theabnormalities scored in the abortus material, whereas innew-borns, more than one third of the abnormalities areof this type.
54. There seems to be an association between maternalradiation history and spontaneous abortions in the sensethat in the work reported, mothers of chromosomallyabnormal foetuses have received higher mean gonadal
doses relative to comparable controls.

55.

The results of both retrospective and prospective
studies on the relationship between maternal irradiationand the incidence of Down's syndrome are conflicting.
Recent work in India showing that the frequency of
Down's syndrome in a population living in an area ofhigh natural background radioactivity is significantly
higher than in controls and than those recorded
elsewhere has not stood the test of a criticalre-examination which took into account the family size,
the number of females in each age group and themortality rate.
56. Recent results of a continuing study of mortality
rates among children born to the A-bomb survivors ofHiroshima and Nagasaki show that no significant effectsof parental exposure can be demonstrated. However, thedata permit the estimation of doubling doses, lower thanwhich can be excluded. These are 46 rad for males and
125 rad for females under the radiation conditions
during the bombings. For chronic low-level exposures,
based on mouse data, it has been estimated that thegametic doubling dose for males is about 138 rad and forfemales, over 1000 rad.
57: . Techniques are now available for specificallyst~~ human chromosomes Y, 1 and 9 and studying
non·diSJunction in sperm samples. However, no radiation
studies have thus far been reported.

A.

DOMINANT LETHALS

1. Introduction
59. The 1972 report of the Committee considered insome detail the induction of dominant lethals in miceand in other experimental mammals. In most of theexperiments reviewed in the report, dominant lethality
had been measured by the pre-natal method, i.e., byexamination of the uterine contents of females atsuitable stages of pregnancy, counting the numbers ofcorpora lutea, living and dead implanted embryos andassessing the amount of mortality occurring before andafter implantation.

2. Dominant lethals in male mice
60. Although no substantially new information has
accumulated since the publication of the 1972 report, atleast as far as male mammals are concerned, the paper ofSearle and Beechey ( 510a) has helped to throw light on
the best choice of an index dominant lethality where themutagenic treatment also induces germ-cell killing.
61. The reasoning of Searle and Beechey is based on
their study involving exposure of adult male mice to
200 rad of acute x irradiation and mating them atintervals to females over the next 9 weeks. T-estis weights
fell to 44 per cent of normal during the 5th week andwere still subnormal when observations were terminatedin the 10th week. Epididymal sperm counts fell in the
4th week (after a peak in the 3rd) and were very low in
the 6th and 7th weeks, though still above zero. This
reduction was correlated with high, frequencies of
pre-implantation loss in females mated to these males
and dissected on the 12th day of gestation (table 16).
Examination of eggs 1-2 days after ovulation showedthat there was a sharp decrease (from the normal level ofalmost 100 per cent) in the fertilization index; it wastherefore concluded that much of the pre-implantation
loss really stemmed from non-fertilization rather thanzygotic death.
62. It has long been suggested that, if a particularmutagen causes intense cell killing with resultant
spermatozoa! depletion, then what appears to bepre-implantation death may really be the expression of alowered fertilization rate (29, 451, 560). The work of
Searle and Beechey provides experimental proof for that
assertion, and they propose that, in these circumstances,an index of dominant lethality basea on post-implantation survival should be used (e.g., the ratio live435

embryos/total implants) rather than the ratio live
embryos/ corpora lutea or the number of live embryos perpregnant female. They show that use of the last two
indices can sometimes lead to a very substantial
overestimate of the true rate of dominant lethality.
However, the index of post-implantation mortality
underestimates the dominant lethal frequencies inspermatozoa and spermatids. Therefore, for a precise
determination of the dominant lethal frequency, it isnecessary to establish the fertilization rate of the ova.
63. In experiments designed mainly to study the
induction of translocations in mouse spermatozoa,
Searle et al. (520) estimated the amount of dominantlethality from the rate of decrease of mean litter sizewith increasing dose. It was found that the relationshipbetween mean litter size and dose was· roughly
exponential and gave a satisfactory fit to the equation
YD = y 0 e-kD, where y D is the litter size at dose D, Yo is
the litter size in controls and k is the rate of induction of
dominant lethals. The estimated value of y 0 was
9.44 ± 0.35 and that of k was (1.28 ± 0.07) 10-3 rad- 1 •The authors stress that the rate of dominant lethals as
estimated above can only be approximate, based as it ison new-born litters.
64. Uonard et al. (285) studied the genetic
radiosensitivity of different mouse strains (RF, BALB/c,
C3H, CBA, C013, AKR/TIAld, C57BL and AKR) using
the rate of induction of dominant lethals in spermatozoa
as the criterion (germ cells were sampled during the firstweek of mating after irradiation). An x-ray exposure of
400 R was used and both pre- and post-implantationmortality were scored. The results showed that (a) the
frequency of fertile matings in most of the strains was
slightly lowered by the radiation exposure; in the RF
and C013 strains, this was enhanced and significantly soin the latter; (b) there were no significant differences
between the numbers of corpora lutea in the control andirradiated series of each strain; (c) the magnitude ofradiation-induced pre-implantation losses varied widelyin the different strains from a low 13 .4 per cent in the
BALB/c strain to a high 52.7 per cent in the
AKR/TlAld strain: likewise, the amount of post-implantation losses also varied from 13 .3 per cent in the
AKR/TlAld to. 39.3 per cent in the BALB/c; and (d) the
total amount of dominant lethality (both pre- andpost-implantation losses) estimated from the expression
100 lo~ (1 -P)- 1 where
Number of live embryos/
number of corpora lutea in the irradiated series

P=l----------------Number of live embryos/
number of corpora lutea in the controls
also varied from about 13 in the RF strain to 43.5 in the
CS7BL strain. In most of the strains, the increased
pre-natal loss is the result of an increase in both pre- andpost-implantation death. A comparison of these datawith those available for rats, guinea-pigs and rabbits(most of the latter were reviewed in the 1972 report: seeparagraphs 22-23, Annex E (I)) would indicate that thevariation between different strains of mice with respect
to radiation-induced dominant lethals is as large as thatbetween the mouse and the other species.
436

3. Dominant lethals in female mice
65. The 1972 report of the Committee considered theearly data of L.B. Russell and W. L. Russell (458) andthose of Edwards and Searle (146) on the sensitivity ofmouse oocytes to dominant lethal damage. In addition,
the results of Lyon and Smith (313) in guinea-pigs andgolden hamsters were dealt with. It was concluded that,as after irradiation of males, most of the induced
embryonic death occurs after implantation. Recently,
Searle and Beechey (513) have published some data on
the induction of dominant lethality in mouse females
after x and neutron irradiation. In the x-ray series,
mature females received acute x-ray doses of100-400 rad and were mated to males either 1 day after
irradiation or 2 weeks later with daily examination forvaginal plugs; in the neutron series, the females were
irradiated with doses of 50-200 rad of fission (0.7-MeV)
neutrons and were mated 1 day later. Pregnant females
were killed on or around the 14th day of gestation for
examination of the uterine contents. Appropriate
controls were maintained. In contemporaneous experiments; meiotic preparations of oocytes were made toassess the amount of cytological damage.
66. The x-ray data are given in table 17. It can be seenthat the survival of implanted embryos to the time of
examination in late pregnancy (as measured by the live
embryo/total implant ratio) declines with increasingradiation dose. Unexpectedly, this decline is much more
rapid when these embryos were conceived in the 3rd
week after irradiation. The increasing dominant lethality
correlated well with the amount of chromosomal
damage found in the meiotic preparations of theoocytes. The dose-effect relationship for post-implantation dominant lethality is roughly linear in both the I stand 3rd-week groups, but in the latter, its level is twice
as high over the 200-400 rad range. In the 3rd week theamount of post-implantation loss rose considerably athigher doses, this rise being associated with increasing
non-fertilization of ova. Therefore, the live embryo/corpus luteum ratio is not a satisfactory index of total
dominant lethality for the 3rd-week group (133). In the
lst-week group, the dominant lethality seemed to reacha plateau at about 200 rad.
67. In the fission-neutron experiments, post-implantation dominant lethality is negligible at 50 and 100 rad
but high at higher doses (table 18). For total dominantlethality, the dose-effect relationship seems curvilinear.
68. Since the females exposed to neutrons were mated
1 day after irradiation, Searle and Beechey compared
their neutron results with those of the lst-week x-ray
data to estimate the efficiency of neutrons relative tox rays in inducing dominant lethals. Because of the lackof clear-cut dose response in the post-implantationmortality data with neutrons, they do not lendthemselves to a ready calculation of the RBE; likewisethe "total dominant lethality" data with x rays also do
not permit an easy comparison with neutron results. The
finding that at a dose of 100 rad (neutrons or x rays) the
total d,ominant lethal frequencies are roughly the same
may suggest that at low doses the RBE may be about 1,
but more data are needed to verify this suggestion.

4. Species differences

69. In the 1972 report, the work of Lyon (307)comparing the x-ray induction of dominant lethals in
post-meiotic male germ-cell stages of the mouse,gumea-pig, golden hamster and rabbit (scored by the
pre-natal method) were discussed. The x-ray doses used
were: 100 and SOO rad (mouse), 500 rad (guinea-pig andrabbit) and 100-300 rad (golden hamster). It was foundthat (a) the pattern of relative sensitivity of the germ
cells in mouse, guinea-pig and rabbit was similar, with
spermatids being more sensitive than mature spermatozoa; in hamsters, spermatids and mature spermatozoa
were about equally sensitive to dominant lethal
induction; (b) in the guinea-pig and rabbit, thefrequency of dominant lethals at 500 rad were lower
than in the mouse; after 200 rad to hamsters, the yieldof dominant lethals in mature spermatozoa was nearly ashigh as after SOO rad to the mouse; for weeks 2-4,
however, the yield in hamsters after 200 rad wasconsiderably lower than in mice; (c) in the mouse,guinea-pig and hamster, a large proportion of deaths
occurred after implantation, whereas in the rabbit, theyoccurred before implantation.
70. In experiments aimed at studying the x-ray
induction of heritable translocations in post-meiotic
male germ cells of guinea-pigs, rabbits and golden
hamsters, Cox and Lyon (122) have collected data on
litter-size reduction (a rough index of dominantlethality) which corroborate the earlier results of Lyon
(307) discussed above, in terms of pattern of relative
sensitivity of the different germ-cell stages and theamount of damage observed in the successive "mating
weeks" after irradiation (1-4 in hamsters and rabbits and1-5 in guinea-pigs). The x-ray doses used in the present
study were: 200 and 600 rad (han1ster), 600 rad(guinea-pig, rabbit). The results demonstrate that (a) in
all the species studied, irradiation of post-meiotic cells
result in reduction in litter size; (b) after 600 rad, thepattern of litter-size reduction and the pattern ofsterility for the matings in the successive weeks are
similar for golden hamster, guinea-pig and rabbit; formatings utilizing irradiated spermafocytes, i.e., week 4
for the hamster and possibly week 5 for the guinea-pig, ahigh incidence of infertility is not accompanied by ahigh reduction in litter size, suggesting that the latter
infertility is only partly due to the induction of
dominant lethals; such infertility could also stem fromreduced fertilizations; (c) in the hamster, the dominantlethal yields (as measured by litter-size reduction) in
weeks 1-4 after 200 rad and in week 1 after 600 rad aresimilar to those of the mouse, but the yields for boththese species are above those obtained for the rabbit andguinea-pig; (d) it can be concluded that the golden
hamster is more sensitive than both the rabbit and theguinea-pig for the induction of dominant lethals by xrays in post-meiotic male germ cells.
71. Cox and Lyon (121) have completed a comparative study of x-ray-induced dominant lethality in guineapig and golden hamster females. The choice of these two
species was dictated in part, by the following
considerations: (a) in all female mammals, the bulk of
the oocytes are in small follicles (primordial follicles),and the response of these stages more accurately reflects
the reproductive performance of the irradiated females;in the guinea-pig, the immature oocytes in primordial
follicles are more resistant than those of the mouse withrespect to cell killing by irradiation, although moresensitive than those of human oocytes (guinea-pig,
LD 5 0 = 500 R; man, LD 5 0 ='== 2000 R (25, 37 6)); (b) incontrast, the oocytes in the maturing follicles in theguinea-pig are more sensitive than those of the mouse:
an exposure of 200 R virtually destroys the larger
follicles in the guinea pig whereas this does not happen
in the mouse; (c) on the evidence available (408) the
golden hamster appears to be intermediate in sensitivity
between the mouse and guinea-pig; (d) it thus appears
that there is no relationship between the nuclear stage ofthe "arrested" oocyte, cell killing, and dominant lethal
induction; for the dictyate oocyte of mouse and hamsteris sensitive, and the condensed diplotene oocyte of theguinea-pig is resistant, to cell killing, yet all three show a
low frequency of dominant lethality; (e) in the mouse,dominant lethal yields are higher in the oocytes sampled
in the third post-irradiation week than in the first (513),
and it would be instructive to inquire whether thepattern observed in guinea-pigs and golden hamsters
resembles that of the mouse and whether any correlation
between sensitivity to killing and genetic radiosensitivity
can be made.

72. Guinea-pigs aged 3-5 months and golden hamsters
aged 2.5-3.5 months were irradiated during middle
dioestrus ( day 6-12 of the 17-day cycle in the guinea-pig
and day 2 and 3 of the 4-day cycle in the golden
hamster) with 100, 200 and 400 rad of x irradiation at ahigh dose rate. Three post-irradiation matings were
employed for the guinea-pig 1st oestrus, 2nd oestrus and3 months) and this number was 4 in the golden hamster(1st oestrus, 2nd oestrus, 1 month and 4 months). In
addition, the effect of maternal age on dominant lethal
yield in the golden hamster was determined in parallelofanimalsexperiments
involvingirradiation
9-11 months old and the study of dominant lethality at
the lst oestrus mating after irradiation. The pregnant
females were killed at appropriate times after matings(3-4 weeks for the guinea-pigs and 10-12 days for
hamsters) and the amount of dominant lethality was
ascertained by the pre-natal method.
73. Table 19 shows that irradiation did not cause any
sterility in the guinea-pig, except at the lst-oestrus
matings (after 400 rad) suggesting that at this dose themature oocytes are more sensitive than the immature(3-month matings) to killing by radiation. In the golden
hamster, there is a marked effect: a large number offemales irradiated with 400 rad and mated 3 months
after exposure wer; sterile (10 out of 13) and had
ovaries that were approximately one third of the normal
size. Histological sections of these ovaries revealed nocorpora lutea or oocytes, suggesting that the sterility wasdue to killing by the radiation of all oocytes wlµch werein either primary or growing follicles at the time oftreatment. Sterility in one month matings after 400 rad
may have similar causes although histological evidence
on this was not obtained. All the other groups of females,both control and irradiated, showed a small amount ofinfertility and this might have been, in part, due to theseasonal breeding habit of this species. These results thus
indicate that the less mature oocytes, i.e., 1- and3-month matings are more sensitive to killing than are
the mature oocytes.
437

74. The data given in table 19 also show that
irradiation of mature oocytes of the guinea-pig with
400 rad lead to superovulatidn, i.e., a 30 per centincrease in the number of corpora lutea per female.
Although this difference was not statistically significant,the amount of increase was very similar to that seen
when mature oocytes of the mouse were treated withthe same dose of x rays ( 455). No superovulatory effects
could be seen for the 2nd-oestrus and/or the 3-monthmatings. In hamsters, irradiation with 400 rad produced
a significant increase in the number of corpora lutea perfemale for the 2nd-oestrus but not for the lst-oestrus
matings.
75. The details of the intra-uterine mortality for both
species are presented in table 20. It can be seen that in
the guinea-pig, in general, the incidence of dominantlethals increases with dose and that the yields for mature
oocytes were above those for the immature oocytes at
each level. In the golden hamster, the situation is nearly
the same.
76. An examination of the components of dominantlethality will show that, in the guinea-pig, bothcomponents increased with dose for the lst-oestrus
matings; for the lst- and 2nd-oestrus matings,
pre-implantation death exceeded port-implantationmortality in contrast to the 3-month matings, where themagnitude of both of these mortalities was relatively
low. In the golden hamster, mortality before and after
implantation increased with dose for the lst- and2nd-oestrus matings and in general, the amount of death
before implantation was somewhat higher than that after
implantation. In both species, some of the pre-implantation death could have been due to the shedding ofdead or dying ova, rather than to true zygotic death.
77. The experiments on the effects of maternal age on
dominant lethality in the . lst-oestrus matings in the .
golden hamster showed that the yields of dominantlethals were higher from older females than from young
females at 100 and 400 rad levels, but not at 200 rad;however, the differences were not significant.
78. Statistical comparison of the dose-effect relationships for dominant lethals in both species showed thatthese were consistent with linearity with no significant
differences between the slopes (model: M = Mo + bD;
bguinea-pig = (1.19 ± 0.25) 10- 3 and bgolden hamster= (1.59 ± 0.18) 10- 3 (M is the mortality, M 0 , thecontrol mortality)). Although statistically a difference
between the two species cannot be demonstrated,
examination of the raw data suggests that the mature
oocytes of the golden hamster may be slightly moresensitive to dominant lethal induction than those of theguinea-pig; in addition, the higher dominant lethal yields
for the 2nd-oestrus matings in the golden hamster are
indicative of a similar situation.
79. As may be recalled, in the mouse, the work of
Searle and Beechey (513) demonstrated that the yieldsof dominant lethals for the 3rd week were generally
higher than those for the 1st week; this pattern is incontrast to that seen in the two species studied by Cox
and Lyon (121), where the yields for the 2nd-oestrus
matings were in general lower than those for the
lst-oestrus matings. A second difference between the438mouse and the other two species is that, in the lst-weekmatings of the former. the total frequency of dominantlethals seemed to reach a plateau at about 200 rad.
whereas in the latter. the data were consistent with alinear increase with dose. Since the x-ray data forpost-implantation mortality in the mouse did not deviate
from a linear relationship with dose, Cox and Lyoncompared the slopes of the regression (on a linear modelfor this component) for the three species. The values ofthe slopes were (in units of 10-4 rad- 1 ):Guinea-pig
3.88 ± 1.18
Golden hamster 6.19 ± 1.18
Mouse
3.57 ± 0.69
80. Thus, although the rate per unit dose for mature
oocytes of the golden hamster appears to be higher thanthat of the other two species, this difference does notreach statistical significance. However, the authors pointout that when total dominant lethality in mature
oocytes of the three species is compared, the yield in themouse after 400 rad is significantly below that of theother two species. All these data considered together
suggest that the order of species in terms of decreasing
sensitivity of the pre-ovulatory oocytes to dominantlethal induction by x rays is: golden hamster, guinea-pig,
mouse.
81. For dominant lethal induction in post-meiotic
male germ-cell stages (as measured by litter-size
reduction), the golden hamster and the mouse appear tohave the same sensitivity (in week 1-4 after 200 rad andin week 1 after 600 rad). Likewise, the rabbit and theguinea-pig have similar sensitivities (after 600 rad,week 1-4). The magnitude of litter-size reduction
however, is higher in the first two species than in the
rabbit and guinea-pig.

5. Summary and conclusions
82. Under conditions where irradiation (or othermutagen treatment) causes germ-cell killing withresultant spermatozoa! depletion, the ratio of dead
implants to total implants is a better index of dominantlethality than that based on the ratio of dead implants tocorpora lutea or the number of dead embryos perpregnant female.
83. There are striking differences between different
strains of mice in the amount of x-ray-induced dominantlethality (spermatozoa! irradiation), which are as large as
those between the mouse and other laboratory mammals
studied in this respect.
84. X irradiation of female mice leads to a reductionin the mean number of implants per female (while thenumber of corpora lutea remain high) and in the ratio oflive embryos to total implants; this reduction is moremarked in conceptions occurring in the 3rd week than in
the 1st week after irradiation; in contrast, in golden
hamsters and guinea pigs, the yields of dominant lethalsfor 2nd-oestrus matings are lower than those for1st-oestrus matings.
85. X irradiation of female mice (100-400 rad) leads
to an increase in post-implantation mortality inconceptions occurring during the 1st and 3rd weeks afterirradiation. The dose-effect relationships are roughly
linear, but in the 3rd-week group, the level of mortality
is about twice as high as in the lst-week group. The total
dominant lethality (pre· and post-implantation) in the
lst-week group seems to reach a plateau at about
200 rad.
86. In guinea-pigs and golden hamsters, the dose-effectrelationships for both post-implantation and total
dominant lethality are consistent with linearity. For the
induction of post-implantation mortality in pre-ovula·
tory oocytes, the order of the three species in terms ofdecreasing sensitivity is: golden hamster, guinea-pig,
mouse.
87. For the induction of dominant lethals in
post-meiotic male germ-cell stages, the mouse and golden
hamster are more sensitive than rabbit and guinea-pig.
88. At a dose of 100 rad, fission neutrons (0.7 MeV)
appear to have the same effectiveness as x rays ininducing dominant lethals in female mice.

B. TRANSLOCATIONS
89. Ever since the air-drying technique for making
meiotic preparations of mammalian testes was developedby Evans, Breckon and Ford (162), the cytogenetic
study of translocations in mammals, especially of thoseinduced by irradiation, has been growing explosively.
During the period 1964-1972, a large number of papers
were published dealing with dose-effect relationships,
dose-rate and dose-fractionation effects, effectiveness ofradiations of different quality, the types of translocations and their effects on fertility etc. These wereextensively reviewed in the 1972 report of theCommittee. The general findings are summarized in theparagraphs that follow (unless otherwise stated, thecomments refer to studies with the mouse):
90. Translocations can be induced by iomzmg
radiation in all stages of spermatogenesis in adults, inembryos in utero and in late oocytes. Radiation-induced
translocations during spermatogenesis have also been
observed in adult male rabbits, guinea-pigs and golden
hamsters.
91. The pattern of radiosensitivity as it emerges fromthe cytological studies closely parallels that from genetic
studies in demonstrating that post-meiotic male germ
cells are more radiosensitive with regard to translocationinduction than premeiotic cells; among the post-meiotic
stages, spermatids are by far the most sensitive.
92. In spennatogonia, translocation induction by x
and fast-neutron irradiation at high dose rates is
consistent with a linear kinetics (up to 600-700 rad withx rays and up to 100 rad with neutrons), after which theyield falls off drastically, giving an overall humped curve;
the results with gamma irradiation at high dose rates
showed that the dose-effect relationship was concave inthe range 56-402 R (suggesting the contribution of aquadratic component to the observed yield), althoughwhen the data were analysed as a whole (5 6-816 R) the
relationship did not significantly depart from linearity.

All these features are very probably the result of
secondary distortions of the primary dose-effectrelationship, which may well have a more marked
square-law component.
93. In rabbit and guinea-pig spermatogonia, the overall
dose-response curve after acute x irradiation is also
humped (as in mice), but the peak incidence occurs at
200-300 rad.
94. The yield of translocations obtained from
spermatogonia is reduced after x and gamma irradiation
at low dose rates, the effect being more pronouncedwith gamma rays; at high doses, protracted neutron
irradiation is more effective than acute irradiation
whereas the reverse is true at low doses: the effects offractionation are dependent on total doses andfractionation procedures. Especially important is theobservation that the fractionation of a total dose of
300 rad of x rays into several small fractions of 10 or
5 rad each leads to a significant reduction intranslocation yields, as compared with the effects of asingle dose.
95. Some translocations induced in spermatogonia cansuccessfully pass through the remaining stages ofspermatogenesis and can contribute to zygotic populations; all the dominant lethality observed after
spermatogonial irradiation can be accounted for by the
segregation of unbalanced haploid genomes from
spermatocytes with translocation multivalents.
96. A marked discrepancy was found between thefrequencies of translocations diagnosed cytologically (inspermatocytes following spermatogonial irradiation) andgenetically (in F I progeny) in that the observed
frequency in the F I is only one half that expected on
the basis of the frequencies observed in primary
spermatocytes: these experiments had been carried out
at an exposure level of 1200 R administered in 2 equal
fractions separated by a period of 8 weeks.
97. Certain translocations ( autosomal) can be fully
viable in the heterozygous state and yet cause malesterility through a failure of spermatogenesis. If suchtranslocations are induced in spermatogonia, they will
not be represented in the effective sperm population andconsequently will not be expected in the progeny offathers whose spermatogonia have beep. exposed toirradiation; this is also true for translocations involving ·the X chromosome.

1. Dose-effect relationships in mouse
spermatogonia(a) X and gamma irradiation
98. Preston and Brewen (419) re-examined the
problem of dose-effect relationship using x irradiation ata high dose rate (50-1200 R; 11 exposure levels). There
was one important methodological difference between
their work and that of other authors: while in most of
the earlier investigations, a single sampling time (ranging
from about 56 to 120 days, depending on theinvestigator, following irradiation with different exposures) was employed, Preston and Brewen staggered the

439

·~
• t

• ~:

.

sampling time so that it coincided with the first wave of
meiotic activity following recovery of the germinal
epithelium. In other words, the time interval between
irradiation and killing of the animals (to makepreparations of the testes) varied with the exposure.
99. Their results are reproduced in table 21. In therange 0-500 R, the data best fit a model thatincorporated both a linear and a quadratic component.
The value for these two components are given by theequationAbove 600 R, however, the yield of translocations
decreased with increasing exposures, leading to ahumped curve over the whole range studied, as has been
observed by several workers previously. The authors
suggest that in earlier work, the use of a single samplingtime might have led to the sampling of different
populations of stem cells at different exposures and thatthese might be responsible for the discrepancies between
their results and the earlier ones with respect to theshapes of dose-response curves up to exposures of
600-700 R.
100. It is worth pointing out here that Wennstrom(605), on the basis of somewhat limited data, had earlier
reached the conclusion that, in the dose range
25-400 rad, the dose-effect relationship for the induction of translocations in mouse spermatogonia wassupra-linear. At all the dose levels, the interval between
irradiation and observation was 2 months. The frequencies of cells with multivalents were (per cent): 0.33
(control), 0.33 (25 rad), 0.66 (50 rad), 2.7 (100 rad),3.7 (200 rad) and 4.0 (400 rad). The numbers of cells
analysed were respectively 1521, 300,300,300,273 and400. These data appear to suggest that, in the particularstrain of mice used by ·Wennstrom (albino mice of the
NMRI strain), the yield peaks at a rather low dose level.
The author did not attempt any mathematicalcurve-fitting.
101. Pomerantzeva, Ramaiya and Ivanov (415) studied
the gamma-ray (1 3 7 Cs) induction of translocations inmouse spermatogonia at three different exposure rates(710, 0.07 and 0.007 R/min). At the highest exposure
rate, the frequencies were (per cent): 3.3 ± 0.7,
5.1 ± 0.7, 4.7 ± 0.8, and 4.2 ± 0.8 per cent at exposurelevels of 300, 600, 900 and 1200 R, respectively. At the
middle rate, the frequencies were lower, being 2.8 ± 0.5
per cent at 300 R, 3.8 ± 1.4 per cent at 600 R and
1.8 ± 0.7 per cent at 900 R. At the lowest rate, thefrequencies were consistent with a linear increase with
exposure (300, 600, 900 and 1200 R), the equation ofbest fit being
Y=(0.225±0.086) 10· 2 +(1.74±0.21) 10" 5 D
where Y is the translocation frequency and D, theexposure. In a subsequent paper, Pomerantzeva, Vilkina
and Ivanov (417)" demonstrated that when the exposure
rate was further reduced to about 0.003 R/min, theresponse over the range of exposures from 100 to 920 R(5 levels) was nearly the same as that obtained with thelowest rate (0.007 R/min) above.
440

102. Searle et al. (516) analysed the available low
dose-rate data obtained with chronic gamma irradiation( 6 °Co or 137 Cs) down to 0.003 rad/min (82, 83, 89,90) (with total doses varying from 100 to 1200 rad) andconcluded that (a) the yield fell with decreasing doserate, with little further decrease in rate of induction
below 0.02 rad/min, and (b) it seemed unlikely that theminimal rate would be less than 1.0 10 · 5 rad -t •(b) Neutron irradiation and RBE estimates
103. Muramatsu et al. (354) have reported the results of
their studies on RBE of 2-Me V neutrons relative to
250-kVp x irradiation for the induction of reciprocaltranslocations in mouse spermatogonia. A wide range ofdoses . was used (48-672 rad of x rays, 8 levels;
24-267 rad of neutrons, 8 levels). The dose-response
curve for the neutrons was linear up to 94 rad beyond· which the yield fell off, giving an overall humped curve,
as had been observed earlier with 0.7-MeV neutrons(29). For x irradiation, the picture was essentially thesame, except that the peak was at 672 rad. The
regression coefficient for the linear part of the neutron
curve was 11.4 10·4 and that for the x-ray curve,
2 .69 1o· 4 • The RBE for 2-Me V neutrons was estimatedas 4.2, the ratio of these values, which is close to that of
3.7 estimated by Searle, Evans and West (518) and thatof 4.4 recorded in the work of Pomerantzeva et al (414)
for 1.5 Me V neutrons.
104. Valentin (596) carried out experiments ontranslocation induction in mouse spermatogonia with
14.5-MeV neutrons at acute dose levels of 75, 150 and
250 rad (dose rate, 1.7 rad/min). It was found that one
mouse in the 150-rad series (out of the 11 used)
contributed significantly to the variation between themice (10 out of 16 affected cells were from this mouse,
which in addition carried 2 translocations in one of thecells scored). The frequency of affected cells at doses of75, 150 and 250 rad were, respectively, 0.7 ± 0.2 percent, 0.8 ± 0.2 or 0.4 ± 0.1 per cent (depending onwhether the aberrant mouse is included or excluded) and
1.6 ± 0.2 per cent. Between 1700 and 2500 cells werescored for each dose point in addition to 4700 cells incontrols, where no translocations were found. The
author points out that the dose response was consistent
with linearity in the range studied, although if theaberrant male is excluded, the data give a better fit to acurvilinear relationship. Since no comparable x-ray data
were obtained in her experiments, Valentin has refrained
from making any RBE estimate except to point out that,in comparison with the results of Searle, Evans and West(518) with ·o.7-MeV neutrons, the 14.5-MeV neutrons
used in her study were presumably not as efficient andthat the RBE value may be much lower than 3.7.
105. Bajrakova et al. (22) examined the effects ofirradiation with 4.1-MeV neutrons {from a Pu-Be source)delivered at a low dose rate of about 0.08 rad/h on the
induction of translocations in male mice. They foundthat the dose-effect relationship up to 52 rad (3 levels)
was linear with an increase of about 3.3 10-4translocations per rad, beyond which the curve
saturated. The frequencies at the two high doses usedwere 1.91 ± 0.45 per cent, at 98 rad, and 1.65 ± 0.40 per

cent, at 150 rad. Since no comparable X· or gamma-ray
experiments were conducted, the RBE value cannot be
estimated. However, in comparison with the results of
Pomerantzeva et al. ( 415) with chronic gamma
irradiation, 4.1-MeV neutrons (also delivered chronically), appear to have an RBE of about 20.

2. Dose rate
I 06. It may be recalled that the earlier results of Searle
et al. (517) discussed in the 1972 report (para. 66)
demonstrated that, at the 600-rad level, a lowering of
the gamma-irradiation dose rate· from 83 rad/min to
0.02 rad/rnin led to a concomitant lowering of the yieldsof translocations and that the frequency at 0.02 rad/min
was about one ninth of that at the higher rate. The
results of Pomerantzeva et al. (415, 417) discussed
earlier in this report (para. 10 I) corroborate the abovefinding and extend the data to more exposures and still
lower exposure rates. They show that (a) when thee](posure rate is reduced from 710 R/min to
0.007 R/min, the translocation yields are lower by atleast an order of magnitude (range of exposures,30().1200 R); {b) over this range of exposures, the data
are consistent with a linear exposure-frequency relationship (see paras. 101, 102); and (c) a further lowering of
the exposure rate to 0.003 R/min (range, 100-920 R)
does not lead to any additional sizeable reduction in thefrequencies over that already observed after irradiation
at 0.007 R/min.
107. In more recent experiments, Searle et al. {516)demonstrated that when a gamma-ray dose ( 6 °Co) of
1128 rad was administered to male mice over a 28-weekperiod (0.004 rad/rnin) the frequency of translocations
per sperrnatocyte was l.70 ± 0.4 per cent, corresponding
to a rate of induction· of 1.40 1o-s rad -i . This rate isquite similar to that of Pomerantzeva et al. (415),
1.74 ± 0.21 10-5 R - i . Relative to the yields after acutex irradiation (163) and after acute gamma irradiation(511), the results of Searle et al. {516) at 0.004 rad/min
show that the efficiency of these is only about 0.05 and
0.01, respectively.
108. Van Buul and Roos (72) irradiated male mice with
400 R of x rays or gamma rays at exposure rates of
60 R/min and 130 R/rnin respectively and compared theyields of translocations obtained with those observed
after a similar exposu~e of gamma rays delivered at rates
of 1.92 R/min and 0.03 R/min. The data show that theyield after 400 R of acute gamma rays is slightly lower
than that after x rays (abnormal cells, 6.6 per centcompared with 8.6 per cent; translocations, 7-.0 per centcompared with 9.0 per cent; number of cells scored,
1400 and 1200) but not significantly so. With gamma
rays at 1.92 R/min, the yield fell to one half of thatobserved after acute gamma irradiation {3.0 per centabnormal cells or 3.2 per cent translocations; 1300 cells
scored) and at 0.03 R/min, to 1.9 per cent (1400 cells· scored). It is of interest to note that the magnitude of
reduction observed here (by a factor of 3.5) is lower
than that recorded by Searle et al. (517) at a dose levelof 600 rad (83 rad/rnin compared with 0.02 rad/min;12.1 per cent to 1.4 per cent), where it is by a factor of
about 9.

109. In the study of Valentin described earlier (596),
the effects of 250 rad of 14.5-MeV neutrons administered at rates of 1.67 rad/rnin and 0.01 rad/min werecompared. The frequency of abnormal cells with either
dose rate was nearly the same (1.6 ± 0.2 and
2.2 ± 0.3 per cent with high and low dose rates,
respectively), thus showing no demonstrable dose-rate
effect at this dose and with the range of dose rates
employed.
110. In the work of Bajrakova et al. with 4.1-MeV
neutrons (22) discussed in paragraph 105, no irradiations
were carried out at high dose rates, and consequently thequestion of whether a dose-rate effect is present withthat kind of neutron cannot be answered. However,their data show that at high exposures of 98 and 150 rad
delivered at 0.0013 rad/mm (80 mrad/h), the frequenciesof translocations remained at approximately the samelevel as after 52 rad. This finding is in marked contrast
to that with 0.7-MeV neutrons {518) administered atabout the same dose-rate as in the experiments of
Bajrakova et al. {22), where a markedly hightranslocation yield {21.7 per cent at 214 rad) wasrecorded.
3. Dose fractionation
111. As was mentioned earlier, fractionation of theradiation exposure affects the yield of translocations
recovered from irradiated spermatogonia and thisdepends on the magnitude of the doses and thefractionation procedures used. For convenience, thevarious results obtained will be considered under theheadings "short intervals" {less than 24 h), "medium
intervals" {24 h to a few days) and "long intervals" (afew weeks). The earlier results reviewed in the 1972
report will be briefly summarized to facilitatecomparisons. Unless otherwise stated x irradiation wasused.

(a) Short intervals
112. Leonard and Deknudt {282) studied the inductionof translocations in mouse spermatogonia by using an
x-ray exposure of 500 R divided into two equal fractions
separated by time intervals from 60 min to 24 h. It was
found that after a single 500-R exposure, the incidenceof sperrnatocytes carrying translocations was 8.1 per
cent; with an interval of 2 h between the fractions, thefrequency fell to 5.7 per cent, rose to 8.8 per cent at
4 h, fell again to 4.4 per cent at 7 h, rose to 8.4 per centat 16 h, with some suggestion for a further fall to 6.8 percent at 24 h. In the study of Searle et al. (515), in one
experiment, when.a total dose of 300 rad was deliveredin two equal fractions separated by a 30-min interval,
the frequency of affected spermatocytes was 8.1 percent compared ·with 14.5 per cent observed after theunfractionated exposure. (As the authors themselves
point out, the latter frequency is higher than the one
expected at this dose (7 .5 per cent) on the basis of theirearlier studies.) With a 2-h interval, the frequency rose to
10.8 per cent, a just significant increase. In the next
experiment, there was again an initial fall from 8.8 percent (unfractionated) to 6.6 per cent (60 min) and a riselater to 8.1 per cent a~ 6· and 8-h intervals; none of thesechanges was statistically significant.
441

113. In the experiments of Preston and Brewen (420),
an unfractionated exposure of 500 R gave a yield of
17 .2 per cent; with intervals of 30, 90 and 150 rninbetween the two 250-R fractions, the yields appeared to
decline to 16.2 ± 1.8 per cent, 14.0 ± 1.5 per cent,12.5 ± 1.4 per cent, respectively. In further experiments
reported in the same paper, the authors investigated theeffects of fractionation of a 1000-R exposure (anexposure which is on the descending part of thedose-effect curve) into two equal fractions separated byintervals of 3, 6 and 18 h. The yield of translocations
following a 1000-R single exposure was 5.2 per cent;following fractionation however, the yield increased to
17 .1 per tent for a 3-h interval and reached a maximumof about 41 per cent for an interval of 18 h when it was
equal to the yield expected on the basis of additivity ofyields of two separate 500-R exposures. On the basis ofthese results and the finding that the yields decreased
with 48- and 72-h intervals to 23.8 and 19.S per cent,respectively, the authors suggest that the pattern of
response observed in the above experiments could be
explained in terms of the killing of cells in the sensitive
stage of the cell cycle by the first exposure, resulting in apartially synchronous population being irradiated by thesecond exposure. The stage of the cell cycle in which the
population exposed to the second fraction is expected to
be in, will vary with time between the two fractions,assuming that over the time period used in theseexperiments, the cells are still partially synchronous.
Preston and Brewen predicted that with a time intervalof, say, 96 h one would expect the yield to return tothat for I OOO R. The results obtained by Cattanach andMoseley (92) indeed show that the expectation is
fulfilled (para. 118).
(b) Medium intervals

114. The early expe9ments of Lyon and Morris (311)
demonstrated that the yield of translocations after two
500-rad doses to spermatogonia (24-h interval), was notenhanced above that extrapolated from lower single
doses, although it was above that for a single 1000-rad
dose, which was beyond the maximum of the humped
dose response curve. In further work with similar
fractionation procedures, Morris and O'Grady (345)
adduced further evidence which showed that the yields
after fractionated exposures involving total doses from100 to 600 rad were remarkably close to those for single
ones (i.e., no enhancement); at hlgher doses (up to
1400 rad) the yields continued to increase approximately linearly after fractionation and were consistent
with the additivity of yields of the two respective
fractions. These observations are in marked contrast to
those made by W, L. Russell (471), whlch showed thatafter fractionation of an exposure of 1000 R (twofractions, 24 h apart) the yield of specific locus
mutations .was much greater than expected byextrapolation from lower single doses. The suggested
explanation for the enhancement of specific locus
mutations was that the first radiation dose synchronized
the cell divisions of the surviving spermatogonia in sucha way that at the time of the second dose 24 h later,
they were particularly sensitive to gene mutations.
Morris and O'Grady speculated that the difference in
response between specific lcous mutations and translo-

cations could be understood if it is assumed that in thelatter case, the cells were synchronized at a stage withrelatively unchanged sensitivity to chromosomal damage(see the 1972 report (589) for details of the data).
115. The work of Wennstrom (605), in which theeffects of fractionation of a 200 rad dose into two,
three, four or six fractions (24-h interval between
fractions) also demonstrated that the yields with thesedifferent regimes were not significantly different fromthe one obtained after a single dose of 200 rad. (Thefinding that, with four or six fractions there was nochange in the yield relative to that of the single dose is atvariance with that of Lyon et al. (315), whlch showedthat with a 300-rad total dose split into five daily
fractions of 60 rad, there was a reduction in frequency
from 6.3 to 3.4 per cent.)
116. Van Buul and Leonard (71) examined the extent
to which the magnitude of the first fraction of a split
dose affected the subsequent response of the cells 24 hlater to the second fraction. In these experiments, malemice were irradiated with a total x-ray exposure of
600 R delivered either singly or in two equal fractions orin two unequal fractions of 100 and 500 R or SOO and
100 R. Translocations were scored 70-120 days after thecompletion of the radiation treatments. The results
showed that when the irradiation was delivered either
singly or in two equal fractions, the frequencies ofabnormal cells were not significantly different (7 .6
compared with 8.4 per cent), as had been found byother workers. With the 100 + 500 R regime, thefrequency was 10.2 per cent (1600 cells scored), whereas
when the order of the exposures was reversed, it was
4.9 per cent {1700 cells scored). These data suggest thatthe net result of interaction between the postulated cellsynchronization by a first fraction of a dose and thealteration of the sensitivity of the surviving spermatogonial cell population is dependent on the magnitude
and sequence of the exposure fractions.
117. In a more recent paper, Cattanach, Heath andTracey (93) adduced further evidence for the thesis thatwith 24-h intervals, the size and sequence of the
exposure fractions determine the radiosensitivity of thestem cells to translocation induction. They found thatwhen I OOO R was administered as 100 R followed by
900 R, the recovered translocation yield (22 per cent)was similar to that whlch can be obtained byextrapolation from lower exposures and also that of a
500 R + 500 R, 24-h fractionation. However, when the
900 R preceded the 100 R, the response was much lower(7 .4 per cent), yet still hlgher than that produced by asingle 1OOO R treatment (4.5 per cent). The same orderof effectiveness was observed for the length of the sterile
period. From these results the authors have concluded
that 24 h after the initial exposure (a) the surviving stem
cells are more sensitive than formerly, both to killingand genetic damage, and (b) they are no longer
heterogeneous in their radiosensitivities so that increasing yields of genetic damage may be obtained withincreasing exposures.
118. Cattanach and Moseley (92) irradiated males witha single x-ray exposure-of 800 R or in two fractions of
500 and 300 R (in that order), separated by intervals

442

L.

'I'

ranging from 24 h to 12 days. Translocations werescored in spermatocytes descended from the irradiatedoonia, 100 days after the completion of the irradiation
freatment. The data showed that the 24-h fractionation
increased the translocation yield significantly above thatof the acute treatment (18.3 per cent compared with
7 .9 per cent) but not higher than expected on the basisof additivity of individual fractions; with a 48-h interval,
the frequency was 16.0 per cent, still being consistent
with the expectation of additivity. On the other hand,with intervals of 3-12 days, the translocation yields weremuch lower and little different from that obtained after
acute exposure. These latter results are consistent withthe prediction made by Preston and Brewen (420) on
the basis of their experiments in which a 1000-R
exposure was given in two fractions separated by periods
ranging from 3 h to 3 days (para. 113).

1•
1119. In the work of Cattanach et al. (93) discussed inparagraph 117, evidence was also obtained showing thatthe subadditive translocation yields recorded with the
800-R treatments (500 R + 300 R, and in that orderwith intervals of 3-12 days) was maintained with further
intervals of up to 15 days and that additivity wasregained by the end of the third week. Sterile period
data indicated that with these intervals (of up to
15 days), the germinal epithelium had recovered
sufficiently from the first fraction for spermatogenesis
to restart before the second fraction was given. These
results permitted the authors to conclude that (a)3-15 days after the first exposure, the surviving cells
proliferate rapidly to repopulate the testes and at thistime they are either resistant to genetic damage (thoughstill relatively sensitive to 14lling) or are sensitive toboth, but with the two events being correlated, and (b)the onset of spermatogenesis is associated with there-establishment of a heterogeneity in radiosensitivity
among the stem cells.
120. Earlier work from Lyon's laboratory (315) had
shown that when repeated small radiation doses aredelivered to mouse spermatogonia, the yield oftranslocations was less that that after the same total dose
given in a single exposure. Two main explanations wereconsidered: (a) each individual small dose has less effectthan the comparable fraction of a large single dose, and(b) repeated irradiation in some way changes thespermatogonial cell population so as to either decrease
the sensitivity to induction or increase the elimination of
mutational changes. The evidence obtained for (b) was
the following: a dose-response curve was plotted forvarying numbers of 10-rad doses (total dose -620 rad).
The form of the curve suggested that later doses were
having less effect than earlier ones and hence wasconsistent with the idea that the sperrnatogonial cell
population was becoming more resistant with repeatedirradiations. If this were true, one would expect that if asingle exposure to a large dose of irradiation were given
after a succession of repeated small doses, it would haveless effect than if the procedure were reversed.
121. Lyon, Phillips and Glenister (317) tested the above
possibility in experiments by comparing the yields oftranslocations obtained with a dose of 300 rad of gamma
rays to spermatogonia administered _before, 24 h after,or 8 days after exposure of the animals to 30 daily dosesof 10 rad each. The results showed that under conditions
when the 300-rad single exposure was given 24 h after
the 30 X 10-rad exposure, the yield was significantly
lower (7.3 per cent) relative to those from the other two
regimes (9.4 per cent: ~00 rad+ 30 X 10 rad; 9.7 per
cent: 30 X 10 + 300 rad, 8 days later). That is consistent
with the hypothesis that the repeated irradiation had
temporarily increased the resistance of the spermatogonial cell population to translocation induction. The
authors consider the above interpretation tentative.

(c) Long intervals
122. So far, there have been only four reports on theeffects of dose fractionation on translocations induced
by x rays in spermatogonia where the interval between
the doses was of the order of several weeks. The first ofthese relate to the work of Ford et al. (170), who foundthat when an exposure of 1200 R was administered in
two equal fractions separated by a period of 8 weeks,
the yield of translocations was high (being 41.6 and32.5 per cent in two different experiments) but nothigher than what would be expected from additivity ofyields, although much higher than what had been foundfollowing single acute exposures of similar magnitude.
Similarly, Pomerantzeva et al. (415) found that when atotal exposure of 900 R was given in three equal
fractions separated by 4-week intervals, the frequency oftranslocations was 9.1 per cent, this being roughly threetimes that after a single 300-R exposure (3.3 per cent)and about twice that after a single 900-R exposure(4.7 per cent).
123. More recently, Pomerantzeva and Ramaiya (412)
made a study with fractionated gamma-ray exposuresdelivered to mouse spermatogonia (900 R in three equal
fractions) and varied not only the interval between the
exposure fractions (1 to 4 weeks) but also the interval
between the completion of the irradiation and thesampling of the spermatocytes (12 to 48 weeks).
Considering first the effect of sampling interval,
increasing it from 12 to 28 weeks led to no significant
change in translocation frequency (9.3 ± 1.2 per centcompared with 10.0 ± 1.4 per cent; pooled data:
9.7 ± 1.0 per cent). However, when the interval was
extended to 48 weeks, there was a slight decrease in thefrequency(7 .3 ± 0.8 per centcomparedwith
9.7 ± 1.0 per cent). The latter finding is qualitatively
similar to that recorded earlier by Evans et al. (163),
namely, that after a single dose of 800 rad the frequency
fell from 20.5 per cent with an 11-week interval to12.8 per cent with a 30-week interval. (See table 11 in
the 1972 report, Annex E.)
124. Concerning theeffects of interval between

exposure fractions on the yield of translocations,
Pomerantzeva and Ramaiya found that, with a 7-day
interval between the fractions (12-week samplinginterval), the frequency was 8.6 ± 0.8 per cent and did
not change with a 28-day interval (9.3 ± 1.2 per cent). In
either case, the yield was lower than expected on thebasis of additivity (yield after a single 300-R exposure:
5.2 ± 0.6 per cent). The first of these findings is similar
to that of Cattanach et al. (93 and paragraph 119)
whereas the second one is not: Cattanach et al. noted443

that with a total exposure of 800 R administered in two
unequal fractions of 500 and 300 R, additivity wasregained 21 days after the completion of the radiation
treatment, whereas this has not been the case in thework of Pomerantzeva and Ramaiya. One possible
reason for the discrepancy between the two sets ofresults may be that, since both the total exposures andthe. fractionation regimes are different, the pattern ofrecovery of the germinal epithelium under these
conditions is also different (see paragraph 125).
125. In their experiments (500 + 500 R), Preston andBrewen (420) found (see also paragraph 113) that withan 18-h interval, the yield was equal to that expected on
the basis of additivity (40.7 per cent compared with
2 X 17 = 34 per cent) but dropped to 23.8 per cent witha 48-h interval and remained at approximately the samelevel up to an interval of 4 weeks. By 6 weeks, additivity
was once again regained (32 per cent compared with
2 X 17 = 34 per cent).
126. In other experiments reported in the same paper,
Preston and Brewen irradiated male mice with repeated
400-R x-ray exposures up to a total exposure of 2800 R,
the interval between successive exposures being 8 weeks.
The expectation was that under such conditions, theinterval between one exposure and the next would belong enough for the cells to re-assort back to a normal
cycle, with cells present at all stages, and consequently
additivity of yields should be obtained. The data
presented in table 22 show that the expectation isindeed realized. The time elapsed between irradiation(after different numbers of exposures) and the samplingof spermatocytes was adjusted to sample the first wave
of meiotic cells following the recovery and repopulationof the geminal epithelium, as emphasized in an earlier
publication (419). This varied from 6 weeks following
four 400-R exposures up to 17 weeks following the last
400-R exposure of the 2800-R total exposure. As will beobvious, the interpretation for the observed effect in thiskind of experiment, involving long intervals between
exposures, is different from that advanced for similareffects observed with fractionation regimes involving
24-h intervals.
4. Interaction with chemicals
127. The 1972 report presented the data of Ehling(148, 152, 153) and of Pomerantzeva and colleagues(209, 411) on the effects of pre-treatment with different
chemicals, such as chloramphenicol, mitomycin C,
aminoethylisothiourea (AET), 5-methoxytryptamineand cysteamine, on radiation-induced dominant lethals.
Similar studies have now been carried out to examine
the effects of chemical pre-treatment on radiationinduced yield of translocations in spermatogonia.
Leonard and Deknudt (283) who studied the effects of
AET pre-treatment, found that the compound exhibitedno protective effect and explained the failure of AET tomodify the rate of translocation induced by x irradiationon the basis of some germinal selection or, more likely,
the stage specificity of AET (Ehling's finding was that aprotective action was manifest only in early spermatid5).
In a subsequent paper, Leonard and Deknudt (284)demonstrated that a mixture of reduced glutathione(GSH), AET, mercaptoethylamine (MEA), cysteine and444

serotonin creatinine sulphate or 5-hydroxytryptamine(5-HT) did have a protective effect: the frequency ofabnormal spermatocytes in the "radiation alone" series
was 8.1 per cent and that in the "mixture plus
radiation", 5.9 per cent (500-R exposure to spermatogonia). Using Adeturon (ATP salt of AET), Bajrakova(21) found that this chemical did offer some protection
against radiation-induced translocations in spermatogonia. The frequencies of abnormal cells afterradiation alone or radiation plus chemical are,respectively, 2.6 per cent and 1.6 per cent (150 R),
4.8 per cent and 2.5 per cent (300 R) and 6.1 per centand 2.6 per cent (450 R). Between 800 and 1000 cells
were scored for each data point.
128. In a further elaboration of earlier work (411),
Pomerantzeva and Vilkina (413) examined the effects ofpre-treatment with cystearnine on gamma-ray induction(100, 300 and 600 R) of translocations in spermatogonia. No protective effects were found. However, inparallel experiments on dominant lethal induction in
post-meiotic and meiotic stages, the pre-treatment led toa reduction in induced dominant lethality after 300 R.
The effect was negligible after 100 R and completely
absent after 600 R.
5.

Comparison between cytologically observed andgenetically recovered frequencies of reciprocaltranslocations

129. Despite the phenomenal progress that has been
made in the cytological screening for radiation-induced
reciprocal translocations induced in spermatogonia,
there is a great paucity of experiments designed
specifically to examine the relationship between thefrequencies obtained in spermatocytes and thoseobserved in genetic tests of F 1 male progeny. In the only
paper thus far published, Ford et al. (170) determined
this relationship at a high exposure ( 600 R + 600 R,
separated by 8 weeks; adult males were irradiated andthe frequencies of translocations in spermatocytes
derived from irradiated spermatogonia were determined,
and in parallel experiments the frequency of heritabletranslocations in the F 1 progeny of similarly irradiatedmales was also ascertained). It was found that thefrequency of transmissible reciprocal translocations was
only about one half of that expected on the basis of thecytological data. The interpretation was that thisdiscrepancy' could arise if some selection process
operated on translocation-carrying diploid (rather than
haploid) genomes between the meiotic metaphase andfertilization.
130. For better use of the latter kind of data in hazard
evaluations, information on whether or not therelationship found at high exposures will hold at lower
levels and the extent to which this is likely to bemodified under other conditions of radiation exposure(low dose rate, fractionation etc.) is of paramountimportance. To rectify this deficiency in our knowledge,
Generoso et al. (186) and Brewen et al. (56) haveembarked on an extensive study aimed at determining,
for a series of acute exposure levels (150, 300, 600 and
1200 R of x rays), the frequencies of translocations inspermatocytes and those of translocation heterozygotes
in the F 1 male progeny, in parallel cytological andgenetic tests.

131. The data available until now are given in table 23.
It can be seen that (a) the frequency of translocation
heterozygotes (genetic tests) increases with exposure up
to 600 R (although the difference between the yields
after 150 R and 300 R is not significant), followed by amarked falling off at 1200 R: this humped characteristicof a exposure-frequency curve is in general agreementwith the cytologically scored reciprocal translocations:(b) at exposure levels of 300 R and above, the ratio oftranslocation recovery in the genetic tests to cytologically determined frequency is roughly one eighth, as
was found earlier by Ford et al. (170): and (c) at thelower exposure level of 150 R, this ratio is one quarter(although not significantly different from one eighth inview of the still relatively small number of F 1 males
tested). Thus at the exposure level of 150 R, there is asuggestive indication that, on the basis of the conditions
assumed by Ford et al. (170), the rate of recovery ofbalanced translocations among progeny might be thatwhich is expected from the cytological frequency.

tIi132. In a recent study in which male mice were given
fractionated gamma-ray exposures (3 X 300 R, separated
by 28-day intervals between fractions), Pomerantzeva,
Rarnaiya and Vikina (416) found that the frequency of
cytologically diagnosed translocation heterozygotes
among F 1 males ( derived from irradiated gonia) was
2.8 ± 0.9 per cent (9/319). Although no direct determination of the translocation frequency was made in theirradiated males themselves, such data collected by thesame group of workers in another study (para. 123)
under similar radiation conditions may be used tocompute the ratio of the frequency of translocation
heterozygotes in the F 1 to the frequency oftranslocations in irradiated males. This ratio, 0.28
(2.8/10.0), is not significantly different from either 0.25
or 0.125, in view of the small number of F 1 males
screened.
6. Summary and conclusions
133. Since the publication of the 1972 report of theCommittee, a sizeable amount of information has
accumulated from studies on the induction of reciprocaltranslocations. Most of this has come from continuing
studies with mice.
134. In contrast with the results from earlier work inwhich the frequency of reciprocal translocations inducedin spermatogonia (and scored in spermatocytes) by
x irradiation at high dose rates was found to show alinear dose-effect relationship, the results of Preston andBrewen (and those of Wennstrom to some extent) showthat the data best fit a model which incorporates both alinear and a quadratic component. In the work of
Preston and Brewen, the interval between irradiation andkilling of the mice was varied (depending on the dose) insuch a manner that the time of sampling of the
spermato.cytes coincided with the first wave of meiotic
activity following recovery of the germinal epithelium;in earlier work, most often, a single sampling time(irrespective of the dose and depending on thelaboratory) had been used.
135. With chronic gamma irradiation delivered at a rateof 0.007 R/min, the exposure-frequency relationship islinear over the entire exposure range investigated (up to

1200 R). Further lowering of the exposure rate to
0.003-0.004 R/min does not lead to any significant
reduction in the rate of induction. An analysis of all theavailable data obtained with chronic gamma irradiation
at low dose rates down to 0.003 rad/min (with doses
varying from 100 to 1200 rad) shows that the minimalrate of induction is unlikely to be lower than 1.0 10 -s
rad -i. At these very low dose rates, the efficiency ofchronic gamma irradiation is at least an order ofmagnitude lower than that of acute gamma irradiation or
acute x irradiation.
136. Irradiation of mouse spermatogonia at high dose
rates with 2-MeV neutrons is about four times as
effective for translocation induction as similar irradiation with 250-kVp x rays. Neutrons at 14 MeV appear to
be much less efficient in this respect. The dose-effectrelationship for chronic neutron irradiation (4.1 MeV) is
consistent with linearity up to 52 rad, after which asaturation effect is observed. Relative to chronic gamma
irradiation, similar irradiation with 4.1-MeV neutrons isabout 20 times more efficient. No dose-rate effect canbe demonstrated with 14-MeV neutrons in the range
0.01-1.67 rad/min.
137. Fractionation of the irradiation exposure affects
the yield of translocations recovered from irradiatedspermatogonia in a way that depends on the magnitudeof the radiation dose and the fractionation regimes
employed.
138. With total doses below 600 rad (i.e., those on theascending part of the humped dose-effect curve),
irradiation in two equal fractions separated by intervalsof up to about 2 h leads to a decrease in the yield oftranslocations (relative to single doses). This might bedue to two reasons: (a) the change in the characteristicsof the spermatogonial population caused by the first
fraction of the dose (for instance, the killing of cells incertain phases of the spermatogonial cell cycle and theconsequent synchronization of the remaining cells), thedegree of which may vary with the dose and (b) the
decreasing possibility of interaction between chromosome lesions produced by the first fraction and thesubsequent one. The former possibility seems, however,most unlikely.
139. When the interval between the fractions is equal toor more than the maximum time that seems to berequired for rejoining of the chromosome lesions
produced by the first fraction (2.5-4 h), the total yield isapproximately equal to the sum of the yields ofindividual fractions, as expected. The limited data
beyond the 4-h interval between fractions in short-term
fractionation studies suggest that there is a further dropin the frequency (7-h interval) followed by a tendency
for an increase (16-h) and then a decrease (24-h). Such acyclical response may be interpretable on the assumption that with the different fractionation intervals, thepartially synchronized cells receiving the second fraction
may be in sensitive or resistant phases of the cell cycle.
140. With a high total dose such as 1000 rad (which ison the descending part of the humped dose-response
curve), administered in two equal fractions and withtime intervals ranging from 30 min to 72 h, again, there
is a cyclical pattern of response: but this is a different
one. With a 30-min interval the yield is close to that of445

the total single dose of 1000 rad. With increasing
intervals, the frequency increases up to a maximum withan 18-h interval when it is equal to the yield expected on
the basis of additivity. The yield subsequently decreases
and after 72 h, falls back to the level expected from asingle 500-rad exposure. With extended intervals of up
to 4 weeks, the increase is only gradual, and by 6 weeks,
additivity is once again regained.
141. With unequal fractions (500 R + 300 Rand in thatorder), additivity of yields is first observed with a 24-hinterval (intervals shorter than this were not used)followed by a drop and a slow rise in the yield (withintervals ranging from 3 to 15 days), and by the end of
the third week, additivity is once again regained. These
results are in general agreement with those described in
the preceding paragraph.
142. The
interpretationof
these
results(paras. 140-141) might be that (a) the spermatogonial
stem cells have different sensitivities in the different
stages of the-cell cycle; (b) the 500-rad fraction kills themost sensitive cells, leaving a partially synchronizedpopulation which passes to a cell cycle stage in which
the cells are sensitive (18 h); (c) the subsequent decline
in yield indicates a progression into a more resistantstage; and (d) by about 6 weeks after the first fraction of
the dose, the cells have become re-assorted to a normal("asynchronous") population.
143. In experiments involving a total irradiation
exposure of 2800 R administered in seven equal
fractions of 400 R and separated by 8-week intervals,
additivity of yields is expected and in fact has beenfound.
144. With unequal fractions separated by 24-h intervals
500 R + 100 R,
500 R + 300 R,(100 R + 500 R,
100 R + 900 R, and 900 R + 100 R), the recovered
translocations yields are dependent on the size andsequence of the exposure fractions. With a small fraction
preceding the large one, the yield is high and is equal tothat extrapolated from lower exposures. With thereversed sequence of exposures, the yield is low, buthigher than that after a single acute exposure. Theseobservations are interpreted on the assumption that 24 hafter the first fraction, the surviving stem cells are in amore sensitive stage than befor~·, both to killing and totranslocation induction and that they are less heterogeneous in their radiosensitivities at the time theyreceive the second fraction (the degree of synchrony
being dependent on the magnitude of the first fraction).
145. When total dose as large as 300 rad is split into
several small fractions (30 of 10 rad or 60 of 5 rad) andadministered over daily intervals, the frequency oftranslocations recovered is much lower, being only about
one quarter of that after the single qose. This finding has
been explained· on the assumption that the spermatogonial population becomes progressively radioresistantunder conditions of repeated irradiation. The possibility
was tested in experiments in which a 300-rad gamma-ray
dose followed an exposure regime involving 30 daily
doses of I O rad each and vice versa. The yield under the
first set of conditions was lower than under those of thesecond ..

446

146. Pre-irradiation injection with chemicals such as
Adeturon or a mixture of reduced glutathione, AET,
MEA, cysteine and serotonin creatinine sulphate has aprotective effect with regard to the induction oftranslocations in sperrnatogonia. However, cysteamine
has no effect.
147. A comparison of the frequencies of cytologicallyobserved reciprocal translocations and genetically
recovered, ones (following spermatogonial irradiation)
has shown that at exposure levels of 300 R and above,
the ratio of translocations recovered in the genetic tests
to cytologically determined ones is about 1 to 8; at alower exposure of 150 R, this ratio is 1 to 4, though thisresult is not significantly different from 1 to 8. In
another study involving a total exposure of 900 R
delivered in three fractions separated by 4-week intervals
and cytological examination of the parental males andtheir F 1 sons (thus no semi-sterility tests), the ratio of
the frequency of translocation heterozygotes in the F 1
males to the frequency of translocations in the parental
males was 1 to 4, but again, the result was not
significantly different from 1 to 8.

7. Other male germ-<:ell stages
148. Searle et al. (520) have reported on the kinetics ofdose response for the induction of reciprocal translocations in mouse spermatozoa. In these experiments,
adult male mice were given· gonadal doses of 0-1200 rad
acute x-irradiation (nine levels) and mated the same day.
The 531 sons conceived within a week after irradiation
were tested for fertility and their testes examined
cytologically for chromosome aberrations in spermatocytes. A total of 55 of the 57 diagnosed as semi-sterileand 35 out of 40 of those diagnosed as sterile were
judged to be heterozygous for one or more reciprocaltranslocations. There was a close agreement between
fertility diagnosis and the presence or absence ofreciprocal translocations. Numbers of 0, 1, 2, ... translocations per mouse showed a good fit to a Poisson
distribution, in contrast to previous finding with
spermatogonial irradiation. Again, in contrast to findings.after spermatogonial irradiation, the translocation
frequency after spermatozoa! irradiation steadily rosewith dose and showed no decline at higher levels.
149. The equation of the straight line that fit the data
best wasY= (4.78 ± 0.43) 10-4 D

where Y is the yield and D the dose. Although the fit tolinearity was good (P = 0.57), it was noted thatdeviations of observed values from expectations were all
negative at low doses (50-400 rad) but positive at thehigher doses of 1OOO and 1200 rad; this suggested thatthe true relationship was curvilinear. The equation ofbest fit to a quadratic model was
Y = (2.25 ± 1.03) 10-4 D + (3.09 ± 1.26) 10- 7 D 2(P= 0.84)

a.11d to a power law model wasy = (3.03 ± 3.98) 10-s n(l.41 ± 0.20)(P= 0.87)

150. Data on the types and relative frequencies ofaberrations induced in spermatocytes by different x-ray
exposures (50-400 R) have been reported by Wennstrom(605). They show that when testes preparations weremade 1 day after irradiation of the mice (to sample
spermatocytes treated at the diplotene stage), thefrequency of cells with multivalents increased nonlinearly (Y= 0.2805 10- 2 + 0.746 10- 6 D 2 ); this was
also true of chromatid breaks and fragments. In one
experiment in which the time interval between
irradiation and killing of the mice was varied from 2.5 h
to 7 days, maximum yields of all aberrations wereobtained with the 1-day interval (3.3 per centmultivalents, 3.7 per cent chromatid breaks and 11 percent fragments) followed by a decline thereafter. The
decrease for multivalents at the 3- and 7-day intervals(sampling of sperrnatocytes irradiated at early and latepachytene stages, respectively) was not significant.
151. In a similar study, Tsuchida and Uchida (579)
found that in preparations made 1 day after irradiation
with 300 R of gamma rays, the frequency ofmultivalents was 6 per cent; those of chromatid breaks
and fragments were respectively 6.4 and 10.5 per cent.
Five days after irradiation (sampling of mid-pachytene
stages) the frequency of multivalents was nearly thesame (7 per cent) whereas.those of chromatid breaks andfragments increased (15 and 10 per cent). The overallfrequency of abnormal cells was 20.7 per cent with the
I-day interval and 32 per cent with the 5-day interval.
152. Although most of the studies on translocationinduction in male mice had been concerned with theirradiation of adult males, some studies have been
carried out using males of other ages. Fazylov andPomerantzeva (164) reported that the sensitivity of thegerm cells of new-born male mice was only one third of
those in the adult (as judged by cytogenetic analysis of
spermatocytes) and that the exposure frequency
relationship was linear in the range 20-400 R. Ivanov andLeonard (245) irradiated male mice soon after birth orwhen 90 or 450 days old with x-ray exposures of 100,
200 or 300 R (100 R/min). One hundred days after the
exposure, the animals were killed and meitoic
preparations of the testes were made. The results that
Ivanov and Leonard obtained however. were at variancewith those of Fazylov and Pomerantzeva: there were nodifferences in sensitivity between gonocytes andspermatogonia at any of the exposure levels. Data similar
to those of Ivanov and Leonard have also been collectedby Brewen and Preston (53) (50-300 R; five levels);again there were no significant differences in sensitivity
between new-born and adult male mice.
153. Another finding of Fazylov and Pomerantzeva(164) relates to their work on irradiation of 16-day-old
foetal males (in utero irradiation; translocations scoredlater in spermatocytes of adults). At the 20-R level, thetranslocation frequencies were higher in foetal than in
adult males; at exposure levels of SOR or more, thereverse was true. The authors concluded that the reason
for the lower mutation frequency in foetal males wasthat there was a positive correlation between mutational
response and radiosensitivity to cell killing and that at
exposures of 50 R and above, only gonocytes with alower mutational response survived. They did not,however, take full account of an important feature of

their data. namely, the likely occurrence of clusters.
Clusters of translocations in a few males could have
explained the higher frequency of translocations theyfound following the 20-R exposure, thereby invalidating
their statistical analysis and their conclusion (526).
154. In similar work but with irradiation of 13.5-dayold male foetuses (this stage was chosen because at day
13.5 of gestation, the sex cords are just developing) at
x-ray exposure levels of 100, 200 and 300 R, Ivanov etal. (246) failed to obtain any evidence for translocationinduction. Tsuchida and Uchida (578) also reported
essentially similar results after 150 R of gamma
irradiation to 12-day-old male foetuses (in utero
irradiation). The aberrations scored included multivalents, chromatid breaks and fragments. The dataobtained by Brewen and Preston (53), however, are atvariance with the above in showing that translocations
can be induced in the dividing gonocytes present in the
13.5-day-old foetus. The frequencies were: 0.24 per cent
(50 R), 1.4 per cent (100 R), 0.90 per cent (150 R),
2.8 per cent (200 R) and 3.4 per cent (300 R). These
results demonstrate that the gonocytes in the 13.5-dayold foetus are about one half as sensitive as those of
adult spermatogonia, at least at exposure levels of
150-300 R.
8. Female mice155. While a number of studies have been carried out inrecent years on the induction by radiation oftranslocations in male germ cells of the mouse, ourknowledge of similar effects in female germ cells hasremained meagre. The reasons for this contrast havebeen more connected with technical difficulties thanwith any expectation that the level of translocationinduction would be negligible. In the 1972 report,
mention was made of the early work of L. B. Russell andWickham ( 463) and of the preliminary results of Searleand Beechey in Harwell. In the former experiments(carried out before the technical advances which madecytological examination possible), after maternal x-ray
exposure to 400 R, I out of 320 F 1 male progeny was
judged to be a semi-sterile offspring and thus presumably
heterozygous for a reciprocal translocation. However, a
few others were sterile and so could have carriedtranslocations.
156. Searle and Beechey (513) have now completed
their experiments, and in addition, two papers by
Gilliavod ang Leonard (192, 193), one by Tsuchida and
Uchida (579) and another by Brewen, Payne and Preston(58) have appeared in the literature; besides, somepreliminary results of Krishna and Generoso (2 67a) havealso become available. In the first experiments of
Gilliavod and Leonard (192), female mice were
irradiated with SO or 200 R of x rays (whole-body, highdose rate) and caged individually with unirradiated males
for one year. The F 1 male progeny were killed whenmature and meiotic preparations of the testes were
made. In parallel experiments, female mice irradiated atsimilar x-ray exposure levels were killed 24 h after
irradiation and meiotic chromosome preparations of theoocytes were made. Although such preparations of theoocytes themselves showed the presence of translocation
configurations in some first meiotic metaphases (2 out

447

of 50 in the 50 R series and O out of 50 in the 200 R
series), no translocations could be diagnosed in the
spermatocyte preparations of the F 1 males examined(101 sons in the 50 R series and 78 sons in the 200 R
series).

i' i

I

,11.·
1

I

11

, I
I

157. In their subsequent paper, Gilliavod and Leonard
reported on cytological observations on control andirradiated oocytes at exposure levels of 25, 50. 100 and
200 R. The frequencies of translocation configurations
observed (R-IV and/or CH-IV) were (per cent): 0(0/101, 0 R); 3.2 (2/63, 25 R); 3.7 (4/108, 50 R); 1.0(1/100, 100 R); and O(0/85, 200 R). At the 100-R level,
there were 6 oocytes bearing chromosome fragments.
The authors attribute the finding that the frequency ofoocytes carrying translocation configurations declines
already at I 00 R (in contrast to the observations ofSearle and Beechey (513) to be described later) to therelatively small numbers of dividing oocytes that could
be examined, although one cannot rule out sensitivity
differences in the strains used by the two groups ofinvestigators (C57BL in the experiments of Gilliavod andLeonard and F 1 females from C3H/HeH X 101 /H in thework of Searle and Beechey).
158. In the experiments of Tsuchida and Uchida (579),
a comparison was made between the radiosensitivi1y ofthe dictyate oocytes and of spermatocytes in diplotene
and mid-pachytene. Gamma-irradiated (300 R) female
mice (2-3 months old) were killed 1 and 5 days after
irradiation and the oocytes collected cultured in i•itro;
they were then processed for the scoring of aberrations
at metaphase I. (No hormonal treatments were administered to stimulate oocyte maturation or superovulation.)
The results showed that there were no significant
differences in the frequencies of abnormal cells at thetwo time intervals (18.9 per cent compared with 21.6
per cent; number of cells analysed, 366 and 352). At thefirst interval, the frequencies of chromatid interchanges,
fragments and chromatid breaks were respectively 4.4,
7 .4, and 10.7 per cent. At the second interval, thecomparable frequencies were 6.3, 9.1, and 8.8 per cent.
Thus, in terms of both the overall frequencies ofabnormal cells and of the kinds of abenations, thedictyate oocytes were found to be somewhat lesssensitive than mid-pachytene spermatocytes (see paragraph 151).
159. Brewen, Payne and Preston (58) irradiated female
mice 2-3 months old with x rays and used hormonal
treatments to induce superovulation. Attention was
focused on (a) correlating the yield of aberrations withthe time interval between irradiation and ovulation, (b)studying the relationship between exposure and theyield of aberrations at the time interval in which highest
frequencies of aberrations were observed. and (c)estimating the frequency of translocation heterozygotes
in the F 1 progeny of irradiated females from cytogenetic
observations on irradiated oocytes. Data on the firstproblem are given in table 24. It can be seen that thefrequency of interchanges is low at the 1-, 3- and 5-day
intervals, increases 6-fold at the 7-day interval andfinally reaches a peak at the 14-day interval followed bya decline thereafter. The observation that very few
interchanges are recovered with 1-, 3- and 5-day intervals
is in accordance with the results of Gilliavod andLeonard (192, 193; 1-day interval). The variation in448

yield of aberrations as a function of time between
irradiation and ovulation agrees well with the variationin sensitivity to dominant lethal induction over the sametime period (458,513).
160. Results that bear on the exposure-frequency
relationship obtained in the above study are given intable 25. Only the 14-oay interval was used. It can beseen that the frequency of chromatid interchanges
increases faster than linearly with the exposure,indicating a significant two-track component. An
examination of whether or not the distribution of cellswith various numbers of interchanges fit a Poisson
distribution revealed that the fit was good at all the
exposures except at 300 R where there was an excess ofcells with two interchanges and a deficit of those with
one interchange. The authors attribute this deviation to
the low numbers of cells scored.
161. At the 300-R level, 50 metaphases were stained for
C-bands and analysed. Of the 11 interchanges found, 5
had two centromeres on one chromatid plus an acentric
fragment and were classified as asymmetrical interchanges. The other 6 were classified as symmetricalinterchanges since each chromatid had a single
centromere. Thus, these very limited preliminary data
indicate that the two interchange events are recovered at
the 14-day interval.
162. The 400-R data were used to estimate theexpected proportion of oocytes carrying a balanced
translocation (that will be transmitted) as well as theamount of dominant lethality that will be generated.
The assumptions used were the following: (a) symmetrical and asymmetrical interchanges occur at about thesame frequencies: (b) there is no preferential segregationof exchange or non-exchange chromatids into polar
nuclei; (c) the inclusion of any deleted chromosome,dicentric chromosome, or duplication deficiency in themature ova will result in dominant lethality. Of the
100 meta phase-I oocytes analysed, 36 had no visible
aberrations; 18 had a single isochromatid break or,
rarely, a chromatid :deletion; 19 had a single interchange;
6 had a single interchange plus a single deletion; and 8
had two interchanges. The remaining 13 cells contained
three or more aberrations (exchanges and/or deletions)and were not considered in estimating the frequency oftransmissible translocations; however they were included
for calculating dominant lethality.
163. The estimated frequency of transmissible balanced
translocations was 1.8 per cent; if the same calculations
were made for the 300-R data, the predicted recovery
rate was 0.6 per cent, in very good agreement with theobserved rate recorded in the genetic tests of Searle andBeechey (513): 4 out of 680 progeny of irradiatedfemales (0.6 per cent) given 300 rad 1-42 days prior toconception (para. 166). The predicted dominantlethality at the 400-R level was about 43 per cent,consistent with the figure of 35 per cent postimplantation mortality (although somewhat lower thanthat of 59 per cent total dominant lethality) recorded in
the experiments of Searle and Beechey (table 17) after
400 R given to females 21 days before conception.
164. The experiments of Searle and Beechey (513) were
designed to study the induction of reciprocal translocations in oocytes. In one set of experiments, maturefemales were irradiated with 300 rad of x rays andmated after irradiation. Up to two litters were produced
before sterility ensued. The sons were tested for fertility
by mating to outbred females, and the method of Carter
et al. (84) was used for fertility diagnosis. The daughters
were crossed to males and those giving small litters (lessthan 9 in the first. less than 12 in the first two) wereallowed to continue breeding, further tests for fertility
being performed in their offspring. The production of 12
or less new-born in three litters was considered aspresumptive evidence for semi-sterility. The diagnosis oftranslocation heterozygosity (or XO condition) in thesetests was cross-checked by making appropriate chromo•
some preparations. As in the experiments of Gilliavod
and Leonard (193). meiotic preparations of the oocytesof irradiated females (100 and 400 rad) were also madeand screened for exchange configurations, fragments etc.

165. The second set of experiments involved exposure
to 0.7-MeV fission neutrons (100 and 200 rad;49-55 rad/min). Mature females were irradiated and bothsons and daughters were tested for fertility as before. In
addition, sons and daughters of females exposed in utero
to fission-neutron irradiation in an earlier study of Searleand Phillips (514) (108.5 rad with 20.5 R gamma
contamination; exposure period of about one week topregnant females between days 0.5 and 11, post-coitum;oogonial irradiation) were also tested for fertility by thesame methods as described for the x-ray experiments.

166. The results showed that O out of 386 sons offemales given 300 rad of x rays showed evidence ofsemi-sterility or translocation heterozygosity, but 9 outof 294 daughters were diagnosed as semi-sterile becauseof the small size of their litters. Further tests led to goodevidence for translocation in 3. In the others thesemi-sterility was not· proved heritable. At least o~e ofthese probably carried a translocation but the inductionof XO females or other causes seemed more likely to be
responsible for the low litter size in the rest. The
frequency of translocations that can be estimated fromthe total data (sons and daughters combined) is 4 out of
680, or 0.6 per cent, at 300 rad. Correcting for the
spontaneous frequency (3/1443 when contdbution byboth males and female gametes is considered or
1.5/1443 for females; see Liining and Searle (302)) andassuming linearity, this would correspond to a rate of
0.16 IOC-4 rad- 1 per gamete. This rate is roughly one?alf .or. that estimated in the male for spennatogonial
irradiation.
167. Examination of oocytes at metaphase I <!•iring thefirst and third week after irradiation with I 00 and

j

r

'II

400 rnd of x rays revealed both multivalents (some of
the ring quadrivalent type) and fragments. The
aberration frequency in oocytes rose with dose and, atthe 400-rad level, with time after irradiation. reaching amaximum of 10 per cent multivalents and 22 per centfragments in the third week. As was mentioned earlier
under ··ctominant lethals in female mice" the increase inlevels of chromosomal damaoe with dose and time after
irradiation was reflected in "dominant Jethals after thesame radiation-conception intervals and doses.
168. In the neutron experiments (513), no evidence forthe induction of translocations either in maturingoocytes or in oogonia was obtained, although there wereindications for X-chromosome loss. However, sample
sizes were small.

169. In the work of Krishna and Generoso (267a), a
total of 288 female mice 11-17 weeks old received
300 R of x irradiation and were divided into fourgroups. Females from the first group were caged withmales immediately after irradiation, while fem ales fromthe other groups were caged with males on the 5th, 11 th
and 15th day after irradiation. They were kept together
until the females ceased to produce young. With theradiation exposure used, 24 females from the first groupand 12 females from the second (caged on the Sth day
post-irradiation) produced two litters, while females in
the other two groups produced only one litter.

170. Both male and female progeny are being tested fortranslocation heterozygosity. The currently available
results from tests of 800 male progeny show that 4 (O.S
per cent) were partially sterile and 2 (0.25 per cent),
completely sterile. The four partially sterile males werecytologically confirmed as translocation heterozygotes,
and tests on the sterile males are not yet complete. Even
if comparisons are restricted to partially sterile males
only, these results indicate a significant induction ofheritable translocations in dictyate oocytes that wererecovered in male progeny ( 4 in 800 compared with 1 in
4392 in controls). These results may be compared withthose of Searle and Beechey (513 and para. 166), who
failed to find any translocation heterozygotes in
386 sons of female mice given 300 R. However, there is
no statistically significant difference between the two
sets of data (P = 0.2 by Fisher's exact test).
·

9. Other species
171. In the 1972 report, the data of Lyon and Smith(313) on translocation induction in pre- and postmeiotic germ-cell stages of golden hamster, guinea-pig
and rabbit were presented and compared with thoseobtained in the mouse. The results showed that (a)translocations are induced in the spermatogonia in all
the experimental species although the dose-effectrelationship differs from that in mice; (b) in both rabbits
and guinea-pigs, the overall dose-response curves are
humped, as in mice, but the peak incidence occurs
around doses of 200-300 rad {compared with
600-800 rad in mice); (c) the pattern of translocationinduction in the post-meiotic male genn cells of thegolden hamster (after 200 rad x irradiation) resembles
that of the mouse showing (in the limited data thenavailable) that s.pennatids are more sensitive thanspermatozoa.
172. More data have now become available not only forthe three species mentioned in the preceding paragraph,
but also for the Chinese hamster and the marmoset. In
addition, the available cytogenetic data derived fromstudies on translocation induction in human sperma~
togonia have also been published.
(a) Post-meiotic cells
173. Cox and Lyon (122) irradiated adult male golden
hamsters with 200 or 600 rad of x-irradiation and ma,edthem to females for four successive weeks; similarly,449

.
adult guinea pigs and rabbits were exposed to,respectively 600 rad and 100 to 600 rad of x rays andlikewise mated to females. The male offspring collectedfrom these matings were cytogenetically screened for thepresence of translocations; in the case of the golden
hamsters irradiated with 600 rad, the F 1 female progeny
were also kept and tested for the presence oftranslocations by examination of their male progeny.
174. After a dose of 200 rad to golden hamsters, theyield of translocations increased from week I to week 3
and fell in week 4 (1.5, 2.0, 6.4 and 3.9 per cent; thenumber of animals tested were, respectively 66, 50, 47
and 51 ). After 600 rad, there appeared to be a similar
pattern but with higher yields, but the number ofoffspring obtained from matings in weeks 3 and 4 wasvery low. The pattern was roughly the same in tests offemale F 1 progeny. In the guinea-pig too, there was asimilar pattern of translocation induction after 600 rad,but again the number of animals tested was too small tomake accurate estimates. In the rabbit, althoughtranslocations were found, too few offspring were
tested, and precise estimates of the pattern of sensitivity
change are not possible. Averaging the data over all
weeks and doses, Cox and Lyon established a tentative
rank order of sensitivity of the post-meiotic cells of thedifferent species, taking into account the earlier mouse
data. This is as follows: mouse> rabbit >guinea-pig>
hamster.
175. Comparing these translocation data with those on
dominant lethality collected in the same experiment (bythe litter-size reduction criterion), the authors foundthat (a) the yields of both translocations and dominantlethals induced in spermatozoa were higher in the mousethan in guinea-pig; (b) in the golden hamster, thelitter-size reduction was as high as in the mouse, but thetranslocation yield appeared lower. These comparisonssuggest, as was mentioned in the 1972 report, that atpresent there are no sure grounds for extrapolating fromone stage or type of genetic damage to another and fromone species to another (see paragraphs 23 and 105,Annex E, 1972 report (589)).

(b) Spermatogonia
176. Our knowledge on the sensitivity of spermatogonia
of mammals other than the mouse to the radiationinduction of reciprocal translocations has considerably
expanded since the publication of the 1972 report, inwhich the available data on guinea-pigs, rabbits andgolden hamsters were reviewed. Additional information
for those three species and new data for the rat, Chinese
hamster, rhesus monkey, marmoset and man have now
become available.
177. In work which is essentially an extension of thatreported in an earlier paper (313), Lyon and Cox (309)
investigated the response of the spermatogonia of theguinea-pig, rabbit and the golden hamster to the
induction of reciprocal translocations. Adult malegolden hamsters, guinea-pigs and rabbits aged 3, 6 and9 months respectively, were given single acute x-ray
exposures in the dose range l 00-600 rad (88 rad/min;part-body exposures). After the irradiation, the animals
were left for varying periods of time (3-7 months for the

golden hamster, and 3-9 months for the guinea-pig andrabbit) to allow for recovery of spermatogenesis, after
which cytological preparations were made of the testesof these animals.
178. The data that bear on dose-effect relationships are
summarized in table 26 along with those of Brewen and
Preston (54) for the guinea-pig and Chinese hamster. (Inthe latter case, young male guinea-pigs were whole-body
irradiated and Chinese hamsters were given part-body
irradiation; no details of when the testes preparations
were made are given.) It can be noted that, in theexperiments of Lyon and Cox (309) the frequency oftranslocations in guinea-pigs and rabbits increased withdose up to 300 rad and then declined sharply giving an
overall humped curve, as in the mouse. (A similar
increase is observed in the data of Brewen and Preston
for the guinea-pig and the Chinese hamster, but the data
at higher doses are not yet .available.) The kinetics ofincrease in the dose range up to 300 rad is consistent
with linearity. In the golden hamster on the other hand.
although the mean translocation frequencies at all doselevels are higher than in the controls, they are notsignificantly different from each other, presumably due
to a severe distortion of the primary dose-response
curve. The net result is a flat-topped curve without any
distinct peak. It may be noticed that in this species, theyields of translocations at 500 and 600 rad are higher
than those in rabbit and guinea-pig.
179. Using the model y = a + bD, the authors estimatedthe values of the slopes (the b values) 1 2 up to 300 rad
in the rabbit 'llld guinea-pig and up to 200 rad in the
golden hamster and found that these were significantly
higher in rabbits than in the other two species (definite
translocations:rabbit,(1.48 ± 0.13) 10-4 rad -i
compared with (0.94 ± 0.07) l 0-4 rad -I in guinea-pigs
and (0.93 ± 0.09) 10-4 rad- 1 in the golden hamsters;
translocations:rabbit,definiteplus _J>Ossible( l. 7 0 ± 0.14) 10
rad- 1compared
with(l.10±0.10) 10-4 rad- 1 in guinea-pigs and golden
hamsters).
180. Lyon and Cox (309) compared their data. withthose obtained in the mouse by other authors (163, 280,353), who also found that the dose-effect relationship
up to doses below the peak of the humped curve wasconsistent with linearity; that showed that irrespectiveof whether the data were expressed in terms oftranslocations per cell or proportion of cells withtranslocations, the slopes of the mouse lines were higher.
except for the comparison of rabbit data with the mouse
data of Leonard and Deknudt (280). In the latter
situation, the slopes (rabbit. mouse) were not significantly different. On the basis of all these, it can beconcluded that in the dose range up to 300 rad, the rankorder of sensitivity is golden hamster e: guinea-pig <rabbit<; mouse. 13
1 2 TI1e slopes estimated are for translocations per cell;"possible translocations" refers to cells containing a configuration which resembles a multivalent, in size and shape but inwhich the individual ·chromosomes are not distinct; "definite
translocations" are those in which the identification isunequivocal.
1 3 Theauthors'conclusion:
goldenhamster<guinea-pig< rabbit< mouse.

450

--

!81. Toe results of Brewen and Preston (54) for theguinea-pig (table 26) show that at comparable exposures,
their frequencies are higher than those of Lyon and Cox(309): the frequencies in Chinese hamsters are roughly
similar to those recorded for the mouse by Preston andBrewen (419).
182. Gilliavod and Leonard (191) compared theinduction of translocations in mouse and rat spermatogonia after an x-ray exposure of 300 R and foundsimilar yields in both species. Lyon et al (314) and van
Buul (70) have recently published some data on the
induction of translocations in the spermatogonia of therhesus monkey (Macaca mu/atta). The experiments of
Lyon et al. were carried out on two colonies, one
maintained in Birmingham, England (United Kingdom)
and the other in Rijswijk (the Netherlands), whereas
those of van Buul were on the latter colony only. The
animals were irradiated (testicular exposures) unilaterally or bilaterally with x-ray doses of 100, 200 and
300 rad (about 63 rad/min for the English monkeys and
30 rad/min for the Dutch monkeys). The recovery of thetestis from radiation injury was monitored by volume
measurements and biopsies at different intervals after
irradiation. The data are presented in table 27.
183. It can be seen that (a) there are between-colony,
between-monkey and between-testis. variations in the
response to the induction of translocations; (b) when thepooled data of Lyon et al. as well as those of van Buul
are considered, the dose-effect relationship suggests a
peak at about 200 rad, but the differences in frequencies
between the different doses are not statisticallysignificant; and (c) the frequencies of translocations areclearly lower than those observed in the mouse (seetable 21 for the latter). The authors noted that monkeys
of both colonies showed seasonal variation in sperrnatogenesis and in addition, some monkeys of the
Birmingham colony were not in breeding condition. The
effect of this seasonal variation on the yield oftranslocations is not known but in any case could
explain some of the variation in response observed
between the different monkeys.
184. Brewen et al. (57) have now published theircomplete results on translocation induction in the
spermatogonia of the marmoset and man. Mature
marmosets were exposed to testicular irradiation withacute doses of 25, 50, 100, 200 and 300 rad of x raysdelivered at high dose rates. At various intervals after
irradiation (depending on the dose), bilateral castration
was performed and preparations were made by themethod of Evans, Breckon and Ford (162) with somemodifications. Biopsy material was obtained from ninehuman volunteers who had received testicular irradiation
with x rays at doses of 78, 200 or 600 rad (225). As in
the marmoset, the interval between irradiation andsampling varied,· depending on the dose. Cytologicalpreparations were made using the same methods as those
employed for the marmoset.
185. The results are given in table 28, where mouse data
are also presented for comparisons. It can be seen that in
the marmoset, peak yields are obtained at about 100 rad
and the human data do not exclude such a possibility.
Under the assumption that the sensitivity of the sperrna-

togonia of both species is approximately the same and thatthe yield increases with dose up to 100 rad (and using bothsets of data at and below 100 rad), the authors estimatedthat the average rate is 7.7 10-4 rad- 1 per cell. A
weighted regression analysis performed on the human andmarmoset data {the 78-rad point for man and the 25-, 50and 100-rad points for the marmoset, all considered
together), assuming that the regression passes through theorigin at zero dose, gives a good fit (P = 0.2) to theequation Y = (6.94 ± 0.92) 10- 4 D; however, the dataalso fit (P = 0.1) the model Y =(7.64 ± 3.94) 10-4 D (0.0089 ± 0.05) 10- 4 D 2 , the negative quadratic component not . being significantly different from zero(P = 0.9) (495). For this reason, the estimate of risks isbased on the linear model.
186. It should be pointed out that the primate data,taken as a whole, show considerable heterogeneity(tables 27 and 28). Translocation frequencies obtainedin the Rhesus monkey are much lower than those atcomparable doses in man and marmoset, while there are
discrepancies at the 100-rad level between the two setsof results on the Rhesus monkey. Yet in all theseexperiments, peak yields of translocations are found atlower dose levels {100-200 rad) than in the mouse. It is
impossible to tell at present whether the marked
differences in yields between the man and the Rhesus
monkey (which are more closely related than man andthe marmoset) are due to fundamental differences inradiosensitivity (akin to those already established withinrodents) or just artefactual. in view of the limited
amount of material and possible differences intechniques or irradiation and scoring (314).
187. Under these circumstances, it has been decided touse data from man and the marmoset (which are in close
agreement) for the purpose of risk estimation. It should
be stressed, however, that the amount of direct
information on man is still very limited (table 28), sothat any estimate derived from it must be regarded as
only approximate. It should also be noted that thisinformation would suggest that human germ cells areabout three times as sensitive as those of the mouse withrespect to translocation induction in spermatogonia,
while the latest information (41) suggests approximately
equal sensitivity with respect to the induction ofdicentrics in lymphocytes in these two species.
( c) Fractionation effects
188. Experiments similar to those carried out in themouse to study the effects of dose fractionation on the
response of spermatogonia to the induction of reciprocaltranslocations have now been conducted in two othermammalian species, the golden hamster and theguinea-pig (308). The fractionation regimes chosen weresimilar to those already investigated in detail in themouse. Male guinea-pigs were irradiated with a total doseof 400 or 600 rad of x rays given in 2 equal fractions
separ.ated by either 24 h or 8 weeks; in another series, a
total dose of 600 rad was administered in 12 fractions of
50 rad at weekly intervals. In parallel experiments,
golden hamsters received 400 rad divided into 2 equal
fractions separated by either 24 h or eight weeks. In
addition, total doses of 400 or 600 rad were delivered in451

8 (or 12) 50-rad fractions delivered a week apart. The
time interval between irradiation and cytologicalexamination
depended on · the species and thefractionation procedures employed, but were similar to
those used for single doses (309).
189. The results obtained are given in table 29 alongwith data collected at pertinent single exposures tofacilitate comparisons. Considering first the data on 24-h
fractionation, it can be seen that in both species
the yields are higher than after single doses ofcomparable size. Statistically, the yields after fractionated exposures did not deviate significantly fromthose predicted by extrapolating the curves for single
exposures (up to 200 rad in hamster and up to 300 rad
in guinea-pigs) to higher doses. The estimated slopes forboth the guineaig and golden hamster are the same.
(0.93 ± 0.07) I O rad -i. These results show that thesituation after fractionation is the same as after
unfractionated exposures. (The authors, however,considered that the guinea-pig was more sensitive than
the golden hamster after single exposures up to 300 rad.
and therefore the situation after fractionation wasconstrued as being different.)
190. Turning now to the data obtained with fractionation at 8-week intervals, it can be seen that theyields are again higher than after unfractionated total
doses of comparable size; however, at both the doselevels, 400 and 600 rad, the yields are lower than thoseobtained after 24-h fractionation, i.e., the yields arelower than those expected from additivity of the effectsof the dose fractions. These differences (24 h comparedwith 8 weeks) are significant only for the guinea-pig at
the 600-rad level. It may also be noticed that in theguinea-pig, the yield after 2 X 300 rad (8-week interval)
is nearly the same after 2 X 200 rad (8-week interval),
which in tum is similar to that in the golden hamster
under the same conditions.
191. The finding in the mouse ( 420) that successive
400-R x-ray fractions to mouse spermatogonia (up to a
total of 2800 R, separated by 8-week intervals) resulted
in yields consistent with the additivity of effects ofindividual fractions favour the view that the 8-weekinterval is long enough for the spermatogonial cells toreturn to normalcy. In contrast, in the guinea-pig andthe golden hamster, the translocation data as well as
those on the lengths of the sterile periods observed in the
8-week fractionation series support the notion that suchnormalcy is not restored in these species within thisinterval of time.
192. In both the guinea-pig and the golden hamster, theresults of multiple small fractions show that thetranslocation yields are much lower than those expectedby extrapolation of the dose-response line for single
exposures (up to 300 rad) to higher doses. In comparing
these results with those obtained in the mouse withsimilar fractionation regimes, it should be borne in mind
that although the total doses are the same (600 rad), in
the case of the mouse, this dose point is still on theascending part of the dose-response curve, whereas in theother two species, it is beyond the dose of peak yield. In
the mouse, the yield after fractionation (12 X 50 rad) isroughly half that obtained after a single unfractionated452

dose of 600 rad (316); in the golden hamster, the yield
after 12 X 50 rad is roughly twice that after the single
dose of 600 rad; and in the guinea pig, after similar
fractionation (600 rad) the yield is about six to eighttimes higher than after the unfractionated dose. Thus itappears that the response after fractionated small doses
is dependent on whether or not the total dose
admini~tered is on the ascending or the descending partof the curve. Further data for the mouse using total
doses greater than that which gives the maximum yieldfor single exposures administered in small fractions asabove are required to confirm this suggestion.
10. Types of translocation and their effects
on fertility and viability(a) The mouse
19 3. From earlier work reviewed in the 1972 report it is
known that (a) certain autosomal translocations recovered from treated post-meiotic male germ-cell stages
may be fully viable in the heterozygous state and yetlead to male sterility through failure of spermatogenesis;(b) such failure may not be specific to a particular stage
or cell type but occurs with variable incidence
throughout the meiotic process and possibly at earlier
steps in the germ-cell sequence and (c) all the known
X-autosome translocations also cause sterility with
failure of spermatogenesis and small testes.
194. Lyon and Meredith (31 O) found that in translocation lines where males were sterile, virtually all ofthe quadrivalents were chains rather than rings, incontrast to the "semi-sterile" lines in which almost two
thirds were rings. The same tendency can be found in
the data of Leonard and Deknudt (281) on translocationinduction by post-meiotic irradiation. Searle (505), whoanalysed the data of Leonard and Deknudt (281) for
single translocations, found that in those males with few
or no spermatozoa, the frequencies of chain configurations (quadrivalent or trivalent) were almost twice thatin males which had no obvious shortage of spermatozoa.
Furthermore, there was a positive correlation between
the severity of the sterility effect and the frequency ofchain quadrivalents, the latter being 99 per cent when
the sperm count was nil (507). In the F 1 males that were
derived from post-meiotic germ-cell irradiation andwhich had enough sperm, the proportion of multivalentassociations of the chain type (44.9 per cent) was
decidedly higher than that found after spermatogonial
irradiation (23.6 per cent), as calculated from previous
results of Leonard and Deknudt (280). Thus there isevidence for a selective process which effects thefrequency of chain quadrivalents and also for aqualitative difference between the types of translocation
observed after post-meiotic and pre-meiotic irradiation.
195. Cacheiro et al. (73) made histological andcytological analyses of the testes of 42 sterile sons ofmales treated with ethyl methanesulphonate (EMS),
butylated hydroxytoluene (BHT) plus EMS, or 200 R of
x rays (treated germ cells: spermatozoa and spermatids).
A very high percentage (36 out of 42) of these males
carried chromosome aberrations and a majority of them
carried translocations. While translocations are impli-

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cated to explain partial sterility (which results in thedeath of the offspring of the carrier) and complete
sterility (where the effects are on the carrier himself) thedata of Cacheiro et al. (73) show that this difference is
not due to single versus multiple translocations. The
evidence indicates that the difference between translocations that cause sterility and those that cause partial
sterility may be correlated with the position of the breakpoints as revealed by cytological evidence: translocations
that cause sterility in males appear to be those in which
at least one of the breaks occurs close to one end of achromosome (position effect). A similar suggestion,
namely that breakage in or near centromeric heterochromatin is associated with male sterility, has been
made by Searle (507).
I 96. These suggestions have now been confirmed, as
shown by the recent results of Cacheiro et al (74). In
this work, 30 sterile F 1 sons of x-irradiated males werestudied. 12 of which were derived from irradiatedspermatids (158 F 1 males tested) and 18 from treatedspermatogonia ( 4286 F 1 males tested). Cytologicalanalyses were carried out in mitotic metaphases from
dividing spermatogonia (orcein staining), kidney cultures(quinacrine and Giemsa banding techniques) and in
meiotic cells at diakinesis wherever spermatogenesis
proceeded to that point or beyond. Of the 12 sterilemales derived from spermatid irradiation, 10 were foundto carry reciprocal translocations as determined bybanding (with 7 of these also revealed by conventional
staining through the presence of a small marker
chromosome): 2 of these had Y-autosome translocations
and two others had two autosomal translocations each.
Except where the Y was involved, at least one of the
breaks in each male was close to the centromere (c) or
telomere (t): in fact, all but two of these males had
c/t·type translocations. Wherever diakinesis could bestudied, it was found that only a small number ofmultivalent configurations were of the ring-IV type, in
keeping with the breakpoint locations near chromosomal
ends. Such c/t-type translocations often lead to long andshort marker chromosomes and seem especially liable togive rise to viable tertiary trisomies which are usuallysterile in the male (though not in the female) and may
have specific abnormalities (310, 507).
197. In sterile sons derived from irradiated spermatogonia, however. only 4 out of 18 had translocations
and, although at least one break was near a chromosomal
end, none was of the c/t type. It thus appears thatstenlity here may be predominantly due to causes otherthan reciprocal translocations with near-end breaks;
possibly small deficiencies or point mutations could playa role.
198. It is worth pointing out in this context that Jacobs
er al. (248), who analysed the breakpoints of structural
rearrangements in man (lymphocyte cultures, quinacrine
and/or Giemsa techniques), noted that within chromosome arms there appeared to be an excess of breaks inthe terminal regions, an excess of c/t translocations
where ascertainment was through a balanced carrier, anda possible excess of terminal/median translocations
where ascertainment was through an unbalanced carrier.
The authors, however, were careful to point out that
there might be observational biases. Similar observations

showing an excess of breaks in the terminal region ofchromosomes have been made by other human
cytogeneticists (278, 580).
199. Although most of the translocations causing
sterility in mice have been recovered from treatment ofmales. especially of post-meiotic stages, there is now
evidence that such translocations could also be obtained
from irradiation of oocyte stages of female mice (513).

{b) Other species
200. Among experimental mammals, most of theinformation thus far available on properties of reciprocaltranslocations has been collected from studies with mice.
The work of Cox and Lyon ( 123) extends suchinformation to guinea-pigs, golden hamsters and rabbits.
These investigators made a cytological study oftranslocations induced by post-meiotic male germ-cell
irradiation in the F 1 ( and in some cases in the F 2 ) sonsof irradiated males. It was found, as in the case of themouse (see review by I..eonard, 279) that the frequencyof spermatocytes displaying multivalent configurations
varied with the translocation, but the average percentage
appeared to depend on the species, the latter being
57 per cent in the case of the hamster and 83 per cent in
the case of the guinea-pig: too few rabbits were
examined to make meaningful estimates of the kind or
the average frequency of multivalents. Chain quadrivalents were more abundant than ring quadrivalents atmeiosis for the guinea-pig and the hamster, in contrast to
the mouse. An attempt was made to estimate the size ofchromosomes involved in the various translocations fromthe size of the multivalent configurations in each of theanimals. It appeared that in the hamsters. the longest,
one of the shortest, and the X, as well as the long.
medium and short chromosomes. had all been involvedin translocations, although there was some suggestionthat more exchanges tended to take place between thelonger than the shorter chromosomes. This is expected if
the longer chromosomes present a larger target for theradiation and if the effect of x rays is random. In theguinea-pig, the situation was roughly similar.
201. One possible explanation for the greater abundance of chain quadrivalents in hamster and guinea-pig
translocation carriers would be that a greater proportionof breaks were near the ends of the chromosomes thanin the mouse; if true, the cause could be in the greaternumber of chromosome arms (and hence chromosomeends) in these species than in the mouse or in the greater
range of sizes of the chromosomes.
202. In all three species, as in the mouse, translocations
were found which caused male sterility. due to partial orcomplete failure of spermatogenesis, although mosttranslocations caused semi-sterility. In the hamster,
chain quadrivalents were more abundant than ring
quadrivalents of two of the sterile males. and in anothersterile male only chain multivalents were obtained.
However, translocations which lead only to chain
multivalents are not necessarily associated with sterility
here, since two other males for which all thequadrivalents were chains proved to be fertile. It is likelythat sterility in the hamster is associated with the size of453

the chromosomes involved, since the translocations in
the four sterile males all involved the longer
chromosomes, but more data are needed to substantiate

this viewpoint.
203. In the guinea-pig, the situation was nearly thesame: only chain quadrivalents Were observed in thethree sterile males; however three of the seven fertile
males also carried only chain quadrivalents. Thus thepresence of only chains in this species, as in the hamster,
is not always associated with sterility. In contrast to thehamster, sterility here does not appear to be correlatedwith the size of the chromosomes involved in thetranslocation. In the rabbit, two of the fourtranslocation carriers were sterile; one of the fertile males
carried two translocations and the other, one. Both ofthem, as well as the two sterile males, carried~ranslocations which involved the longer chromosomes.
Hence, there is no evidence here of any association
between size of chromosomes involved and sterility.
204. It is instructive to compare these results with thoseavailable in man. Only a very few human reciprocaltranslocations with known breakpoints have beenstudied at meiosis: however, in those which nave been
described, a preponderance of ring-IV configurations wasfound in three male translocation heterozygotes showing
a nonnal spermiogram (98, 99,100, I66)while chain-VI
configurations were found in the majority of spenna·
tocytes from a sterile translocation heterozygote (99).
Unequal bivalents, indicative of telomeric breakpoints,
occurred in one patient who was a t(Cp- Eq+)
heterozygote, and he too was sterile through spennatogenic breakdown (99).

(c) Embryonic mortality in the progeny oftranslocation heterozygotes
205. If the chromosomes involved in a translocation
between non-homologous chromosomes segregate randomly at anaphase-I of meiosis, four classes of gametes
are produced in the proportion 1: 1:2 of balanced
normal, balanced-translocated and unbalanced. Thus,
about 50 per cent of the zygotes derived from a matingof a balanced translocation heterozygote and normal
animal will die. As the number of translocations per cellincrease, the proportion of unbalanced gametes will also
increase (see for instance table 1 in Ford et a/., 170).
Mice heterozygous for a single translocation are
therefore expected to be semi-sterile whereas thoseheterozygous for two or more translocations would be
expected to show a much higher order of infertility, it
not outright sterility. due to death of unbalanced
embryos.
206. In their translocation experiments involving
irradiated mouse spermatozoa, Searle et al. (520)compared the embryonic lethality in the progeny ofmales with or without detectable types of chromosomal
aberrations (mainly translocations). As the data given intable 30 shows: (a) the mean number of live embryos
per female in the cytologically abnormal category is lessthan one-half that in the normal category: (b) the mean
number of implants is also markedly reduced by a clearlysignificant amount. That suggests that some pre-implantation loss is occurring, which may be in part due
to reduced fertilization of eggs and in part due to early
death of some very unbalanced zygotes, The embryonicsurvival (in terms of live embryos per female) in thosewith one reciprocal translocation is 42.4 per cent of thesurvival in those without detectable abnormality(3.08/7 .3). Since a value of 50 per cent is expected withnormal disjunction, the observed figure suggests anaverage level of adjacent-2 disjunction of around 15 percent (519). However, this must be regarded as an upper
limit, for the comparative embryonic survival in terms of
the ratio live embryos/total implants is 50.1 ± 1.9 percent (ratio 369/806 for translocation carriers divided by
the ratio 6161/6743 for normal males).
207. Cox and Lyon (123) have presented similar data
for the golden hamster and guinea-pig. They found that,in general, both the frequency and the time ofembryonic death in the progeny were the same as in themouse. In the hamster (table 31), although with sometranslocations, the number of implants per female waslower than in the controls; on the average, the difference
was relatively small (10.3 compared with 11.1).
However, there was a large increase in the number ofsmall moles among the translocation series, suggestingthat for all these males, the death of unbalanced zygotes
was occurring predominantly in the early postimplantation period. Thus, the golden-hamster resembles
the mouse in this respect (170, 245). Inspection oftable 31 will also reveal that among females mated tosome males (Nos. 3, 4 and 13) there is a slight reductionin the riumber of implants which could be due to some
excess pre-implantation loss. In 6 of the 7 animals with
one translocation, the frequency of post-implantation
loss was close to SO per cent which is as expected if
chromosomal disjunction is nonnal and unbalanced
zygotes die; in the single male with two translocations,the post-implantation death was 75 per cent, which
again is as expected. The remaining male with one
translocation (male No. 8) had a low proportion ofembryonic deaths, 34.9 per cent, and the mean numberof live embryos in females mated to him was 6.9, or 75
per cent of the control value.

208. In the guinea-pig. the data are limited, most of theresults pertaining to 3 of the 7 males tested. In general,the number of implants per female was similar to that incontrols, but there was a marked increase in small molessuggesting that here too, the death of unbalanced
translocation products occurs mainly in the earlypost-implantation period. For male No. 6, postimplantation death was 50 per cent, but for Nos. 4 and9, it was significantly less than 50 per cent. These latter
results and similar ones obtained in the golden hamster
can be explained on the assumption that one of theunbalanced translocation zygotic types is viable,
although other interpretations cannot be excluded. In
the mouse. at any rate, there is evidence for the survivalof some unbalanced zygotes (89, 310, 334, 335).

11. Summary
209. After spermatozoa! x irradiation in mice. thefrequency of translocations increases with dose and does
not show a decline at high dose levels; this finding is in454-

contrast to that after spermatogonial irradiation, where
the frequency increases with dose up to about 700 rad
followed by a decline thereafter.
'.210. There does not appear t.o be any significant
differences in sensitivity to the induction of translocations between new-born and adult male mice.
Contradictory data have been reported with regard to
the induction of translocations in the germ cells present
in male foetuses 12-13.5 days old, some showing thattranslocations can be induced and others showing thatthey are not. In those experiments in which positive
evidence has been obtained, the data show that the germ
cells in the foetuses are about one half as sensitive as
adult spermatogonia.
211. Studies on the induction of translocations infemale mice have gained momentum during the last few
years. The development of techniques for culturingoocytes has facilitated this line of inquiry. Oocytes
derived from irradiated females 14 days after irradiation
show the highest response in terms of aberration
recovery: the frequency of chromatid interchange~
induced in oocytes increases faster than linearly with
x-ray dose. The frequency of genetically recoverable
translocations (after irradiation of females) predictedfrom the kinds and number of aberrations observed
cytologically are in accordance with one another, at leastat the 300-R level.

i'

212. In terms of both the overall frequencies ofabnormal cells and of the kinds of aberrations,rnid-pachytene spermatocytes (sampled 5 days after
irradiation) are more sensitive than dictyate oocytes if
the same sampling interval is used; however, if thesensitivity of the latter at the 14-day interval is used, thereverse is true.
213. For the x-ray induction of translocations in
post-meiotic male germ cells, the mouse is more sensitive
than the rabbit, the latter more sensitive than theguinea-pig. and the guinea-pig more sensitive than the
golden hamster. After spermatogonial irradiation, therank order of sensitivity is mouse> rabbit> guinea
pig ~ golden hamster.
214. Human spermatogonia and those of the marmoset
appear to be roughly three times as sensitive as those of
the mouse for the x-ray induction of translocations. In
contrast, the spermatogonia of the Rhesus monkey
appear to be less than one half as sensitive as those of
the mouse. The primate data as a whole shows
considerable heterogeneity. In using the data frommarmoset and man for hazard evaluations, theCommittee wishes to stress the uncertainties involved; itfurther notes that the estimate so derived can only be
regarded as approximate.
215. In the guinea-pig and the golden hamster, as in themouse, the effects of dose-fractionation on the yield oftranslocations (after sperrnatogonial irradiation) aredependent on the time interval between the fractions
and the size of the fractions.
216. In translocation lines where the males are sterile(derived from irradiation of post-meiotic male genn cells
in the mouse). there is a predominance of chain

quadrivalents in contrast to the semi-sterile lines, which
are characterized by a predominance of ring quadrivalents; in addition. there is a positive correlation
between the severity of. the sterility effect and thefrequency of chain configurations, the latter being 99
per cent when the sperm count is nil.
217. With banding techniques, the breakpoints involvedin the translocations that were recovered have been
localized. In male-sterile translocations obtained frompost-meiotic germ cell irradiation, at least one of the
breaks was close to the centromere (c) or telomere (t)and the majority of the translocations were of the c/t
type. In sterile sons derived from irradiated spermatogonia, however, this was not the case.
218. Apart from the qualitative difference between
translocations recovered from irradiated post-meiotic
versus spermatogonial cells, there is also a quantitative
difference in that, in semi-sterile sons, there are morering than chain quadrivalents after spermatogonial
irradiation, whereas the reverse is true after post-meiotic
germ-cell irradiation.
219. In the golden hamster and the guinea-pig, intranslocation lines in which male semi-sterility is found(derived from post-meiotic germ-cell irradiation), chain
quadrivalents are more abundant than ring quadrivalents,
in contrast to the situation observed in the mouse (seeparagraph 216 above).
220. In the golden hamster, guinea-pig and rabbit,
although most transloca.tions recovered from postmeiotic germ cell irradiation cause semi-sterility inmales, there are some which cause male sterility: in thehamster, sterility is not necessarily associated withtranslocations leading to chain multivalents, and thesituation is the same with the guinea-pig. In the hamster,
sterility seems to be associated with the size of thechromosomes involved in the translocation (involvementof longer chromosomes) but this is not true in theguinea-pig or rabbit.

C. INVERSIONS
221. Roderick (436) and Roderick and Hawes (438)have published the results of a continuing study
concerned with the identification, recovery and genetic
properties of chromosomal inversions in mice. (Earlier
work by the authors was reviewed in the 1972 report(589) in paragraphs 126-132 of Annex E.) So far,
26 paracentric inversions have been induced (by x raysor other mutagenic agents), out of which 18 seem to be
simple autosomal ones, with almost no deleterious effecton viability. In animals homozygous or heterozygous forany two inversions on different chromosomes, there wasno significant lowering of reproductive performance.
However. there were two instances in which the animals
heterozygous for two inversions on different chromosomes were sterile (437).
222. Using inversion ln(l)lRkonchromosome l(earlier
designation, In(I3)1Rk), which has now been demonstrated to be about 43 cM long (3 .5 t,er cent of the
genome), Roderick (436, 437) has uncovered two455

recessive mutations in a sample of 400 chromosomes
tested. One of them causes lethality at various stages ofembryonic development and hours after birth. The othermutation may be allelic with the leaden (In) locus.
223. In the course of an experiment designed tomeasure sex-chromosome loss after x-irradiation of
spermatogonia, L.B. Russell et al (454) found, within
the presumed XM O (i.e., paternal loss) class, a female
that had a submetacentric chromosome in a complementof 39. Further genetic and cytological work showed thatthe submetacentric segregated independently from the
39-chromosome (XO) condition, indicating that theabnonnal chromosome was wholly autosomal. Meiotic
preparations from animals heterozygous for thesubmetacentric failed to vield multivalent configurations
and had only 20 bivalents. Since the short arm of themetacentric is of considerable length, multivalentconfigurations would be expected, if the abnonnal
chromosome were the result of a reciprocal translocation. A pericentric inversion was, therefore,suggested by this finding. The suggestion has beententatively confirmed by the results of cytologicalbanding studies which indicate that the affected
chromosome is No. 8. A breeding stock has now been
established, and homozygotes have proved to be viable
and fertile. Current attempts are directed at introducing
chromosome-8 markers in order to determine the effectsof the presumed inversion on recombination. The
submetacentric may prove to be a useful cytologicalmarker chromosome.
224. Evans and Phillips (161) recently reported on thefinding of an X-chromosomal inversion among thedescendants of a male that had been exposed to a
fractionated dose of x irradiation (12 X 50 rad separated
by weekly intervals; spennatogonia). It was foundassociated with Bare-patches (Bpa), a mutation which
proved to be sex-linked and male-lethal (509). The
original Bpa/+ females produced amongst their progeny
considerable numbers of XO daughters, all of whichwere of the rare OXP type. This enhanced capacity to
produce XO progeny was subsequently shown to be
separable from Bpa and was given the symbol Fxo (410).
The presence of Fxo also suppressed crossing overbetween Bpa and Ta or Bio, and it was suggested that a
structural change of the X-chromosome might beinvolved; this suggestion has now been confirmed
cytologically in Bpa Fox/++ animals and shown to beabsent from Bpa+/++ animals. Consequently, the symbol
Fxo has now been withdrawn and replaced by In(X) 1H
in accordance with the standardized nomenclature forthe mouse (117).

the inversion and is separable from it by a double
crossover ( one exchange on either side of Bpa ). The
frequency of such separation has been estimated to beabout 8 per cent. The authors believe that while theoccurrence of a double crossover would reduce theefficiency of the use of the inversion in the detection of
sex-linked lethals. the inversion may still prove useful inexperiments aimed at estimating rates of induction withmore accuracy than has hitherto been possible.

D. TANDEM DUPLICATION
226. In the course of studies on induced mutations
involving the mouse haemoglobin loci, L. B. Russell etal. (460) found the daughter of an x-irradiated female(SEC/Rl, Hbah /Hbab; Hbb 5 cch /Hbb 5 cch) and an
unirradiated male (101/RL Hba 3 /Hba 3 ; Hbbd C/HbbdC)
whose haemoglobin was not of the usual type by thecriteria of electrophoretic pattern (fast-moving band
relatively fainter), solubility (low), and crystal pattern.
The presumed mutant was also of small stature. The
abnormal haemoglobin pattern was not transmitted inbackcrossing to SEC/Rl (although the small size was),
but was transmitted in backcrossing to 101/Rl, together
with the small size. Crosses of the presumed mutant toan albino stock (Hbbdc/Hbbdc) yielded offspring whichwere cch/cch in coat colour (instead of the expected
cch/c), possessed the abnonnal haemoglobin phenotype,
and were of small size. Subsequent cytological analysis
by the use of quinacrine banding clearly showed achromosome 7 which was approximately 20 per centlonger than normal and in which there was a repetitionof the bright E band and adjacent sub-bands (D and F).
The combined genetic and cytoiogical findings indicate atandem duplication within chromosome 7 which involves a segment including at least the Hbb and c loci.
The abnormal haemoglobin phenotype is consistent withthe presence of two doses of cch and one of c. Thistandem duplication. the first recorded in experimental
mammals, provides a valuable new tool in mouse
genetics, for example, in the study of gene-dosage
effects, and has the advantage that involves the c locus
region, which is well characterized as a result ofcomplementation analysis and also is involved in a
number of X-autosome translocations.

.

E. LOSS OR ADDITION OF CHROMOSOMES
1. Sex-chromosome losses(a) Spontaneous rates
225. Preliminary observations suggest tha't thisX-chromosomal inversion is a long one, covering about
85 per cent of the physical length of the X-chromosome;the genetic data obtained are consistent with a length
greater than 48 cM (the map distance beiween themarkers spf and Bio). Strong crossover suppression
occurs between Bpa and Ta, between Bpa and Bio andbetween Bpa and spf as well. The latter result indicatesthat there is crossover suppression on both sides of Bpa.
In addition, recombination between Bpa and either ofthe markers Ta or spf leaves In(X)IH on the unmarked
chromosome. This evidence implies that Bpa lies within456

227. L.B. Russell (450) has recently reviewed theresults that bear on the spontaneous incidence and rates
of induction of numerical sex-chromosome anomalies inmammals. In this paper. among other things, data on
spontaneous frequencies which have accrued from thecontrols of various mutagenesis experiments. from thetreated groups of mutagenesis experiments where theanomalous type must have originated in the untreatedparent and from the routine maintenance of stocks thatcarry X-linked markers have been compiled. They showthat for maternal X-chromosome losses (scored as OXPt

exceptions), the overall average based on 49 176 females
in these various sets (excluding those in which thepre-existing XO condition was not ruled out) is 0.053
per cent. The corresponding figure for paternal
X-chromosome losses (scored as XMO exceptions), as
judged from a comparison of the frequencies of XM 0
and OXP exceptions (in crosses where these could be
simultaneously scored), is 5-10 times higher.
ii·!

(b) Induction in male genn cells
228. The complete results of the sex-chromosome loss
experiment carried out by L. B. Russell and Montgomery (reported in a preliminary form in the 1972
report) have now been published (450, 457). In thisstudy the incidence of sex-chromosome anomalies after
x irradiation of mouse spermatogonia and spermatozoa
was studied. A 600.R x-ray exposure was used, delivered
either singly or in two fractions, 100 R and 500 R,
separated by a 24-h interval. The breeding scheme
involved crossing irradiated or control ( 101 X C3H)F 1
males to females homozygous for the dominant
sex-linked gene, Greasy (Gs); this permitted thephenotypic detection of paternal or maternal sexchromosome losses (by the occurrence of XG 5 /0 or
0/X+ female progeny, respectively). paternal nondisjunction (XG 5 /X+ /Y progeny) and certain translocations. All exceptional progeny were examined
cytologically and through breeding tests. Mothers ofpresumed XO progeny were likewise tested. It turned
out that in 9 out of 14 cases of 0/X+, there was apre-existing XO condition indicating the importance ofperforming such tests.
229. The results obtained confirmed the conclusions
reached in the 1972 report, namely, that (a) there wereno significant differences between the effects of single or
fractionated exposures; (b) after spermatozoa! radiation(pooled data of the single and fractionated exposure
series), 2 exceptional females were recovered among
421 offspring (0.48 per cent); there was none in thecontrols. The induction rate (on the assumption of
linearity) is therefore 0.8 10- 5 R -, per gamete; (c)after spermatogonial irradiation, the frequency of
sex-chromosome loss was 0.20 per cent (16/8155),
which is not significantly different from that in controls(0.24 per cent (12/4994)).
(c) Induction infemalegenn cells
230. In the 1972 report. the data of W. L. Russell et al.
(489) on the induction of X-chromosome losses infemale mice irradiated with 400-R gamma rays at
exposure rates of 80 R/min and 0.6 R/min were
presented. They showed that the frequency wassignificantly lower at the lower exposure rate. These
results have now been confirmed and extended to alower dose rate of 0.006 R/min and besides, additionaldata, hitherto unpublished, have become available (450).
These are summarized in table 32.
231. It can be seen that (a) the exposure-frequency
relationship for the induction of X-Iosses (in maturing
dictyate oocytes sampled up to 6 weeks after acuteirradiation) is non-linear, (b) there is a continuous dropin the frequency with the lowering of the exposure rate,and (c) the interval effect which had earlier been
documented for the induction of specific-locus mutations is even more pronounced for the induction of
X-losses: in mice irradiated with 400 R at 0.006 R/min,the exceptional progeny are almost exclusively recoveredfrom conceptions that occurred during the first 6 weeks
followed by a steep decline to control levels thereafter.
These data thus confirm the earlier observations withreference to specific locus mutations and extend them toyet another measure of genetic damage. From thestandpoint of hazard evaluations. these findings are ofgreat importance: (a) the rate of induction at low
exposure rates, such as 0.006 R/min, is extremely low(even after a high total exposure); (b) there is a marked
interval effect: ( c) after relatively low exposures, such as
50 R. delivered at high exposure rates, the rate ofinduction is negligible.

2. Non-disjwtction
232. In recent years, there has been a growing interestin the study of non-disjunction. The reasons are (a) the
increasing realization that non-disjunction is an important cause of spontaneous abortion in man (autosomal
trisomies and X-monosomy together constituting over
20 per cent of karyotyped abortuses and over 70 per
cent of all those that are chromosomally abnormal (45,127 and tables 12 and 13)): (b) the findings that about
0.4 per cent of live-born children carry sex-chromosomal
and autosomal numerical anomalies (table 11); and(c) the fact that, with the exception of maternal age, theroles of factors affecting the frequency of non-disjunction (including the effects of radiation) are notunequivocally established in our species.
(a) Mouse(i)Male genn cells

233. Spontaneous non-disjunction of sex-chromosomes
at meiosis in the male mouse is an exceedingly rare event(450, 457, 462). L.B. Russell and Saylors (462) and
L.B. Russell (452) used a breeding scheme which would
allow the genetic detection of XXY mice as males
heterozygous for the sex-linked dominant gene Tabby(Ta). Only one such exceptional mouse was recoveredfrom irradiation of spermatocytes (200 R of x rays) in
6214 classified offspring, compared to none in controls.
234. In a cytological investigation with male mice
carried out by Ohno et al. (384), every one of the 1460
second meiotic metaphase divisions examined contained
either a single X or a single Y and not one instance ofabnormal segregation which might lead to the formationof XY or O sperm was noted. In a recent cytologicalstudy. Szemere and Chandley (558) examined theeffects of radiation on non-disjunction in male mice.
Mice of the random-bred Q strain were irradiated with
100 or 200 rad of x rays: half of the irradiated animals
were killed 5 days after irradiation (to sample
metaphase-II cells treated at the pachytene stage of themeiotic pro phase). the remainder being killed at 12 days
(to sample cells treated at pre-leptotene). In the 200-rad457

series, one group of males was killed 43 days after
irradiation to sample cells treated as early spermatogonia. Estimation of the irradiatiqn-to-killing intervals wasmade using timings established earlier for the Q strain ofmice (261). Additional experiments at 100 rad involved
mating of irradiated males to females, first after 4 weeks
for a 1-week period (to sample cells treated as earlyspermatocytes) and subsequently remating them foranother week (to sample late spermatogonia). The
pregnant females were killed and dissected when the
foetuses had reached 9 days of gestation. It was thoughtthat the foetal age chosen would permit the detection ofat least some of the trisomic offspring (168).
Appropriate controls were maintained.

young, x-irradiated young, non-irradiated aged andx-irradiated aged, respectively. The number of foetuses
examined in the different groups was 149, 11 I, 156 and43, respectively. While the difference in the incidence ofchromosome abnonnalities between non-irradiated andx-irradiated young adults was not significant, there
appeared to be a definite increase in the incidence ofaneuploid foetuses in the x-irradiated aged as comparedwith the x-irradiated young. The authors concluded thatthe incidence of aneuploid foetuses in aged mothers isfurther increased by 5 R of x irradiation. Of the 7
aneuploids in the aged group (irradiated) there were
4 trisomies, 1 double trisomy and 2 mosaics (39/40 and
40/41).

235. The estimates of non-disjunction frequencies weremade on scores obtained at metaphase-11 in control andirradiated groups as the ratio of the number of cells
containing 19 and 21 dyads to the number of cells
containing 19, 20 and 21 dyads. For each dose andtreated stage, 200 cells were analysed. The results
showed that in controls there were no hypoploid or
hyperploid cells, in agreement with the data of Ohno et
al. mentioned earlier. In the x-irradiated groups,
aneuploid cells were found, particularly in cells treatedas pre-leptotene spermatocytes, 12 days prior to scoring.
The non-disjunction frequency for this treated stage was
4.5 per cent at JOO rad and 6.0 per cent at 200 rad; thefrequencies for other stages were relatively lower. The
foetal karyotyping gave no evidence .for pure trisomics
although some mosaics were present. In addition, and of
special interest. was the finding of two triploid (3n = 60)
foetuses in the treated group; both were from matings inweek 5 and could have therefore arisen following theirradiation of early spermatocytes.

238. Gosden and Walters (197) have reanalysed theabove data statistically for evidence of interaction
between age and x irradiation using three procedures:(a) comparing the appropriate function of the percentages with its standard error; (b) partitioning thethree degrees of freedom into an age, irradiation andinteraction effect; (c) examining the proportionate
increase apparently due to irradiation. None of thesetests showed any significant difference of the kindclaimed by Yamamoto et al. (627). i'he other objection
which Gosden and Walters raise to the conclusion of
Yamamoto et al. pertains to the fact that the authors
have used as controls the earlier published results
detailed at the beginning of this section; such a
procedure would be open to question in view of possible
differences in environmental conditions.

(ii)

Female germ cells

236. Yamamoto et al. (626) examined the maternal
age-dependence of chromosome anomalies in unirradiated mice. The procedure was briefly as follows: female
mice (strain CF ·1) were mated at ages of 3-5 months(control), I 1-13 months, and 14-16 months to young
adult males. On day 10.5, the pregnant mice weresacrificed and the foetuses recovered and analysed
cytogenetically. The proportion of aneuploid foetuses
was 2/149 in the control, 5/117 in the 11-13 monthgroup and 5/39 in the 14-16 month group. In the younggroup, both the aneuploids were mosaic monosomies
and in the two, the breakdown was 3 trisomies,
1 monosomy and 6 mosaics. The incidence ofaneuploidy in the two "old groups" considered together
was significantly higher than in controls. However, acomparison of the control with the 11-13 month groupalone did not reveal any significant difference infrequency whereas that of the control with the
14-16 month group showed that in the latter it was
higher.
237. In a subsequent study, Yamamoto et al. (627)
irradiated female mice of different ages with 5 R of
x rays and conducted a cytological study similar to theone described in the preceding paragraph. Chromosome
analyses revealed that the number of aneuploid foetuses
was 2 (1.3 per cent), 4 (3.6 per cent), JO (6.4 per cent)and 7 (16.3 per cent) in the groups, non-irradiated458239. Uchida and Lee (582) made use of the recent
advances in the technique of culturing mouse oocytes invitro to obtain preparations of meiotic chromosomes (inmetaphaseof oocytes derived from irradiatedfemales. C3H X ICR/Swiss F 1 hybrid females aged 3 and6 months were exposed to 10, 20 and 30 R ofwhole-body gamma irradiation; the irradiated females
and their paired controls were sacrificed and the ovaries
removed within one week of radiation exposure. The
oocytes were teased out of the ovaries and thosecontaining a germinal vesicle were incubated in foetal
calf serum for 18-23 h to obtain cells in metaphase II
and subsequently processed for cytological examination.

rn

240. In a total of 15 713 viable oocytes collected, ll51
and 1054 metaphase-II oocytes (in the irradiated andcontrolgroups,respectively)
were
analysable(428 oocytes at 10 R, 368 at 20 R, and 355 at 30 R).
Since there were no significant differences between the
two age groups used, the results were combined. Six ofthe irradiated oocytes had an extra chromosome (i.e.,
21 chromosomes), Two of these were in the 10 R series,
three in the 20 R series and one in the 30 R series. No
hyperploid cells were found among the controls.
Assuming that for each oocyte with 2 I chromosomes. acomplementary cell with 19 chromosomes must havebeen produced (and formed the polar body) and barring
preferential segregation of one type of abnormal productinto the polar body, there should be an equal number ofhyperploid polar bodies with their complementary
hypohaploid oocytes. The total number of nondisjunctional products should therefore be closer totwice the observed frequency of hyper-haploid oocytes,
i.e., 12 (I per cent) among the irradiated oocytes. Thernumbers of non-disjunctional events in the sample arestill too scanty to permit comparisons between the
different exposure levels. From the work of Uchida andLee. it would therefore appear that non-disjunction canbe induced in young females by in viva radiation
exposure.
241. Uchida and Freeman (581a) have now performed
another experiment on the same lines as that of Uchida
and Lee described above, but with the use of older
female mice, aged 12 months instead of 3-6 months. The
frequency of hyperhaploid metaphase-II oocytes was
significantly higher than in controls and was also higher
than in young females. The incidence of non-disjunction
among aged controls was 0.6 per cent, increasing to
2.7 per cent on irradiation, as compared with a rate of
1 per cent in irradiated young females. The authors
consider that these results support the suggestion thatthe risk of producing trisomic offspring among humans
is increased with exposure of the abdomen to diagnostic
x rays. They point out that 9 out of 11 epidemiologicalstudies have shown an increase in non-disjunction withradiation exposure, although in some series theseincreases were not statistically significant.
242. In a genetic study on the effects of maternal age
on spontaneous and x-ray-induced (200 R) sexchromosome non-disjunction and loss in the mouse,
L.B. Russell and Montgomery (457a) compared the
response of mice about 3 months old with that of miceabout 9 months old. The experiments, which are still
continuing, have so far generated no unequivocal
instances of maternal non-disjunction, although there
has been one case of paternal (spontaneous) nondisjunction. In a new series, also still in progress, inwhich the females are even older (11.5-12 months at thetime of irradiation and/or mating), no cases of maternal
non-disjunction have so far been recovered ( 457a, 467).
243. Luning, Eiche and Uming (303) conducted somepilot experiments with the CBA strain of mice to check
whether or not· exposure of females to low doses of
x rays induce non-disjunction in oocytes; should this be
the case, then the nullosomic and disomic gametes that
result from non-disjunction should lead to the
production of aneuploid foetuses. Such foetuses, except
possibly some trisomies, are expected to die in utero andthe frequency should be ascertainable under properconditions. The experimental scheme included (a)irradiated and control females of various ages, with andwithout a history of having produced a few litters prior
to the commencement of the experiments; (b) acuteexposures of 2, 4, 8, 12, 16 and 32 rad to females as well
as fractionated doses (4 X 4 rad; 2 X 8 rad at 1-weekintervals), (c) exposure of foetuses at various times
before parturition; and (d) intervals ranging from O to
182 days between radiation exposure and mating. The
females were killed 11-20 days after mating for uterine
examination.
244. The results obtained in this study show that (a) in
the irradiated series, there is no excess intra-uterine
death at a more or less late stage in development and(b) the age of the females at the time of mating has aconsiderable effect on the intra-uterine death rate(varying from about 8 per cent in young females to25-30 per cent in older females) in both the irradiatedand control series. It should be pointed out that
conclusion (a) cannot be considered a definitive one; inspite of the variety of pilot tests performed, the absolute
numbers in any one series are relatively small and thismakes it difficult to detect small increases in intrauterine death.

(b) Hybrids between the house mouseand tobacco mouse
245. The diploid chromosome complement of thetobacco mouse. Mus poschiavinus, consists of only
26 chromosomes, 14 of which are metacentric (206) incontrast to the all-acrocentric complement of 40 in thehouse mouse. The fundamental numbers are the same
(2n = 40), however, and both meiotic (572) and mitotic
studies ( 628) with the two species and their F 1 hybrids
have demonstrated that the chromosomes are, in fact.
homologous. It is believed that the tobacco mous~
metacentrics were derived from acrocentric chromosomes of the house mouse by Robertsonian fusion/translocation. During the last few years, stocks of mice
have been constructed each carrying a different
metacentric chromosome derived from M. poschiavinus.
These are being extensively utilized to examine
systematically the involvement of specific chromosomes
in meiotic non-disjunction and as a good model system
to gain an insight into the conditions leading toaneuploidy in mammals, including man.
246. The F 1 hybrids between the house mouse and thetobacco mouse are fully viable, but have greatly reduced
fertility. Studies on first meiotic division cells haveshown that the seven metacentrics regularly form
trivalents with the 14 homologous M. musculus acrocentrics. The finding that in the F 1 hybrids more than
one half of the metaphase II figures are geneticallyunbalanced suggests that anaphase-1 disjunction isdisorderly leading to gametic aneuploidy, which in tum
leads to the production of aneuploid inviable zygotes(572). Tettenbom and Gropp (572) suggested that thenon-disjunction observed in the hybrids could result
either from genetic heterozygosity per se or fromstructural heterozygosity leading to trivalent formation.
247. Cattanach and Moseley (91) found that in males,
each of the 7 tobacco mouse metacentric chromosomes
when carried heterozygously with the house mouseacrocentrics led to non-disjunction. but the frequency
was not the same for each chromosome. In addition,
considerable heterogeneity existed between individuals
heterozygous for any one metacentric and thenon-disjunction frequencies did not appear to becorrelated either with chromosome size or centromere
position. The authors also obtained some evidence of
non-disjunction inmetacentric homozygotes, small testis
size and reduced sperm production among homozygotes
and crossover suppression between the metacentrics andthe homologous acrocentrics in heterozygotes. On thebasis of these data, they concluded that a large part of
the non-disjunction observed when the tobacco mousemetacentrics are carried heterozygously with the house
mouse acrocentrics may result not from structural
heterozygosity per se, but rather from other genie or459

minor chromosomal differences which reflects the factthat the chromosomes are derived from two different
species.
248. The results obtained by Ford and Evans (169) and
Gropp. Giers and Kolbus (204) in the backcross progenyof single metacentric heterozygotes are essentially thesame: depending on the metacentric involved. heterozygous males caused different rates of non-disjunction
and the estimates based on metaphase-II counts and on
karyotying of pre- and post-implantation embryos werein agreement with one another. The work of Gropp,
Giers and Kolbus (204) also demonstrated thataneuploidy (trisomy) of the zygotes was considerably
more frequent in the progeny of heterozygous females
than in those of heterozygous males and has led to thesuggestion that this disparity might be the result of ahigher non-disjunction rate in female gametogenesis. The
authors have entertained the possibility that mechanismsof selection against unbalanced male germ cells might
operate on a small scale. Ford and Evans, however,suggest that in their work there was no evidence of
selective elimination of spermatids or spermatozoa with
unbalanced genomes up to the third day of gestation.
249. So far. all the trisomic conditions reported in thissystem cause pre-natal death, which in some of themoccurs at an early stage (day 10-13) while in others, at alater one (day 14-16), and the developmental profiles
differ considerably according to which individualautosome is involved (169. 203, 204, 205). Consistent
with this, Ford and Evans found a considerable
reduction in the number of trisomic embryos between
the earlier gestational period (8-11 days) and the later
period (12-15 days). Taken together. these observations
with primary trisomics contrast with the findings withpartial (tertiary) trisomics (in the house mouse) which
may be viable and not infrequently fertile (40, 154,
310). One further finding relates to the observation thatvery few monosomic zygotes survived to be identified
subsequent to implantation in the M. muscu/us X
M. poschiavinus heterozygotes for any of the metacentrics thus far tested (169).

(c) A method to measure non-disjunction in thelaboratory mouse using Robertsonian translocations
250. Lyon et al (319) have recently devised a method
for measuring non-disjunction in mice using Robertsonian translocations of the tobacco mouse. Since a highfr~quency of non-disjunction occurs spontaneously innuce heterozygous for Robertsonian translocations (seethe ?receding section), it was thought that it should bepossible to measure this by a method similar to that used
for. measuring adjacent-2 disjunction in mice withreciprocal translocations. In the latter method, animals
heterozygous for the translocation and homozygous fordiffere?-t alleles of a marker gene are mated together.
Offspnng homozygous· (rather than typically heteroz~~ous). for the marker arise through adjacent-2
dis1unct1on (or non-disjunction) and their frequency canbe recorded {519).
251. In the scheme of Lyon et al., homozygotes for theRobertsonian translocation are crossed to homozygotes
for the chosen genetic market, and the offspring

460

intercrossed. The intercross progeny are tested for thepresence of the transiocation by biopsy of the testis,spleen or ear skin. By this means a stock homozygous
for both the translocation and the marker is constructed.
Animals from this stock are crossed to homozygotes forthe marker. but not carrying the translocation. All theoffspring from these matings will then be heterozygous
for the translocation and homozygous for the genetic
marker and are used in the experiments. They are matedinter se and the young are observed at birth, 7-14 days
later, and at weaning for the presence of recessive markers.
All such marker-carrying young are tested for fertility
and their karyotypes are determined by biopsies of ear,skin or corneal preparations.
252. Using this method, Lyon et al., estimated that thenon-disjunction frequencies ranged from Jess than 5 percent to about 15 per cent in the different crosses. In
addition, the finding that all the marked young were
heterozygous for the Robertsonian translocation suggests that the gametes involved are derived from
non-disjunction at the first meiotic division. A search forgenetic or environmental factors affecting the frequencyof exceptional progeny revealed that with one
Robertsonian translocation, the frequency increased
steadily with increasing maternal age whereas with another, the maternal age of the marked young was not
significantly below that of the total progeny.
(d) Microtus oeconomus
253. Another mammalian species which may bepotentially useful in the study of non-disjunction,
specifically of the sex-chromosomes, is the northernvole, Microtus oeconomus. The use of C-banding
techniques have shown that in this species with a diploid
chromosome number of 30, while all the 14 pairs ofautosomes exhibit tiny dots of centromeric heterochromatin. the X-chromosomes have large blocks ofthese and the Y-chromosome is C-band positive along its
entire length ( 636).
254. In an exploratory study, Tates, Pearson andGeraedts (570) found that in testes preparations
processed for C-banding, cells having a single X or Y,
two Ys, two Xs and XYs can be identified in early andmid-term spermatids (some of the spermatids were larger
in size and appeared to be polyploid). In a subsequent
study, Tates (569) irradiated males with 50, 100 and
200 R of x rays and made preparations 1, 2. 4, 8 and
12 days after irradiation. Such a procedure would enable
the sampling of cells irradiated as spermatocytes andpossibly as spermatogonia (in the later interval series).
There is definite evidence for the induction of
non-disjunction even at the lowest level of exposure
used, but the calculation of the exact frequencies iscomplicated by the occurrence of great variability
between animals within exposure levels, and within andbetween the different sampling intervals. The author isat present collecting more data.

3. Summary
255. Additional data on the x-ray induction of
sex-chromosome losses in mouse spermatogonia thathave accumulated since the publication of the 1972

I
•

.'

~

.j

report support the conclusion reached earlier. namelythat there is no evidence for an increase in the frequency
at which exceptional XO progeny are recovered (relativetocontrols)
after
spermatogonial
irradiation(600-R level). After spermatozoa! irradiation however,the frequency is significantly higher than in the controls.
256. X-chromosome losses can be readily induced byirradiation of mouse females at high dose rates; there is amarked dose-rate effect in that a lowering of the
exposure rate from 80 R/rnin to 0.6 R/min down to0.006 R/min (total exposure of 400 R) results in a
progressive reduction in the frequency. This continuous
reduction in frequency with lowered dose rates parallels
the results obtained for the induction of specific-locus
mutations; in addition, the frequency ofX-chromosome
losses in conceptions occurring later than a few weeks
after irradiation is not significantly higher than in thecontrols. Thus there is evidence for the operation of aninterval effect for this end-point of genetic damage as
well.

'

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II

257. Studies on non-disjunction have been recemng
more attention during the past few years. In male mice,the results show that non-disjunction can be induced byirradiation of pre-leptotene spermatocytes. In female
mice, although there are indications for the radiationinduction of non-disjunction in oocytes, the data do not
permit reliable risk estimates to be made.

t
258. Use has been made of the presence of the sevenpairs of metacentric chromosomes in the tobacco mouseto construct mouse stocks carrying known metacentricchromosomes to study the process of non-disjunction in
more detail. Although up ~o now no radiation
experiments have been carried out with these stocks, thestudies have provided interesting insights into how thefrequencies of non-disjunction vary depending on whichmetacentric is involved, the time of death of thedifferent trisornics and their developmental profiles.
259. A new method has been developed to measurenon-disjunction in mice using Robertsonian transloca tions of the tobacco mouse. This is likely to provevaluable in studying radiation-induced non-disjunction inmice.
260. Attempts are being made to study non-disjunctionof the X and Y chromosomes in the males of thenorthern vole, Microtus oeconomus, in which techniques
are now available to score for non-disjunctional
spermatids derived from sperrnatocytes and sperrnatogonia.

F. POINT MUTATIONS
1. Specific-locus mutations in male mice
261. The complete results of the work of Selby(discussed in the 1972 report in a preliminary form,
Annex E, paragraph 174) on the x-ray induction of
specific locus mutations at the seven loci in the germ
cells of male mice of different ages (new-born, and age

groups from 2 to 35 days 14 after birth at irradiation)
have now been published (526, 527) (table 33). These
results, with only a slight increase in the number ofprogeny tested (over those presented in the 1972 report)
confirm the conclusions reached earlier: (a) for
new-born mice, the rate is 1.37 10-7 R -I per locus,
which, statistically, is significantly lower than that of
2.91 10- 7 R -i per locus for spermatogonia in adults(300-R level for both). The former rate does not
significantly differ from the one reported by Carter et al.
(194) (1.83 10- 7 R- 1 per locus, 200-R level) for
17 .5-day-old foetal males; (b) none of the mutation rates
in the other age groups was significantly higher than thatof the adult, although this conclusion does not rule out
the possibility that individual age groups may havemutation rates somewhat different from that of theadult.
262. The combined mutation rate for the nine agegroups of young males (day 2-35), 2.63 10-7 R- 1 locus.
is significantly higher than that of the new-born males,
1.37 10-7 R -i per locus, and almost the same as that ofsimilarly irradiated adults. An examination of the data
for the different age groups suggests that the change inmutational response may have occurred by day 8; day 8
has the second-highest mutation frequency of all ninegroups. All three age groups before it have lower
mutation frequencies than the adult. The mutation
frequencies on days 2 and 4 are the lowest found among
the nine groups, both being very similar to the rate
found for new-born males. The point estimates for thedata are arbitrarily grouped according to their indicationin table 34, in which estimates for new-born and adultare also shown for comparison.
263. The incidence of clusters of specific-locus
mutations following irradiation of new-born males was
significantly higher than the cluster incidence reported
by W. L. Russell for similar irradiation of adults. This
presumably indicates the survival of relatively fewer
reproductive cells following irradiation of the day-0
testes. In the other age groups, clusters were found only
on day 21 (two clusters of two mutations each). In
new-born males, although tltere were suggestive indications for differences in the distribution of mutations
among the loci studied (relative to that in the adults) the
differences were not significant: for the other age
groups, the distribution appeared similar to that inadults.
264. Cattanach and Moseley (92) investigated theeffects of irradiating mouse spermatogonia with a total
exposure of I OOO R of x rays delivered in two equal
1 • Between birth and 35 days of age, the testis undergoes
many developmental changes. and by 35 days, the cell
population resembles -that of the adult. Widmaier (609) foundthat primary spermatocytes were commonly observed 7-8 days
after birth in the mouse strain he studied. Some spermatids were
present by 20 days and at 25 days, almost all tubules contained
secondary spermatocytes and spermatids. At 30 days, thegerminal epithelium was fully developed. Widmaier concluded
that the first cycle of the seminiferous epithelium begins on dayof birth in the mouse and that the cycle proceeds essentially as
described by Oakberg (368). Oakberg (369) has shown that thetime required for type-A spermatogonia to develop into mature
spermatozoa averages 34.5 days in the adult male. However,spermatogenesis has been sho\\<11 to proceed more rapidly injuvenile rats than in the adult (236).

461

fractions separated by 4 and 7 days; the yields ofspecific-locus mutations (at the loci) were comparedwith those obtained in the experiments ofW. L. Russell(472) with single 1000-R exposures, two 500-R exposures separated by 2 and 24 h and two exposures of
600 R and 400 R; 15 weeks apart. These are shown intable 35 from which it can be seen that (a) neither the 4nor the 7-day interval results in yields which are similar
to that of the 24-h fractionation, (b) the frequencies arehigher than after the single acute exposure of 1000 R,
and (c) the data for the 4- and 7-day fractionation
periods are consistent with the expectation of additivityof yields such as that seen in the long-term fractionation
experiments of W. L. Russell (472), 600 R + 400 R,> 15 weeks apart.
265. One of the early attempts to screen forbiochemical variants induced by irradiation of the mouse
was that of Feinstein et al. (I 65) who devised a rapid
semi-quantiative screening method for blood catalase; inthe progeny of irradiated male mice (derived from
W. L. Russell's experiments), the authors observed two
with low blood catalase in a total sample of
12 OOO mice.
266. A programme designed to study the radiationinduction of haemoglobin variants in the mouse,
initiated a few years ago at Oak Ridge is continuing andhas produced some interesting results ( 461, 492). The
work consists of screening blood samples of F I progeny
from experiments in which either 101 or SEC strain
mice are x irradiated and mated to non-irradiated miceof the other strain. The parental strains differ from eachother for alleles at the Hba chromosome 11 and Hbb locichromosome 7. The haemoglobin characters of F 1
offspring analysed included electrophoretic pattern,solubility and crystal pattern. Blood samples were also
checked for possible alterations in serum albumin andred-cell lysis.
267. Of a total of 8621 F 1 's so far analysed for thesecharacters, 6918, 875 and 828 were derived from
irradiated spennatogonia, post-spermatogonial stages andoocytes, respectively. Five haemoglobin variants havebeen found. Two of these appear phenotypically to bealpha-chain deletions or inactivations, the third a sterile
male (with spennatogenic block in pachytene), a resultof an independent translocation T(3;16), the fourth atandem duplication (para. 226) involving Hbb, and thefifth presumably carries two No. 7 chromosomes fromthe 101 mother and none from the irradiated SEC
father. No serum albumin mutants were detected andfour anaemic F 1 's probably did not transmit theirabnormality. The authors note that the per-locus
mutation rate based on the 3 Hba mutants (which may
be either small deficiencies or intragenic changes) are notout of line with earlier specific-locus results, althoughconfidence limits are still very wide.
268. Work similar to that outlined above, but using nineelectrophoretically detectable markers (Es-1, Es-3,
Gpd-1, Gpi-1, Id-I, Mod-1, Pgm-1, Dip-I and Hbb) iscurrently underway at Research Triangle Park (321 a,
591, 592). Irradiated DBA/2J mice (500 R + 500 R
gamma rays, 24 h apart,· spermatogonial irradiation)
were crossed to C57BL/6J and the F I progeny screened
by electrophoretic methods for variants. Thus far,462

4 mutants (two of which are haemoglobin variants) havebeen found in 2600 progeny. Work is continuing.
269. In two recent papers, Kohn and Melvold (262) and
Kohn, Melvold and Dunn (263) have summarized theresults of their x-irradiation mutagenesis studies in miceusing the histocompatibility (HJ system. Before
describing the results, it will be useful briefly to review
the system and the methodology. This system iscomprised by a group of co-dominant histocompatibility
genes that are located throughout the genome and on
whose action the acceptance or rejection of dermal
grafts depends. For operational reasons, the H loci are
divided into two classes which can be distinguished fromone another in the F 1 hybrid of the 86 and C lines thatare employed (B6 =C57BL/6Kh: C =BALB/cKh). The
class-I loci, 30 in number 1 ~ have different alleles in theparental lines and are therefore heterozygous in the F 1
hybrid. The class-II have similar alleles in the parental
lines and are therefore homozygous in the hybrid. The
number of class-II loci is unknown.
270. The H-test involves five major steps (264, 266):(I) mating of selected parents. one of which can betreated with a mutagen or x irradiated (Co X B69); (2)skin-graft testing of the F I progeny; each animal
exchanges one graft with each of two other animals in a"reciprocal circle"; (3) identification of suspectedmutants; (4) backcrossing of each of these to a parental
line; (5) graft-testing of the resultant backcross progeny
to establish the transmission of the suspected mutation.
271. The mutations are classified on the basis of theirgraft-rejection patterns as "gains" (appearance of a new
antigenic specificity, i.e., grafts donated by the putative
mutant rejected), "losses" (loss of a specificity, i.e.,
grafts placed on the putative mutant rejected) and "gains
and losses" (one specificity replaced by another, i.e.,
reciprocal rejection occurs between the putative mutant
and other animals). Class-I mutations are distinguishedfrom class-II by their loss or gain-a11d-loss phenotypes
for loci on autosomes and X-chromosomes: class-II
mutations could produce only gains unless the
Y-chromosome was involved.
272. Male mice (C-line) were x irradiated (60-65 rad/min; 350, 500, 650 and 800 rad acute x irradiation;unequally fractionated doses of 500, 650 and 800 rad)and mated to the B6 line. The F I progeny ( derived from
irradiated spermatogonia) were graft-tested, the mutations identified and subsequently verified as mentioned
in the preceding paragraph. The results showed (262)that among a total of 13 614 F 1 progeny, including
11 279 experimental (all radiation groups) and
2335 controls, a single class-I mutation was recovered,
which was of the "gain-and-loss" type (in a series inwhich the male parents were irradiated with afractionated dose of 300 + 500 rad, 24 h apart). The
mutant rejected skin from the C parental line but
accepted skin from the B6 maternal line. No class-I
mutations were detected in the controls. Four otherclass-I mutations that were detected (I loss and 3
gain-and-loss) had occurred in the B6 alleles, i.e., in theunirradiated maternal gametes.
1 5 Minimum number of loci at which the B6 and C strainsdiffer (19).

1,273. In tests for class-II mutations (263), a total of
36 mutants was found, of which 8 were in the controls
and 28 in the different irradiated groups. Neither thefrequency of mutations in any of the irradiated groups
nor the total frequency of mutations recovered wassignificantly different from the control frequency.
274. The failure to obtain evidence for radiationinduction of histocompatibility mutations is attributedby the authors, not to the failure of the system to detectmutations, but rather to the very low mutability of theseloci (relative to the seven or the six loci used in the Oak
Ridge and Harwell studies) after radiation exposures.
They surmise that this can happen if x-ray-induced
histocompatibility mutational lesions are more efficiently repaired or if spermatogonia carrying x-rayinduced histocompatibility mutations fail to result in
viable progeny. Obviously, further research is needed. It
would be especially of interest to determine if similar
results would be obtained after·spermatozoal irradiation.

".,•

275. In a subsequent paper, using target theory
considerations, Kolm (265), estimated the sizes of genes
involved in mutation experiments with specific loci inthe mouse and in other organisms. The author's main
conclusions were: (a) mutation rates tend to be much
lower than predicted by target theory: (b) selection
and/or repair are major factors that determine the rates;and (c) the mouse seven-loci test, which provides aprincipal data-base for the standards of human radiation
protection, may not provide adequate overall representation of the mutability of the mammalian genome.
However, since the author himself points out "estimatesof gene size by target theory most likely will beincorrect, depending on the magnitude of balancing of anumber of factors'\ it does not appear profitable to enter
into a detailed discussion of this paper. Furthermore, inthis report, the Committee has used the specific-locus
data from the mouse only in the context of assessing theeffect of various physical and biological variables onmutation rates, in the sense they were originallyintended to be used (480) .

2. Dose-rate effects

become available, and the same trend towards higher
mutation rates at very low dose-rates is observed; (c)when, using all the available data, the induced mutation
rates are plotted against logef (where I is the exposurerate), some kind of curvilinear relationship is indicated.
Statistically, the data fit well a model in which themutation rate varies continuously with the exposure rateand in which there is a dose,rate I min at which themutation rate is minimal. Th,e maximum likelihood
estimate of Imin is about 0.03 R/min (95% confidence
limits, 0.002 and 0.09); (d) although the quadraticmodel used gives a good fit to the data, the model ofzero slope below exposure rates of 0.8 R/min also fits
the data; {e) an examination of whether the true
relationship is not a smooth curve but rather two
independent straight lines, one with a negative slope over
the range of 0.001 and 0.02 R/min, and the one with apositive slope thereafter, reveals that the data also fit
this model.
278. In the opinion of Lyon et al., the biological basis
for two separate mechanisms that might conceivably
operate ( one on either side of Im in) may be related to
the number of cell cycles, duration of the irradiation andcell population changes. A relevant consideration may bethat the position of Im in is such that at exposure rates
below it, the period of irradiation necessary in order togive the total dose extends over many cell cycles,
whereas above Im in, the total exposures are delivered
within one cell cycle. Published results of Oakberg and
Clark (375) suggest that at a rate of 0.001 R/min, thesurvival of type-A spermatogonia is much higher than at
0.009 R/min and above. At 300 R, the survival of
type-A spermatogonia expressed as experimental/control
ratio is 0.372 at 0.009 R/min and 0.846 at 0.001 R/min.
The pattern of survival at 0.009 R/min resembles that at
high exposure rates (600-R level).
279. The essence of the argument then is that cellkilling may be a plausible explanation for the dip in the
curve with relatively little depression of the mutation
rate due to cell killing at 0.001 R/min and lower rates.
At environmental levels of radiation. one might
therefore expect a response more similar to the one at
0.001 R/min than at 0.009 R/min (see also Oftedal(383) for a discussion of the problem).

276. Lyon, Phillips and Papworth (318) published apaper in 1972 in which they re-examined the publisheddata on dose-rate effects with low-LET irradiation for
the induction of specific-locus mutations in mouse
spermatogonia besides some new data from Harwell
hitherto unpublished. Based on earlier results, theconclusion was that in adult spermatogonia, the maximal
effect of reducing the exposure rate is already obtained
at 0.8 R/min (namely a reduction of the yield to about
30 per cent of that at high exposure rates), such that afurther reduction in exposure rate has no measurable
effect. The paper of Lyon et al. (318) raises questions
about this conclusion.

280. The implication of the analy-is of Lyon et al. forhazard evaluation is this: on the basis of Russell'sinterpretation that the mutation rate remained constantat exposure rates below 0.8 R/min, it was reasonable tosuppose that it would still remain constant at
environment radiation levels (0.1 R/year or 1.9 10-7 R/min).
This means that for low-dose conditions applicable toman, the assumption that the induction rate is one thirdof that seen at higher dose rates is justified. On the otherhand. if the efficiency of the irradiation increases at very
low dose rates, the rate used in risk evaluations shouldneed upward revision.

277. The arguments are as follows: (a) in Russell'swork, the mutation rate per unit of exposure at an
exposure rate of 0.001 R/min is actually higher than at
0.009 R/min. suggesting an inverse relationship between
mutation rate and exposure rate, but the difference is
not statistically significant: (b) from work at Harwell,
new data (recorded in the paper) at low dose rates have281. The paper of Lyon et al. has been re-examined by
W. L. Russell ( 467, 481 ). This reanalysis bears out thefollowing conclusions: (a) at rates below 0.8 R/min. the
Oak Ridge data for induced mutation rates do show an
upward trend: however, the confidence intervals are
sufficiently large such that they fit the model of zero
slope; {b) the Harwell data at low dose rates show a less463

pronounced upward trend if Harwell controls are used tocorrect for spontaneous rates; furthermore, the confidence limits for the Harwell data are also wide, indeed
very wide, for the 0.001-R/min point. Thus there is nofirm evidence for a negative slope at lower dose rates.
282. Additional data that have since become available
seem to lend support to the earlier conclusion of
W. L. Russell that at exposure rates below 0.8 R/min tomouse spermatogonia there is no further significant
reduction in mutation rate and that at low exposure
rates (0.8 R/min or lower) the induction rate is about
one third of that obtained at high dose rates. In
experiments specifically focusing attention on the
problem of determining mutation rates at very low
exposure rates, W. L. Russell and Kelly (486, 487)
irradiated male mice with a total gamma-ray exposure of
300 R (1 37 Cs) at rates of 0.0007 R/min and0.0056 R/min (delivered over a IQ.month and a 38-day
period, respectively). Two factors that were notconsidered in earlier experiments have been controlled in
the current series: (I) in the earlier work, animals of allgroups entered the experiment at about the same age.
Thus, those that required the longest exposure time were
irradiated over a more advanced age than the rest: (2)their matings did not start until they were considerably
older.
283. In the new experiments, the animals exposed at
0.0056 R/min for 38 days were divided into four groups.
The exposure period of these groups was distributed at
approximately 3-month intervals of age to cover the age
range involved in the 0.0007-R/min group that was
exposed for over 10 months. All groups, including
controls were not mated until the 0.0007-R/min group
had received its exposure.
284. The data thus far available show that at a rate of
0.0007 R/min there are 11 presumed mutations in
42 020 offspring and at 0.0056 R/min 19 _presumed
mutations in 74 842 offspring. The frequencies arehighly significant above the controls and differ fromeach other by less than one mutation from that expectedon the null hypothesis of no difference between the twogroups. The tests are continuing.
285. In studies initiated to complement those of
W. L. Russell and Kelly (discussed in the preceding
paragraphs) on spermatogonial stem cell survival,
Oakberg and Palatinus (378) demonstrated that there
was no reduction in the stem cell population after 300 R
of 1 3 7 Cs gamma rays given to mouse spermatogonia at a
rate of 0.0007 R/min, whereas it was 37 per cent ofcontrols after the same exposure delivered at
0.0056 R/min. Thus, in these experiments, a change incell killing has not been accompanied by a change inmutation frequency. contrary to the arguments presented in paragraphs 278-279.

3. Specific-locus mutations in female mice(a) Dose-fractionation effects
286. In earlier studies (reviewed in the 1972 report),
W. L. Russell {476, 480) had found that a single
exposure of SO R of x rays to female mice gave a lower

mutation rate per unit exposure (conceptions within thefirst seven weeks after irradiation; maturing oocytes
samples) than those of 200 or 400 Rand that the yield
after 8 X 50 R (75-min interval between the fractions)
was 8 times that of a single exposure (i.e .. much lower
than the effect of a single 400-R exposure; see the 1972
report, Annex E. table 16).
287. Lyon and Phillips (312) have now investigated themutational response of maturing oocytes to irradiation
with a total dose of 200 rad given in 20 fractions of
10 rad each, over a period of five days ( 10 rad at a time,
four times a day; interval between fractions in a day,approximately 2 h) or over four weeks {l O rad per day
for five days in a week (Monday through Friday) 24-hinterval between fractions). The effects observed werecompared with those in females irradiated with single
doses of 200 rad, of which there were two groups. One
group was mated soon after irradiation and the otherafter a week's delay. All x irradiations were delivered tomice 9-10 weeks old at about 52 rad/min. The offspring
conceived within seven weeks (and later) after theirradiation were scored for mutants at the seven loci of
the PT stock. There were no controls.
288. The results showed that there were no significant
differences in mutational yields between the two
single-exposure experiments (although in one, mating of
the irradiated mice was delayed for a week) and between
the two fractionation regimes (although in one, theirradiation was delivered in five days and in the other,over four weeks). Therefore, the data of the two acutegroups were pooled and, likewise, those of the two
fractionation groups were pooled: these data are given intable 36. Inspection of table 36 will reveal that (a)among the offspring conceived within seven weeks after
irradiation, the mutational yield after single exposures is
9/34 813 (a rate of I.85 10- 7 rad- 1 per locus) and after
fractionation, 1/39 887 (a rate of 0.18 10-7 rad- 1 perlocus); and (b) if the comparisons are restricted to theprogeny conceived within the first three weeks after
irradiation, the corresponding figures are 7/21 578 (arate of 2.32 10- 7 rad -i per locus) and 1/20 398 (a rateof 0.35 10- 7 rad- 1 per locus). Thus, there is a reduction
in mutation frequency by factors of about 10 or 7,
depending on which progeny groups are included inthese comparisons.
289. The rates given above have not been corrected forcontrols. 1 6 If this is down. the induction rates will beeven lower. For instance, the induction rate obtained atthe 200-rad level (even by correcting with the minimal
spontaneous rate) is lower than that of W. L. Russell at
the same exposure: for the first seven-week conceptions.
W. L. Russell obtained a rate of 3.95 10- 7 R- 1 perlocus, which is twice that which can be estimated fromthe data of Lyon and Phillips (1.75 10-7 rad- 1 perlocus). The latter authors suggest two possible reasons

I

I
t'

1 'The Oak Ridge control data currently stand at three
independent mutations (one of which was a cluster of six)among 166 826 progeny. This, combined with the earlier data of
Batchelor et al. (28) (no mutations among 37 813), bring thetotal number of progeny scored to 204 639. Based on themethod used in the 1972 report (Annex E, paras. 145-146) a
minimal and an upper rate of spontaneous mutations can be
estimated. n,ese are, respectively, 2.1 10 ·• per locus per gameteand 5.6 10 ·• per locus per gamete.

464

L..

~

for this discrepancy: (a) the stocks of mice used(originally descended from those of Russell) have cometo differ in the twenty-year period since they were
founded, and (b) there was a difference in the dose rates
used: about 90 R/min in W. L. Russell's experiments andabout 53 rad/min in those of Lyon and Phillips.
L. Russell (467) has pointed out that when his
oocyte data are split up into those pertaining to "older"and '-younger" females the discrepancy between his data
and those of Lyon and Phillips is very much reduced.

w.

(b) Dose-rate effects
290. One of the difficulties in measuring the effect of
protracted irradiation on mutation frequency inmaturing oocytes of mice is that the length of radiation
exposure time necessary to accumulate a sizeable dose
may approach the duration of the oocyte stage being
studied. This was recognized in the earliest work on
mutation induction in females by W. L. Russell. When helater discovered (475a) that the mutation frequency
apparently drops to zero for conceptions occurring morethan six or seven weeks after irradiation, he pointed out,
at that same time, that the lowness of a total mutation
frequency in offspring collected during a period ofseveral weeks after the end of a chronic irradiation
exposure could have resulted partly" from an effect oflow dose rate on maturing oocytes and partly from a
portion of the dose having been given to oocytes in
immature stages, more than six weeks before conception. However. it was shown that a very low mutation
frequency from chronic irradiation, compared withacute, would be demonstrated, even for maturingoocytes, by restricting the comparison to conceptions
occurring within a short time after accumulation of the
dose.

,,'

.

,
:I

:

',Ii

•

'.
'I

291. In order to utilize data collected from conceptions
occurring over a longer period, and still restrict themeasurement to maturing oocytes, it is necessary tocompute the proportion of the dose received while theoocytes are in maturing stages. This was done, in a crude
approximation, in computations used to arrive at thefigure of 1/20 which was accepted in the 1972
UNSCEAR and BEIR reports as the ratio of effects ofchronic to acute irradiation in maturing oocytes.
292. W. L. Russell ( 483a) has now presented previously
unpublished data which have enabled him to make more
precise estimates of the effects of protracted irradiation
on maturing oocytes. The data shown in table 37, werecollected to determine as sharply as possible the timeinterval after irradiation at which mutation frequency
drops, and to find out if this is a sudden or gradualchange. No mutations were obtained in conceptions
occurring more than six weeks after irradiation, and thefrequencies in the fifth and sixth weeks showed no
decline compared with earlier weeks. Thus the drop in
mutation frequency with time after irradiation is very
sudden. Russell also points out that, on the basis ofthese and other data, offspring conceived in the firstweek after irradiation appear to have a lower mutation
frequency than those conceived during week 2-6 after

irradiation.
293. On the basis of the above results, an ''effective
dose" can be computed for offspring conceived atvarious intervals after a period of protracted irradiation.
By "effective dose" is meant the portion of the total
dose that was received six weeks or less before ovulation.
Russell applied this analysis, computing a weighted meaneffective dose, to his own experiments and to the data of
Lyon and Phillips (312) and Carter (84a), as shown intable 38. A mutation rate for protracted irradiation of
mature and maturing oocytes was then determined byfitting a weighted least-squares regression line to thefrequencies for all six irradiation experiments and thecontrol. Actually, four regression slopes were calculated,
one set of two based on all the data and the other set
excluding an experiment that used only old females.
Each set had two fits, one for each of the two different
estimates of the control mutation frequency.
294. The mutation rates estimated in this way for
low-level irradiation of mature and maturing oocytes,
when compared to the mutation rate for his 400-R acuteirradiation data, yield ratios of 1/18, 1/24, 1/29 andI /46. The ratios might have been even lower if the
proportion of oocytes that received most of their dose in
the first week before ovulation (when mutational
sensitivity is somewhat less) had been as high in thechronically irradiated animals as it was in the acutely
irradiated ones. In any case, with the more precise
estimation of the effective doses than was available forthe preparation of the 1972 UNSCEAR and BEIR
reports, and with the addition of the new data of Lyon
and Phillips, it turns out that the Committees'
statements of a ratio of 1/20, for the relative
effectiveness of chronic to acute irradiation, did notunderestimate the risks from low-level irradiation of
mature and maturing oocytes.
295. In his paper, Russell also compared the specificlocus mutation rates obtained for mature and maturingoocytes with the mutation rate for chronic irradiation of
spermatogonia. The mutation rate, for the same sevenloci, in mouse spermatogonia irradiated at dose rates of
0.0009 R/min and below, was calculated in the review
paper by Searle (506) to be 6.59 10-s R- 1 per locus.
The four rates for low-level irradiation of mature andmaturing oocytes estimated by ;ussell are only 0.17.
0.27, 0.33 and 0.44 times as effective, and only in thehighest of these is the induced rate in oocytes
significantly above the control rate. Thus the ratio ofeffectiveness to the spermatogonial mutation rate could
be zero. Similarly, the ratio of effectiveness to acuteirradiation given in the previous paragraph could also be
zero.
296. With regard to the validity of extrapolating
mutation rates in mouse immature arrested oocytes tohuman immature arrested oocytes, which has beenquestioned because of marked differences in thesensitivity of these oocytes to cell killing, Russell
indicates that his data have an indirect bearing on thisproblem. The mutation frequency is lower for the mostmature oocytes, those which produced the offspring forweek 1 in table 37, than for the less mature oocytes that
produced the offspring listed for weeks 2-6. The fully
mature oocytes are less sensitive to killing than the lessmature ones. Thus here there is a positive correlation
between killing and mutational sensitivity. That is incontrast to the negative correlation found for immaturearrested oocytes, which give no evidence of mutation465

induction but which are highly sensitive to killing. It
appears from the lack of consistent correlation, that
mutation induction and killing are independent events.
In support of this view, Russell cites the new data of Cox
and Lyon (121), on x-ray induction of dominant lethal
mutations in mature and immature oocytes ofguinea-pigs and golden hamsters. In both species, theyfound lower mutation yields from immature than frommature oocytes, despite the fact that the immature
oocytes of the guinea-pig are less sensitive to killing thanthe mature ones, while the reverse is true for the golden
hamster, which, therefore, resembles the mouse. Thus, asthese authors state, there is "no general pattern ... ofcorrelation, either positive or negative, in the sensitivityof oocytes to killing and to dominant lethal induction".
297. In Russell's view, there would now seem to be Jessreason than before for rejecting an application of dataon the mouse immature arrested oocyte to the humanimmature arrested oocyte. In the immature oocytes, thespecific-locus mutation frequency in the mouse and thedominant lethal frequency in guinea-pig and golden
hamster are all low compared to the frequencies inmature oocytes. The fact that this is so, despite thetremendous differences in sensitivity to cell killing, andin chromosomal morphology, in the immature oocytesof these species, weakens the contention that thesensitivity to cell killing of the mouse immature oocyte
is an argument against using it to predict the mutational
response of the human immature oocyte.
298. Russell concludes that if, on the side of caution,
one continues to consider the possibility that the humanimmature arrested oocyte might be as mutationallysensitive as the most sensitive of all oocyte stages in themouse, namely the maturing and matuJe oocytes, then
one can use his estimates of mutational frequency fromlow-level irradiation in 'these stages. The rates range from
0.17 to 0.44 times that in spermatogonia, but it should
be kept in mind that in three of the four estimates thefrequencies are not significantly above control values.
Thus. even in the event that the human immaturearrested oocyte does not respond like the mouse arrested
oocyte, but more like the most sensitive oocyte stages in
the mouse, it seems likely that the genetic hazard ofradiation in the female will still be Jess than in the male.

4. Mutation processes at low and high radiation doses
and dose rates: criticism of a current model
299. Since the discovery of the dose-rate effect with
low-LET irradiation for the induction of specific-locus
mutations in mice in 1958 (491), the question ofwhether the phenomenon mainly involves the repair of
only two-track mutational events or primarily the repairof one-track events has been repeatedly discussed (473.
475, 477, 479); In their 1958 paper, W. L. Russell et al.
pointed out that one-track events can show a dose-rate
effect (and a supra-linear dose-effect curve at high dose
rates) if the repair system is damaged or saturated athigh doses and at high dose rates. For various reasonsthat have been enumerated in detail (473, 475, 477,
479; also discussed in the 1972 report, Annex E,
paragraphs 182-195) the hypothesis of repair of
one-track mutational events has been favoured.
466

300. In 1967, Wolff (617) listed several arguments(based essentially on dose-rate effects and RBE
considerations) which in his opinion lend support to theview that mutations scored in specific-locus experiments
are predominantly chromosome rearrangements that
result from two-track events. More recently, this viewhas been modified in papers by Wolff (618) and
Abrahamson (3) and Abrahamson and Wolff (4a) inwhich the mutationed lesions are assumed to be a
mixture of one- and two-track events. Attention will befocused on the last paper since this contains the mostdefinitive account of these authors' views.
301. Abrahamson and Wolff fitted the specific-locus
data obtained in the mouse to an equation of the form
Y = C + aD + {ID2 , where Y is the expected yield of
mutations, C = control rate, D = the dose of sparsely
ionizing radiations, and a and (3 the linear and quadraticcoefficients, respectively. For the oocyte data, the a and(3 terms were estimated by a least-squares regression
analysis of the data obtained at 50 R, 200 R and 400 R(acute x-ray exposures). The estimated values, if acluster of six mutants was counted as one mutation.
were
C=I.6710- 6 ,a=l.2610- 7
R- 1 . andp = 1.14 10-9 R-2 • From these, the expected number ofmutations in other experiments was computed. For
sperrnatogonial data, Wolffand Abra.ham$on used all the
low dose-rate data available (below 1 R/min to derive a,and this was then used with the 300-R acutex-irradiation data to derive (3. The values so obtained
were
C=8.310-6 ;a=6.8810- 8
R- 1
and(3 = 6Si 10- 10 R- 2 • These values were used to calculate
expected frequencies of mutations in the otherexperiments.
302. Considering first the oocyte data, the predicted
and observed mutation freque11cies were similar except
in three low doi;e-rate experiments (in which exposureslasted for 12, 20 and 31 days, respectively) and in anacute irradiation experiment from Harwell (312) inwhich the yield of mutatiqns was lower than expectedfrom Oak Ridge data. Abrahamson and Wolff suggested
that the low observed frequencies in the chronic
exposure experiments "are the consequence of some ofthe oocytes having. been irradiated during the Jessmutable stages rather than resulting from repair of
pre-mutational damage in a static cell population". The
authors made some "crude corrections" to allow for thispostulated admixture with less sensitive stages, whichbrought the expected numbers into reasonable agreement with the data.
303. Turning now to the sperrnatogonial data for acuteirradiation, many discrepancies were found between
observed and expected values. However, these weremainly in single or fractionated irradiation experimentswith individual exposures of more than 500 R and witha general pattern of less than expected yields. The
authors attributed these discrepancies to differential cellkilling and selection, in line with previous views (e.g.,ref. 470).
304. The main implications of this reanalysis for theevaluation of radiation hazards are the following: withsparsely ionizing radiations at low doses and low dose
rates, it is the linear component that predominates, andthe magnitude of the risk of induction of mutationaliidamage will be determined by this. For males. where thehazard evaluation is in fact based on the observed linear
dose-effect relationship (506,617) at low dose rates overa wide range of doses, the above reanalysis adds nothing
new. For females however, the risk (on some methods ofcalculation) will be expected to be higher than what had
been hitherto assumed by UNSCEAR (589) and BEIR
(34).
305. The above model and, especially, the modifications of the mouse data which the authors perform to
fit their model have been criticised by W. L. Russell( 482, 483, 483a). With regard to the model itself.
Russell points out that the data also fit his hypothesis. inwhich the mutational lesions themselves are one-track
events, but multiple tracks are involved in damaging or
saturating the repair process. The data will fit still othermodels. Therefore, Russell argues that the fit of the data
to the model proposed by Abrahamson and Wolff does
not prove their hypothesis to be superior to otherhypotheses which also give acceptable fits. He contends
that other criteria which he has discussed elsewhere(473, 475, 477, 479) provide more cogent evidence on
the na·ture of the mutational lesions.
306. Russell's strongest objection is against themodification of the mouse oocyte data carried out by
Abrahamson and Wolff. The two fits preferred by theauthors fail to give good agreement with the low
dose-rate experiments considered by them, essentiallythe data of Carter and Russell's 258-R and 400-R data
shown in table 38. Abrahamson and Wolff recognize this
and, in an extensive discussion, try to explain away thediscrepancy by claiming that the effective doses in thechronic irradiation experiments are v.ery low, much
lower than the values calculated by Russell (paras. 293294 and table 38). Russell claims that errors arose when
they tried to compute the effective dose on the basis ofnumber of oestrus cycles and an oocyte maturation
scheme which, according to Russell, seems to have nobasis in reality. Thus they compute that the effective
dose in the Carter experiment would be only one half orless of the dose given. The irradiation time in thisexperiment was 12 days, and all matings occurred within
two weeks of the end of irradiation. In view of the datain the table and the well known fact that maturation andloss of oocytes occurs at the same rate in breeding andnon-breeding females (370a), Russell finds it hard to see
how any of the oocytes in Carter's experiment could
have received less than the total dose during thematuring stages that are sensitive to acute irradiation. It
is also noted by Russell that the two mutations in the283-R experiment in table 38 were from conceptions
occurring in the 5th week after the end of irradiation, aperiod long after the time when Abrahamson and Wolff
would have had the effective dose down to zero.
307. Although Abrahamson and Wolff include the new
Lyon and Phillips data for a single 200-R dose of acuteirradiation, they omit any consideration of thefractionated exposures. Russell states that if Abrahamson and Wolff had computed their expected value(assuming cluster= O) for this set of data, it would havebeen 9.5 mutations expected where only 1 was ogserved.
With a correct adjustment for effective dose they would
still have predicted 7 .8 mutations.

308. Abrahamson and Wolff. on the one hand, admitthat their corrections for effective dose are crude, but on
the other hand, conclude "it is reasonable to assume thatthe wide discrepancies [between their theoretical
expectations and the actual data] are caused by some of
the radiation occurring during less mutable stages".
Since the data shown in table 38 have already beenadjusted for this factor using Russell criteria, it is ofinterest to see how much discrepancy is still left between
his corrected observed values and the Abrahamson-Wolff
prediction.
309. Of the two Abrahamson-Wolff fits preferred bythem, the slope of one (cluster= 0) exceeds the slopes ofthe four regression lines derived by Russell (paras. 293294) by multiples of 4.8, 6.5, 8.0 and 12.7, and theslope of the other Abrahamson-Wolff line (cluster= 1)
exceeds the slopes of the Russell lines by 4.3, 5.7, 7.1
and 11.2. Russell calculates that both Abrahamson-Wolff
slopes are significantly above all the slopes of the actual
data with a high degree of confidence. The lowest t value
in these comparisons is 7 .84, where the critical value oft
for 5 per cent probability is 2.57.
310. Thus the Abrahamson-Wolff derivation from acuteirradiation of the mutation frequency theoretically
expected, on their hypothesis, for low-level irradiation
exceeds the observed value by a factor o{ 4-13. Russell
concludes that their approach is not a reliable one for
estimating hazards.

5. Nature of specific-locus mutations
311. The 1972 report considered in some detail thework of L.B. Russell (453), who analysed. by means ofcomplementation tests, the mouse chromosome 9 region
between and surrounding d, se and sv (recombination
frequency d-se, 0.16 per cent; se-sv, 2 per cent). This
analysis revealed the existence of 16 complementationgroups spanning eight or nine functional units. It was
found that (a) there was a strong effect of the irradiatedgerm-cell stage. as well as the type of radiation, on thelocus-spectrum. i.e., on the relative frequencies of events
involving d, se or both, and on the involvement of single
functional units as against that of two or morefunctional units; (b) the frequency· of mutations
interpreted as aberrations (deficiencies) ranged from
13.5 per cent in most x- or gamma-irradiatedspermatogonia to 42:3 per cent in post-spermatogonialstages and 65.6 per cent in oocytes; and (c) therecombinational length of most of the aberrations wasvery small, 75-80 per cent of them spanning less than
two crossover units. Even in the groups that had a hightotal frequency of aberrations (post-gonial stages andoocytes), no more than 23 per cent of all mutations
exceeded this length and the frequency was for x- andgamma-irradiated spermatogonia (excluding the 24-h
fractionation group).
312. The type of analysis discussed in the preceding
paragraph has now been extended by L. R. Russell toanother locus, the c (albino locus), located in linkage
group I, chromosome 7 (213); the mutations used in thepresent work, as in the earlier one on d-se were derived
from the specific locus experiments of W. L. Russell and467

co-workers and were recovered in the heterozygous statewith the cch allelle carried by the tester -stock. Since all
but one of the 103 completely, and 12 partially, tested
mutants are distinguishable in colour from cch, testing
consists of crossing the original mutant to cch Jech andthen intercrossing the heterozygotes, for tests on
viability. and in the case of lethals, time of death.
313. The results available thus far show that (a) among
the 9 control mutants, 8 were viable and l subvital in
the homozygous condition; (b) among 50 mutants
derived from x- or gamma-irradiation experiments(spermatogonia), 35 were viable, 3 subvital and 12 lethal
in the homozygous condition (there was no signifi.canteffect of dose rate, fractionation or of x irradiationcompared with gamma irradiation in the distribution ofmutants in the three classes); (c) neutron irradiation of
spermatogonia appears to_ produce a shift in the sensethat now 7 out of 19 tested mutants are lethal, 1subvital and 11 fully viable; and (d) the data for the
post-spermatogonial and oocyte stages are limited andthe numbers in the subgroups too small to compare thespectra between gamma or neutron irradiation, but, outof a total of 25 mutants tested, 12 are viable in thehomozygous condition and the rest are lethal.
314. L.B. Russell et al (454a, 464, 465) carried out
complementation and deficiency-mapping tests on a
total of 30 independent lethal mutations involving the clocus. It was found that these lethal mutants groupthemselves into at least seven complementation groups.
The authors suggest that at least seven functional units
must be postulated to account for all the interaction
effects on a linear basis. The complementation groups
have been designated as A, A', E, B, C, D, and F. Theirproperties and their relationship to the c locus andadjacent markers used in the complementation study canbe diagrammed as follows:
Mod-2

lP

Pre-Ntal death

G-6-P11e
Neo-nat•I deathL,ter invilbility

Albiniim

M1lic fflZVffll

Pre-naul duth

Pr~NuJ death

A

A'

B

C

D

(G-6-Pase: glucose-6-phosphatase deficiency; Mod-2:
mitochondrial malic enzyme (malate oxidoreductase
decarboxylate ); tp: taupe; sh-1: shaker-I).
315. Five of the c-lethal mutants (A-group) do not
involve Mod-2 (only I cM distant from c).However, thefinding that complementation for neo-natal death canoccur without complementation for albinism andseparate from complementation for various kinds ofpre-natal death suggests that even these mutants may besmall deficiencies involving several cistrons. Since theneighbouring marker tp is not involved, their maximum
length must be less than 3 cM. Among provendeficiencies, the two longest were at least 4 but less than
8 crossover units in length ( 454a). The remainder of thec-lethal mutations were at least I but probably much lessthan 6 cM long. It is likely that the 66 viable c-locus
mutations found are not deficiencies.
316. Four of the 30 c-locus lethal mutants have beenstudied cytologically using both quinacrine-mustard andGiemsa-banding (454a). In one of these, a B complementation-group mutant, no loss of cytological material was
detectable. The two F-group mutants (deficient for sh-1
as well as c) were found to lack most of one of the major
bands of chromosome 7 (the E band), one being slightly
468

longer than the other. One of the D-group mutants lacks
an amount of chromosome 7 that is almost equivalent inlength, but is located more proximally, overlapping with
the F-group mutants with regard to cytologicalsub-band E 1. This serves to locate the c-locus cyto-logically within El and its flanking markers, sh-1 and tp
in E2 ( or E3) and proximal to D 1, respectively. Thisfinding, and evidence from cytological studies of otherchromosome-? aberrations, indicate that relative cytological lengths in that region of the genome may notcorrespond closely with relative genetic lengths (454a).
317. Again, on the basis of complementation tests,
Erickson et al. (160) and Gluecksohn-Waelsch et al.
(195) concluded that all six lethal albino alleles thatthey examined which included three of Russell's, could
be overlapping deficiencies of various sizes at and aroundthe albino locus. The fact that no complementation
could be observed in combinations of c 2 5 H with any of
the other five permitted these authors to infer that c 2 5 H
may be the biggest deletion of all.
318. Using Q- and G-banding methods, Miller et al.
(338) demonstrated cytologically that the above
inference is in fact true: in heterozygous animals, thetwo No. 7 chromosomes could be distinguished from one

another, and the one carrying c 25 H was found to be
7.6 ± 1.5 per cent shorter than its homologue, providing
a direct estimate of the size of the deletion. Since thetotal genetic length of the mouse genome is about
1250 cM (589) and since chromosome 7 makes up about
5 .4 per cent of the haploid genome, a deletion of 7 .6 percent of this chromosome might be expected to be about
S cM long (1250 X 5.4% X 7.6%); this value is in therange of length based on the available genetic
recombination data. However, there are indications thatcytological length may not be a good indicator ofgenetic length for this portion on the genome (seeparagraph 316).

6. Autosomal recessive lethals
319. Since the publication of the 1972 report, somenew data on recessive lethals in mice (spontaneous andradiation-induced) have become available (298, 299,
300, 416). In addition, several specific problems andstatistical difficulties involved in the design andevaluation of experiments on autosomal recessive lethals
have become apparent, such as the presence anddetection of pre-existing lethals in inbred strains and in
the control and experimental groups derived from them,
the basis and magnitude of fluctuations from generation
to generation in the frequency of heterozygotes forrecessive lethals, and what steps need to be taken to
make a proper assessment of spontaneous and induction
frequencies of these lethals (269, 299, 493).
320. In a recent paper on testing for autosomal
recessive lethals, Lfming ( 198) elaborated further on themethodology that he described earlier (296), with areconsideration of several aspects that are involved.
Among these may be mentioned (a) the identification ofindividual lethal heterozygotes, (b) the identification offamilies carrying pre-existing recessive lethals by
analysing 10 or more F 1 brothers, and (c) the varianceof the rate of heterozygosity for recessive lethals in
inbred strains in successive generations and its relevancefor estimates of the mutation rate per genome.
321. The general scheme for individual and familial
identification of recessive lethals can be diagrammed asfollows:

P1 generation

dX9

F I generation

c5 X 999 (at least 10 F I sons,I.

F 2 generationjper parental crossto be tested)

999 X F I father (backcross)

Uterine contents of pregnant females are
examined to assess the amount of pre-natal
mortality; identification of heterozygosity forlethals dependent on the rate of intra-uterinemortality, which is expected to be higher incrosses where a lethal is involved, than in thosewhere it is not.

If an F I male is heterozygous for a recessive lethal, thenSO per cent of his progeny will carry the same lethal.

therefore, in a backcross involving father-daughter
mating, the expected mean mortality is 12.S per centover and above the normal mortality of 8 per cent, or a
total of 100-(0.875)92 = 19.S per cent. In practice, thisinvolves the identification of each individual case as
belonging to a distribution with a mean mortality of 8 or
19.5 per cent, the precision of which is dependent on
the number of implants examined per backcross.
322. The model presented by Liining (298) enables theidentification of lethal heterozygote·s and lethal-freeanimals in the backcross test of F I males to theirrespective daughters. According to the model, when aminimum of 50 implants (50-60: mean, 53) are analysedfor each backcross of the F I male to his daughters, thosewhich gave 9 or more dead are judged to beheterozygous for a recessive lethal; those crosses which
resulted in 4 or less dead are considered lethal-free. An
increase of the number of implants analysed increases
the accuracy of the above procedure to identify lethal
heterozygotes and lethal-free animals.
323. It is known that in inbred strains of mice there arelarge variations in the spontaneous frequencies ofautosomal recessive lethals between one generation andthe next. The earlier analysis by Liining and Searle (302)of three sets of control data (controls for radiation
experiments) revealed considerable differences in frequencies between them, and the authors estimated thatthe mean rate was 2.9 10-3 with an upper 95%
confidence limit of 6.5 10- 3 per gamete. In a
subsequent paper, Li.ining (299) examined the control
data accumulated over the years 1964-1971 in hislaboratory (and which pertain to four different series,
including the three that were considered by Liining andSearle) and concluded that the mean rate was 5 10-3 per
gamete with an upper limit of 1 10-2 • In relatively small
experiments involving three strains of mice (CBA, C3H
and 101) and their hybrids, Lyon (306) likewise foundvariability in the frequencies of autosomal recessive
lethals and stated that "allowing for sampling error, theresults are not incompatible with the hypothesis that in
all three strains, recessive lethal genes arise with afrequency of 0.1 per gamete" (an approximate upper
limit).
324. Haldane (211) first drew attention to the fact that,
although spontaneous recessive lethals arise at a finite
rate, the average frequency of heterozygotes for lethals
can vary within a strain depending on how the parents
are selected to prepetuate the strain and dealt with the
problem from a theoretical standpoint. In the case wherea pair of full-sib parents heterozygous for the same
recessive lethal leave three quarters as many offspring as
the others not carrying the lethal. he showed that the
average accumulation of lethal heterozygotes is equal to9.3 µ where µ is the mutation rate per genome. In theextreme case where the number of offspring fromheterozygous parents is not reduced at all, the averageaccumulation would be equal to 12.7 µ. At the otherextreme. where the heterozygous parents leave nooffspring, Lyon (306) showed that the averageaccumulation would equal 6.0 µ. Thus, depending on thebreeding scheme. the average frequency of lethal
heterozygotes in an inbred strain would be between 6.0
and 12.7 µ. These calculations. however, are based on469

the assumption that the strain in question is constitutedby an ''infinite" number of breeding pairs, a theoreticalsituation. In practice, due tq economic and spatial
reasons, a given strain is propagated with only a limited
number of breeding pairs. This restriction will cause
drastic fluctuations in the frequency of heterozygotes
for lethals in the strain when sampling is done atdifferent periods of time, as has actually been observedin the experiments discussed by Liining and Searle (302)
and by Liining (299).

breeding pairs and/ or an increase in the number of litters
per pair leads to a decrease in the variation and thenumber of lethal-free generations also becomes reduced.

325. Ryman (493) conducted a computer-simulation
study to investigate the magnitude of temporal variationin the frequency of heterozygotes for autosomal
recessive lethals in inbred strains, a problem to whichlittle attention has been devoted so far. The simulation
studies were performed with special reference to themouse with the following assumptions: (a) the numberof breeding pairs for strain continuation is 10, 20 or 50;(b) each pair produces 1 or 5 litters in every generation;(c) the minimum number of live-born young from a pairwhose offspring can be selected to continue the inbred
strain is 6 or O (I-litter situation) and 30 or O (5-Jitter
situation) {The offspring not used to form breedingpairs in the strain were "surplus" and used for"experimentation", and the frequency of lethal
heterozygotes was determined in the "surplus" group.);(d) the sex ratio is I to 1; (e) the mutation rate of
autosomal recessive lethals per generation and the
pre-weaning mortality are Ll.5 and 16 per cent,respectively, these values being the long-term averages
for the inbred CBA strain in the Stockholm Laboratoryof Radiation Genetics; (f) lethal homozygotes die before
birth.

(b) Since heterogeneity within an inbred line
may be considerable, the use of an inbred strain does not
automatically guarantee a homogeneous genetic background;326. The computer was programmed to trace thetemporal pattern (over 200 or 500 generations) in thefrequencies of heterozygotes for lethals under thedifferent conditions mentioned above and to estimate
the means and variances. The results showed that, withthe 200-generation trials with "surplus" animals, there
were large and sudden changes in the frequencies oflethal heterozygotes (0-40 per cent) under conditions
when the number of breeding pairs per generation was
10. This variation became much less (0-12 per cent)when the latter was 50. The means over all generations
and their respective variances were 0.046 ( 4.6 per cent)and 0.00573 with 10 breeding pairs and 0.038 (3.8 percent) and 0.0006 with 50 breeding pairs. The estimatedvariance of the differences between consecutive
generations was, as would be expected, much higher
under the first set of conditions than under the second(0.00878 compared with 0.00092).
327. The results of simulations for 500 generations(with different maximum number of breeding pairs forthe continuation of the inbred strain) revealed that witha low number of breeding pairs (such as 10), the varianceof the mean frequency of lethal heterozygotes, as well asthat between successive generations. is quite large,
irrespective of the number of litters per pair and ofwhether selection against small litters is practised or not.
Furthermore, the number of generations in which there
were no lethal heterozygotes ("lethal-free'' generations)
was also appreciable. An increase in the number of328. The practical implications of these findings are thefollowing:(a) Since the maintenance of an inbred line is
not an end in itself. great care must be taken in choosing
an optimal breeding regime in relation to the type ofexperiments that are proposed to be conducted:(c)Estimates of spontaneous or induced rates ofmutations to recessive lethals will be reliable only whenthe experiment is repeated at different time intervals or
when pre-existing mutations are identified and taken
into consideration:.
(d) lfthe strain is not very large, the replications
are expected to yield statistically heterogeneous results
and it is justifiable to pool the results obtained with agiven strain at different time intervals to get a realistic
estimate of mutation rates, as was done earlier by
Liining and Searle (302) and by Liining (299).

329. New data on the x-ray induction (500 R acute) ofautosomal recessive lethals in mouse spermatogonia
come from the experiments of Uining and Eiche (300)and those of Pomerantzeva et al. (416). Uining andEiche used the technique outlined in paragraph 310 andfound that the rate of induction (after correcting for the
spontaneous rate of 0.5 10-3 per gamete) was 0.9 10-4
rad -I per gamete. This is in complete agreement withthe earlier estimate made by Lnning and Searle (302)
based on three different sets of data (see the 1972 reportfor details). In the work of Pomerantzeva et al. (416) a
total exposure of 900 R of gamma irradiation was
administered to male mice in three equal fractions
separated by 4-week intervals, and the frequency ofautosomal recessive lethals was determined. In one of
the two experiments, the induced frequency was
22.65 per cent (or 2.5 10-4 R- 1 ) and in the other, 5.4
per cent ( or 0.6 10-4 R -I). Pooling the controls andlikewise pooling the radiation data, the induced
frequency can be estimated as 9.93 per cent whichcorresponds to a rate of ! . I I 0-4 R -I • As will beobvious, this rate is quite close to that recorded by
Liining and Searle (302) and by Liining and "!:iche (300).

7. Dominant mutations
330. Since the publication of the 1972 report. somenew data have become available from studies on
dominant mutations in mice. These pertain to the workof Selby and Selby (528), who investigated the
gamma·ray induction and transmissibility of dominant
mutations affecting the skeleton. Before considering theresults of Selby and Selby. it is profitable to examine thebackground of this kind of work and place it in theperspective of earlier work done in this area.

470..........

33 I. The rationale was, and continues to be, based on
the following considerations:(a)
For the evaluation of genetic radiation
hazards to a population, it is desirable to obtain more
information on the frequency of induced dominant
mutations;(b} Information on dominant visible mutations
which has been generated as a by-product of mutation
rate studies with specific loci continues to be limited;(c) The skeleton is a complex system formed
over an extended period of development and differentiates in specific ways in various parts of the body toform permanent structures. It is therefore subject tomodification by gene action at very many loci;{d) Consequently, studies on the , radiationinduction of dominant genetic changes affecting theskeleton are useful because they uncover a portion,
perhaps a substantial one, of the overall genetic damage
induced by irradiation.

332. In their first studies, Ehling and Randolph {147)showed that, in skeletal preparations of F I progeny ofirradiated male puce (x rays and neutrons; pre- andpost-meiotic germ-cell stages), it was possible todistinguish between the effects of newly induced skeletal
variation and that which is normally present in thestrains employed. Thus, they demonstrated the feasibilityof using skeletal abnormalities as a criterion ofradiation-induced, presumed dominant genetic damage
and set the stage for further work ( 149, 150). The
abnormalities were classified into Class l (those that
occurred only once in the whole experiment) and Class 2(those that occurred more than once). The further
subdivision of these classes can be diagrammed asfollows:
Class 1

Class 2SingleAxial
skeleton

Multiple
Appendicularskeleton(.

I

Bilateral

Axial
skeleton

AppendicularskeletonI

Unilateral

333. Since the sample sizes in these experiments weresuch that not more than one mutation would be
expected from any particular gene locus, it was thoughtthat the classification into Class 1 and Class 2 would
provide for the optimum separation of the existing
natural variation from that caused by newly occurring
genetic changes. The results showed that there was astatistically significant increase in the incidence ofClass 1 abnormalities after irradiation; among these,
multiple abnormalities (those with at least one Class 1
abnormality plus others. regardless of whether Class 1 orClass 2) and single bilateral abnormalities of theappendicular skeleton were considered to provide themost sensitive indicators of mutational damage. There
was no significant increase in Class 2 abnormalities after
irradiation.

334. In the earlier experiments of Ehling (149, 150),
since the F I mice were killed for making skeletal
preparations at four weeks of age, breeding tests of thepresumed mutants could not be carried out. Subsequently, however, Ehling (151) designed experiments
to permit such breeding tests. the F I offspring being
sacrificed only after they had produced a litter; 3 out of
5 presumed mutations were found to be transmitted tothe second and later generations. In similar work carriedout 'by Tutikawa (quoted in the Committee's 1972
report) in which 11 presumed dominant skeletal
mutations were observed, 2 which showed externallyvisible effects were given breeding tests and both were
found to be transmitted. Since the tran~mission of only
a few skeletal mutations had been studied at the time ofwriting the 1972 report and the transmission· pattern ofa majority of them did not permit an unequivocal
conclusion of dominance. the Committee stated, "Itseems probable that many of the presumed dominant
mutations may be heterozygous manifestations ofrecessive mutations".
335. In their work Selby and Selby (528, 528a) focused
attention on the problem which had hitherto not beenstudied in depth, namely, that of the transmissibility ofthe presumed dominant mutations affecting theskeleton. Male mice received a fractionated gammairradiation exposure (100 R + 500 R, separated by 24 h;
60 R/min; the choice of this radiation regime wasdictated by Ehling's finding ( 150) that this gave maximal
response for spermatogonial irradiation). The F 1 sonssired subsequent to the sterile period were processed for
examination of their skeletons after they were allowed
to produce progeny. Thirty-seven of the 2646 F I males(1.4 per cent) were judged to have dominant mutations
that caused one or more rare skeletal abnormalities: 31of these were shown to be mutants by breeding tests andthe remaining 6, having no progeny, were counted as
mutants based only on criteria .supported by the data.
The frequency of 1.4 per cent recorded in this study issimilar to the 1.8 per cent (5/277) obtained by Ehling(150) under similar radiation conditions.
336. In breeding tests, Selby and Selby found that thedominant mutations affecting the skeleton showedvariable expressivity and incomplete penetrance formany or all of the effects that they caused. A number ofthem severely affected viability. With the experimental
procedure used, both incomplete penetrance anddecreased viability would have caused an underestimateof the mutation rate. Thus, some of the F 1 's would not
be counted as mutants because they do not show theeffect of the mutation they carry and others would not
be counted because the mutation they carried caused
their death before they could be examined.
337. In comparing the results of Selby and Selby (528)with those of Ehling (149, 150), the following
comments are in order. Ehling expected that a critical
determination of which of the skeletal abnormalities are
actually heritable would reveal that his criteria for
detecting presumed mutations would, on the one hand,miss some mutations altogether and, on the other hand.
wrongly classify some abnormalities as mutants when infact they were not. The degree to which these errors
might occur was not known at the time Ehling471

conducted his studies, but it was hoped that they would
not be large. The finding of only one presumed mutationin the control group of Ehling (1 / 1739) indicated thatthe second kind of error could not have contributed
substantially to the large number of presumed mutations
observed in the experimental groups. The work of Selby
and Selby (528) indicates that, as expected, the use of
Ehling's criteria would lead to some false positives andsome false negatives when estimating the mutation rate.
It is difficult to estimate, from the Selbys' data, theextent of the two kinds of error, but it appears that theymight have approximately balanced each other, since
there is close agreement between the total rates obtainedby Ehling and the Selbys.
338. The mutation frequency in the experimental seriesof Selby and Selby is 1.4 per cent. No controls were run,since the main thrust of their work was to find out if thegeneral types of skeletal abnormalities classified aspresumed mutations by Ehling's criteria would in fact betransmitted to descendants. However, the authors pointout that, although a reliable estimate of the spontaneous
mutation frequency needs to be made, Ehling's results
indicate that this frequency is very low and would
probably not appreciably affect the assumption that 1.4
per cent is close to the induced rate. The authors further
conclude that, for the reasons given in paragraph 324,
taking 1.4 per cent as the induced rate is more likely tobe an underestimate than an overestimate of the true
value.
339. While it is true that the number of dominant
mutations of spontaneous origin present in any
generation must be considerably higher than the
spontaneous mutation frequency per generation, owing
to accumulation in the ancestral mouse populations, it isimportant to realize that the experiments of Ehling andof the Selbys were designed to exclude most, if not all,such accumulated mutations in the mutation frequencies
recorded in the F 1 • For example, in Ehling's
experiments any abnormalities caused by mutations
accumulated in the stocks should have occurred morethan once in his large samples. Thus, they would havebeen classified as Class 2 abnormalities, and the animals
containing them would have been considered non·mutants. In the Selbys' . experiments, skeletal examination of the parents, as well as the sibs, of the mutant
individuals was performed and supported the conclusionthat most, and perhaps all, of the reported mutations
were of new origin.
340. In humans, partial trisomies and monosomies areoften associated with skeletal abnormalities (see, forinstance, references 208 and 349). Although it cannot be
ruled out that some of the skeletal abnormalities in themouse may have resulted from partial trisomies andmonosomies. it is unlikely that such chromosomal
aberrations made an important contribution to thenumber of mutations reported, because most of theseaberrations are male sterile and derive from spermatozoa! rather than spermatogonial irradiation (507).
Thirty-one of the 37 mutants reported in the Selbys'
experiment proved to be fertile.
341. Many of the anomalies found in the above study
are similar to rare dominant and irregularly inherited
dominant conditions in man. As will be recalled(chapter I), regular dominant and irregularly inherited
dominant diseases (together with multifactorial ones andcongenital anomalies) constitute a very substantial
proportion of human disorders. Studies on the effect ofradiation in inducing mutations affecting the skeleton ofmice may thus lead to an independent and direct
estimate of the rate of induction by radiation of
dominant mutations in man.
342. Bartsch-Sandhoff (27) studied the incidence ofskeletal abnormalities in mouse embryos 19 days oldderived from germ cells of irradiated males ( 600 R of
1 3 7 Cs gamma irradiation). This work was undertaken toverify the presumption that some of the skeletal
mutations may be incompatible with post-natal survival
and would not have been represented in thefour-week-old mice examined in the earlier experimentsof Ehling and Randolph (147) and Ehling (149, 150). It
must be stressed that not all Class 1 abnormalities infour-week-old mice are presumed dominant mutations as
determined by Ehling, and that only the total frequencyof Class 1 abnormalities is comparable with the data of
Bartsch-Sandhoff.
343. The small sample sizes in the Bartsch-Sandhoff
investigation permit only limited quantitative conclusions. The incidence of Class 1 anomalies for theirradiated postspermatogonial stages (9/371) is significantly higher than that for all unirradiated controls(5/1190 in 19-day foetuses: 14/1739 in four-week-old
animals), and not significantly different from theincidence (18/569) scored in four-week-old mice for thesame irradiated cell stages. The frequency after
spermatogonial irradiation (4/343) does not differ
significantly either from the controls or from thecorresponding frequency in four-week-old mice
(13/597). However, there was an important qualitative
conclusion. In the 19-day foetuses there were several
new abnormalities hitherto undetected in four-week-old
animals, some of which were so drastic that they could
conceivably have caused death before four weeks of age.
344. Comparing the results of the Bartsch-Sandhoff
study with those obtained by the other investigators, it
can be concluded, on the one hand, that Ehling and the
Selbys would not have seen those skeletal abnormalities
which, although apparent in foetal stages, were too
severe to survive to the ages studied by them. On theother hand, most of the anomalies scored by Ehling and
the Selbys would not have been detected in the 19-day
foetuses, owing to the limited degree of ossification
present at that stage. Thus the total load of dominantskeletal abnormalities induced by radiation is likely to
be greater than the number which can be detected at any
one particular stage.
8. Summary and conclusions
345. In the germ cells of new-born male mice,mutations at seven sfecific loci are induced by x rays at
the rate of 1.37 10- R -I perlocus, which, statistically.
is significantly lower than the 2 .91 10 - 7 R -I per locus
recorded for spermatogonia in adults (300-R level inboth). In young males 2-35 days old, the rate is
2.63 I O-, R -i per locus.

472

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dntlfliStered to male mice in two equal fractions
3 ennatogonial exposures) separated by intervals of 4(s~ 7 days. the frequencies of specific locus mutations:inbserved are s1"mil ar to those seen m. Iong- t erm~ractionation experiments (interval between fractions
bout 15 weeks) and are consistent with the additivity~f yields from two separate fractions. These frequencies,
however, are higher than those after a single exposure of
JQOO R and lower than -that after two fractions of
500 R separated by a 24-h interval.
347. The limited data that have been collected on theradiation induction of biochemical variants using nineelectrophoretically detectable markers in the mouseshow that such mutations can be induced inspermatogonia. Likewise, studies on the induction of
haemoglobin variants (at two loci) by x-irradiation ofmice (spermatogonia, post-spermatogonial stages andoocytes) have yielded positive results: a total of fivehaemoglobin variants have thus far been found in asample of over 8000 F 1 animals screened.
348. In mouse spermatogonia, the rate of x-ray
induction of mutations in a large group of histocompatibility loci is very much lower than that at the sevenspecific loci.

349. The reanalysis of the data on dose-rate effects forthe induction (by low-LET irradiation) of specific locus
mutations in mouse spermatogonia by Lyon andcolleagues raised the possibility that at dose rates below
0.03 rad/min the mutation rate might be higher thanthat at 0.8 rad/min. Recent work that is being carriedout by W. L. Russell at Oak Ridge to test this (at rates of
0.0007 R/min and 0.0056 R/min, 300-R gamma-ray
exposure) shows, however, that in over 40 OOO and74 OOO F I progeny so far scored at these two exposure
rates, the mutation frequencies are the same.
350. Histological studies carried out by Oakberg andPalatinus demonstrate that the survival of type A5
spermatogonia (stem cells) is practically unaffected
when a gamma-ray exposure of 300 R is delivered to thetestis at a rate of 0.0007 R/min, whereas it is reduced to37 per cent of that in controls when the irradiation isdelivered at 0.0056 R/min. This result, viewed inconjunction with the mutation data reported in thepreceding paragraph, suggests that the cell-killinginterpretation of Lyon et al. proposed to explain thepossible differentiaJ yield of mutations at low and lower
dose rates may not be tenable.
351. In female mice, fractionation of an x-ray dose of
200 rad into several small fractions leads to a very
pronounced reduction (an order of magnitude or more)in the yield of specific-locus mutations relative tounfractionated irradiation. These data, along with theearlier data on chronic irradiation of the same oocyte
stages, indicate that the mutation rate in mature andmaturing oocytes is lower than that of spermatogonia.
The results therefore support the conclusion that, even if
the mutational sensitivity of the human immaturearrested oocyte is as large as that of the most sensitive
oocyte stages in the mouse (the mature and maturingones), the genetic hazards. under most conditions ofhuman exposure, will be Jess in the female than in the
male. At the same time. there appears to be stronger
evidence than before for assuming that the immaturearrested oocyte in the human may. like the same stage inthe mouse. have a mutational sensitivity which, at most.
is much less than that of the male.
352. Abrahamson and Wolff have attempted to fit thedata on radiation-induced specific-locus mutation
frequencies observed in mice to a simple quadratic
equation. For chronic exposures of mature and maturingoocytes, the quadratic fit gave much higher expected
frequencies than those actually observed. The authors
attributed these poor fits to an admixture ofmutationally less sensitive immature oocytes into thesampled population. However, W. L. Russell has shown,in several separate experiments, that the duration of thesensitive stage is longer than that assumed byAbrahamson and Wolff and that, therefore, theirargument is not tenable. Thus, the mutational response,even of this sensitive stage, is low under conditions ofprotracted irradiation, and is not as high as theoretically
predicted in the fit of Abrahamson and Wolff. In any
case, this stage is not likely to be the best model forassessing the mutational response of the humanimmature arrested oocyte .
353. The analysis of the nature of specific-locus
mutations (spontaneous and radiation-induced) which
had earlier been carried out for mutations at the d-se
region have now been extended to the c locus. A
majority of the c locus mutations derived from x- or
gamma-irradiation experiments (sperrnatogonia) arehomozygous viable. Complementation tests haverevealed that the lethal mutants group themselves into
seven complementation groups.
354. Recent work on mice with spontaneous rate oforigin of autosomal recessive lethals and their induction
by x irradiation has produced results which confirm
those presented in the 1972 report of the Committee:
they show that the spontaneous rate is about 0.5 10-2
per gamete and, at the 500-R level, the rate of induction
is 0.9 10-4 R -i per gamete. ·In addition, computersimulation studies designed to in\lestigate the magnitudeof temporal variation in the frequencies of heterozygotes
for autosomal recessive lethals in inbred strains haveshown that the frequencies can vary over an enormous
range in the different generations and that the use of an
inbred strain does not automatically gwqantee genetic
homogeneity. Because of this, in experiments aimed atdetermining spontaneous or induced frequencies ofrecessive lethals, care must be taken to ensure that thestrain (from which the animals are derived forexperimentation) is maintained with an adequate
number of breeding pairs in every generation andfurthermore. the experiments ought to be repeated at
different time intervals.
355. Substantial progress has been made in the study ofradiation-induced skeletal mutations in the mouse. The
data show that the frequency of presqmed mutations
detected in the earlier work of Ehling is closely
paralleled in studies by Selby and Selby in which the473

actual inheritance of skeletal abnonnalities of a similar
type was detennined by breeding tests. Another findingof considerable importance is that most of the mutations
have pleiotropic effects with various degrees ofpenetrance.

G. EFFECTS OF INCORPORATEDRADIOISOTOPES
356. In its 1966 report (587) the Committee briefly
considered the genetic effects of internally depositedradioisotopes such as 90 Sr (in mice), 14 C and 3 H (astritiated thymidine and tritiated deoxycytidine (inDrosophila)). and in the 1969 report (588) someattention was given to the incidence of chromosome
aberrations in humans occupationally exposed toradioactive substances. Since then, interest in the studyof effects of radioisotopes has increased, in part
stimulated by the growth and anticipated expansion of
the nuclear power industry. Results of experiments withmice on the genetic and cytogenetic effects of 2 3 9 Pu
and 3 H have now become available, and these will be
discussed in the following paragraphs.

(b) Induction of post-implantation loss through2 3 9 Pu treatment of male mice
359. Uining and Frolen (301) and Liining, Frolen and
Nilsson (304) have reported on the induction of
dominant lethals in male mice following the injection of
plutonium salts. In the first series of experiments (Eland E2), in which the males received 2 3 9 Pu nitratesolution (intravenous injection). there was no evidenceof any significant increase in intra-uterine mortality overthat observed in controls {I O255 and 7216 implants
were analysed. in the experimental and control groups.
respectively). The situation was different however, whena solution of the citrate was used (experiments E3, E4
and ES). In these studies. the effects were examined over
the range 0.05-0.5 µCi per mouse. After injection, eachmale was mated to three females each week for up to
24 weeks. Appropriate controls were run. The pregnant
mice were dissected 18 days after the commencement of
the matings for examination of their uterine contents.
The number of living and dead implants was recorded:
the latter group was further divided into early deaths (nofoetus discernible) and late deaths (dead foetuses). In
certain groups, the pregnant mice were allowed to go toterm and the F 1 male progeny were used in dominantlethal and semi-sterility tests.

1. Plutonium-239(a) Distribution of 239 Pu in the mouse testis
357. Green et al. ( 199) studied the distribution of
intravenously-injected 2 3 9 Pu (as a solution of the citratesalt) in the mouse testis. Four or 12 weeks after injection(a mean activity of 88 µCi per mouse, 3.2 mCi/kg bodymass), the mice were killed and their testes suitably
processed for radiochemical measurements and autoradiography. It was found that the amount of 23 ~Pu in
the testes was roughly the same with the two intervals
used (mean amount, (280 ± 45) pCi/g of testis and(350 ± 38) pCi/gm of testis after 4 and 12 weeks
respectively). The results confirmed the earlier findingsof Ullberg et al. (585) in demonstrating that the
distribution of 23 9 Pu in the testis was inhomogeneous:in autoradiographs, about 90 per cent of the plutonium
seemed to be deposited within the intertubular spaces
and in the peritubular tissues surrounding them. Of the
12 OOO alpha tracks counted, 42 per cent lay above
the peritubular membrane or adjoining the layer of cells
containing the spermatogonial stem cells, 47 per centlay entirely above the intertubular spaces, and 11 percent lay entirely. within the peritubular membranes, but
excluding those in the first group.
358. On the assumption that the testis consists of aclosely packed series of cylinders of uniform diameter(the experimentally determined mean diameter was
206 µm), the authors calculated that the 10-µm shell oftissue surrounding each tubule and containing thespermatogonial stem cells constituted J 7 per cent of thewhole testis mass, yielding an estimated doseinhomogeneity factor of 42/ l 7 = 2.5, i.e., the radiationdose rate to spennatogonial stem cells is higher by afactor of 2.5 than the average for the testis as a whole,calculated directly from the total amount of 2 3 9 Pu
deposited.
47.4

360. The data on the induction of dominant lethals inplutonium-injected males are given in table 39. The
figures given are pooled over successive weeks. It can beseen that in the plutonium-injected groups, there is asignificant excess of intra-uterine mortality, and this isunrelated to the amount of injected plutonium per male(range, 0.05-0.5 µCi). When the results of all theplutonium series (E3, E4 and ES) were combined(ignoring the amounts of Pu injected) and the pattern ofmortality over the successive· weeks was examined(figure 2 in reference 304). it was · found that themortality increased from an initial value of about 9 percent (first week) to about 12-13 per cent during the fifth
week, after which the changes were small andnon-significant. The sperm utilized during the first five
weeks would have been in post- and peri-meiotic stages
at the time of injection while those utilized subsequently
would have been in earlier stages. It thus appears thatthe increased mortality observed during the first five
weeks is due to the induction of dominant lethality in
post- and peri-meiotic stages of spenn development.
361. Another interesting finding that emerged from theanalysis of the weekly records of mortality in theplutonium series was that there was a trend for an
increasing proportion of late deaths in later weeks, one
which was opposite to that found by Bateman (29) with
x irradiation (200 R). The causal basis for the increase inlate deaths is unknown at present.
362. Dominant lethal tests performed on F I males siredby fathers which received plutonium injection (andderived from matings during the ninth, fourteenth andsixteenth weeks) showed that here again there was (a) an
increase in intra-uterine mortality relative to controls and(b) a trend for an increase in the proportion of latedeaths.

363. In studies primarily aimed at comparing thecy tooenetic effects of protracted exposures to alpha"·239p u and to gamma rays from 6oc o,particles
fromSearle et al. (516) collected some data on the inductionof dominant lethals. In the plutonium series, male mice
were intravenously injected with 23 9 Pu citrate so1ution( 4 µCi/kg body mass, or about 0.1 µCi per mouse) andkept for 21. 28 or 34 weeks. The estimates of theabsorbed gonadal dose and dose rates (based onradioactivity counts and testis mass determinations at
the end of the exposure periods) were 13 ± I rad(0.090 rad/day),18 ± 2 rad(0.095 rad/day)and18 ± I rad (0.088 rad/day). respectively. The means
were 17 ± 1 rad and 0.088 rad/day. (The fraction of
injected plutonium actually retained in the testis was
w1changed from 21 to 34 weeks at 0.027-0.028 percent.) In the gamma-ray series, the exposure period was
28 weeks, with an accumulated dose of 1128 raddelivered at a rate of 0.004 rad/min.
364. In both series, the irradiated males were mated tofemales during the last four weeks of exposure, fordominant lethal tests. In the gamma irradiated series,
there was good evidence for the induction of dominantlethality. with a marked increase in both pre- andpost-implantation losses, relative to controls. The
induced frequency of post-implantation losses was
12.9 per cent. In the plutonium experiments, there wasno significant heterogeneity between the amounts ofembryonic lethality for the three exposure periods. The
pooled data on post-implantation mortality suggested aslight non-significant increase, relative to controls. If thedata are taken at their face value, the frequency of
induced dominant lethality is 4.6 per cent.

367. It was found that the fractiQn of injected
plutonium retained in the testes changed very little overthe period of the experiment: therefore. the radiationdose was taken to be the product of the assay-derived
dose rate (~.0002 rad/min) and the_ time interval
between injection and killing. The estimated doses andthe results of cytological analysis of the spermatocytes
are summarized in table 40. It can be seen that (a)although the frequency of cells with fragments washigher in each experimental group than in the controls,
the difference between the overall fragment frequency
after treatment, 3.4 ± 0.7 per cent. and the control
frequency, 1.3 ± 0.7 per cent, did not quite reach asignificant level (P = 0.07): {b) there is a significant
increase in the frequency of cells with quadrivalentconfigurations in the plutonium series: while most ofthese were rings and chains of four arising from thechromosomal reciprocal translocations in spennatogonia(as in earlier low- and high-LET radiation studies). a few
were similar to those observed after x irradiation ofoocytes in female mice (513) and were considered to be
the result of chromatid interchanges in spermatocytes
during meiotic prophase: and (c) the yield of quadrivalents declines at the longest exposure period of
18 weeks.

(c) Cytogenetic effects of 2 3 9 Pu in male mice

368. The data on reciprocal translocations were used by
Beechey et al. to obtain a rough estimate of the RBE ofalpha particles relative to chronic gamma and neutron
irradiation (0.7 MeV). For this purpose, only the data
pertaining to the 6- and 12-week periods were used ( the18-week data were excluded since it was thought thatthe decline in frequency might be connected withpreferential killing of the sensitive spermatogonial cells).
To estimate the period of spermatogonial exposure, theauthors subtracted 13 days from the actual exposure
period, the former being the time taken for the germ
cells to pass through meiosis to metaphase-I. When the"amended" doses of 10 and 25 rad (instead of 14 radand 30 rad; see_ table 40) are used and linearity of
response is assumed, the rates of translocation induction
at the 6- and 12-week intervals are, respectively.
1.010-3 rad- 1 and 1.910-3 rad- 1 • with a mean of
1.45 10-3 rad -i of the average testis doses. However,Green et al. (199) found that the alpha particle dose tospermatogonial stem cells was 2-2.5 times that to thetestis as a whole in their experiments, after 239 Pu
injection. Assuming a factor of 2.5 in the present
experiments (with similar experimental conditions tothose of Green et al.), one obtains a rate of induction of
0.58 10- 3 rad- 1 of spermatogonial alpha particle dose.
This is very similar to that of 0.53 10 - 3 translocations
per rad found after chronic exposures of mousespennatogonia to fission neutrons of mean energy
0.7 MeV (518), which suggests that these two high-LET
radiations have similar RBEs.

366. In their first studies, Beechey et al. (33)
investigated the cytogenetic effects of intravenously
injected 2 3 9 Pu ( 10 µCi/kg body mass in 1% trisodium
citrate solution, about 0.3 µCi per mouse). The mice
were killed 6, 12 and 18 weeks after injection and theirtestes were used for radiochemical determinations (lefttestis) and cytogenetic analysis (right testis). Control
mice injected with the citrate solution alone were
processed likewise.

369. In the next set of experiments from the samelaboratory (details of which were given in paragraph 363), Searle et al. (516) made a direct comparisonof cytogenetic damage induced by chrortic exposures to
2 3 9 Pu alpha particles and to gamma rays, with areduction in the amount of plutonium injected and withlonger exposure periods than in the previous study {33).
As can be seen from an inspection of table 40, in theplutonium series, there were no significant differences in365. Since nearly all the induced dominant lethality inthe irradiation series would be expected to arise in the
meiotic and post-meiotic stages, the frequencies
mentioned above can be divided by the gonadal dose
received in this maturation period of 28 days (meandoses of 161 rad in the gamma series and 2.5 rad in theplutonium series). The rates obtained are: 8.6 10-4
rad- 1 per gamete (gamma) and 1.9 10 1 rad- 1 per
gamete (alpha). At face value, these results thus suggestthat irradiation with alpha particles from plutonium isabout 22 times as effective as chronic gamma irradiation
for the induction of dominant lethals in the germ-cellstages mentioned. It should be noted that thealpha-particle dose of 2.5 rad is that to the testis as awhole. The dose to the germ cells at risk may bedifferent from this because of dose inhomogeneity
resulting from preferential deposition of plutonium in
the interstitial tissue (199) but cannot be calculated atpresent.

475

frequencies of translocations between 21, 28 and
34 weeks of exposure. The estimated mean spermatogonial dose (not taking into · account the inhomogeneous distribution of plutonium in the testis) was
15.5 rad and the corresponding mean frequency oftranslocations, 0.74 ± 0.18 per cent. With gainma rays
(1055 rad to spermatogonia), the frequency was
1.7 ± 0.4 per cent. The induction rates, estimated bysubtracting the mean control frequency (for both theplutonium and the gamma series), were 3.4 10- 4 rad -Iof alpha particles and 1.4 10 -s rad -t of gamma rays(linearity of response assumed). Thus it appears that
irradiation with alpha particles from 2 3 9 Pu is about 24
times as effective as chronic gamma irradiation fortranslocation induction.
370. Other data reported in the paper of Searle et al.
(516) pertain to induction of chromosome fragments,
the rate for which was estimated as 1.2 10- 2 rad -t ,which is over twice as high as the rate of 4.6 10- 3 rad -t
which can be estimated from the results of Beechey etal. (33) with shorter exposures. Relative to chronic
gamma irradiation, the ratio of effectiveness is about 24.
In the same experiment, an alpha-gamma ratio of 22 wasfound for dominant lethal induction (see paragraph 365). Thus. three indices of cytogenetic damage
gave very similar alpha-gamma ratios of 24, 24 and 22,
from which a mean value of 23 can be derived. However.
the frequencies of sperm-head abnormalities were notsignificantly different from that in controls in theplutonium series. whereas in the gamma series, they weresignificantly higher (17.1 per cent compared with 3.9
per cent).
371. The above estimates of relative effectiveness ofalpha particles and gamma rays for the induction of
cytogenetic damage are based on the average dose to thetestis. which is likely to be different from the doses to
the germ cells at risk because of the known
inhomogeneity in plutonium distribution in the testis( 199). The authors felt that it was unwise to try tocorrect for this because (a) no estimates of themagnitude of the inhomogeneity were available except
for sperrnatogonial stem cells and (b) even for these stem
cells. the 2-2.5 correction factor estimated by Green etal. (199) might not apply under the much more
protracted exposure conditions of the present experi·ment. In particular, there were signs of aggregation of
the plutonium deposit (198), which might decrease theeffective alpha particle dose to the stem cells and thus
could help to account for the much lower rate oftranslocation induction by alpha particles found in thepresent experiment (3.4 10- 4 rad -t) than in theprevious one of Beechey et al. (33), namely, 1.45 10- 3
rad- 1 . In any event. the present results confirm the higheffectiveness of 2 3 9 Pu alpha particles in inducing
translocations, even with very protracted exposures.
2. Tritium (3 H)(a) Induction of dominant lethals in mice
372. Carsten and Commerford (81) and Carsten andCronkite (80) have published the results of their studies
on the induction of dominant lethals in mice
(random-bred, Hale-Stoner-Brookhaven strain) fed with476

tritiated water (HTO). The HTO test animals werefirst-litter mice resulting from breeding of eight-week-old
animals that had been maintained on HTO (3 µCi/ml)
since weaning at four weeks of age. The control animals
were first-litter mice taken from the colony andmaintained on tap water. From the second generation
animals. four experimental groups were established fordominant lethal tests. Group l consisted of animals
where both the male and female were on HTO. Group 2
females received HTO. males, tap water. In group 3, thesituation was the reverse of that in group 2. and group 4
received only tap water (both males and females). At
eight weeks of age, in each group. each male was matedto five females for a 5-day period, and 15 days after the
mid-point of this breeding period, the females were
killed and their uterine contents examined for assessing
dominant lethality.
373. The results, based on 366 pregnant females in thecontrols, 764 in group 1, 315 in group 2. and 316 ingroup 3, clearly demonstrated that dominant lethals are
induced by HTO in both sexes. Significantly fewer viableembryos were found when either both mating partners
or only the female was maintained on the tritium
regimen. Similarly. when both the partners were on
tritium, the incidence of early death (dark mole) issignificantly higher than in the control group. Treatmentof the males only gave similar effects, but these were notsignificant. When post-implantation mortality (early plus
late deaths in the authors' terminology) is used as thebasis for comparison, the increased mortality due to
HTO in groups 2 and 3 is of the same magnitude in both
sexes, and in group 1 (both sexes on HTO) the effect isnearly twice that in groups 2 or 3. Current experiments
are directed at repeating these studies with a lower
concentration of 1.0 µCi/ml.

(b) Induction of specific-locus mutations
in male mice
374. Cumming et al. (128) have completed the firstseries of experiments on 3 H-induced specific-locus
mutations in mice, providing the only data available onsuch gene mutations in any mammal. In view of possiblelevels of tritium release,. not only from existing nuclear
installations but also from contemplated controlled
thermonuclear reactors. these data are of great relevance.
A total of 14 groups of males was used. Two groups
were injected with 0.75 mCi, and the 12 others with
0.50 mCi, of tritiated water per gram of body weight.
The results demonstrate that beta radiation from thedecay of tritium can induce specific-locus mutations inspermatogonia as well as in post-meiotic stages:
16 mutations have been recovered among a total of
20 626 offspring derived from germ cells irradiated as
spermatogonia and 11 in 7943 offspring from irradiatedpost-meiotic stages. The mean absorbed dose to thespermatogonial cells has been estimated to be 700 rad
and that to post-meiotic cells, 430 rad. These data thus
permit mutation-rate estimates of 1.58 10- 7 rad- 1 perlocus for spermatogonia and 4.60 10- 7 rad- 1 per locus
for the other stages. These rates are within the statisticallimits of what would have been expected from acomparable external dose of x or eamma irradiation. The
point estimate of the RBE f;r post-spermatogonialstages is close to 1, with fairly wide confidence intervals;

I

that for spermatogonia is slightly above 2, withconfidence intervals that include 1. There are someindications that the distribution of mutants among theseven loci may differ from that produced by
gamma rays: noteworthy is the observation that only
one of the mutations was at the s locus (the expectation
would be about 5 or 6). In more recent studies.
currently in progress at Oak Ridge. Cumming and
W. L. Russell (129) are engaged in collecting more
extensive data on tritium irradiation, focusing attention
on the induction of mutations in spermatogonia.

(c) Induction of chromosome aberrations in human
lymphocytes by tritiated water ( HTO)

375. Hori and Nakai (233) and Bocian et al. (39) havereported on the induction of chromosome aberrations inhuman lymphocytes exposed to tritiated water in vitro.
Exposures were carried out by the addition of whole
blood to the culture medium containing tritiated water.
In the work of Hori and Nakai, the concentration oftritium ranged from 1 10- 6 µCi/ml to 1 10- 2 µCi/ml,
and the cells were exposed during their entire period in
culture ( 48 h). Bocian et al., used two regimens: in one("acute exposures" in the authors· terminology), thelymphocytes were exposed for a 2-h period prior to PHA
stimulation (range of concentrations, 1.71-14.36 mCi/ml), after which they were washed and cultured (53-hcultures); in the other ("protracted series") the cells
were exposed during 53 h (concentration range,
0.063-0.51 mCi/ml).
376. The results indicate that with protracted exposures(48 or 53 h) the aberrations produced were mos~ly of
the chromatid type. such as gaps. deletions andfragments, and there were relatively few chromatid
exchanges. In the concentration range used by Hori andNakai, the dose-effect curve for the number of breaks
induced was quite complex at low concentrations. In thework of Bocian et al. and with the range ofconcentrations they used, the frequency of chromatidaberrations increased linearly with dose. A quantitative
comparison of the frequencies between the two groupsof authors is, however, not possible because each groupused only one (but different) fixation time, and in
addition. the ranges of concentration were different.
377. In the 2-hour exposure experiments of Bocian etal., chromosome-type aberrations were found to be
induced (dicentrics, centric rings, terminal andinterstitial deletions). The data for dicentrics plus rings,
as well as those on deletions, gave a good fit to a linear
plus quadratic model. Using the data obtained inx irradiation experiments (acute doses of 50-300 rad).
Bocian et al. have estimated that the RBE for the
induction of dicentrics plus centric rings is about 1.2.

3. Summary and conclusions
378. During the past few years. there has been agrowing interest in the study of the biological effects ofradioisotopes. particularly of 239 Pu and 3 H. A numberof genetic and cytogenetic studies that have so far been

carried out in mice demonstrate that these isotopes arecapable of inducing dominant lethals, chromosome
aberrations and point mutations (for the last category.
only the effects of 3 H have been studied).
379. Autoradiographic studies have shown that in mice.
intravenously injected 23 9 Pu (as citrate solution) is
inhomogeneously distributed in the testis and is largely
localized in the interstitial tissue outside and between
the seminiferous tubules. A consequence of this is thatthe alpha-irradiation dose rate to the spermatogonial
stem cells is 2-2.5 times greater than the average for thetestis as a whole.
380. When 2 3 9 Pu-injected males are mated to females.
there is a significant excess of intra-uterine mortality
relative to controls and the effect persists in matings up
to five weeks after injection (post- and peri-meiotic
stages sampled). In addition, the effect appears to beunrelated to the amount of 2 3 9 Pu injected (in the range
0.05-0.5 µCi per mouse).
381. Dominant lethal tests performed on F 1 males siredby fathers which received plutonium injection (andderived from matings during the ninth, fourteenth andsixteenth weeks) showed that here again there was anincrease in intra-uterine mortality relative to controls.
382. Relative to chronic gamma irradiation. alphaparticles from 2 3 9 Pu seem to be more than 20 times as
effective in inducing dominant lethality (postimplantation) in meiotic and post-meiotic stages.
383. In male mice exposed to alpha particles from
2 3 9 Pu (intravenously injected citrate solution) for aduration of 6-34 weeks. reciprocal translocations (inspermatogonia) and chromosome fragments (in spermatocytes) are induced. Relative to chronic gamma
irradiation, alpha-particle irradiation from 239 Pu is morethan 20 times as efficient for the induction of theseeffects. This finding is similar to that recorded for the
induction of dominant lethals in meiotic stages. These
calculations do not take into account the inhomogeneous distribution of 2 3 9 Pu in the testis.
384. Male and female mice fed on tritiated water. show.
in dominant lethal tests, an increased amount of
intra-uterine death.
385. In specific-locus tests, mutations have been foundto be induced in male mice fed with tritiated water. The
data currently available suggest that the rate ofinduction per unit dose of irradiation with beta particles
from 3 H is about the same as that of x irradiation. The
estimates are 1.58 10- 7 rad- 1 per locus for spermatogonial mutations and 4.60 10- 7 rad- 1 per locus forpost-spermatogonial stages. These estimates have wide
confidence limits. There is some evidence that the
distribution of mutants among the seven loci may bedifferent from that after x irradiation.
386. In human lymphocytes exposed to tritiated waterin vitro, both chromosome- and chromatid-type
aberrations are induced, depending on the concentrationof 3 H and the duration of exposure.
477

H. THE RADIATION RESPONSE OF SPERMA·TOGONIAL CELLS AND ITS RELEVANCE TO
THE INTERPRETATION OF GENETIC EFFECTS
OF RADIATION
387. In the 1972 report, the model of stem cell renewal
proposed by Oakberg (372) for the mouse and by Huckins(239) for the rat and the model of Clermont andBustos-Obergon (113) for the rat were discussed.
According to the Oakberg-Huck.ins model, stem-cell
spermatogonia occur as single isolated cells in contrast to
the groups of differentiating spermatogonia which form"chains" of cells connected by cytoplasmic bridges.
Renewal of stem cells occurs by the division of some A5
cells; other divisions of As spermatogonia result in theformation of paired cells and constitute the initial stepin differentiation. Further divisions of the pairs result in
irregularly aligned spermatogonia which transform
morphologically into the chains of A 1 spermatogonia.
All A1 cells divide into A2 cells, then into A3 cells, andso on. Division of ~ cells results only in the formationof cells of the intermediate type. There is no evidence
that the A-type spermatogonia of any type are formed
from ~ cells. The model can be diagrammaticallyshown as follows:

As - singles~ A-paired (Apr)-+ A-chains (aligned)(Aai)-+A1 -+ A2 ~ A3 -+ A4 -+ intermediate spermatogonia-+
type-B spermatogonia-+ primary spermatocytes
388. The As spermatogonia are the most resistant of the
spermatogonial types; they are the only survivors after
x-ray exposures of 150 R or more and are responsible
for the repopulation of the germinal epithelium. The
studies of Huckins (237, 238, 239), Oakberg (372, 373)
and de Rooij (439) have shown that some of the As cells
have a long cell cycle (about 8.5 days in the mouse (372,373)); it also seems that most have a much shorter cycle,
only slightly longer than 26-28 h characteristic ofdifferentiating spermatogonia (238, 374). The relationship between As cells having these two average cycle
times has not been explained.
389. On the other hand. the "two-stem-cell" modelof Clermont and Bustos-Obergon (113) assumes that
there are two populations, one of which is the
non-cycling Ao (reserve stem cells, activated only if themore mature classes of cells become depleted) and theother, a renewing cycling population of A1 - ~spennatogonia. The ~ cells were given the role ofpivotal cells, forming either more differentiated In(intermediate spermatogonia) or less differentiated A1
spermatogonia as in the following scheme:(Activated Ao in emergency)

/A'\
A,

A2

~~.)
t

Type.a spe:rmuogonia

J

Primiiry sperma1ocy1e1478390. In recent experiments, Oakberg and Huckins (377)
used 3 H-thymidine labelling in conjunction with x irradiation to study further the problem of germ-cell
repopulation in the testis of the mouse. more
specifically, to examine whether the ~ cells form A1
cells and intermediate spermatogonia (as the model
outlined in the preceding paragraph predicts), or
whether confirmatory evidence can be obtained for theirearlier findings that As cells are the active stem cells of
the germinal epithelium. Both sectioned material (as in
the earlier work of Oakberg) and whole mounts wereused to compare the results of the two techniques. The
activity of 3 H-thymidine used was 17.5 µCi/0.25 ml(intraperitoneal injections, either one or six at 12-hintervals), and the x-ray exposures were 150 R and
300 R (90 R/min).
391. The prediction was that, if the model of Clermontand Bustos-Obergon were valid, only A1 - ~ cells should
be labelled (and subsequently be killed by irradiation)and the Ao cells should not be labelled. If the
Huckins-Oakberg model were valid, since As cells are theonly survivors after x-ray exposures of 100 R or more,
only these should be labelled. It is worth pointing outthat data demonstrating labelled A sperrnatogonia 5 days
after an x-ray exposure as high as l OOO R had been
available for some time (370) and preliminary resultsfrom long-term labelling studies in control mice revealed
heavily labelled cells as long as 10 days (more than one
cycle of the seminiferous epithelium) after 3 H-thymidine injection. On the basis of these results, it has beensuggested that the ~ spermatogonia are the active stem
cells of the serniniferous epithelium (37 I).
392. The results obtained using both methods (sections
and whole mounts) demonstrated the survival of
A spermatogonia labelled with 3 H-thymidine prior toirradiation, and the survivors were almost exclusively As
spermatogonia. The data are consistent with the
hypothesis that the stem cells are indeed in continuous
cycle, and that the total cycle time of some As cells islong. There was no evidence for a non-cycling reserve
stem cell(~ population).
393. De Ruiter-Bootsma et al. (447) examined thesurvival of spermatogonial stem cells in the mouse after
irradiation with I-Me V fission neutrons. Groups of malemice (CBA strain) were irradiated with neutrons withacute dc;>ses of 200-410 rad (7 levels at 10 rad/min). For
purposes· of estimating the RBE, two other groups ofmice were exposed to 900 and 1200 rad of 300-kVp
x rays at 30 rad/min. The number of surviving cells after
irradiation was estimated in histological preparations of
the testeS' of the mice killed 11 weeks after irradiation
by determining the fraction of repopulated seminiferous
tubules in cross-sections. This fraction. called the"repopulation index'' (RI) 1 7 was assumed to be directly
proportional to the number of surviving stem cells.
394. It should be pointed out that the above method of
estimating stem cell survival is different from the one
11 The repopuJation index after a given irradiation dose D,
is defined as Rl(t) = 100 Ng(t)/L (per cent), where N is thenumber of surviving stem cells, g(t) represents the growth
function (mean clone length as a function of time t), and L is thetotal length of all scminlferous tubules In a testis.

used by Oakberg and others. Subdividing the morphologically undifferentiated A-type spermatogonia into
three classes. A-isolated. A-paired and A-aligned.
Oakberg (372) observed that the isolated cells were theleast sensitive. their numbers being reduced to about
58 per cent of control value when measured 72 h after
irradiation with 100 R of x rays, while the numbers of
the other undifferentiated types were drasticallyreduced. De Ruiter-Bootsma et al. point out that bycounting the number of cells in cross-sections of thetestis (as Oakberg has done), difficulties are encounteredin distinguishing the isolated cells and the otherspermatogonia. To avoid this coinplication, the authors
use the colony-forming unit 1 8 as the stem cell andemploy RI as a reliable measure of survival of A5
sperma togonia.
395. The results showed that the dose-response curvefor stem cell survival had an exponential slope with a Doof 85 rad, indicating the rather low radiosensitivity ofthe stem cells. A comparison of the values of RI fromthe neutron and the x-ray experiments revealed that
under the experimental conditions used, neutrons havean RBE of about 4.4. It also approximates the RBE of
6.5 for fission-neutron (1-2 MeV) doses of 18-101 radand of 4.7 for 172-258 rad reported by Oakberg (370).
396. In a subsequent paper, de Ruiter-Bootsma et al.
( 448) have reported additional data on the radiation
response of the spermatogonial stem cells in the mouse
testis. Both neutron irradiation ( I Me V, 50-410 rad,10 rad/min, single exposures) and x irradiation(200-1200 rad. 30 rad/min, single exposures) were used.
The mice were killed at four different intervals after
irradiation (3, 5, 8 and 11 weeks) and the numbers ofsurviving stem cells were determined using the colony
method discussed earlier, as well as direct colony counts.
The formula for calculating RI was slightly modified asfollows: RI(t. D) = 100 Ng(t, D)/L(D) (per cent).
According to this formula, at a given time t, RI is alinear function of N if either both the growth function
g(t) and the total tubular length L are independent of
the degree of stem cell depletion or they vary in thesame proportion.
397. The main results are the following: (a) for all
post-irradiation intervals studied, RI is exponentiallyrelated to the neutron dose, except when its value
exceeds 90 per cent; this has been explained by theauthors by assuming that the clones which are
distributed at random over the tubules, by growing inlength, have an increased chance of meeting each otherresulting in clonal growth inhibition; (b) the mean D 0
value of the stem cell survival curves in this period is
81 ± 2 rad, pointing to the existence of a highly
radioresistant population of stem cells; (c) there is a
1 'Commenting on the technique used by Ruiter-Bootsma
et al. (447), Oakberg (367) has drawn attention to the possibility
that the colony method can lead to errors if surviving stem cells
are not randomly distributed, especially if longer intervals areused. His preliminary data suggest that survival at high doses isdependent upon the stage of the cell cycle of the seminiferous
epithelium i.e., specific areas of the tubule) and, furthermore,that the number of tubules with more than one survivor per
cross-section is greater than would be expected. This could leadto a change in the mechanics of repopulation along the innerwall of a cylinder.

good correspondence between the RI values and mean
numbers of clones counted per testis; ( d) the age of theanimals does not influence stem cell sensitivity once theanimal is adult, although clones grow at a slower rate inolder animals; (e) the x-ray curve for stem cell survival isexponential from 400 rad onwards and the value of Do
is 242 ± 7 rad; (f) the RBE of fission neutrons isdose-dependent and decreases from 5.5 at a surviving
fraction of 0.3. to 4.1 at a fraction of 0.01: (g) theirfindings permit an estimate of about 1800 stem cells
present in the normal unirradiated testis. 1 9

1. Effects of dose fractionation
398. In the paper of de Ruiter-Bootsma et al. discussed
in paragraph 393, results on the effects of neutron-dose
fractionation on stem cell survival are also given. In thesestudies, male mice received 350 rad of neutrons
delivered in two fractions of I 50 and 200 rad, in thatorder, with intervals ranging from 4 to 48 h. The data
show that fractionation decreases the number ofsurviving stem cells; the extent of such a decreasedepends on the time interval between the fractions. The
radiosensitivity of the stem cells increased sharply
between 4 and 8 h after the first fraction. The authors
sunnise that, if this decrease in cell survival is due to a
progressive decrease in Do values of the stem cells after
irradiation. then the first dose apparently triggers thecells to pass into a very sensitive phase of their cell cycle
within a period of ~8 h. (The rise in translocation
frequencies in the short-tenn fractionation experiments
discussed in paragraphs 112-113 may thus be explainedon the basis of the increased sensitivity of the stem cells
to the induction of reciprocal translocations.)
399. Rudnicki and Trojczuk (445) and Rudnicki and
Kochmanska-Twardowska ( 446) investigated the effectsof x-ray dose fractionation (8 X 25 rad compared with
1 X 200 rad. 8 X 50 rad compared with I X 400 rad,2 X 50 rad, 5 X 20 rad and 9 X 10.1 rad compared withl X l 00 rad) on survival and re population kinetics of thedifferent spermatogonial cell types. The interval between
the dose fractions was 24, 72 or 96 h. The findings arethat (a) the different cell types have differential
sensitivity to killing. (b) dose fractionation leads to a
detectable delay in the rate of repopulation of germinal
epithelium and ( c) the survival is approximately equal
after single and fractionated irradiation and appears to
be independent of the number of fractions and theinterval between them.

2. Effects of dose rate
400. To evaluate the possible role of spennatogonial
cell survival on mutation frequencies at different dose
rates, Oakberg and Palatinus (378) irradiated male micewith 300 R of x rays (at 94 R/rnin) or with 300 R of
1 3 7 Cs gamma rays at rates of 0.0056 and 0.0007 R/min.
One group of mice was killed immediately after
1 P From the relationship In ND= 1n N0 - (D/D ) where0
ND is the number of clones after a dose D, N 0 is the number ofcells in an unirradiated testis, and D 0 is the 37% survival dose.

479

removal from the gamma field; a second group was killed
24 h later in order to observe initiation of cell
repopulation. Cell counts for these intervals were thesame for gamma-irradiated mice, and the data werepooled. Mice exposed to x rays were killed five days
later when cell survival is minimal. The testes were thenprocessed for making histological preparations.
401. The results showed that (a) after 300 R of acute
x rays, only 16 per cent of the stem cells (relative tocontrols) and only a few differentiating spermatogonia
survived; (b) after gamma irradiation at 0.0056 R/min,the survival was 37 per cent for As and 24 per cent forA 1 cells; the remaining spermatogonial types showed adepletion to about 5 per cent of control, indicating amarkedly higher radiosensitivity of these stages: and(c) at 0.0007 R/min, the number of stem cells was
unaffected with survival values for A 1 ·A.i spermatogonia
ranging from 81 to 97 per cent.
402. On the basis of these data. the authors conclude
that (a) the mutation frequency at 0.0007 R/min ismeasured on a cell population which is maintaining itself
at normal levels: {b) after 300 Rat 0.0056 R/min {which
reduces the As spermatogonia to 37 per cent of control),the mutation frequency appears to be the same as after
0.0007 R/min (para. 284 ); and ( c) after 300 R of x rays
at 94 R/min ( where cell survival is only 16 per cent), themutation frequency is the highest of all ( 1972 report,
Annex E, table 14); this result is the reverse of that
expected if there is a positive correlation between
sensitivity to cell killing and sensitivity to mutationinduction under the radiation conditions investigated.
Other mechanisms, such as repair, are indicated as theprimary factors in the reduced frequency of mutations
observed at low dose rates.
403. Hsu and Fabrikant (235) conducted a study to
examine the cellular response and cell population
kinetics during spermatogonial cell renewal in the mouse
testis exposed to continuous gamma irradiation (1 37 Cs)
at 1.8 rad/day (=0.00125 rad/min) and at 45 rad/day(=0.031 rad/min). Labelling with 3 H-thymidine was used
for the analysis of cell-population kinetics. Mice were
killed at different times during irradiation and theirtestes were processed for histological examination andautoradiography. In addition. the pattern of postradiation recovery of the seminiferous epithelium wasinvestigated in mice which were exposed to irradiation at
45 rad/day for two weeks (accumulated dose, 630 rad).
The main results are given in the following paragraphs.
404. Depending on the dose rate, continuous irradiation
alters the cell-population kinetics in the seminiferous
epithelium and more damage occurs at the higher dose
rate. At 1.8 rad/day. the type-A population considered
as a whole (As and A 1 -A4 ) shows an initial decrease
within the first two days of irradiation followed by a
new level of steady-state growth at about 80 per cent of
the control level. This is essentially a reflection of thedecrease in the A 1-A 4 cells. since the As cells remain atnear-control levels during the entire seven weeks ofcontinuous irradiation. Among the A 1 -A4 spermatogonia there is a gradient in radiosensitivity. thereduction in cell numbers increasing from A 1
through~-405. In the 45-rad/day group, there is a marked
decrease in the numbers of type-A spermatogonia (toabout 50 per cent) in the first three days {accumulated
dose. 135 rad) followed by an increase to normal values
by the second week, then a fall and a rise again(60-70 per cent) in about three to five weeks. The As
cells alone undergo a reduction in numbers to nearly
50 per cent of controls during the first three days ofcontinuous radiation: from day 4 up to about 10. thisfigure is about 80 per cent of controls and from then on,
up to week 3 (when the studies were terminated) control
levels are reached. The proliferating type-A cells,
depending upon their individual radiosensitivities, reachsteady-state levels at various levels below controls.
406. At the lower dose rate, in the In cell population,
there is an initial (compensatory) rise in cell numbers on
day 1, after which normal levels are attained for two
weeks, followed by a slight decline to approximately
95 per cent of control values. At the higher dose rate,
there is a steep decline in cell numbers and no cells arefound in the seminiferous epithelium at the end of thefirst week of exposure.
407. At the lower dose rate. there is also a decrease in
the type-B population, which reaches steady-state levels
at about 80 per cent of controls by the third week ofradiation exposure. At higher dose rates, the decline issteeper, so that by day 5 of exposure, this compartment
is completely depleted.
408. Examination of the recovery pattern after
irradiation (45 rad/day for two weeks; accumulateddose, 630 rad) shows that the repopulation of theseminiferous epithelium commences with increased cellproliferation in the As cells and is supported by thefinding of an increase in the As mitotic index; the waveof renewed cell proliferation moves from As cells
through to the A.i cells. By day 14 after irradiation, In
and type-B cells reappear. and their cell numbers reach acompensatory peak between 25 and 50 per cent above
normal cell numbers, but subsequently stabilize at
normal levels. By eight weeks, the curves showing thepercentage of labelled mitoses for type-A, In and type-B
cells show cell-cycle parameters similar to those forunirradiated controls.

409. All these findings of Hsu and Fabrikant (235) arein very good agreement with those of Oakberg (243,310, 372). Oakberg and Clark (375), Oakberg andPalatinus (378), and Oakberg and Huckins (377). They
demonstrate that at a dose rate of 0.00125 rad/min,
which is twice the lowest rate employed by Oakberg andPalatinus, the As spermatogonia maintain themselves atcontrol levels and that the temporal sequence ofrecovery commences with the As cells.
3. Stem-cell killing and sterile period(a) Irradiation of adult male mice
410. It is known that when adult male mice areirradiated, a period of sterility develops several weeks
after treatment and that it is a direct consequence of
spermatogonial-cell killing. The length of the sterile

480'period therefore provides a good indirect measure of cellkilling. A relationship between sterile period and dose
was indicated in several studies carried out in the 1930s,
but until the recent studies of Sheridan (533) withfractionated exposures. no precise measurements had
been reported.
411. Sheridan irradiated male mice of the CBA strain
with either single x-ray exposures of 600 or 800 R orwith fractionated exposures of 300 + 300, 400 + 400
and 500 + 500 R, separated by time intervals of 24, 48,
72 or 144 h. he found that the mean number of days
between irradiation and return of fertility was prolonged
after fractionation (24, 48 and 72 h) relative to single
treatments or with an interval of 144 h. He interpreted
tltis observation as indicating that the shorter intervals
used (24. 48 and 72 h) lead to a greater amount of cellkilling.
412. Cattanach and Moseley (92) and Cattanach (90)made a thorough study using the length of the sterile
period as a measure of spermatogonial killing by
irradiating F 1 hybrid ( C3H/HeH X 1OIH) mice with awide range of single and fractionated x-ray exposures. It
was found that the sterile periods were much shorter
than those reported by Sheridan at equivalent exposures,
even allowing for the fact that Sheridan reported hisdata in terms of the mean, rather than the median, as
Cattanach did.
413. Cattanach (90) found (table 41) that after single
x-ray exposures, the dose response curve for therecovery of fertility was irregular; the median day ofreturn to fertility increased little from 300 to 400 R;
there was then a slight increase to 500 R, but from 500
to 600 R, there was a dramatic increase in response.
Beyond this level, there was little change up to 800 R,
but the 900-R and 1000-R treatments gave rapidly
increasing lengths of the sterile periods. Quite clearly,
the exceptional point is that for the 600-R level: were it
not for this, a simple exponential relationship would beobtained. However, five replicate experiments werecarried out at this level, and all the results wereconsistent; consequently Cattanach feels that there islittle doubt that the plateau is a true phenomenon. The
fact that at 600-800 R, a plateau is obtained is notwithout significance: it is at these exposure levels thatthe yields of translocations and specific-locus mutations
begin to decline and then fall giving humped dose-effectcurves. The coincidence between the hump in thedose-effect curve for genetic damage and the plateau in
the sterile period supports the conclusion thatthroughout the exposure range investigated, the sterile
period is a direct consequence of the stem-cell killing.
414. Cattanach believes that the most readily acceptable explanation for the shape of the cell-killing curve isthat it has two components. these representing two cell
populations with different sensitivities to radiation
killing. Since on the basis of Oakberg's data (372), allspermatogonial cells other than the stem cells should be
eliminated by even the lowest dose (300 R) employed
here, it may be concluded that the postulated sensitiveand resistant cells must exist within the spermatogonial
stem cell population itself. The two populations could
represent two distinct stem cell types and this wouldfind support in the histological observation that within
the stem cell population, there are cells with quite
different cell cycle times. Alternatively. the two
populations could represent stages in the cycle of asingle stem cell type. By either interpretation, theexistence of two cell populations of different radiation
sensitivities could provide the basis of the heterogeneityof the spermatogonial stem cell that has been proposed
as an explanation for the humped dose-effect curves forgenetic damage (190, 382, 470). Thus. if the genetic
damage stems plimarily from the population moresensitive to killing, then a drop in yield would be
expected when killing increases beyond a certain point
and at the highest exposures, genetic damage may only
be recoverable from the·more resistant cell population.
415. There is doubt on the extent to which the
600-800 R plateau in the length of the sterile period(para. 413) is really connected with stem-cell killing as
Cattanach (90) has suggested. Oakberg's data (370) on
survival of type-A spermatogonia gave a good fit to asimple exponential curve. as did the stem cell data of deRuiter-Bootsma et al. in the 400-1200 rad range, as
discussed earlier (paras. 396-397). Factors other thanstem-cell killing. such as the mechanics of stem-cell
repopulation, may well influence the shape of thedose-effect curve, for return to fertility, although it isdifficult to see why they would modify the time
recovery of fertility so specifically over a narrow rangeof doses.
416. The data obtained from the fractionation experiments (24-h interval between fractions) show that theeffect on the sterile period varies according to the total
exposure (table 41). Thus at the 400-R level, fractionation results in a sterile period not different fromthat with the single exposure, but at 600-R,
fractionation reduces the sterile period irrespective of
the relative magnitude of the two fractions. The
opposite situation was then found at the 700-R level
where fractionation enhanced the sterile period abovethat for the single exposure: at 1000 R, fractionation
gave either the same response as the single exposure orcaused some enhancement. The uncertainty about theeffect of fractionation at the 1000-R level derives fromthe great variability in the response: while this may becharacteristic only of high exposures, there is reason tosuspect that the relative magnitude and the order offractions may be important. When 100 R follows 900 R.
the median day of return to fertility (108 days) is notvery different from the 1000-R single exposure level(102 days) but when the fractions were given in thereverse order, this number is 135.
417. In spite of these complexities. two general
conclusions can be made: (a) fractionation at highexposures causes more, rather than less, stem-cell killingthan does single treatments. This demonstrates that the
decline in yield of genetic damage with higher single
doses does not derive from extensive cell killing per seand supports the idea that \vith this form of treatment,
genetic damage and cell killing tend to occur in the same
cells so that at higher doses, more cells with genetic
damage are killed (382). The corollary of this is that.
with fractionation, the cell killing brought about by thesecond of two fractions must tend to occur in different
481

cells from those in which the genetic damage had been
induced by the first fraction: (b) the cell killing responsewith fractionated treatments rises regularly withincreasing exposures, as does genetic damage, so that theirregularity of the single-exposure curve which indicatedthe existence of two cell populations is eliminated. This
would again suggest that whether considering genetic
damage or cell killing, the two fractions tend to hit
different cells. Further interpretation of the data is notpracticable, but it is probable that the diverse response
obtained with fractionation, relative to that with thesingle treatments, stems from an alteration of therelative sensitivities of the two stem-cell populations.
The results of Oakberg (372) show that differential cell
survival occurs within the long-cycling A5 population.
418. In a subsequent paper, Cattanach and Moseley(92) explored the effects of 500 + 300 R, 500 + 500 R
and 300 + 300 R separated by 2 h to 12 days (800-R
series), 4 and 7 days (1000-R series), and 24 h, 3, 7 and
10 days (600-R series) between the two fractions(x irradiation). In some experiments, the yields oftranslocations and/or specific-locus mutations were alsomeasured in addition to the determination of the lengthsof the sterile periods.
419. In the 800-R series, with intervals of 16 h to
4 days, fractionation resulted in an increase in the sterile
period, well beyond that of the acute exposure (from a
median of 71 to one of 81 days); with longer intervals.
there was a gradual decrease, reaching a minimum with a
10-day interval (median, about 55 days). In the
1000-R series, the 4-day interval gave a sterile period
somewhat shorter (95 days) than that of the 1000-R
acute (102 days); the 7-day interval led to a drop (about
82 days), but the results of the individual tests
overlapped. In the 600-R series, fractionation reduced
the sterile period from about 67 days (acute) to about
52 days (fractionated), and there was very little changewith fractionation intervals longer than 24 h.
420. Although the translocation and specific-locus datacollected in the fractionation experiments of Cattanachand Moseley (92) are discussed in other sections, it isworth pointing out here that there is no correlation
between genetic damage and cell killing (as measured by
the length of the sterile period) with different
fractionation intervals. However, the translocation andspecific-locus mutation experiments give, qualitatively if
not quantitatively, a similar variation in response tovariation in the treatments. Both indicate a peculiar stateof cells surviving the first fraction, such that they may
be particularly sensitive to genetic damage 24 h later.
The translocation data suggest that this sensitive state
may last up to two days and b,oth types of experimentdemonstrate that this state does not exist beyond thistime.
(b) Irradiation of young mice
421. In a recent paper on x-ray induction oftranslocations in young male mice, Cattanach, Murray
and Tracey (94) have presented some results on theincidence of sterile period. In the first two series
involving whole-body exposures of 300 and 500 R(acute), 5-day-old, 10-day-old and adult mice were used.
482

The younger age-groups were selected on the basis of
Selby's results (527), which suggested a change from animmature to an adult response by the eighth day. In thethird series, the exposure was 1000 R (part-body), andin this the response of 3-day-old mice was also studied.
The results are summarized in table 41.
422. It can be seen that with 300- and 500-R
treatments, the 5-day-old mice did not exhibit any
obvious sterile period. As a group, they could beconsidered to become fertile as they reached maturity,and the fertility remained until the time of sacrifice: it
was not of short duration. With adult mice irradiatedwith 300 and 500 R. the results are in good' agreementwith all previous tests (median day of return to fertility,
51 and 53, respectively). The response of 10-day-old
mice is similar to that of adults.
423. After 1000 R. the response was different: in the
5-day-old group, I out of the 10 became fertile onreaching maturity, 5 showed some clear indication of asterile period and the remaining 4 appeared to bepermanently sterilized. The irradiation of 3-day-old micegave similar results. Fertility in both groups, whenregained, was highly variable: litter-size was often quite
small, litter production was irregular and 4 of the 14'"fertile" males became sterile after· producing only a few
litters. All these findings contrast sharply with the
response following adult irradiation. The response ofanimals treated with 1000 R when 10 days old tended to
be intermediate between those of the younger and adultgroups.
424. The lack of any clear sterile period following theirradiation of 3- or 5-day-old mice might suggest that
their germ cells are perhaps more resistant to radiation
killing than those of the adult. However, histologicalstudies with rats irradiated when 4-5 days old haveshown that the germ cells of the immature animal arehighly sensitive to radiation killing (241, 287. 532). The
difference from the adult lies in the fact that thesurviving cells can still proceed directly into sperrnatogenesis and lead to the appearance of sperm at sexual
maturity (241, 532). It is likely that a similar situation is
obtained in the mouse, and this provides an explanation
for the lack of clear sterile periods following irradiationof immature animals and points to oligospermia being
the cause of the variable fertility observed with the most
severe treatment.

(c) Ocher species
425. In studies on the induction of translocations in the
spermatogonia of the golden hamster, guinea-pig and the
rabbit, Lyon and Cox (308, 309) also examined thelength of the sterile periods after different x-radiation
doses delivered either singly or in two or more fractions
with various time intervals between the fractions. A
comparison of the results obtained with those collectedby Cattanach (90) for the mouse clearly show that after
single exposures the golden hamster, guinea-pig andrabbit took longer to return to fertility than the mouse;
among the three, the golden hamsters were the mostsensitive. After 600 rad. the guinea-pig appeared to havea somewhat shorter sterile period than the golden

hamster, but the confidence limits were wide. Since inthe golden hamster the duration of spermatogenesis is
35 days, as in the mouse, and since the sterile period
includes a component due to multiplication of surviving
stem cell sperrnatogonia and another for the duration of
sperrnatogenesis (after adequate spermatogonial repopulation has occurred) the longer sterile period in thisspecies must indicate a longer period spent insperrnatogonial repopulation. This might mean thateither the spermatogonial killing is more severe in the
golden hamster or the dynamics of their recovery is very
different.
426. In the guinea-pig and rabbit, spermatogenesis lasts
longer than in the mouse, durations of 49 days in theguinea-pig and 52-60 days in the rabbit being estimatedfor spermatogenesis plus epididymal transport. Therefore. for equal killing, longer sterile periods would be
expected than in the mouse, but only 1-2 weeks longer(since sperrnatogenesis plus epididymal transport in themouse takes about 42 days). In fact the sterile periods
were more than 1-2 weeks longer and hence, again there
is a suggestion either for more severe spermatogonial
killing or for slower rates of recovery in the guinea-pig
and hamster than in the mouse. Thus. althoughdifferences in recovery cannot be ruled out. it seems
probable that spermatogonial killing is more severe in
the guinea-pig and hamster than in the mouse and ismost severe in the hamster.
427. In the golden hamster, with a 2 X 200 rad (24-hinterval), the sterile period was longer by about a month
relative to that after the acute dose (4.5 ± 0.5 months
compared with 3.5 ± 0.5 months); in the guinea-pig
treated with a 2 X 300 rad (24 h) the sterile period wasmuch longer. and at the time cytological examinations
were made, the testes were very small ( one third normalsize) and suggested that the animals were unlikely torecover fertility; in contrast, after a 600-rad single
exposure, fertility was invariably recovered after
4.5 ± 1.5 months. Thus the increased length of thesterile periods in both the species indicate thatspermatogonial killing was severe after the fractionatedexposures. As will be recalled, in the mouse, at thesedose levels. there was either no change in the length of
the sterile period (relative to that after acute exposures)or a slight lowering with fractionation. The data witheight-week fractionation also suggest that in both thespecies, the spermatogonial population did not return tonormal at the end of the second fraction. With multiple
fractions, the sterile periods were slightly shorter thanafter single doses, but owing to repopulation of thetestes during the course of the treatment, it is notpossible to interpret this in terms of spermatogonial-cellkilling.

4. A model for stem cell heterogeneity in theadult and immature mouse testis
428. The existence of heterogeneity in radiosensitivity
among spermatogonial cells to the mutagenic and killing
effects of ionizing radiation had long been inferred fromgenetic and cytongenetic studies and direct evidence had
been provided by histological studies of the irradiatedtestis. It is also known that the A5 spermatogonia are themost resistant of the spermatogonial cell types (and theonly survivors after x-ray exposures of 150 R or more)
and are responsible for the repopulation of the germinal
epithelium. Although the exact basis of stem cell
heterogeneity is not fully resolved, two current ideas are
the following: (1) two or more distinct stem cell types
with differing radiosensitivities exist (90, 92, 93), andsome basis for this is provided by the histologicalevidence from both rat and mouse that stem cells withdifferent cell cycle times are present in the testis (seeparagraph 388); (2) heterogeneity simply reflects cellstage sensitivity differences within a single cell type (90,92. 93. 420). The model of Cattanach et al (94). which
in tum is based on the model of cell cycle devised bySmith and Martin (541) argues that the heterogeneity inradiosensitivity stems from variations in cell cycle times
that normally occur in the stem-cell population (see also
reference 96).
429. The model of Smith and Martin divides the cell
cycle into two parts. One, termed the B phase, isregarded as a deterministic and highly co-ordinatedprocess: it corresponds to S, G2 , M and part of G 1 • The
other part, contained within G 1 is called the A phaseand is thought of as an indeterminate, static condition inwhich the cell is not actually progressing through the cell
cycle. Cells may remain in the A phase for any length oftime, but leave and enter the B phase exponentially withrespect to the time they have spent in the A phase inmuch the same way as radioactive material will decay at
a rate determined by its half-life. The model also
predicts that the interrnitotic times of sibling cells
should also be exponentially distributed. It might benoted that a long A phase together with thedeterministic part of G 1 constitutes the so-called G0
period for slowly dividing or non-dividing cells.
430. Application of this model to the sperrnatogonialstem-cell populations of the adult mouse suggests thatthe existence of long (8.5-day) and short (""' 28-h)
cycling cells (374) may perhaps be an oversimplification. A range of cell cycle times should be
expected and these would not be characteristic ofdifferent cell types, but represent only cell-to-cell andgeneration-to-generation variations in the time a single
cell type spends in the A phase. On the basis of this, theheterogeneity in radiosensitivity of the spermatogonial
stem cells is a reflection of the existence of the A phasein addition to the stages of the B phase of the cell cycle.
Cells in a long A phase might be most resistant toradiation damage since, like the non-dividing gonocytes(primordial cells) of the new-born mouse, they would
have a great time available for mutational/lethal repair
before the onset of S. Cells in a short A phase, leaving an
A phase of any length or in the B phase might be moresensitive to radiation damage and yield higher levels ofgenetic damage at doses compatible with significant
levels of survival. (However, this interpretation seems to
be at variance with the observations of Huckins (237),
which suggest that fast-cycling A,. spermatogonia areprobably already differentiated.)
431. The above line of reasoning applies to spermatogonial stem-cell populations under normal conditions.
483

However, within a few days of germinal depletion byirradiation exposure, a quite different situation may
exist. Genetic and sterile period data indicate that thesurviving, formerly more resistant cells are stimulated
into proliferating activity, this being maintained until
stem-cell repopulation is essentially complete (90, 92,
93, 420). During this period, cell cycle times. must be
much shorter than under normal conditions and, on themodel of Smith and Martin, this should be achieved byshortening or eliminating the A phase, and this might be
expected to lead to a greater radiosensitivity. This may
be true for cell killing (92, 93), but the yield of genetic
damage from proliferating cells is actually reduced (92,93,420).
432. Evidence against the hypothesis of Cattanach et al.
outlined in the preceding paragraphs has recently beenpresented by Oakberg (367a, 377a), and the relevant
data are summarized in table 42. This shows no evidence
for alterations in the cell-cycle properties of As
spermatogonia labelled prior to irradiation with
exposures of 100, 300, 500 and 600 Rand scored 207 hafter irradiation, even though survival was reduced to 9
per cent of control by 600 R. Differential cell survival
was observed after a single I 000-R exposure and after
two 500-R fractions given 24 h apart, yet no apparent
effect on cycling properties occurred since relative
positions of the different exposure groups (in per centlabelled cells) was maintained over the 207-414 hinterval. (The lower value for controls at 414 h arises
from differentiation into Apr and Aa 1 cell types.)433. Turning now to the response of the testis of young
mice, the observations of Cattanach et al. (94) are: (a)the lack of any clear sterile period following theirradiation of 3- or 5-day-old mice; (b) their reduced
adult testis weight; (c) lower translocation yields than in
adults with a decline in yield at the highest exposure of1000 R, which is more pronounced than in adults (about
1 per cent in 3-day-old mice, 0.3 per cent in 5-day-old
mice, and 4.2 per cent in adults). A consideration of thedevelopmental processes that occur in the testis within
the first few days of birth suggest an explanation for thedifferent response of the immature mouse. Histologicalstudies with rat and mouse testis (32, 240, 241, 341)
have established that only non-dividing gonocytes arepresent at birth. but within the next 2-3 days, large
division figures m·ay be seen and the. first generation of
spermatogonia appear. These lie centrally within the
sex-cords. but, at about 6.5-7 .5 days in the rat (240) andevidently much earlier in the mouse (95), these movetowards the limiting membrane, where proliferative
activity is initiated. It is not known however, whether
these mitotically active sperm,atogonia are stem cells or
the more differentiated types that appear during
spermatogenesis. Quite probably, both are represented.
434. Thus, it _is cle~r that spermatogenesis must be
undenvay in the 5-day-old mouse, for the animal isfertile by at most six weeks of age (the time taken forthe duration of spemiatogenesis, including spermtransport to the ejaculate). In addition, tl1ere may be abuild-up of stem cell numbers over the first week or twoof life. Proliferative activity by the stem cells could
alone account for the low translocation yields. The
situation may be considered analogous to that of the484

adult testis several days after an initial radiation
exposure (92, 93, 420). In both situations, mostspermatogonial stem cells may have a short cell cycle
and yield low levels of genetic damage. It might also be
expected that under these conditions, not all cells would
be proliferating. Some would be longer cycling and thus,
liable to be in the relatively radioresistant A phase. The
presence of such cells in the generally proliferatingstem-cell population of the immature testis would
account for the heterogeneity in radiosensitivity
indicated by the decline in yield of genetic damage at
the highest exposure. Alternatively, tllis decline ofgenetic damage at the highest doses could be attributed
to differential survival of stages in the mitotic cycle, justas in the adult.
435. Finally, it bears mentioning here that for the adulttestis, the model of stem cell heterogeneity proposed by
Cattanach et al. (94) is. in several conceptual aspects,
strikingly similar to that independently advanced by
Preston and Brewen ( 420) although the latter authors
did not, at least explicitly, relate their model to that ofSmith and Martin.

5. Summary and conclusions
436. Recent histological and labelling studies carriedout with x-irradiated mouse testes have provided
confirmatory evidence for the conclusion that the As
spermatogonia are tlle active stem cells of theseminiferous epitllelium and that they are in continuous
cycle. There is no evidence for a non-cycling reserve
stem cell.
437'. ~fter acute irradiation with fission neutrons, thedose-effect relationship for spermatogonial stem cell
survival (50-410 rad) has an exponential character with a
Do value of around 80 rad; after acute x-irradiation, thecurve is also exponential from 400 to 1200 rad, with
Do= 242 rad.
438. The RBE of fission neutrons relative to x rays forstem cell survival (acute irradiation) decreases from 5 .5
at a surviving fraction of 0.3, to 4.1 at a fraction of 0.01.
439. Fractionation of a neutron dose of 350 rad into
two fractions (150 + 200) with intervals ranging from 4
to 48 h leads to decreased survival of spermatogonial
stem cells; the radiosensitivity of the latter increases
sharply between 4 and 8 h after the first fraction.
440. After an x-ray exposure of 300 R delivered at a
rate of 94 R/min, the survival of As spermatogonia is
reduced to 16 per cent of that in controls; withgamma-ray exposures of 300 R delivered at rates of
0.0056 and 0.0007 R/min, the relative survival of the As
spermatogonia was reduced to 37 per cent with the firstregime and unaffected with the second. The mutation
frequencies (specific-locus mutations) under the firstregime (acute x rays) is higher than those under thelatter two. These observations suggest that under these
conditions, there is no correlation between spermatogonial stem-cell killing and mutation rate.

441. Under continuous gamma-irradiation conditions at
a dose rate of 1.8 rad/day for seven days, the A 5
sperrnatogonial cells show no reduction in number; when
the dose rate is increased to 45 rad/day. the number ofAs cells is reduced to 50 per cent of that of controls
during the first three days of continuous irradiation;from day 4 to 10, this figure is 80 per cent, and fromthen on. control levels are reached.
442. Examination of the recovery pattern after
continuous irradiation with gamma rays at 45 rad/day
for two weeks shows that the repopulation of theseminiferous epithelium commences with increased cellproliferation in the A 5 cells and is supported by thefinding of an increase in the As mitotic index.
443. The duration of the sterile period in irradiatedmale mice is a good but approximate measure of theamount of spermatogonial stem-cell killing. With acute
x-ray exposures ranging from 300 to 1OOO R, therelationship between exposure and the median day ofreturn to fertility gives a good fit to an exponential
relationship if the data obtained at levels of 600-800 R(where a plateau is observed) are excluded.
444. The length of the sterile period following
fractionated x-ray exposures varies with the total
exposure. the size and sequence of the fractions, and thetime interval between the fractions. Thus, for instance.
fractionation at high exposures (with 24-h intervals)causes more, rather than less, stem-cell killing than dosingle treatments. When 100 R follows 900 R, themedian day of return to fertility (108 days) is not very
different from the 1000-R single exposure (102 days),
but when the fractions are given in the reverse order, thisnumber is 135. In experiments where the fractionationinterval varied from 2 h to 12 days, the length of thesterile period also varied; for instance, in the 800-R
series (500 + 300 R), with intervals of 16 h to 4 days,
fractionation led to an increase in the length of thesterile period (relative to that acute exposure), and withlonger intervals there was a gradual decrease reaching aminimum with a 10-day interval.
445. X irradiation of 3- and 5-day-old male mice (300
and 500 R) did not lead to any clear sterile period; theadult testis weight of these mice was reduced and lower
translocation yields were obtained (relative to adults);
with a higher exposure of 1OOO R. the response wasvariable: some mice became permanently sterilized.
some showed clear indications of sterile period, andsome became fertile (but in these, litter size, litter
production etc., were quite variable). the translocation
yields were also severely reduced.
446. After single x-ray exposures to males, the golden
hamster, quinea-pig and rabbit took longer to return tofertility than the mouse; of the three, the golden
hamsters were the most sensitive. With fractionatedexposures, the sterile period in the golden hamster(2 X 200 rad, 24-h interval) was about a month longer
than after acute exposure; in the guinea-pig(2 X 300 rad, 24 h interval). the situation was qualitatively the same. The data after multiple small fractions
showed that the length of the sterile period was slightly
shorter.

447. A model to account for spermatogonial stem-cellheterogeneity in the mouse has been proposed by
Cattanach and co-workers, which in several respects issimilar to that proposed by Preston and Brewen. The
heterogeneity is attributed to a variation of cell cycle
times within a single stem cell type. Those in the Jong
G 1 (A phase) would be the most radioresistant. having
greater time available for repair before the onset of S;
those with a shorter A phase, leaving the A phase or inother stages of the cell cycle (S, G2 and M) would be
more radiosensitive and contribute most to the total
yield of genetic damage at lower x-ray exposures(<600 R). Under normal conditions most cells will be in
G 1 , and after acute irradiation most of the genetic
damage will be recovered from these stem cells.
However, under fractionated irradiation conditions. for
instance when cell proliferation takes place ( dependingon the interval), such cells must spend a much higher
proportion of their time in S and G2 , and the recovery
of both translocations and point mutations from thesestages should approximate only one half of that from G 1
due to chromatid segregation. Likewise, yields fromshort-cycling cells such as those in the immature testis
will be lower.
448. An alternative explanation has been proposed by
Oakberg on the basis of the data of Huckins. whichsuggest that the short-cycling As spermatogonia arealready differentiated. Furthermore, it appears that only
the long-cycling A cells survive exposures of 300 R or
more. As repopulation begins, short-cycling cells
reappear, but they all will have been derived fromsurvivors of the long-cycling As spermatogonia. The
heterogeneity therefore arises from differences attributable to a stage in the mitotic cycle of the long-cyclingstem cells.

I. TIMING OF OOCYTE DEVELOPMENT
IN THE ADULT MOUSE

449. The timing of oocyte development in the adultmouse is an essential prerequisite to our understandingof the dramatic change in radiation-induced mutation
rate; in addition, such knowledge is very important torelate specific oocyte (and follicular) stages to specificpost-irradiation litters. With this objective in mind,
Oakberg and Tyrell (379) have initiated an autoradiographic study in which adult eight-week-old female miceare given two injections of 50 µCi of N{acetyl- 3 H)D-glucosamine 16 h apart (a total of 100 µCi per female).
Mice are killed at intervals ranging from l h to 10 weeks
after irradiation and the ovaries appropriately processed.
The progressive appearance of unlabelled oocytes(unlabelled in the zona pellucida) and concurrently
developing follicles was used to obtain an estimate ofabout 35 days for a follicle of 20-30 cells to reachmaturity and be ovulated; this period of time is not longenough to account for the number of litters conceived
after 50 R. However, more recent work (367) suggeststhat 45-46 days are required for all stage 3b follicles toreach ovulation. Stages 2 and Ja appear to grow quite
slowly, and this may explain the fertility response after
acute exposure of adult female mice to 50 R of x rays.
485

J.

THE INDUCTION OF CHROMOSOME ABERRATIONS IN SOMATIC CELLS, THE ARM NUMBER
HYPOTHESIS, AND ITS RELEVANCE TO THE
EVALUATION OF HAZARDS FROM THE INDUCTION OF RECIPROCAL TRANSLOCA TI ONS

1. Introduction: the arm number hypothesis
450. The literature on the induction of chromosmeaberrations in somatic cells, mainly in human peripheral
blood lymphocytes. was last reviewed comprehensively
in the Committee's 1969 report (588). In 1972, theComrnitee had at its disposal some new data that had justbecome available from the comparative study of Brewen
and colleagues 20 on the x-ray induction of asymmetricalinterchanges (dicentrics) in lymphocytes of six different
mammalian species (man, marmoset, wallaby, pig,
Chinese hamster and mouse). The data showed that(a) in all the species except the wallaby, in the range of
exposures up to 400 R, the exposure-frequency
relationship for dicentric induction gave a good fit to themodelwhere Yi is the yield in the jth species, bi and cl. thelinear and quadratic coefficients, and D, the radiation
exposure. In the wallaby, the best fit was to the model
Y = cD 2 ; (b) the combined dicentric data of all the
species gave a satisfactory fit to the model

Yj= (Nrl) (bD + cD2 )where N; is the effective chromosome arm number ofthe species and the other symbols are defined in (a);(c) the above models accounted for a large amount ofvariation in the frequencies among the species, but not
all of it; (d) man, with an effective chromosome arm
number of 81, was found to be· twice as sensitive as themouse. with an arm number of 40; and. (e) with regard
to the production of deletions, which was also studied,the arm number did not seem to influence the yields,
and man and the mouse had approximately equalsensitivity.
451. Assuming that the probability of forming areciprocal translocation is the same as that of forming a
dicentric (as shown by Heddle's results in Vicia faba(224)), Brewen et al. concluded that "dicentric
production in these organisms is, at best, a direct
measure of reciprocal translocation production and thatman is twice as sensitive as the mouse in the dose range
measured". Both UNSCEAR and BEIR (34, 589)
considered the above line of reasoning valid and assumed
that human spermatogonia are twice as sensitive as thoseof the mouse for the induction of reciprocaltranslocations. Their risk estimates for this class ofgenetic damage were based on this assumption.
2.

Relationships between the frequencies of dicentrics
in lymphocytes, reciprocal translocations in spermatocytes, and heritable translocations in F 1 progeny

452. Subsequently, Brewen and Preston (52, 54) madea direct comparison of the yields of dicentrics after
2 0 The results were published in 1973 (55).

486

irradiation of lymphocytes and of reciprocal translocations in spermatocytes after spermatogonial irradiation in the mouse, Chinese hamster. marmoset andguinea-pig. The results showed that the model of a linear
relationship between the dicentric yield and the effective
chromosome arm number (which has come to be called
the ''arm number hypothesis'') is applicable totranslocation induction in spermatogonia as well;
however. the absolute frequencies of reciprocal translocations in sperrnatocytes were roughly one fourth of
those of dicentrics in lymphocytes.
453. The earlier published data of Ford et al. (170) had
shown that, in mice, the frequencies of heritabletranslocations recovered in the F 1 progeny of irradiatedfathers (spermatogonial irradiation) were about one
eighth of those expected on the basis of direct counts inspermatocytes of the irradiated males themselves (alsospermatogonial irradiation). Considering all these results
together, Brewen and Preston (52, 54) proposed that(a) a method is now at hand to make at least apreliminary estimate of germ-cell radiosensitivity froman extrapolation of that of somatic cells and (b) the
frequencies of heritable translocations after spermatogonial irradiation are predictable from those of dicentrics
in lymphocytes (i.e.. frequency in F 1 progeny =dicentric frequency in lymphocytes X 1/4 X 1/8 =1/32).

3. Further work on the arm number hypothesis
454. Since the hypothesis looked plausible and, if valid.
would permit a meaningful use of data collected withlymphocytes in the context of hazard evaluations, thework of Brewen and colleagues catalysed research. The
work that has been carried out during the last few years
in this area (see reference 494 for a recent review) bearon the different steps in the extrapolation sequence.
dicentrics in somafic cells -+ reciprocal translocations insomatic cell-+ reciprocal translocations in sperrnatocytes-+ reciprocal translocations in the F 1 progeny. The
evidence thus far accumulated suggests that the arm
number hypothesis is now less strong than it was at thetime it was formulated and the procedure of
extrapolating from the sensitivity of somatic cells to thatof germ cells in fraught with uncertainty.

(a) The relationship between dicentrics andreciprocal translocations in lymphocytes455. Until recently, most of the data on the inductionof aberrations in human peripheral blood lymphocytes
were collected from experiments that were designed for
purposes other than for testing whether dicentrics andreciprocal translocations are induced at equal frequencies (88, 231, 497, 504). There is a suggestion of anexcess of dicentrics in these studies. The recent work of
Buckton (66), in which she used G· and R-banding to
identify the break-points in human lymphocyte
chromosomes, shows. however, that after an x-ray doseof 200 rad, the frequencies of dicentrics are equal tothat of reciprocal translocations involving two chromosomes (G-banding: 100 cells, 29 dicentrics and 31 trans-locations: R banding: l 00 cells, 26 dicentrics and
27 translocations: conventional staining: I 00 cells,
33 dicentrics and 26 translocations).

(b) Induction of dicentrics in lymphocytes ofdifferent mammalian species
456. Extensive data that have recently become available
on the radiation-induction of dicentrics in peripheral
blood lymphocytes of species other than thoseinvestigated by Brewen and colleagues 2 1 have shownthat the arm number hypothesis is by no means valid for
all the species; exceptions have been found in several
primate species and two carnivores (the beagle dog andthe cat). In addition, the comparative study of de Boeret al. (41) involving, among others, mouse and human
lymphocytes has shown that at x-ray dose levels of 100
and 200 rad (100 rad/min), the frequencies of dicentrics
are equal in both species:
Dose level (rad)
200

JOO

Cells scoredDicentric frequency(%)

Mouse

Man

Mouse

Man

800
12.3

200
10

500
39.6

200
34

The major technical difference with respect to themouse work is that de Boer et al. found that, 36 h after
the initiation of the cultures, the irradiated-stimulated
lymphocytes were in the first division and used this as
their harvesting time. In the work of Brewen et al.,however, longer culture times ( 60-63 h) were used. The
sensitivity of the lymphocytes of the tobacco mouse,
which was also investigated in the study of de Boer et al.
at the l 00-rad level. was found to be the same as that ofnormal mouse lymphocytes.
457. In the rhesus monkey (Macaca mulatta, arm
number 84), van Buul (70) noted that the frequencies
were lower than those in man at x-ray dose levels of 100,
200 and 300 rad, although the difference reachedstatistical significance only at the highest dose (26.6 percent in the monkey compared with 39.0 per cent inman). However, in this study, the absolute frequencies
of dicentrics in man were much lower than thoseobtained in other work (see Brewen et al. (55), forexample).
458. The results of Muramatsu and Matsuoka (352)with lymphocytes of the cynomolgus monkey (Macaca
fascicu/aris, arm number 83) showed that their sensitivity relative to sensitivity of human lymphocytes was
0.79. Hirai and Nakai {229) noted that in the
cynomolgus monkey, the squirrel monkey (Saimiri
sciureus, arm number 77) and the slow loris (Necticebus
cougang. arm number 99 ), the frequencies of dicentrics
after 100. 200 and 300 rad of gamma rays were roughly
similar. The lymphocytes of the two carnivorous species,
the beagle dog (Canis familiaris, arm number 79) and the2 1 Brewen er aL had observed that in the marmoset, thedicentric yields were consistently higher than expected on thebasis of relative ann numbers.

domestic cat (Felix catus, arm number 71). studied by
Muramatsu and Matsuoka (352) manifested much lower
sensitivities than human lymphocytes {16 and 22 per
cent. respectively, of human lymphocytes). To this list
may be added the work of Sasaki et al. (501) withhuman lymphocytes. These investigators found that, at adose of 160 rad, approximately twice as many exchangeaberrations (dicentrics plus rings) were induced in thelymphocytes of individuals who had trisomic constitutions ( trisorny 21, trisomy 18, trisomy D etc.) as inthose of normal individuals.
459. In contrast to the above findings, the work of
Scott and Bigger (502) with potoroo lymphocytes (armnumber 24) has shown that the frequencies of dicentrics
observed were in agreement with the expectation basedon the arm number hypothesis. The data of Leonard etal. (286) for the lymphocytes of the goat (Capra hircus,
arm number 60), the sheep (Ovis aries, arm number 60)
and the pig (Sus scrofa, arm number 64) showed that, up
to 300 R (x rays). there were no striking differences
between the dicentric frequencies of these three species.
After 400 R however, higher yields were obtained withthe pig and goat lymphocytes (76.3 ± 4.4 and88.5 ± 4.5 per cent) than in those of sheep(57.5 ± 3.7 per cent). Although the sensitivity of thelymphocytes of the cow (Bos taurus, arm number 64)
was also assessed in the work of Uonar!l et al., thefrequencies between replicates were so wi~ely different
that a firm conclusion cannot be made. The averagefrequencies recorded up to 300 R were not very
different from those in the other three species, and at
400 R they were similar to those in sheep.
460. With rabbit lyiµphocytes, the reported results arecontradictory: Scott and Bigger (502) found that afterx- or neutron-irradiation, the observed frequencies ofdicentrics were less than one half of those in human
lymphocytes. The x-ray data of Muramatsu andMatsuoka (352) and the gamma-irradiation data of
Sasaki {498) for the rabbit are in line with those of Scottand Bigger. In contrast, Bajerska and Liniecki {20) foundthat the frequencies of dicentrics in rabbit lymphocytes
were similar to those recorded for human lymphocytes
in an earlier study {291 ). There were several technicaldifferences between the experiments of Bajerska and
Liniecki and those of the others, so that here again. nofirm conclusion can be reached.

(c) Induction of dicentrics in mouseand human fibroblasts
461. In assessing the radiosensitivity of another type ofsomatic cells, namely fibroblasts, Sasaki ( 499) foundthat (a) mouse embryonic fibroblasts irradiated in vitro
at the fourth and sixth subculture generations
manifested the same sensitivity to the induction ofdicentrics as comparable human material irradiatedunder similar conditions. In addition, the frequency ofdicentrics recorded were very similar to those in an
earlier study of Sasaki and Tonomura (500) at
100-500 R with human lymphocytes: {b) fibroblasts
from mice irradiated as embryos or as new-boms wereless than one half as sensitive as those mentioned under(a), but the frequency increased to about 60 per cent for487

4-day-old mice and to 90 per cent for 11-day-old mice;and (c) human embryonic fibroblasts (from embryos
9-11 weeks old) had the saml! sensitivity as that offibroblasts in advanced in vitro passages (see (a) above).
462. In other experiments briefly mentioned in his
paper, Sasaki (499) noted that in the cultured fibroblastsof the rat (62 arms) and the African green monkey(Cercopithecus aethiops, arm number 120) similar yields
of dicentrics were obtained. despite the wide differencein the chromosome arm numbers.

(d) Induction of reciprocal translocations in spennatogonia of different mammalian species
463. Work on translocation induction in spermatogoniaof rats, guinea-pigs and rabbits have revealed no apparent
correlation of the relative yields with arm numbers.
Thus, after 300 R of x irradiation, the frequencies oftranslocations in the rat and the mouse were the same
( 191 ). The findings of Lyon and Cox (309) show that in
the range of l 00-300 rad of x rays, the mouse, with
40 chromosome arms, is the most sensitive, followed by
the rabbit (80 arms). guinea-pig (64 arms) and golden
hamster (80 arms). Essentially similar results wereobtained with post-meiotic germ cells of the above four
species. In the rhesus monkey, van Buul (70) and Lyon
et al. (314) found that reciprocal translocations were
induced at much lower frequencies than in the mouse.

(e) Relationship between dicentrics (lymphocytes)
and translocations (spemiatogonia)
464. It may be recalled (paras. 452-453) that the resultsof Brewen and Preston (54) in the Chlnese hamster,mouse, marmoset and guinea-pig 2 2 suggested that there
appeared to be a definite quantitative relationshlp
between the frequencies of dicentrics in lymphocytes
and those of reciprocal translocations in spermatocytes,
namely, that the latter were about one fourth of theformer. The complete data recorded in the paper ofBrewen, Preston and Gengozian (57) for the marmoset
and man 2 3 now raise questions about the validity of thisrelationshlp, as can be seen from an inspection oftable 43. In this table data for the rhesus monkey andthe rabbit are also given. It will be clear that therelationshlp between the dicentric frequencies (inlymphocytes) and reciprocal translocations (in spermatocytes) is dose- and species-dependent. Also worth
noting is the finding that the radiosensitivity of humanspermatogonia is more similar to that of the marmoset
than to that of the rhesus monkey.

(f) The relationship between the frequency of translocations observed in spermatocytes and in the F 1 progeny
465. The new data of Generoso et al. (186) and of
Brewen et al. (56) on the relationship between thefrequency of translocations recorded in spermatocytes
2 2 The guinea-pig and marmoset results were incompleteat the time the paper (54) was published.
.
"Preliminar)' data on human testicular irradiation given
in reference SI were not inconsistent with a two-fold higher
sensitivity of our species relative to the mouse for the inductionof translocations.

488(after spermatogonial x irradiation) and that in the F 1progeny have raised the possibility that the latter may be
one fourth of the yield in spermatocytes at an exposurelevel of 150 R. one that is consistent with theoretical
expectations. This is in contrast to the observations of
the above authors at 300, 600 and 1200 R and theearlier ones of Ford et al. at 1200 R. where the ratio was
one eighth. The limited data of Pomerantzeva et al.
(416) (see paragraph 132) suggest that at a gamma-ray
exposure level of 900 R (delivered in three equal
fractions at 28-day intervals), the above ratio may
be one fourth. although not significantly different fromone eighth.
4. Problems, perspectives, summary and conclusions
466. The information presented in the preceding
paragraphs supports the view that the arm number
relationship is not adequately documented to be used atpresent to predict from lymphocyte data of one species
the expected frequencies of dicentrics in lymphocytes ofanother, nor can it be reliably used to estimate thefrequencies of translocations in spermatocytes of one
species from those of another. Furthermore, thetwo-fold hlgher sensitivity of human lymphocytes to the
induction of dicentrics (relative to the mouse) found by
Brewen et al. could not be confirmed. The available
human data (testicular irradiation), although consistent
with a two-fold higher sensitivity of the human testis totranslocation induction relative to mouse. do not ruleout a difference as hlgh as four-fold. Besides, the primate
data considered as a whole show striking species
differences.
467. The research interest generated by the work of
Brewen and colleagues and the work that has followed
have exposed several interesting facets of the problem of
the induction of exchange-type aberrations. whether it
be in somatic or germinal cells. Among these may bementioned (a) the variatior.3 in aberration yields
depending on fixation times arising as a consequence ofthe heterogeneity of the radiosensitivity of thelymphocytes even though they are at the same stage ofthe cell cycle; (b) differential spermatogonial killing in
the different species, the nature and kinds of reciprocaltranslocations induced in spermatogonia and theirdifferential effects (leading to different degrees ofunderscoring in spermatocytes, for instance): (c)differences in radiosensitivity between chromosomes andbetween parts of chromosomes and differences in thecapacity for accurate DNA repair: and (d) the e:x."tent towhlch aberration induction is influenced by the amountand/or distribution of heterochromatin and the organization of the chromosomes and heterochromatin in the
nuclei of cells or cell stages irradiated.
468. In their human lymphocyte experiments.
Searbright (504), Holmberg and Jonassen (231 ),Caspersson et al. (88) and Ayme et al. (16) found thatchromosome breakage was non-random and that the
break-points were located preferentially in the so-called
R bands (bands of weak fluorescence). In contrast,
Buckton's results (66), while not contradicting thefindings of non-random breakage of the chromosomes,
show that chromosomes rich in R-band material are notpreferentially damaged by irradiation at all and that

about 30 per cent of the breaks that are induced occur
neither in a G-positive nor in a negative band. but on theinterface between them (which certainly does notconstitute a third of the chromosome material). She
argues that most of the investigators have used either G
or Q bands for identification purposes and the majorityof identifying features on the chromosomes isconstituted by the positively stained bands (389).
Because of this, if one positively stained band isdetermined to be present and the next positive band to
be absent, there will tend to be a bias for locating the
break-point in the associated negative band. Her
technique in which the same cells are G-banded followedby R-banding appears to permit greater resolution of thelocation of break-points. In any case, the observations
that breakage is non-random are valid, although themechanism underlying it is not known. This nonrandomness may have important implications for theformation of chromosome aberrations.
469. Conclusive cytogenetic evidence on the radiosensitivity of our species for translocation induction (atleast in males) will have to come from irradiated humanmaterial. In the meantime, interspecific cytogenetic
studies with several experimental mammals (including
primate species) will continue to serve a useful purposein providing us with insights into the kinds of problems
that we are likely to encounter at the human level, sothat the right questions can be asked.

Ill. NATURE OF RADIATION-INDUCEDDAMAGE AND THE PHENOMENON OF
REPAIR
I. Introduction
470. The 1972 report of the Committee considered insome detail the developments in the molecular andcellular radiobiology of damage to DNA induced by uv
light and ionizing radiations and the processes of repair
associated with it. The past few years have witnessedconsiderable progress in the study of uv damage in DNA
and its repair in prokaryotic systems and an extension
and application of this knowledge and know-how toeukaryotic systems and to damage induced by ionizing
radiation and a number of chemicals. A comprehensive
and up-to-date appraisal of the current state of the art inthis area is given in the proceedings of the recent Squaw
Valley Conference (343). Much of the progress madestems from the continued refinements of the existing
methods and the development of more powerful andmore sensitive ones: these have led to the isolation andcharacterization of a number of enzymes involved in the
repair of DNA damage and the exploitation of theunique properties of such purified specific lesionrecognizing enzymes in further studies (392).
2. Progress in the enzymology of repair
after uv irradiation
471. Ultraviolet and chemical treatment, particularly
with prokaryotes, has led to the elucidation of major
enzymatic reactions underlying both photoenzymaticand excision repair. An E. co/i photoreactivating enzyme
has been purified and characterized (551). A number of
specific lesion-recognizing enzymes involved in excision
repair (after uv or chemical treatment) have been
purified and characterized including a further delineation of their roles (49. 101, 102, 103, 194, 207, 227.
392, 423, 590). Among these may be mentioned
endonucleases specific for pyrimidine dimers, apurinic
sites. uracil residues etc. (see references 207 and 392 forrecent reviews). Advances in the study of postreplication repair in bacteria have demonstrated. among
other things, that uv-induced pyrimidine dimers remain
in high molecular weight DNA after DNA replication(181. 182, 449) and t,hat they are transferred (by aprocess which may involve genetic exchange) into all
daughter strands during post-replication repair (182).
Evidence is rapidly accumulating for the existence ofstill another type of repair process in £. co/i-called the··sos repair" -which is induced or activated following
damage to DNA, one which is ''error-prone" ("mutationprone") (424,603,615).
472. The continued efforts to elucidate repair pathways
in£. coli and other prokaryotes have greatly aided in the
search for similar ones in a number of eukaryotes. The
existence of three repair mechanisms-photoenzymatic.
excision and post-replication repair-is now documented
in several higher organisms (213), including mammaliancells cultured in vitro (388). Attempts to isolate andcharacterize the photoreactivating enzyme from humanleucocytes (550, 552) and from murine and humanfibroblasts (553) have been fruitful. Sutherland et al.
(554) have recently demonstrated that fibroblasts frompatients with xeroderma ·pigmentosum contain low
levels of the photoreactivating enzyme (0-50 per cent of
normal in the four different strains tested). With the
exception of £. coli, the excision repair process has
perhaps been most extensively investigated in human
diploid fibroblasts. The impetus came from thediscovery of Cleaver in 1968 ( 110) that the molecularbasis for one of the xeroderma pigmentosum syndromes(XP) in man is a defect in an early step in excision
repair. Subsequent research revealed that XP is much
more complicated than was originally imagined. (Seereference 112 for a recent review.)
473. Employing somatic cell hybridization techniques.
it has been possible to demonstrate the presence of fivedifferent complementation groups (A to E) associatedwith defects in excision repair in XP cells (43, 138, 139,
140, 258, 435). There is also evidence for the
involvement of defects in post-replication repair in XP(276). Tanaka et al (568a) found that whenuv-irradiated XP cells of complementation groups A to E
are incubated with endonuclease V of bacteriophage T4,
the ability to perform unscheduled DNA synthesis wasrestored in all of them. These results suggest that thoseXP cells might have a common defect in the first step ofexcision repair. Motelmans et al. (347) recently showedthat crude extracts of normal human diploid fibroblasts
and of human peripheral blood lymphocytes excise
thymine dimers from purified uv-irradiated E. coli DNAor from the DNA presumably present as chromatin incell-free preparations of cells that has been labelled with
3 H-thyrnidine. Extracts of XP cells from complementation groups A, t and D also excise thymine dimers from489

purified DNA, but extracts of gr~up-A_ cells do notexcise dimers from the DNA radioactively labelled,
unfractionated cell-free preparations. These findings
suggest a biochemical defect in cell-free extracts thatmimics a defect known to exist in intact XP cells.

repair enzymes peculiar to each species not only remainfunctional in the resulting multinucleate heterokaryons,
but are free to act on pyrimidine dimer-containing sites
in foreign DNA contributed by the fusion partner ...
with absolute impartiality" (393).

474. Buhl and Regan (67) reported the presence of sites
sensitive to dimer-recognizing endonuclease from Micrococcus /uteus in newly made DNA from uv-irradiated XP
cells; this fmding and one reported by Meneghini and
Hanawalt (333) are similar to those recorded by Ganesan(182) in bacteria, suggest that DNA synthesis after
uv-irradiation does not necessarily leave gaps opposite
the dimers, raise the possibility of a recombination
mechanism in which segments of dimers containing
parental strands are inserted in daughter stands.
However, in work with uv-irradiated XP cells, a Chinese
hamster cell line, and chick embryonic cells (all of theseare defective in the excision of dimers), using a protocol
similar to that of Buhl and Regan (67), Paterson,
Lohman and Sluyter (396) obtained no indication ofendonuclease-susceptible sites in the daughter DNA. The
technique employed by Paterson et al. would havedetected the presence of as few as 15 per cent of the
dimers initially induced in the parental DNA.

478. The use of uv-irradiated human viruses as asensitive test to detect the various defects in DNA repair
have already yielded interesting results (131. 425). For
instance, uv-irradiated adenovirus-2 assay procedures
have revealed that every xeroderma pigmentosum strain
tested has a DNA repair defect, including thosepreviously judged by other methods to have normal
DNA repair.

475. Work with XP prompted the search for similar
repair defects in other human diseases but thus far has
met only with limited success (see also paragraph 502).
Thus, for example. in fibroblasts from a patient withF anconi' s anaemia ( characterized by a progressive
aplastic anaemia, increased frequencies of spontaneousand radiation-induced chromosome aberrations and ahigh incidence of cancer) normal amounts of unscheduled DNA synthesis were observed at early times after
uv-irradiation: however, the excision of dimers assayedat 24 h was depressed compared to normal cells at doses
over 15 J/m2 (254-nm radiation). Excision seemed
normal at early times and, at low doses, at late times( 418). It may be that at high doses the basic defect is
not in the repair system but in other cellular properties
that result in the turn-off of the repair system at highdoses and long times (530).
476. Some success has been achieved in showing thepresence of putative dimer-specific endonuclease and adimer-excising exonuclease in extracts of human cells(18, 142). Work in this area of enzymology of repair in
mammalian cells has just begun.
4 77. Cell-fusion techniques have also been successfully
used at the interspecific level: the experiments ofPaterson et al. (397, 398, 399) and Paterson andLohman (306) focused attention on a further characterization of repair mechanisms present in human and chickfibroblasts and an elucidation of the efficiency withwhich repair enzymes unique to each species can operate
on uv-damaged foreig_n DNA in multinucleate humanchick heterokaryons artificially derived from both
fibroblastic lines. Advantage was taken of the fact thatprimary human cells lack photoenzymatic repair but
possess excision repair machinery. whereas chickfibroblasts possess photoenzymatic repair but notexcision repair, to cope with uv-induced pyrimidine
dimers. The results provide good evidence that "after
artificial fusion of human and chick fibroblasts, DNA

3. Ionizing radiation:
nature of the lesion produced and its repair
479. In contrast to the great strides made with the uv
work, a brief overview of which was given in thepreceding paragraphs, the progress in our understandingof the precise nature of the lesions induced by ionizing
radiations and the repair processes operating on thisdamage must be considered modest. Ionizing radiations
induce single- and double-strand breaks in the DNA,
cross links, apyrimidinic and apurinic sites, as well as
damage to the base and sugar moieties. The elucidationof the relationship between changes induced in the
biological activity of DNA by ionizing radiation andspecific structural modifications or lesions has therefore
been difficult. Most of the available information isconcerned with the production of single- and doublestrand breaks, their yield, rejoining and biologicalsignificance; some of it was reviewed in the 1972 reportof the Committee.
480. The physical reconstruction of radiation-induced
single-strand breaks has been shown to occur in a
number of organisms and systems investigated in thisrespect. In E. coli, this process of repair is mediated byat least two distinct mechanisms: rapid repair involving
DNA polymerase I and a slower. presumable recombination. repair involving the recA gene product (574,575). In the radioresistant bacterium Micrococcus
radiodurans, radiation-induced double-strand breaks also
are repaired (132, 257), and evidence for such repair isaccumulating in other systems as well (116, 242, 269,
432). Recent studies on a radiosensitive mutant ofMicrococcus radiodurans have shown that it was
deficient in the initial fast-rejoining reaction and thesecond phase proceeded more slowly, concomitantlywith greater·degradation of the DNA (42). The work ofGentner (187) has provided evidence for the presence ofa level of Mg-dependent DNA polymerase in the mutant
that was only 2 per cent of normal. A good coverage ofthe efficiency of the production of double-strand breaks
and the dynamics of repair of these lesions in
Micrococcus radiodurans is given in Burrell and Dean

(69).
481. It has been known that extracts of Micrococcus
luteus (394, 610), E. coli (568) and human cells (50)contain endonuclease activity which selectively attacks
defects other than strand breaks in gamma-irradiatedDNA (the so-called ·•gamma lesions"). Using crude

490

1

l

protein extracts of 12-day-old chick embryos as thesource of the test endonuclease in in vitro assays,
Paterson and Smith (395) recently identified gamma
lesions in the DNA of human diploid fibroblasts. Under
anoxic conditions, the incidence of gamma lesions
approximates 12 times that of single-strand breaks
concomitantly produced. Substitution of the chickextract with a crude extract of Micrococcus luteus yields
the same number of lesions, suggesting that theendonuclease activities involved have identical, or atleast closely overlapping, substrate specificities.
Although there is some indication that both the type of
gamma lesions and the yield depend upon the irradiation
conditions (563), the chemical identity of the
gamma lesions is not known with certainty. Two
possibilities exist, namely apurinic sites and ringsaturated radiolysis residues of pyrimidines (see below).
Since neither the calf thymus endonuclease (Bacchetti
and Benne, 17) nor E. coli extracts act on apurinic sites,
ring-saturated pyrimidine residues are the more likely. In
fact, the work of Hariharan, Cerutti and colleagues
strongly supports this conclusion (reviewed by Cerutti(96)).
482. Early evidence for the contribution of DNA basedamage to the lethal effects of ionizing radiation camefrom work with bacteriophages in which the formationof single- and double-strand breaks as a function of doseand radiation conditions could be accurately measured
and related to the loss of infectivity. The involvement ofsome form of base damage in the inactivation process
was inferred when Jess than one double-strand break was
introduced per genome per biological hit (177, 178,
597). While a considerable amount of work has been
done on the radiation chemistry of free nucleic acidbases, especially of thymine, not much is known as towhether or not the major reactions observed in thesemodel systems under non-physiological conditions are
representative of reactions occurring in situ in the living
cell. The recent experimental demonstration in Cerutti'slaboratory of the formation of thymine damage
following irradiation with ionizing radiation (gammarays) in both bacterial and mammalian systems and theprogress made in the enzymology of repair of theselesions therefore represent a very significant forward
step in molecular radiobiology with ionizing radiations.
While the assay procedures used permit the identification only of damage to thymine, additional damage
undoubtedly occurs at adenine, guanine and cytosine
moieties, and consequently it has been concluded thatbase damage is a major type of damage induced byionizing radiation in bacterial and mammalian cells (96).
483. Hariharan and Cerutti (214, 215) have developed
two highly sensitive radiochemical procedures for thedetermination of gamma-ray-induced thymine damage in
DNA both in vitro and in vivo. The · first assay
determines the radiochemical reactivity of the thymine
methyl group and the second, ring-saturated radiation
products of thymine of the 5-hydroxy-6-hydroperoxydihydrothymine type (the latter will hereafter beabbreviated to t'). In a series of papers, evidence waspresented showing that (a) thymine damage is one of the
major types of gamma-ray-induced damage in bacterial(214,215,556, 557) and mammalian cells in situ (219,440); (b) excision repair of such products occurs inbacterial and mammalian cells (97. 215, 219); (c) crudeconcentrated extracts of E.coli (216, 218) and isolated
mammalian nuclei (219,326,441) are able to selectively
remove the t' residue from gamma-irradiated poly[d(A·T)] or Os0-1-oxidized poly [d(A·T)] or bacteriophage PM2 DNA: (d) although the identity of theendonuclease recognizing t' products in £. co/i is not
fully established, possible candidates appear to beendonuclease II (196) and the endonuclease recognizing
depurinated DNA (598); (e) the second step in theexcision repair of t' (namely the exonucleolytic
degradation of the damaged DNA region) is accomplished by 5 1 -+ 3' exonuclease associated with thepolymerase I of E. coli (247); (t) the last step in excision
repair (strand resealing) is accomplished by ligase (216);
and (g) the excision of gamma-ray-induced thymine
damage in mammalian cells used (CHO, HeLa S3) is
considerably faster-between 25 and 35 per cent of thet' removed within 60 minutes of incubation at 37°C
(219)-than that of cyclobutane-type photodimers,
which has been shown to continue for several hours inhuman cells (see Cleaver (111) for a review of the latter).
484. In the context of the excision repair of gamma
lesions discussed in the preceding paragraph, it is worth
noting that Paterson et al. (400) observed a decreased
ability of some ataxia telangiectasia (AT) strains toundergo repair replication following exposure to gamma
rays delivered under anoxic conditions. This finding
implicated defective DNA repair in the aetiology ofradiosensitivity and predisposition to neoplastic transformation, two clinical features of the syndrome. In
leucocytes from these patients, the radiation-induced
chromosome aberrations were found to be enhanced(228, 426). In fibroblasts, the rates of rejoining of single
and double-strand breaks appear to be similar to those innormal cells (571).
485. The results of Remsen and Cerutti (430), on theother hand, show no in vitro deficiencies for the excisionof t' products from exogenous DNA-chromatin
substrates (from sonicates of three AT cell lines tested,
including two studies by Paterson et al. (400)) which
had been gamma irradiated under aerobic conditions.
Remsen and Cerutti point out that differences inexperimental design (in particular the irradiation
conditions) between their work and that of Paterson etal. do not permit a straightforward comparison of theresults. In addition, Remsen and Cerutti used unirradiated cell preparations as the enzyme source whereas
supra-lethally irradiated cells were used by the otherinvestigators. Different lesions are produced in DNA
under aerobic and anoxic conditions, which may beprocessed by different repair pathways. Support for thisnotion comes from work with XP cells, where ionizing
radiation induced normal levels of repair replication
under aerobic but not anoxic conditions (386).
486. The role of DNA repair in the progeria syndrome(a human genetic disease characterized by accelerated
ageing) is not clear and the reports that have appeared
are contradictory. Epstein et al. (158) reported thatprogeroid fibroblasts seemed defective in strand-breakrepair, but Regan and Setlow (429) were unable torepeat this observation and suggested that the basic
defect is not one of repair enzymes, but more likely one491

of permeability or fragility of progeroid cells in culture.
From the findings that several progeric strains manifest
differing and unusual responses to ionizing radiation, afinite repair capacity in repair-deficient strains and that
normal repair apparently returns after SV40 transformation, Little et al. (292) suggest that the control of
the genetic expression of a repair system may be
involved.

4. DNA repair in mouse genn cells
487. In spite of a number of DNA repair studies carriedout with different mammalian somatic cell systems, notmuch similar work has been carried out with germ cells
until recently. The report of Sega (522) in 1974
demonstrating unscheduled DNA synthesis in the germ
cells of male mice exposed in vivo to alk.ylating chemicalmutagen EMS, 24 therefore, is a very significant
contribution. Using the well studied sequence of eventsoccurring during spermatogenesis in particular andadministering (by testicular injections) 3 H-dT. Sega
obtained clear evidence for unscheduled DNA synthesis;
the germ cell stages showing it ranged from early to
middle meiotic prophase stages through early to middle
spermatids. The initiation of thls synthesis (taken to beevidence for repair of chemically-damaged DNA in thesegerm-cell stages) is rapid, beginning within one hour
after injection of EMS. Unscheduled DNA synthesis was
not detected in the most mature germ-cell stages, whlch
are responsive to the induction of dominant lethals, nor
was it found to occur in stages where protamine replaces
the chromosomal proteins {during spermiogenesis).
488. Subsequently, Sega et al. (524) focused attention
on early spermatids and extended the findings with EMS
to three chemical analogues: MMS, PMS and lMS. The
results indicate that (a) with all four there is a linear
increase in DNA repair in early spermatids withincreasing dose of the chemical; only at the hlghest doseof MMS (100 mg/kg) is the repair response observed
hlgher than expected on linearity; and {b) withequimolar doses. the most effective chemical in inducing
DNA repair is MMS followed by EMS, IMS, and PMS.
489. In a further study, Sega (523) has extended thefinding of unscheduled DNA synthesis (with chemicals)
to x-ray damage. The stages showing the repair of x-ray
damage are the same as those found undergoing repair of
EMS-induced damage except that the level of unscheduled incorporation of 3 H dT after x-ray treatment ismuch less, in spite of the fact that the EMS dose used
(25 0 mg/kg) gives a roughly comparable yield of dominantlethals to that given by 600 R, ofx rays (185). Secondly,
the level of DNA repair occurring in early spermatids has
been found to jncrease linearly with increasing x-ray
exposures from 50 to 600 R, and at 1200 R there isevidence for an elevated level of repair. Thirdly,
unscheduled DNA synthesis (after 600 R) is highest
immediately after irradiation and continually decreases
during the subsequent 4 h although some repair is stillmeasurable at this time.
2 • EMS =ethylmethanesulphonate,
MMS =methyl~ethane sulphonate, PMS =propylmethane sulphonate, IMS =1sopropylmethane sulphonate.

492

490. Ono and Okada (386) irradiated male mice
10-14 weeks old (gamma irradiation, I 0-40 krad,
3300 rad/min) and used the testes from these animals to
study single-strand breaks in the DNA and their rejoining
using alkaline density-gradient centrifugation methods.
Microscopic examination of cell suspensions from thetestes revealed that the population was made up of
about 3 per cent spermatogonia, 12 per cent spermatocytes, 70 per cent spermatids, 6 per cent spermatozoa
and 9 per cent unknown. The conclusions drawn
therefore apply primarily to DNA from spermatid-rich
populations. It was found that the number ofsingle-strand breaks was 0.22 per 10 12 daltons of DNA
per rad, one whlch was significantly lower than the rateof 0.6 to 0.7 per 10 12 daltons per rad for mouse liver
and thymus DNA found in an earlier investigation {385).
The authors obtained evidence to show that thisdifference is primarily due to hypoxic conditions of thetesticular cells. A majority of the breaks were found torejoin quickly, the time necessary for rejoining of 50 per
cent of the breaks being about 18 min at 10 krad and
50 min at 40 krad. The remainder of the breaks did notseem to rejoin until about 6 h after irradiation. Ono andOkada suggest that these latter breaks might be of theslow and/or non-reparable DNA scissions, since evenunder "low" dose-rate conditions ( 40 rad/min comparedwith 3300 rad/min) a similar situation was obtained.
491. In a subsequent study, Ono and Okada (387)
refined their methods and were able to study
single-strand breaks and their rejoining in spermatogoniarich and spermatozoa-rich populations. In spermatogonia, single-strand breaks were induced at a rate of
0.42 10 12 daltons of DNA per rad under oxic (aerated)conditions, decreasing to 0.24 per 10 12 daltons per radunder hypoxic conditions. In spermatozoa, the breakefficiency was 0.22 breaks per 10 12 daltons per radunder oxic conditions and changed little under anoxic
conditions. The breaks were efficiently repaired inspermatogonia. 50 per cent of the breaks rejoinino
within about 10-min after l O krad and within abou~
30 min after 40 krad. The slow or non-rejoining fraction
for these doses was 40-60 per cent. In contrast, thebreaks in spermatozoa were not rejoined at all even twodays post-irradiation.
492. Masui and Pedersen (325) examined the repair
capacity of full-grown mouse oocytes after exposure to
uv-irradiation. Fully-grown oocytes were asepticallyisolated from the ovaries of adult mice and cultured inchemically-defined medium. Irradiations were carriedout 2-18 h after isolation and the oocytes were then
immediately incubated with 3 H-dT. Many of the oocytes
underwent germinal vesicle (GV) breakdown within 3 hafter isolation and metaphase I (MI) was reached 4-8 hlater. Oocytes cultured for 16-18 h reached metaphase II(MII) as evidenced by the appearance of the first polar
body (PBI). Autoradiography showed that grain counts
over the GVs or the chromosome set were significantly
higher after uv irradiation than in controls, regardlessof the stage of meiotic maturation at which they were
irradiated. The grain count increased with dose from 30
to 60 J/m 2 , but except for the MI oocytes. there was nofurther increase at 120 J/m 2 • The numbers of grains overthe GVs were approximately an order of magnitude
higher than over the MI and MII chromosomes and thoserover the PBI were much less (relative to MI and MII). It
is thus clear that mouse oocytes can carry out
unscheduled DNA synthesis after uv irradiation and thus
seem capable of excision repair of DNA damage in thesestages.

5. DNA repair in Drosophila
493. In recent years, an increasing interest has been
manifest in studies on DNA repair in Drosophila. In
1969, Valencia and Plaut (594) demonstrated unscheduled DNA synthesis in the salivary glands of x-irradiatedlarvae. Trosko and Wilder (577) showed that thyminecontaining dimers are excised from the DNA of
uv-irradiated tissue culture cells. Boyd and Presley ( 4 7)
showed that repair replication of DNA (in first instar
larvae) was stimulated by treatment with uv. x rays.
MMS and EMS; MMS was found to be ten times aspotent as EMS. With uv, a dose of 20 J/m 2 largely
saturates the level of repair replication; the latter falls
off after x-ray exposures of 80 kR. The authors alsostudied photo repair (as a reduction in repair synthesis
resulting from post-irradiation exposure to photoreactivating light) and found that this occurs afteruv irradiation but not after x irradiation.
494. Other lines of work have focused attention on theisolation of mutagen-sensitive mutants as experimental
material for the systematic analysis of genetic controlsof DNA replication. DNA repair and recombination(542, 543). A number of mutants which were selectedon the basis of hypersensitivity to larval killing by lvIMS
have now been assigned to seven complementationgroups on the X-chromosome (48, 543), and mutants at
one locus have been clearly identified as alleles of apreviously described meiotic mutant mei;'IJ (23),thereby confirming that mutagen sensitivity can be
employed as an effective method for the selection ofrecombination-defective mutants in this organism. In
addition, mutants belonging to certain complementationgroups are sensitive to nitrogen mustard (HN 2 ) andcurrent evidence (48) indicates that there is a correlation
between sensitivity to HN2 , to 2-acetylaminofluorene
and a deficiency in post-replication repair. Several of themutants exhibit sensitivity to gamma rays, although only
the mei-41 mutants are hypersensitive to uv irradiation.
Nguyen and Boyd (362), who studied the mei-9b mutant(mutant alleles at the mei-9 locus which reduce meiotic
reco~bination in homozygous females (77)) using thetechnique employed previously by Boyd and Presley(47), found that it was deficient in repair replication
after uv or x irradiation. This result is complementary totheir observations that homozygous larvae of mei-9
alleles are hypersensitive to killing by x-rays as well as
uv irradiation.

6.

Relationships between the radiation-induced lesionin DNA, its repair, and the production of mutations
and chromosome aberrations

495. Turning now to the question of how the nature of
the uv- or x-ray-induced lesions in the DNA and themechanisms by which they are repaired can account forthe production of mutations or chromosome aberrations, it can be stated that we have relatively more
information available for uv damage. In prokaryotes
where this problem has been intensively studied, there is
no doubt that pyrimidine dimers are an importantsource of mutations, although · the role of otherphotoproducts is less clear. In the yeast Saccharomyces
cerevisiae. there is evidence that pyrimidine dimers are
responsible. for most of the revertants of cycl-9 in
rad+, rad 1 and rad 6 strains (271) (see also paragraph 498). If pyrimidine dimers or the other photoproducts were the direct cause of mutations, every
kind of repair that eliminates these products should
reduce the frequency of q1utations. This however is not
the case. Convincing evidence for the possibility that
under certain circumstances repair may produce
mutations rather than erase them was provided inbacteria by Witkin (613). Her distinction between
error-proof (mutation-proof) and error-prone (mutationprone) repair has been generally accepted. Both types ofrepair remove lethal lesions and increase survival, butwhile the former restores the original nucleotide
sequence and thus removes potential mutations. thelatter tends to insert a wrong base sequence and thus
produces mutations.
496. Photoenzymatic repair is error-proof; the bulk of
excision repair, too, is error-proof and reduces lethality
as well as mutation frequency. Only a small fraction of
mutations induced by uv arise from errors in
pre-replicative repair while the bulk are produced by anerror-prone post-replicative process. Recently, Witkinand others (188. 614. 616) have adduced evidence thatin bacteria the error-prone mechanism requires an
inducible enzyme for its action.
497. There is much less information on the relationshipbetween uv damage and chromosome aberrations. Griggs
and Bender (202) demonstrated that all the lesions
which lead to uv-induced chromosome aberrations in
Xenopus cells disappear on photoreactivation. This isperhaps the only experiment which directly demonstrates a relationship between DNA repair and chromosome aberrations. Following !}V irradiation of XP cells,
Parrington et al. (391) demonstrated that there was a
six- to seven-fold increase in the frequency ofchromosome aberrations (relative to normal cells).
However, the data are only preliminary, with only one
fixation time, namely 30 h. and the increase could be
due to other factors as well (357).
498. The possible role of single-strand breaks and basechanges in generating x-ray-induced mutations wasconsidered in the 1972 report of the Committee(Annex E, paras. 516-525). The recent experimental
demonstration that base damage is a predominantcomponent of x-ray damage in the DNA (in vivo and invitro) and that it is repairable will undoubtedly intensify
the search for correlations between this and mutational
damage in prokaryotic and eukaryotic systems. Whatever
the nature of the primary lesion, its final fate depends
largely on the type of repair to which it is exposed. It isclear that repair mechanisms-some of them identicalwith those acting on uv lesions-enter. in similar ways
into x-ray mutagenesis. In bacteria. the functions of two
repair genes (recA and exrA) that are essential for uv
493

mutagenesis also play a major role in the production of
mutations by x rays (234). A similar situation is
obtained in the yeast Saccharomyces cerevisiae:
Lawrence et al (271) have shown that the highly
specific uv-inducible reversion of cycl-9 (an ochremutant (UAA codon) of the structural gene for
iso-1-cytochrome c) depends largely on the action of therad6 locus. which appears to be involved in the repair ofdamage induced by uv gamma rays and all chemicals
tested. Radl 8, a gene concerned in the same pathway as
rad 6, also has some influence on the specificity. though
the evidence is less decisive in this case. Rad], a genethat functions in ecxision repair. has no effect on thisspecificity.
499. In a subsequent paper (271a), Lawrence andChristensen have presented data which suggest that in
Saccharomyces cerevisiae there is a single error-prone
repair pathway involving the function of at least sevengenes (rad6, rad9, rad18, revl, rev3 and possibly re112
and rad8). The function of two of these (rad6, rev3)
would seem to be essential for uv mutagenesis at all sites
and of all types, while the others appear to act
principally or exclusively during the production ofcertain mutational events. In contrast, all mutations
blocking excision repair exhibit enhanced mutability perunit dose. a result compatible with the idea that thisprocess is largely error-free.
500. Double-strand breaks in the DNA are presumably
responsible for the production of chromosome aberrations. The possibility of single-strand breaks being
converted into double-strand breaks during repair by asingle-strand nuclease has been proposed by Bender et al.
(35) in their general model for chromosome aberration
production by diverse agents. Currently available
information on the sensitivity of cells from patients withdifferent inherited disorders to the induction ofchromosome aberrations by different mutagens (including x rays) and the known defects in DNA repair
mechanisms in these cells has been summarized byNatarajan (357). In the case of AT cells discussed earlier(para. 484) there is evidence for an increased sensitivity
to chromosome aberration induction by x rays inleucocytes, and as may be recalled, these cells may be
deficient in excision repair of base damage under certain
conditions. XP cells and cells from patients with
Fanconi's anaemia and progeria do not appear to show
any increased sensitivity to x-ray induction ofaberrations although they are known to be deficient in
one or another step in DNA repair.
501. Scott, Fox and Fox (503) studied the relationshipbetween chromosomal aberrations, survival and DNA
repair in tumour cell lines of differential sensitivity tosulphur mustard (SM) and x rays. In the first comparison
with a pair of rat lymphosarcoma cell lines (Yoshida)with a pronounced differential sensitivity to killing by
SM (but with the same sensitivity to x rays). theresistant line suffered much less chromosome damage
after SM treatment than the sensitive cells in spite ofequal alkylation of DNA. RNA and protein in both; theamount of x-ray-induced chromosomal damage wassimilar in both. In the second comparison with a pair ofmouse lymphoma cells (one resistant and the othersensitive to _killing by x rays), much less chromosomal
494

damage was observed in the resistant line. although there
were no differences between the two lines in theircapacities for repair replication either after SM or x-ray
treatment. As Natarajan (357) has pointed out. the typeof measurement used for repair replication, i.e.
unscheduled DNA synthesis, gives information only
about the whole repair process which may not all be
related to the induction of chromosome aberrations.

7. Conventional (non-molecular approach)repair studies in Drosophila
502. Several aspects of radiosensitivity and repair
phenomena studied in Drosophila germ-cell stages werereviewed in the 1972 report of the Committee. A
comprehensive treatment of the subject has sinceappeared (496), besides others which focus attention on
the radiosensitivity of embryonic stages (623) and on
aberration induction and segregation in female germ-cellstages (390). In what follows, some of the more recent
work on repair of genetic radiation damage in
Drosophila and that which has a bearing on therestitution of chromosome breaks induced in mature
spermatozoa will be considered.
503. It has long been known that chromosome breaks
induced in Drosophila spermatozoa do not rejoin before
fertilization (256, 350). More recently, Leigh and Sobels(277) showed that compound chromosomes (autosomes)
homozygous for markers on one or the other of thechromosome arms could be recovered after x irradiationof post-meiotic male germ-cell stages. The interpretationof this finding was that these chromosomes originate bya chromatid type of exchange occurring after the
post-zygotic chromosome replication, the implication
being that the chromosome breaks induced in thesegerm-cell stages stay open until this time. Further
evidence substantiating this thesis came from exposure
fractionation studies which showed interaction of breaks
produced in spermatids in male flies (544) (or in malepupae, 48 h old (545)) with those produced in mature
spermatozoa (in inseminated females). There are otherfindings which strongly suggest that the repair
machinery in the oocytes plays a vital role in theformation of induced chromosome aberrations in male
germ-cell stages (see paras. 504-505). It should therefore
be possible. by suitably modifying the genotype of thefemales and/or physiological environment of the oocytes
to influence the magnitude of the genetic damage
induced in the male germ cells. The work that has been
carried out during the past few years in the laboratories
of Sobels and of Wurgler adequately support this line ofreasoning.
504. In a series of studies. Wurgler and his collaborators(68, 622, 625) have collected data which indicate that(a) the rate of recoverable ring-X chromosome losses inmature sperm is dependent on the genotype of thefemales used; (b) such "maternal effects" are demonstrable for radiation-induced dominant lethals, loss of
rod-X chromosomes and translocations; and (c) for all
the female stocks, the rate of recoverable XO males falls
drastically to nearly one-half after the first day. The
authors concluded that (a) some factors which are under
the control of the maternal genome influence thex-ray-induced lesions in the sperm chromosomes after
insemination of the egg, (b) a change in the lesions in thestored sperm occurs during the first 24 h of storage inthe females, and (c) sperm inseminating old, stored
stage-14 oocytes (present in aged virgin females) are
influenced by a maternal factor in a different way or toa different degree than sperm inseminating newly
produced non-stored oocytes. The observation of
Wurgler and Maier (622) that the frequency of
sex-chromosome losses induced in spermatozoa isdependent on the genotype of the females used for thematings and that of Wurgler. Burki and Burki (624) andBurki (68) that it is differentially affected in stored andnon-stored oocytes in the females have been extended
by Clark (109).

510. By means of cell-fusion techniques (human andchick fibroblasts) it has been possible to show that the
repair enzymes peculiar to each species remainfunctional in the resulting multinucleate heterokaryons
and act on pyrimidine dimer-containing sites in foreign
DNA contributed by the fusion partner.

505. Following the earlier work of Proust (421) and of
Proust, Sankaranarayanan and Sobels (422), whichshowed that mating of irradiated males to females
injected with actinomycin D led to an increase in thefrequencies of dominant lethals and a decrease in thoseof translocations and recessive lethals, Mendelson (330,331) and Mendelson and Sobe ls (332) extended this lineof inquiry to the effects of caffeine treatment offemales. It was found that (a) caffeine treatment offemales led to an increase in the frequencies of dominantlethals and sex-chromosome losses and a decrease in
those oftranslocations induced by x irradiation of mature
sperm; (b) different strains of females used manifested
different levels of repair of radiation-induced breaks inmales, with one strain very deficient in this repair
machinery: and (c) the repair system specific for the
repair of chromosome damage induced in the paternal
genome is susceptible to blocking by metabolic
inhibitors such as caffeine.

512. Some ataxia telangiectasia cell strains appear to be
defective in the excision repair of gamma lesions
following gamma-ray exposures under anoxic conditions:after irradiation and under aerobic conditions, they are
excision-repair proficient.

506. In a very recent study, Maddern and Leigh (320)have obtained convincing evidence for the thesis that ahigh proportion (as much as 75 per cent) of the "'total''and "partial" sex-chromosome loss induced by x irradiation of mature Drosophila sperm is a consequence ofchromatid rearrangements arising from chromosome
breaks which stay open until they are replicated. Thisfinding provides a fresh insight into our thinking on themechanism of chromosome loss.

8. Summary and conclusions
507. The past few years have witnessed the accumulation of a substantial amount of information on DNA
damage induced by mutagenic agents and on repair
processes in both prokaryotic and eukaryotic systems.
508. A number of specific lesion-recognizing enzymes
involved in excision repair have been purified andcharacterized. Evidence for the presence of photoreactivating enzymes in human leucocytes, human andmurine fibroblasts have also been adduced.
509. With the exception of E.coli, the excision repair
process is perhaps most extensively investigated inhuman fibroblasts. There is evidence that there are atleast five complementation groups associated withdefects in excision repair in xeroderma pigmentosum
cells. Search for similar repair defects in other humandiseases has met with only limited success.

511. The most significant advance in ionizing radiation
studies is the identification of base (thymine) damage
and the development of sensitive procedures (radiochemical) for detecting it in microorganisms as well as inmammalian cells. These gamma lesions (as they arecalled) are subject to excision repair and an endonuclease acting specifically on such lesions has beenfound.

513. The induction of single-strand breaks in mousegerm cells has been investigated. The results show thatthey are induced at rates of 0.42 per 10 1 2 daltons of
DNA per rad in spermatogonia, 0.22 per 10 12 per rad inspermatids, and 0.22 per 10 12 per rad in spermatozoa(under aerated conditions). A majority of the singlestrand breaks induced in spermatogonia and sperrnatids
undergo rapid rejoining (50 per cent of the breaks
rejoining in 10-18 minutes after 10 krad), while theremainder were of the slow or non-rejoining type. In
contrast, in spermatozoa, the breaks induced do notundergo rejoining even two days post-irradiation.
514. Unscheduled DNA synthesis has been demonstrated to occur following treatment with alkylating
agents and with x rays in meiotic prophase stages and in
spermatids in the mouse; such synthesis also takes placein full-grown mouse oocytes after uv irradiation.
515. There is evidence for the occurrence of unscheduled DNA synthesis in the salivary glands of x-irradiatedDrosophila larvae and for the excision of thyminecontaining dimers in tissue culture cells. In first instar
larvae, repair replication is stimulated by treatment with
uv, x rays, MMS and EMS.
516. In Drosophila as in prokaryotes, sensitivity tomutagens can be employed as an effective method forthe selection of recombination-deficient mutants.
Mutants selected on the basis of hypersensitivity tolarval killing by MMS have been assigned to sevencomplementation groups. Mutants belonging to certain
complementation groups are also sensitive to nitrogenmustard, 2-acetylaminofluorene and appear to be
deficient in post-replication repair.
517. The relationships between lesions induced in DNA,
repair processes and mutations have been worked out inprokaryotes and some simple eukaryotes: afteruv irradiation, there is no doubt that pyrimidine dimers
are an important source of mutations, but there isevidence that certain repair processes may be errorprone. Photoenzymatic repair is error-proof; most495

excision repair is also error-proof. The bulk of mutations
are produced by an error-prone post-replicative repair
process (after uv irradiation).

the total genome (DNA content) for the species ... itsuggests that extrapolation directly from experimentalorganisms to man can be done with confidence."

518. In higher eukaryotic systems (mammalian cells, for525. In a subsequent paper. Conger (118) compiled andanalysed tl1e data on the amount of chromosomal
proteins and RNA in relation to the amount of DNA in
different species. He found that the total mass ofchromatin per unit mass of DNA is not very different
over the span from prokaryotes to higher eukaryotes,
i.e., the total chromosomal mass and expected target size
is proportional to DNA content over this span of species.
That permitted him to conclude that ''the correlation of
induced mutation rate with DNA content per cell over awide range of species cannot be used to establish that
DNA alone is the target for induction of mutations byionizing radiation. The target could be DNA alone; butequally well, it could be total chromatin mass." The Ad
Hoe Panel on Research Needs for Estimating the
Biological Hazards of Low Doses of Ionizing Radiationsof the United States National Research CouncilNational Academy of Sciences ( 431) expressed thisopinion: ''We believe this normalizing of mutation rates
by the amounts of DNA per nucleus is a valid genetic
bridge between lower organisms and man" (see also
Wolff (617)).

instance) there is no direct evidence on the relationshipbetween uv lesions and mutation induction. In Xenopus,
there is some evidence for the involvement ofphotoreactivable uv damage in chromosome aberrations.

519. The recent experimental demonstration that basedamage is a major component of damage induced byionizing radiation in prokaryotic as well as eukaryotic
systems and that it is subject to excision repair will
intensify the search for correlations between these andmutational events.
520. Double-strand breaks induced in DNA are
presumably responsible for the production of chromosome aberrations, but the exact relationship between
these and the known DNA repair processes is not clear.
521. Studies on the repair of x-ray-induced genetic
damage in male germ-cell stages of Drosophila(non-molecular approach) have shown that chromosome
breaks induced in spermatids of pupae 48 h old remain
open and are available for interaction with breaks
produced in spermatozoa.
522. A considerable amount of evidence has beenobtained in Drosophila which strongly suggests that the
repair machinery in females plays a vital role in theformation of induced chromosome aberrations in male
germ-cell stages. This has been demonstrated by suitably
modifying the genotype of the females and byphysiological alteration of the oocyte environment withmetabolic inhibitors.
523. In Drosophila. recent results indicate that a highproportion of the total and partial sex-chromosome
losses induced by x rays in spermatozoa is a consequenceof chromatid rearrangement arising from breaks which
stay open until they are replicated (after sperm entry
into the egg).

IV. INTERSPECIFIC COMPARISON OF
MUTATION RATE ESTIMATES
1. Introduction

524. Recently, Abrahamson et al (4) re-examined theavailable data on forward mutations at specific lociinduced by acute, low-LET irradiation for a wide varietyof organisms from bacteria to mammals and noted thatthe rates per locus per rad varied over three orders ofmagnitude; however, when these rates were adjusted forthe amount of DNA per nucleus, the mutation rates
obtained were all essentially the same, varying by a mere
factor of 3 instead of I OOO. The authors speculated that"the consistency that obtains when the data are adjusted
for the amount of DNA per nucleus for each species
indicates either that it is the nucleus and not the locus
that determines the target size or that, on the average,
the size of a (radiation-mutable) locus is proportional to496526. Kohn (265) made estimates of gene sizes indifferent species based on target theory considerations
and found that (a) in the mouse, the estimates from thedata on the seven-specific-loci tests (300 R, acutex irradiation. spermatogonia) are consistent with expectations, whereas those from the histocompatibility lociare not; and (b) most of the genes ( or groups of genes)
tested in different organisms for forward mutation with
low-LET irradiation tend to behave more like thehisfocompatibility loci than the seven loci in the mouse(see aiso paragraph 275).
527. If the hypothesis of Abrahamson et al (4) is valid,
it \\ill have far-reaching implications, especially for theevaluation of mutational radiation hazards in man.
Schalet and Sankaranarayanan (562), who made acritical analysis of the data used by Abrahamson et al(and of others not cited by these authors but which bearon the issue in question), concluded that (a) the notionof simple proportionality between DNA content andmutation rate cannot be substantiated and (b) thegeneralization that we can now extrapolate from lower
organisms to man with greater confidence must be
viewed with extreme reservations. Since all the arguments
have been given in detail in the above paper, thefollowing paragraphs will be devoted to summarizing themain points only. The papers of Conger (118) and of
Kohn (265) will not be further considered in this review
since, for the former, the basic premise (the existence ofa linear relationship between mutation rate and DNA
content) is weakened (562) and. for the latter, theestimates of gene size based on target theory
considerations are likely to be incorrect.
2. General considerations
528. One very important consideration that should beborne in mind in normalizing mutation rates in different
species to DNA content is whether the mutation rates

themselves relate to a common base line, namely, tochanges within a ··1ocus" or gene to the extent to which
we can define them with precision at present.
furthermore, for most of the organisms, owing to theJack of biochemical and genetic analysis, there is theuncertainty as to whether a locus consists of one gene ormore than one gene, although the terms "locus" andgene are used interchangeably in the absence of a better
alternative.
529. A second important consideration is whether, withthe array of mutation screening techniques used indifferent organisms. the same proportion of all the
induced mutations is detected. The question itself is nota new one and has been raised earlier (see, for example,
reference 358). In fact, the particular technique used for
detecting mutations can be a major determinant of themutation rate estimate. Two examples will suffice toillustrate this point. First, in Drosophila, rosy locus
mutants are detected as brownish eye-colour variants by
conventional tests; in addition to eye-colour variants, .
there are, at this locus, several classes of mutations·which present a normal eye colour and that can bedetected by other techniques (105). Secondly, there a~eother findings, such as those in Saccharomyces
cerevisiae, which show that, at the molecular level,
different forward mutation systems vary widely in theirability to detect the broad spectrum of intragenic
mutational events (539) and consequently, the comparison of mutation rates even in one and the same organism
is complex. not to mention interspecific comparisons.

530. Thirdly, and this is a relatively minor point, someof the DNA values cited in the paper of Abrahamson etal. refer to total cellular DNA. Therefore, these values
include different, but for the present purpose generally
insignificant, proportions of non-nuclear DNA. However,
it is clear that Abrahamson et al. erred in using a DNA
content for E.coli which is about three times the widely
accepted value (0.004 pg) (75, 119). Furthermore, avery recent estimate of the nuclear DNA content for
Saccharomyces cerevisiae has raised the distinct
possibility that the value used by Abrahamson et al. may
also be overestimated by a factor of three (270).
3. Examination of the data used
by Abrahamson et al.

531. The data used by Abrahamson et al. are derived
from experiments and with test systems which differ
widely in their ability to discriminate between changes
within genes as opposed to those involving -several genes
or loci and those which can include gross chromosomal
aberrations, such as losses of chromosome arms or evenentire chromosomes. That means that the effective
radiation·mutable target is not necessarily the same inthe different organisms whose mutational response iscompared, making the grounds for comparison insecure,
if not totally invalid.

532. The rate for the induction of waxy mutations
given by Abrahamson et al. is not a forward mutation
rate but a back mutation rate, does not pertain to threeloci but to a single locus, and does not relate to acuteirradiation but to chronic irradiation with 90 Sr.

Arguments have been presented in the literature (159.
290) for believing that radiation-induced waxy mutations are in fact chromosomal aberrations. Likewise, thedata for tomato used by Abrahamson et al. pertain tochromosomal deletions (60; 61, 434) and some of themutations may represent loss of chromosome arms oreven whole chromosomes.

533. The E. coli data of Demerec and Latarjet (137) on
the resistance to Tl phage used by Abrahamson et al.
were collected at a time when much was not known
about the nature of changes involved in phage resistancein £. coli. Several lines of evidence accumulated sincethen testify to the complexity of this phenomenon.
Phage resistance in £. coli can originate in two unlinked
regions designated as tonA and tonB, the latter of which
is a classical deletion system: £. coli can tolerate
extensive deletions of tonB including the neighbouringtryptophan operon, and such deletions exhibit the tonB
phenotype. Mutations to Tl resistance haYing an
additional auxotrophic phenotype of many types otherthan tryptophan have been reported (64, 65). Some ofthese may have been deficiencies involving the tonA
region while others seem to involve nutritional
deficiencies that are not linked to either tonA or tonB.
TonB mutants and deletions require exogenous iron for
their growth and are inhibited by chromium (210);
surprisingly high concentrations of chromium have beenfound in commercial agar preparations commonly used
to prepare solid media (120). Consequently, therecovery of phage-resistant mutants on solid media
containing chromium in sufficient quantity to inhibit
growth of resistant (but not of sensitive) cells may wellhave contributed to the low mutation rate reported byDemerec and Latarjet (137).
534. In the other organisms-Neurospora, Drosophila,
and the mouse-(the data for which were used byAbrahamson et aL), the level of genetic analysis used todetect and characterize the events scored as specificlocus mutations varies a great deal. Only in Neurospora
can one be certain that the data pertain to intragenic
changes, since here the distinction between intragenic
and extragenic changes has been made by intensive andrigorous complementation tests. In Drosophila, this
distinction is made on the basis of cytological analysis of
the mutants (salivary gland chromosomes), and theabsence of a detectable aberration (within the limits ofresolution possible) is considered presumptive evidence
that one is most likely dealing with a change at the levelof the gene. While the viability of homozygotes for the
mutation (or of hemizygotes in the case of sex-linkedones) can be considered good evidence for intragenic
change, its lack does not necessarily demonstrate thepresence of an aberration, since lethal intralocus changes
are well documented in Drosophila (604). In the mouse,transmission tests (to determine whether one is dealing
with a mutation) and homozygous viability tests areused to characterize the nature of changes. Here again,
while homozygous viability can be construed as goodevidence for an intralocus change, its Jack does notnecessarily demonstrate the presence of an aberration.
For instance. most of the d locus mutations recoveredfrom irradiated (x or gamma) spermatagonia have beenfound to be homozygous lethal, but the evidence fromcomplementation tests suggests that a majority of these
involve intragenic lethal changes at the d locus (453).
497

535. The estimate of 1.28 10-s rad -I per locus of
Alexander (9) used by Abrahamson et al. represents thebest presumptive intralocus mutation rate for Drosophila
spermatogonia. For mouse spermatogonia, the rate ofI. 7 10- 7 rad -i per locus ( used in the 1972 report of theCommittee) has been estimated from the results
obtained in experiments involving two sets of loci (sevenand six respectively, with one locus being common toboth sets) giving equal weight to each locus. It should be
remembered that the overall response of the two sets ofloci differ by a factor of three and within the seven loci(for which the data are extensive) the difference
between the lowest rate (a and se loci) and the highest
rate (s locus) is more than 30-fold ( 488). Schalet andSankaranarayanan (562) used the data presented by
W. L. Russell and L.B. Russell (488) and L.B. Russell
and W. L. Russell (459), in conjunction with those given
by L.B. Russell (453) to estimate a probable intralocus
rate for mouse spermatogonial specific-locus mutations.
The assumptions here are that (a) all homozygous viable
mutations are intralocus changes and (b) most of the dlocus lethals are intralocus events. From these they
concluded that for the seven loci, about 37 per cent of
all spermatogonial mutations are likely to be intralocus
changes. This gives a figure of0.81 10-7 rad- 1 per locus(i.e., 37 per cent of2.2 10-7 ).

4. Other relevant data
536. An examination of other relevant mutation rate
data published in the literature suggests, not unexpectedly, that the average mutation rate is a function ofa given cell stage, a given set of loci, or a given set ofexperimental conditions; there is therefore no soundreason to use one or the other alone as typical of the
response of the species under consideration. This point
can be illustrated by the following examples.
537. In Neurospora, de Serres et al (529) reported an
intralocus rate of (5-6) 10-9 rad- 1 per locus (ad-JA andad-3B loci) under slightly different experimental
conditions than the ones used in the work of Webberand de Serres (602). (In the latter work, the rate wasestimated as 2.7 10- 9 rad -i per locus.) Again in
Neurospora, Hoffmann and Malling (230) found that therate of x-ray induction of forward mutations at the aza
locus was higher than in the ad-3 system, being
3.1 10-s rad- 1 • Although the isolated mutants were
tested for transmissibility, no analysis was made todiscriminate between intralocus and extralocus events. If
the aza-3 locus were to show a proportion of intralocus
mutations similar to that in the ad-3 system (in thelatter, at 10 kR, roughly two thirds were intralocus
events). then the mutation rate becomes 2.0 10-1 rad- 1 •The above data plus those for the ad-3 loci permit an
overall estimate of 8.7 10- 9 or 1.0 10-s rad-• per
locus, depending on whether the ad-3 data used arethose of Webber and de Serres or those of de Serres et al.
(See also the arguments presented in Hoffmann and
Malling (230) for believing that the aza-3 frequency andpossibly even the ad-3 frequencies were obtained underconditions of suboptimal mutant expression.) At facevalue, either of the two rates above is not far removedfromtherate for Drosophila spermatogonia(1.28 10-1 R- 1 per locus). The Drosophila haploid498

DNA content is about 4.5 times higher than that of

Neurospora.
538. In Saccharomyces cerevzszae, Skobkin (540)studied the gamma-ray induction of canavanine resistance and noted that the maximal recovery rate was
1.0 10- 10 rad- 1 per locus or about one fifteenth of thatgiven by Abrahamson et al. Skobkin found that the
selection of mutants, among other things, was sostrongly dependent on the initial number of cells
inoculated into the medium and the concentration of
canavanine used that, under certain conditions, nomutants were recovered. (For details of other complexities encountered in this system, see also references 201
and 637.) Schwaier (564) who studied the x-ray
induction of forward mutations at the adenine loci inthe same organism, reported a rate of 6.0 10-9 rad -i
per locus. In a similar system studied in Schizosaccharonzyces ponzbe, the data of Nasim (356) and of
Loprieno et al (293) permit estimates ranging from
0.4 10- 9 to 1.1 10- 9 rad- 1 per locus per haploid
genome.
539. In barley, the available data for the x- or
gamma-ray induction of eceriferum, erectoides andchloroplast mutations show that there is no discernible
relationship between the mutation rates and DNA
content: in Arabidopsis tlzaliana the mutation rate tothiamine auxotrophy is (1.1-1.5) 10-9 rad- 1 per locus
per haploid genome, which is an order of magnitude
lower than that for Drosophila spermatogonia, although
the haploid content of Arabidopsis is 1.5 times that in

Drosophila.
540. Data collected by W. L. Russell (475, 478, 480) infemale mice (see reference 506 for a recent review) showthat the mutation rate at the seven specific loci (acutex irradiation) for maturing oocytes varies with the dose:in immature oocytes virtually no mutations have been
recovered. These observations are out of line with thepredictions of Abr;ihamson et al on the relationshipbetween DNA content and mutation rate. The nearabsence of x-ray-induction of mutations at thehistocompatibility loci (paras. 272-274) in the mouseprovides an additional case in point.
541. Table 44 summarizes the data (some of whichwere discussed in the preceding paragraphs) that areworth examining in the context of interspecificcomparisons, although it should be realized that here,
too, there are several uncertainties that stem from thelack of detailed genetic analysis, resolution power of thesystem and the paucity of loci examined for someorganisms. Inspection of the table will reveal that anincrease in DNA content is not always accompanied by aproportional increase in mutation rate (compare, forexample, the entries for barley with those for
Arabidopsis or Neurospora). Thus the data do not bearout the reasoning that higher DNA content and higher
mutation rates go hand in hand; it is true of some cases,
but by no means all, and consequently no unifying
interpretation is possible.
542. To explain the ''uniformity'' of mutation rates
among different species that they observed, Abrahamson
et al suggested as one possibility that, on the average,

the size of a radiation-mutable locus is proportional to
the total genome (DNA content) for the species. A
simpler way of stating this is that bacteria and higherorganisms contain the same number of genes, but inthe latter they are larger, with the implication that thelarger the gene, the higher the mutation rate. As theauthors themselves point out, although the inference oflarger genes .. is contrary to the usual genetic expectationthat the structural gene should be of the same size inbacteria as in man, we note that the functional genetic
unit in Drosophila corresponds to a band in the salivary
gland polytene chromosomes. This indicates thatcomplementation groups in higher forms contain much
more DNA than is necessary for the specification of aprotein. It has been postulated that the extra DNA has aregulatory function and that mutations in any one of thecomponents of the functional genetic unit would fail tocomplement mutations in any of the other components."
543. Recent studies of DNA organization in higherorganisms indicate that Drosophila DNA exhibits a quite
different pattern of sequence organization as comparedwith many other eukaryotes including the rat, whichhave higher DNA contents (130, 323). Thus, forDrosophila, there appears to be much greater average
length for the single copy sequence DNA (about
13 OOO nucleotides compared with .800 to several
thousand nucleotides). which is interspersed withrelatively larger middle repetitive sequences (5600 nucleotides average length compared with about 300 nucleotides). It would therefore appear that the Drosophila
pattern may not serve as the best model to speculate
about the size of the radiation-mutable target.
544. In Drosophila there are some data that permit one
to examine whether there is a positive correlation
between salivary band size and mutation rate as might be
expected if the total amount of DNA associated with aparticular band played a major role in determining themutation rate. The basic premise here is that afunctional genetic unit corresponds to a salivary
chromosome band {252). Inspection of table 45, in
which the data that bear on this point are summarized,
will reveal that there is no simple correlation between
band size and mutation rate. For example, lociassociated with bands of the smallest size (g, et and ras)
span the greater-than-one-order-of-magnitude range ofmutation rates indicated by the loci listed in the table;
when the male-lethal et and ras mutations are included
as "gene mutations", the rates for these loci are among
the highest.
545. The inability to detect a predictable relationshipbetween band size and mutation rates is not surprising inview of the detailed cytological analysis of the white (w)locus, which indicates that, for this locus at least, theradiation-mutable part occupies at most only a small
fraction of the DNA present in the band with which it
has been thought to be associated (546). Evidence from
yet another Drosophila system documents a similar
conclusion: Mutations at the rosy (ry) locus inchromosome III can be detected as eye-colour mutants.
The cytological position of the locus has only beendelimited to a region containing no more than five bands(86D8-12). In Lefevre's view (272), whichever of the

bands ry is associated with, it would be of below averagedimensions. (Gelbart et al. (183) believe that the bands
are of average dimensions.) Although the kind of
cytogenetic analysis performed at the w locus has notbeen done here, the ry locus has the advantage in that it
is known to code for the structural gene of the enzyme
xanthine dehydrogenase {XDH). Of primary importanceis the fact that spontaneously occurring electrophoretic
variants of the enzyme are known, and the mutant sites
responsible for electrophoretic differences in XDH havebeen located on the known genetic map of x-ray-induced
null-enzyme eye-colour mutants.
546. Gelbert et al. {184), in a very recent paper, havepresented more decisive evidence than that presented
earlier (183) and have concluded that "every rosy
eye-colour mutant (null XDH) is now included within
the structural element" and that there are "no mutant
sites, either radiation- or EMS-induced, which falloutside of this region". From the work of Rudkin (443,444), it is known that the amount of DNA in the averageband, 3.5 10 4 base pairs. is sufficient to code forapproximately 30 average-size proteins. The XDH
polypeptide requires a coding sequence of (34) 10 3 basepairs (183). The available mutation rate data for
intralocus null-enzyme ry mutants (23 mutants in
170 OOO flies after about 4700 R exposure of spermatozoa) give a rate of 2.9 10-a R- 1 {526, 561), whichfalls close to the upper limit of the range of mutation
rates given in table 45. One is thus left with the firm
conclusion that, at the ry locus. an average orabove-average mutation rate has been elicited fromchanges restricted to the structural portion of the locus,
i.e., the DNA which codes for XDH and represents atarget size of about one tenth of the amount of DNA in
the average size salivary gland chromosome band.

547. In summary. an examination of mutation rates fora number of loci associated with bands of different
dimensions reveals that the relative size of the bands is
not quantitatively correlated with the intralocus
mutation rate. There are therefore no firm grounds forbelieving that the higher average mutation rate in
Drosophila as compared with some lower forms may be
due to the fact that on the average. the amount of DNA
associated with complementation groups in Drosophila is
far in excess of that required to code for single
average-size proteins.
5. Summary and conclusions
548. On the basis of an examination of some data on
forward mutations at specific loci induced by acutelow-LET irradiation in different organisms, Abrahamson
et al. proposed that there is a linear relationship between
DNA content and mutation rate and that thisrelationship could be used to extrapolate directly fromlower organisms to man with confidence and to predictthe mutational response of human genes to irradiation.
549. The data used by Abrahamson et al. come fromexperiments and with test systems which differ widely intheir ability to discriminate between changes withingenes as opposed to changes involving several genes orloci and those which can include gross chromosomal499

aberrations such as losses of whole chromosome arms oreven entire chromosomes. The effective radiationmutable target is therefore not the same in the different
organisms, and consequently the grounds for comparison
are insecure. if not invalid.
550. A consideration of data other than those cited byAbrahamson et al. does not lend credence to the line ofreasoning pursued by them. There are several notable
exceptions, such as the response of the immature
oocytes of the mouse and that of the histocompatibilityloci.
551. In Drosophila, the available evidence indicates that
there is no simple quantitative correlation between thesize of the band in the salivary gland chromosome andthe mutation rates of loci located in them. For instance,at the rosy locus which codes for the enzyme xanthine
dehydrogenase, every null-enzyme mutant (whether
x-ray or EMS-induced) is located within the structural
portion of the gene, which is no more than half (andperhaps as low as 10 per cent) of the DNA in the bandwith which rosy is associated. Yet, the intralocus
mutation rate for null-enzyme mutants at this locus(after x irradiation) is close to the upper limit of
intralocus mutation rates in this organism.
552. In conclusion, the reanalysis of the data on
mutation rates has made it clear that the hypothesis of asimple linear relationship between DNA content and theradiation-induced forward mutation rate in livingorganisms as a whole cannot be substantiated. Even
within the different germ-cell stages of a single species
such as the mouse, there is extreme variation inradiosensitivity with no variation in DNA content. In
view of this, the belief that DNA content can be used as avalid genetic bridge to extrapolate from micro-organisms
through higher species to man is unwarranted. The
diverse processes, including repair, which intervene in
mammals between the induction of mutation and its
expression would. in any event, preclude such an
extrapolation.

"mutants" are now better understood, several new ones
have been uncovered and their effects are actively
pursued at present.

B. HYPOXANTHINE-GUANINE PHOSPHOR·IBOSYL TRANSFERASE (HG-PRT)

1. Gene localization
554. The discovery of a severe deficiency of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT)(521) in patients affected with the Lesch-Nyhan
syndrome (288) catalysed interest in studies on purine
metabolism in man. The Lesch-Nyhan syndrome ischaracterized clinically by symptoms of excessive
production of uric acid and certain neurologicalabnormalities, including mental retardation. Studies ofpedigrees affected with this syndrome indicated that thelocus for HG-PRT is on the X-chromosome and that themode of transmission is X-linked recessive (63,226, 366,
629). With in vitro methods (somatic Cl'l! hybridization)
it was demonstrated that in human fibroblasts, the locifor HG-PRT, G-6-PD and 3-phosphoglycerate kinase(PKG) are linked (329): current evidence indicates that
HG-PRT and G-6-PD genes are localized on the distal
half of the long arm of the X-chromosome and withthe following sequence: centromere, PGK, HG-PRT and
G-6-PD (433, 533). A major review article on HG-PRT
deficiency in man has recently been published (62).
555. Chu (106, 107) advanced the hypothesis that in
Chinese hamsters and perhaps in other mammals as well,
the gene controlling HG-PRT activity is also X-linked.
This hypothesis has now been confirmed for the Chinese
hamster by Westerveld et al. (606), who demonstrated
linkage of loci for PGK, HG-PRT and G-6-PD, and forthe mouse by Epstein (156, 157), Shows, Brown andChapman (536), and Hashmi and Miller (223).
2. Screening methods and originof HG-PRT deficiency

V. SOMATIC CELL GENETICS 25
A. INTRODUCTION
553. The 1972 report of the Committee (589)considered the data then available from studies onmutation induction in mammalian somatic cells. The
events studied are biochemical ones capable ofexpression at the cellular level under appropriate
conditions. Most of the investigations reviewed in thereport had been carried out with an aneuploid(near-diploid) ~hinese hamster cell line (V 79) andstudies with human diploid fibroblasts were justbeginning. During the last few years, the scope ofsomatic celJ genetics has expanded: new cell systems andselection schemes have come into use. While the roles ofsome of the factors that affect the recovery of the' 1 In this section, the terms "mutant" and "variant" will
be used interchangeably in view of the fact that although there isgood evidence for mutational origin of the events studied, it is
not unequivocally proven.

500

556. Normal substrates for HG-PRT are hypoxanthineand guanine which are converted to inosine 5' -monophosphate and guanosine 5'-monophosphate, respect·
ively. Cells having HG-PRT activity can also convert
purine analogues such as 8-azaguanine (8-AG),
6-mercaptopurine and 6-thioguanine to their respective
nucleotides, the incorporation of which results in growth
inhibition or death, indicating that normal cells are
sensitive to these metabolites. The screening systems
employed to detect the HG·PRT variants therefore use
resistance to these purine analogues as markers. It has
been found that some cell variants that are resistant to8-AG have either a defective HG·PRT protein, caused by
a mutation in the structural genes (31 ). or a variant
enzyme with altered kinetic constants and heatsensitivity (531).
557. In his review, De Mars (135) considered in detailthe general question of resistance of cultured humanfibroblasts and other cells to purine and pyrimidine
analogues in relation to mutagenesis: he pointed out that

1(a) genetic changes have been. and may still be. regarded
as reasonable explanations for the origin of baseanalogue-resistant variants although definitive proof forthe mutational origin of these is lacking; (b) arguments
favouring explanations invoking epigenetic mechanisms ofresistance to base-analogues (such as those of Harris(220, 221) and of Mezger-Freed. (336)) are based oninconclusive evidence: and (c) emphasis must be placedon rigorous studies aimed at examining the nature of
events scored for which several possibilities can beenvisaged (mutations in genes determining the structureof the enzyme, mutations in genes regulating theexpression of the structural genes, mutations in genesthat have epistatic functions modifying the expression of
the enzyme phenotypes, karyotype changes, mitotic
recombination, extra-nuclear genetic changes, etc.).

3. Factors affecting the recovery of spontaneousand induced mutants
558. Several factors. such as the analogue used, itsconcentration, cell-seeding density, time of addition of
the analogue to the mutagen-treated cells (expressiontime), the source of serum used in the selective medium,
interclone metabolic co-operation etc., have been foundto influence the recovery and type of mutants obtained(2, 7, 86, 87, 105, 135, 173,380,407,538,575,630,
633, 634, 635). Table 46 summarizes the role of some ofthese factors in the different cell systems currently inuse. While most of the studies referred to above have
employed 8-AG as the selective agent. in recent years. a
preference for the use of 6-thioguanine ( 6-TG) has been
expressed (125, 126. 175, 259, 632). Human HG-PRT
has a much greater affinity for 6-TG than for 8-AG(267).
559. Nikaido and Fox (364). who compared the relative
effectiveness of both these selective agents in two V 79
Chinese hamster cell lines, (V 79A and V 79S) foundthat in both cell lines (a) the frequency of survivingcolonies exposed to either agent was dependent on theinitial plating density; (b) difterent degrees of metabolic
co-operation were manifest and the loss of bothspontaneous and added mutants (the latter in reconstruction experiments) occurred at lower cell-densitywith 6-TG; (c) both analogues were degraded on the
incubation in medium plus serum in t!ie absence ofadded cells; and (d) variations in serum batch had little
effect on the rate of degradation or on the frequency ofcolonies recovered after treatment with 8-AG. They
concluded that, at least in V 79 cell lines, (a) cell killingby 8-AG is less dependent on cell density than that by
6-TC, (b) 6-TG is apparently not more efficient inselecting for HG-PRT-mutants (although 6-TG iseffective in cell killing at much lower doses), and(c) since 6-TG has been shown to be incorporated into
DNA (155) (in contrast to 8-AG which is incorporated
into RNA (342). 6-TG itself may be mutagenic, andtherefore 8-AG is preferable.
560. Thacker et al. (573) however, found that the useof 8-AG as the selective agent yielded highly variable
results with the Chinese hamster cell line V-79-4 andthat different batches of sera in the growth medium
could affect the response considerably. Although dialysisof the serum eliminated some of the variation. it also
reduced the plating efficiency of the cells. Moreconsistent results were obtained with 6-TG, although.
the "mutation fraction" was not completely resistant tothe analogue. The authors noted that the mutants fell
into two classes: one class is HG-PRT- (less than 2 percent wild-type activity). resistant to 5 µg/ml of
thioguanine and sensitive to azaserine 2 6 while the othershows about 15 per cent of wild-type HG-PRT activity,
resistance to 1 µg/ml of thioguanine and some growth inazaserine medium. The two classes of mutants differedlittle in ability to withstand suppression of their growth
on dishes by wild-type cells and performed similarly incompetition experiments where mixtures of mutant andwild-type cells were kept in exponential growth over
several population doublings. Only one of these types(HG-PRr) increased in frequency with radiation dose.
The authors suggested that these results can account for
many of the discrepancies encountered in previous
studies and stressed that characterization of the mutants
is essential to establish the true frequency of resistantmutants. The work of Cox and Masson (125) provides
additional evidence that in human diploid fibroblasts.
too. 6-TG is a better selective agent.
561. Van Zeeland and Simons (631) studied the
problem of the x-ray induction of 8-AG mutants in two
sets of diploid and tetraploid Chinese hamster cells(DON and V 79) and found that the induced mutation
frequencies in both kinds of cells were of the same orderof magnitude and that all mutants showed an almost
complete loss of HG-PRT activity, except that in thetetraploid V 79 cells, 50-100 per cent activity wasretained. Measurements of G-6-PD activity in these cells
revealed that the spontaneous and induced mutants fromthe diploid V 79 and DON. as well as the tetraploid
DON cells. retained the G-6-PD activity of the parental
cells whereas the induced mutants from the tetraploid
V 79 cells had about 35 per cent of the parental G-6-PD
activity. Using 6-TG as a selective agent. the authors
found that the induced ''mutant" frequencies in diploidand tetraploid DON cells and in diploid V 79 cells werein the same order of magnitude, but no mutants could
be recovered from tetraploid V 79 cells in a single step.
Thioguanine-resistant tetraploid V 79 cells could only beobtained from the AG-resistant mutants and some ofthem showed an increase in G-6-PD activity. All theseresults taken together led the authors to conclude thatthe induction of HG-PRT deficiency may not be asingle-step event and that it may be the result of a
two-step process, as has been suggested by them for thehuman diploid fibroblasts (635) and by Freed andMezger-Freed (176) for BUdR resistance in haploid frog
cells.
562. Van Zeeland et al. (635) te.sted the validity of theassumption implicit in selecting for purine-analogueresistant variants in somatic cells (AG-resistance in thiscase), namely, that their origin is independent of theselective agent. Should this be true, then the proportionof mutants with no or very little HG-PRT activity will be
2 'Azaserine is known to inhibit de novo synthesis ofpurines in cultured human cells and cells dependent for growthon de novo purine synthesis (as a result of defective purine
salvage enzymes).lmpaired growth in azaserine medium might.be
expected (180 ).

501

the same at different selective AG concentrations.
DeMars and Held (136) reported that in diploid humanfibroblasts, 90 per cent of the variants selected at
1.2 µg/rnl AG concentration had residual enzyme
activities: in contrast. the experiments of van Zeelandand colleagues showed that mutants selected at 5 or
10 µg/rnl AG were totally deficient in HG-PRT activity.
Further experiments were therefore aimed at resolving
this contradiction.
563. Using human fibroblasts, van Zeeland et al. (635)selected for spontaneous mutants at 1.2 and 5 µg/ml AG
concentrations. It was found that the total mutation
frequencies were the same at both concentrations;however, the mutants selected at the low concentration(the A mutants) were qualitatively different from those
selected at the high concentration (the B mutants):
among the A mutants, the proportion that had very little
or zero enzyme activity was small (3 out of 28) whereas
in the other group, a vast majority fell in this class (20out of 25). This finding strengthened the results
obtained from the direct HG-PRT assays of the mutants
and suggested that AG is not acting merely as a selective
agent. To test this, mutants recovered from both groups
were examined for their stability in the presence orabsence of AG (1.2µg/ml). It was found that 5 mutants
without residual HG-PRT activity (4 A and 1 B)
remained stable over a period of 30-50 days; 7 withresidual activity (5 As and 2 Bs) showed varying degrees
of increase of activity indicating that stability of themutant phenotype in these was dependent on thepresence of AG. On the basis of this and other lines ofevidence collected, the authors proposed that AG
resistance may occur through a two-step process; thefirst step, which is rare and occurs at random. is
AG-independent. In it wild-type cells are predisposed tomutation in a second step, which is AG-dependent.
While the validity of this theoretical model remains to be
proved, it nonetheless suggests that the property ofAG-resistance may not have a simple basis.
564. Until recently, in most of the mutation experiments, the induction of and selection for AG-resistantphenotypes had been carried out within the same dish of
cultured cells, as described by Chu and Malling (108).
With this technique, as the expr_ession time is increased,
the number of presumptive mutant colonies characteristically rises to an optimum and then sharply falls. Thus,
for instance, Chu and Malling observed a maximum
frequency of AG-resistant colonies at 42 h expression
time following treatment of V 79 cells with
10 - 2 M EMS. The usual explanation for the sharp decline
in mutant colony frequ,encies is the metabolic
co-operation between wild-type and mutant colonies,
causing a shift to the HG-PRT+ phenotype (547, 634).
This process limits the time available for expression of
the HG-PRT- .phenotype and may in fact cause aspurious reduction in the maximum observable mutation
frequency. Myhr and DiPaolo (355), who investigated
the reliability of this assay procedure demonstrated thisto be true and showed, with three different chemicalmutagens, that it is necessary to respread the cells after asuitable expression time before adding the selection
medium to obtain maximum frequency of colonies
resistant to 8-AG. (The maximum frequencies following
502

MMS or MNNG 2 7 treatments were about 10-fold greaterthan those obtained when induction and selection of
AG-resistant mutants were performed in the same
culture dish.)565. Fox (173) compared the efficiency of the two
assay procedures (in situ and replating techniques) for
the selection of 8-AG mutants in V 79 Chinese hamster
cells (after x-ray, MMS, EMS and uv treatment) andshowed that a significantly higher frequency of mutants
was obtained in replated experiments as compared with
the in situ situation. The experiments of Abbondandolo
et al (2) adduce further evidence that the identificationof a plateau in expression time by the replating
techniques is important for an accurate quantitativeevaluation of mutation rates. Similar conclusions werereached by Carver et al (86).

4. Dose-effect relationships
566. In the 1972 report, it was pointed out that in V 79
Chinese hamster cells. the dose-effect relationship forthe induction of AG-resistant mutations was non-linear
and that there were dose-rate and dose-fractionation
effects (100-1200 R) {12, 59, 107). The concentrationof AG used for selection varied from 7 .5 µg/rnl in theexperiments of Bridges and Huckle (59) and those of
Arlett and Potter (12) to 30 µg/ml in those of Chu(107). The results of Albertini and De Mars with diploid
human fibroblasts ( unpublished at that time) also
showed that the dose-effect relationship for AG-resistantmutants was non-linear. Albertini and De Mars (7) have
now published their complete results, which show that(a) the average spontaneous rate is about 4.5 10-6 percell generation (1.9 1o- 6 if one large-yield experiment is
excluded) and (b) the minimum estimates of the induced
rates at different dose levels are
Induced rate
(R· 1 per cell)

Exposure(R)

1.13 10- 9
7.14 10-s
6.85 10-s
2.16 10- 7

75
125
150
250

indicating. as in Chinese hamster cells,
dependence of the mutation rate.

a

dose

567. Van Zeeland and Simons (632) used the replating
method for selecting for induced 6-TG mutants in V 79
cells and showed that the optimal expression time isabout 7 days, beyond which the induced mutant
frequency stays constant (in contrast to a dropping-off
of the yield in earlier studies). The exposure-frequency
relationship for x-ray-induced mutants is consistent withlinearity in the range up to 800 R, the mutation rate
being 1.35 10- 7 R -i per cell. Thus, with the improved
method, there appears to be no dose dependence of themutation rate.
568. Cox and Masson (126) investigated the x-ray
induction of 6-TG mutations in human diploidfibroblasts. They found that the maximum yield wasobserved when the cells surviving the irradiation hadcompleted 3-4 doublings (6-7 days growth) in a2

'MNNG = N-methyl-N'-nitro·N-nitrosoguanidine.

rY'non-selective medium and that under these conditions,in the range 50-200 rad, the mutation frequency waslinearly related to dose, the rate being 3.1 10-7 rad- 1
per cell.
569. Knaap and Simons (259) have succeeded indeveloping the mouse lymphoma cell system (L5178Y)
as a useful and reliable test system for studying
induction of mutations to HG-PRT deficiency (thissystem differs from others, such as the fibroblasts. inthat the growth of the cells takes place in suspension
culture). The selective agent used was 6-TG. The results
show that (a) the selection of the mutants is not
influenced by the concentration of the selective agent (1or 5 µg/ml); (b) all of the mutants selected (spontaneous
as well as induced ones) show a complete loss of
HG-PRT activity; (c) the optimal expression time forthe mutants is 6-7 days and appears to be doseindependent; and (d) over the range of exposures from
200 to 400 R, the dose-response curve is approximately
linear with a rate of (1-1.8) 10- 7 R- 1 per cell. After 500
and 600 R, the induction rate is slightly higher. In
agreement with the results of Clive et al. (114 ), Knaap
and Simons (259) also found that 8-AG was unsuitable
as a selective agent in the mouse lymphoma system,
owing to inactivation of 8-AG by a serum factor (630).

C. THYMIDINE KINASE (TK)
570. Another locus that is currently used in mutation
studies is the thyrnidine kinase locus (TK) in mouse
lymphoma cells. It is known that in man, the genecoding for this enzyme is located on an autosome(chromosome 17) (337, 339, 442), and it is presumed
that the situation is similar in the mouse. Like HG-PRT,
TK provides a '·salvage pathway" which can circumventa folate-dependent step, i.e., thyrnidylate synthetase inthis particular case. Thus the same principle employed in
the HG-PRT system applies to the use of selective media
in the TK system. Forward mutations at the TK locus
are detected through resistance to BUdR, IUdR or TdR,
depending on the cell strain used.
571. In the earlier work, Fox (171) used P388 cells (amouse lymphoma cell line) and studied IUdR resistanceafter x irradiation. The selective concentrations of IUdR
used in experiments on dose-effect relationships ranged
from 10 µg/ml to 100 µg/ml. Surviving cc,ionies, both incontrol and irradiated populations (scored after 10 daysof incubation) were tested for heritable resistance to
IUdR. The results showed that (a) after selective dosesof 10 µg/rnl or less, no heritable resistance could bedemonstrated; when higher selective concentrations(20 µg/ml and 100 µg/ml) were used. the number ofvariant colonies increased, although there appeared to be
no consistent increase in efficiency of mutationinduction with increasing x-ray dose; and (b) there was a
linear relationship between inoculum size and thefrequency of IUdR-resistant variants and greaternumbers of resistant colonies were recovered when cells
were incubated for 24 h before plating on the selective
medium.
572. Fox and Anderson (174) isolated IUdR-resistantand TdR-resistant clones from x-irradiation andchemical-treatment experiments with the P388 cell line

and conducted TK assays on them. All were found toshow significantly reduced enzyme activity relative towild-type levels, but none showed zero levels. Fourteenof these clones were tested for thymidine uptake and all
showed a marked reduction in the rate of 3 H-TdR
incorporation into acid-soluble fractions and into DNA.
Measurements of TK activity in 8 TdR-resistant clones
showed that the initial rate of thymidine phosphorylation was not significantly altered in 5 of them;
however, significantly lower amounts of phosphorylated
products were observed in 6 of the 8 clones.
Incorporation of 3 H-dTR was reduced in 9 of the
12 clones tested, and 2 of them showed no corresponding reduction in TK activitY,.
573. In another set of experiments, Fox (172)compared the x-ray induction of IUdR-resistant andTdR-resistant variants in P388 cells and found that thedose-response curves for both of these showed linear andquadratic terms and were roughly similar. In the same
study, Fox also compared the x-ray induction of
TdR-resistant variants in four L5178Y cell lines, LS (themost sensitive), All, AIU and AIV, which differed inradiosensitivity to killing. When induction frequencies
were compared, either at the same dose or at the same
survival levels, the most radiosensitive line (LS) was
more mutable than the others, including P388. It may
be pointed out that the expression times for both IUdRand TdR-resistant variants were similar. the maximum
frequencies being reached at 48 h followed by a slightfalling-off at the longest expression time (96 h) tested.
574. In limited radiation studies with L5178Y, Clive etal. (114) recorded an induced mutation rate of
5.0 10- 1 R- 1 using BUdR as the selective agent (600-R
level). Clive et al. as well as Fox and Anderson (I 74)
have discussed the possible genotypic constitution of therespective cell strains used in their mutation assay
systems. The former authors showed that the original
L5178 Y cell strain is TK +/TK + with spontaneous
mutation rate to BUdR-resistance of 5 10- 11 per
generation (based on one recovered mutant); they
recovered revertant cell lines from the single TK - /TK originally isolated, and a study of their enzyme levels
and mutability indicated that some of these were
TK +/TK-. One such line was used for determining the
x-ray mutation rate given earlier.
575. Fox and Anderson (174) favour the view that theP388 and L5178 cells that they used are TK +/TK - and
TK+/TK+. respectively. The reasoning is based on thefollowing: (a) L5178YS cells have much higher TKlevels, possibly a reflection of a gene-dosage effect, as
demonstrated by Clive et al. (114); (b) L5178YS cells
have a very low frequency of IUdR-resistant colonies (nomutants recovered from 108 plated cells), which alsosuggests that they may be TK+/TIC; (c) if P388 cells are
TK +/TK +, it is difficult to explain the large difference inthe frequency of spontaneously occurring IUdR-resistantcolonies in the two cell lines; this is not the case whenother mutagens (chemicals) are used (172, 174).
576. The nature of the events involved in IUdR
resistance in the P388 line was examined by Fox andAnderson (174) in revertability tests: it was found thatfive out of the seven tested reverted at measurable rates
503

either spontaneously or after mutagenic treatment(HN 2 , MNU, MMS etc.), suggesting that the IUdR
resistant mutants tested could have been the result ofgene mutations at the TK locus rather than deletion-type
events.

580. Morrow et al. (346) studied the effect of EMS. NG
and quinacrine hydrochloride on the induction ofreverse mutations from asparagine auxotrophy to
prototrophy in rat sarcoma cells: EMS was mosteffective. No x-irradiation experiments have so far been
carried out.

D. OUABAIN RESISTANCE
577. Arlett et al. ( 13) have recently compared
the mutagenic effects of gamma rays, uv light and several
chemical mutagens using AG-resistance and systems in
Chinese hamster cells resistant to ouabain (Qua).
Resistance to Qua is different from that to AG and isprobably the result of an alteration of the membrane
ATP-ase involved in Na+ /K + transport such that it is nolonger sensitive to Oua (322). The resistance alleles
behave codominantly in somatic hybridization experi·ments (24). The results of Arlett et al. showed that there
was a broad agreement between the AG and Qua systems
for each mutagen tested, EMS being the most effective
mutagen. Surprisingly however, Qua-resistance could not
be induced by gamma rays, and the authors have as yet
no ready explanation for this negative result.

E. FRUCTOSE UTILIZATION
578. Cox and Masson (124) reported the developmentof another selective system for mutation studies inhuman fibroblasts: this is concerned with the utilizationof fructose instead of glucose as the major energy andcarbon source. HF 10 diploid human fibroblast cultures
were initiated from a male embryonic limb, and cells
from early passage (6-25 generations in culture) were
utilized for the x-ray experiments. Cells were irradiatedin suspension and immediately afterwards plated onfructose medium. It was found that the frequency ofFruct clones (uncorrected for cloning efficiency)
increased approximately exponentially with radiationdose, the rates per survivor being 4 10-s (0-100 rad
range),
1.24 10- 7(0-200'rad),and2.10 10- 7(0-343 rad). The genetic basis of Fruc + variants observed
is however, not yet worked out.

F. BACK MUTATIONS
579. Susuki and Okada (549) studied the reversion
from alanine auxotrophy to prototrophy in mouse
lymphoma cells (L5178Y) both in vitro and in vivo.
following gamma irradiation (I 00-800 rad). (In the invivo experiments, the alanine-requiring cells were
injected into mice (intraperitoneal injections); the mice
were irradiated on the eleventh day after inoculation,
following which the irradiated cells were removed andassayed for reverse mutations.) The expression time waschosen to be two days for cells grown in vitro andfive days for those grown in JJivo. It was found that(a) the cells grown in vitro were more sensitive, both tokilling and to mutation induction and (b) the dose~ffectrelationship was linear for cells irradiated in vivo andappeared faster than linear for the other. The rate ofinduction under in vivo conditions was approximately
110-7 rad- 1 , for the in vitro irradiation, it was higher
by factors of 2.5 to about 4.

G. GENE TRANSFER THROUGH PURIFIEDMETAPHASECHROMOSOMES
581. Following the initial demonstration by McBrideand Ozer (327) that incubation ofHG-PRT- mouse cellswith purified metaphase chromosomes from HG-PRT+
Chinese hamster cells resulted in the appearance of
HG-PRT+ mouse cells in which the Chinese hamster
enzyme was expressed, a number of investigators havecarried out similar experiments. Willecke and Ruddle( 611) extended this work by successfully transferring
the human liG-PRT gene (via isolated metaphase
chromosomes from He La S3 cells) into murine A9 cells
which lacked functional murine HG-PRT activity. Twenty
human isozymes other than HG-PRT, whose genes havebeen assigned to 14 human chromosomes. were found to
be absent in the transformed clones. In addition. thehuman isozymes of G-6-PD and PGK (whose genes arelinked to that of HG-PRT) were also absent. In
subsequent work, Willecke et al. (612) succeeded intransferring two linked human genes which code for the
expression of cytosol thymidine kinase and galactokinase to mouse L cells.
582. Wullems et al. (619, 620) and Degnen et al. (134)published the results of experiments in which both
intraspecific and in~erspecific gene transfers have beenaccomplished. In the experiments of Wullems et al.
(619) for instance, the mutant cell line Wg 3-h (derived
from the Chinese hamster DON cell line by stepwise
selection with increasing concentration of 8-AG andpropagated in the presence of 6 µg/ml AG) with
HG-PRT activity of about 2 per cent ,(compared to the
DON cells) served as the recipient in all experiments; thedonor chromosomes were from the DON line (intraspecies) or from the human He La S3 cell line and thehuman T cell line (interspecies).
583. The results show that (a) incubation of HG-PRTChinese hamster cells with chromosomes isolated from
HG-PRT+ Chinese hamster or human cells resulted in theappearance of HG-PRT+ cells: (b) in the hamsterhamster situation. the frequency ofHG-PRT+ clones wasfound to be dependent on the number of chromosomes
added (5 10-5 and 16.6 10- 5 following incubation with
107 and 108 chromosomes, respectively). In thehamster-human situation (after incubation with 10 7
human chromosomes) the frequency of HG-PRT+ cells
was 6 10- 6 , showing that intraspecies transfer may be
more effective. Furthermore, in the first situation, the
HG-PRT activity of the cells approached the level foundin DON cells whereas treatment with human chromosomes resulted in 2040 per cent of the activity in the
Chinese hamster and 15-25 per cent of that in human T
cell line; and (c) two clones isolated after incubation ofChinese hamster cells with human chromosomes, whichwere tested electrophoretically, showed the pattern of
the human HG-PRT. However. the human X-linked

I

I

504

l

genes codes for G-6-PD, phosphoglycerate kinase anda -galactosidase) were not expressed in those clones,
suggesting, as in the work ofWillecke and Ruddle (611).
that perhaps only a small fragment of the X-chromosome was incorporated into the Chinese hamster cells, afinding similar to that of Willecke and Ruddle (611).
584. In their very recent paper, Wullems et al. (621)presented evidence for the uptake of the human
X-chromosome by human-Chinese hamster cell hybrids,
which lack HG-PRT activity. following incubation withisolated human chromosomes from He La S3 cells. The
activity of X-linked genes other than that for HG-PRT,
such as PGK, alpha-Gal A. G-6-PD, was also found to bepresent, as determined by electrophoresis (the recipient
cells gave no evidence of containing human X-chromosomes). These results therefore suggest that incorporation and expression of He La X-chromosome is accomplished in human-Chinese hamster cell hybrids whichlack X-chromosomes. This is in marked contrast to theresults obtained with other experimental systems
discussed earlier.

H. SUMMARY AND CONCLUSIONS
585. Since the publication of the 1972 report of theCommittee, substantial progress has been made in thefield of somatic cell genetics. A number of selection
systems have come into general use and the role ofseveral factors affecting the recovery of mutants is now
better understood.
586. Most of the data have come from the work on the
induction of forward mutations leading to a deficiency
of hypoxanthine-guanine phosphoribosyl transferase(HG-PRr) in Chinese hamster cells, mouse lymphoma
cells and human fibroblasts. While the purine analogue8-azaguanine (SAG) continues to be used as the selective
agent, in recent years, there has been a preference to use
6-thioguanine (6-TG) instead: the use of the latter has
provided more reproducible results.
587. One of the major technical advances concerns thedelineation of the '·expression time", the time needed
for the mutants to fully express themselves following
their induction, in contrast to earlier studies with
Chinese hamster and human cell lines where theinduction of and selection for mutant phenotypes werecarried out in the same culture dishes, under which
conditions the frequency of mutant colonies rose to a
maximum and then fell. The current use of the replating
technique (where the treated cells are respread on to newplates for optimal mutant expression) has facilitated therecovery of mutants. With the replating technique, it has
been found, for instance, that the optimal expression
time for HG-PRT- mutants x-ray or EMS-induced in
V 79 Chinese hamster cells is about 7 days, beyond
which the induced mutant frequency stays constant. The
same is true for the induction of such mutants in humanfibroblasts.
588. Using the replating technique, it has been shownthat the dose-effect relationship for the x-ray inductionof HG-PRT- mutants is linear in Chinese hamster cells aswell as in human fibroblasts. The rates are, respectively.
1.3510-1 R- 1 percelland3.l 10-7 rad- 1 per cell.

589. In mouse lymphoma cells (15 l 78Y). 6-TG-resistant mutants have an expression time of 6-7 days, andhere too, the exposure frequency relationship isapproximately
linear,being(1-1.8) 10- 7 R -i
(200-400 R); at higher exposures (500 and 600 R) therate tends to be slightly higher.
590. Another locus at which mutation induction insomatic cells is being studied is the thymidine kinaselocus (TK): all the studies so far have been carried out
with mouse lymphoma cells. Forward mutations at thislocus are detected through resistance to BUdR, IUdR or
TdR, depending on the cell strain used. For BUdR
resistance in L5I78Y cells, the x-ray induction rate is
5.0 10- 7 R -i per cell ( 600-R level).
591. Resistance to ouabain (result of alteration of themembrane ATP-ase) is another potentially usefulforward mutation system in Chinese hamster cells; x rays
have been found to be ineffective in inducing
ouabain-resistant mutants. although several chemicalmutagens are capable of inducing such mutants.
592. Attempts to induce back mutations from alanine
auxotrophy to prototrophy in mouse lymphoma cells
and from asparagine requirement to prototrophy in rat
sarcoma cells have met with success.
593. Techniques are now available for effecting genetransfer in somatic cells using purified preparations ofmetaphase chromosomes. Both intraspecific transfer(Chinese hamster) and interspecific transfer (mouseChinese
hamster,human-mouse,
human-Chinese
hamster) of HG-PRT genes have been demonstrated.
Donor genes can be selected for in the recipient cell
population by independent electrophoretic, chromatographic or immunological criteria. The frequency ofchromosomal gene transfer is about an order ofmagnitude higher than the rate of reversion of recipient
cells but can also lie within the same range. Evidence forthe presence of donor chromosomes was negative inthese studies, suggesting that the transferred chromosomal segment must be very small.
594. In contrast. in more recent experiments successhas been obtained in transferring a presumably wholehuman X-chromosome using metaphase chromosomes.
from human He La cells as the donor to humanChinese hamster cell hybrids as the recipient. Thesestudies offer interesting possibilities for examining the
problem of gene regulation in somatic cells.

VI. EVALUATION OF GENETIC
RADIATION HAZARDS IN MAN
A. A COMPARISON OF THE MAIN CONCLUSIONSOF THE UNSCEAR AND BEIR REPORTS
595. The UNSCEAR (589) and BEIR (34) reportsbrought into focus the status of knowledge in the area ofradiation genetics and, using this knowledge as a basis,made quantitative evaluations of genetic radiation
hazards to man. Tables 47 and 48. which are reproduced505

from these reports, summarize these estimates. Some of
the major conclusions reached in these reports have since
been reviewed and compared (3.60, 383. 495, 508, 509).
596. The general approach to the problem of hazard
evaluation has been essentially the same in both thereports; they have the following aspects in common:(a) The germ cell stages most relevant are
spermatogonia in males and oocytes in females:(b) Due to the paucity of direct human data, theresults obtained with experimental mammals, primarily
the mouse. form the main basis for hazard evaluations:(c) Since most of the genetically significant
radiation dose received by the human population isdelivered either as small doses at high dose rates or as
greatly protracted exposures, these are the radiation
conditions under which the response of the human germ
cells have been assessed;(d) The rates of induction of different kinds ofgenetic damage used for hazard evaluation purposes are
all based on the response of the mouse germ cells under
the radiation conditions stated in (c) above. Appropriate
corrections. if any, are made to apply them to man. The
rates of induction are assumed to be linear with dose.
For the induction of one kind of chromosomal damage,
namely that of reciprocal translocations, human germ
cells were assumed to be twice as sensitive as those of
the mouse.

597. The two reports differ from one another in certain
respects. The UNSCEAR report predominantly used the
direct method to express risks, namely in terms of theamount of damage ~xpected per gamete per unit dose ofradiation: however, some limited attention was paid toquantify risks in relation to the natural incidence- ofhereditary diseases in man (the doubling dose method).
The BEIR report mainly used the doubling dose method
to quantify risks, although it stressed the fact that.. there are so many facets to the problem that several
ways of estimating the risk are more useful than any
single one in arriving at the best policy decisions".
598. Although in both reports the increases in theamount of hereditary diseases in man (with the doublingdose method) were estimated using the spontaneous
incidence figures as given in the 1962 and 1966UNSCEAR reports (587, 596) (which in turn weremodified from the original ones given by Stevenson(566)). the actual doubling doses were arrived at indifferent ways. The 100-rad doubling dose used by
UNSCEAR was based on those estimated by Lilning andSearle (302). These authors showed that, for different
kinds of genetic damage induced in mouse spermatogonia by high acute x-ray doses, the point estimates
for the doubling doses ranged from 16 to 51 rad.
averaging about 30 rad; with chronic exposures or withacute x irradiation at very low doses, it was inferred(based on different lines of evidence) that the rate ofinduction will be reduced by a factor of 3-4 and hence
the doubling dose under these conditions was estimatedas approximately 100 rad for males. Lilning and Searle(302) gave no ·doubling dose estimates for oocytes sincevery little information on spontaneous rates in females
has been obtained.

599. BEIR. on the other hand, used a range of
20-200 rad as the doubling dose. calculated in thefollowing way: the average rate of induction of recessive
specific-locus mutations in the mouse (unweighted
average of the rates for mouse spermatogonia andoocytes) under conditions of low-dose. low dose-rateirradiation was taken as 0.25 10- 7 rad- 1 per locus. The
spontaneous rate of mutations for human recessive genes
was taken to be in the range of (0.5-5) 10- 6 per locus.
Dividing the spontaneous rate by the rate of induction,
one gets a doubling dose range of 20-200 rad. This range,
which, as just mentioned was estimated from spontaneous rates of human recessive gene mutations andinduced rates of recessive specific-locus mutations in themouse was assumed to be applicable to the different
categories of genetic disease in man. BEIR consideredthat (a) the mouse induced mutation rate used above
might be on the high side because these loci were tosome extent preselected for mutability; (b) the rate ofinduction of dominant visible mutations in the mouse
was at least a.1 order of magnitude lower than that forrecessive specific locus mutations; and (c) the mortality
data pertaining to the children of Hiroshima andNagasaki offered strong evidence for believing that the
doubling dose is unlikely to be as low as 20 rad ofchronic irradiation.

r600. The UNSCEAR report gave two estimates for therisk of induction of recessive point mutations per
genome using the direct method, namely. one based on
the specific-locus mutation rate in mouse spermatogonia(0.5 I 0- 7 rad -i per locus) multiplied by the assumed
number of "genes" that make up the human genome(30 OOO), and another based on data on the induction of
autosomal recessive lethals in mouse spermatogonia(since a majority of specific-locus mutations behave as
recessive lethals). There was roughly a 40-fold difference
between the two estimates (1500 mutations per
106 gametes following I rad of paternal irradiation(specific-locus data used) versus 36 recessive lethals per
10 6 gametes per rad (recessive lethal data used)) .
601. The estimated size of the human genome(30 OOO genes) was based on that of the mouse genome(25 OOO genes); the latter figure was indirectly derived
from results on complementation analysis of a very shortsegment of the mouse genome (d-se region, 0.16 map
unit, 1/7500 of the mouse genome) and in fact referred
to "functional units" rather than genes or loci. Since
there is evidence from complementation tests that someof the events scored as specific-locus mutations involve
more than one functional unit, and since specific-locus
mutations are scored after birth at weaning age andautosomal recessive lethals, pre-natally, it was thoughtthat these factors could explain the discrepancy between
the two estimates (1500 point mutations per 10 6
gametes per rad versus 36 recessive lethals per 10 6
gametes per rad).
602. The UNSCEAR report used, again, a multiplicative
method to estimate the rate of dominant mutations per
genome (the assumed per-locus rate times the assumed
number of loci that determine dominant traits in man).
The BEIR report used, as mentioned earlier, thedoubling dose method (20-200 rad) to estimate theexpected increases in both recessive and dominant
genetic diseases.

506_......._

T
603. The UNSCEAR report gave no quantitiative
estimates for expected increases in congenital anomalies,
constitutional and degenerative diseases due to radiation
exposures. The reason was that with the direct method
employed (where mouse rates were used). suchcalculations are not possible. Although the calculations
can be done with the doubling dose method, theCommittee refrained from giving quantitative estimates
because of uncertainties regarding the genetic basis ofsuch diseases. The BEIR report, on the other hand, didprovide such figures and stressed the uncertainties
involved.
604. The UNSCEAR report gave separate estimates ofrisk for irradiation of males and females and pointed outthat in females, because of inadequate or no
information, no estimates could be given for recessive
lethals, dominant visibles and skeletal mutations. The
BEIR report estimates are for irradiation of both sexes;
the rates used (in conjunction with the doubling dose
range of 20-200 rad) were either unweighted averages of
the rates in males and females (when known, as in thecase of specific-locus mutation and X-chromosome
losses) or were based on the conservative assumptionthat the rate in females is the same as in males (as is thecase with translocations).
605. Estimates of hazards given in the UNSCEAR
report related to the induction of the different kinds ofgenetic damage per rad of parental exposure, and the
expected effects in F I per I 0 6 conceptions. The BEIR
report figures relate to the estimated increase among 10 6
live-born individuals whose ancestors had received
radiation at the rate of 0.17 rad/year (5 rad per 30-year
reproductive generation). The first-generation effects
were arbitrarily estimated as one fifth of that expected
at equilibrium (for dominant diseases) or as one tenth ofthat at equilibrium (for congenital anomalies etc.). For
chromosomal and recessive diseases the effects were
found to be negligible.

B. CURRENT STATUS
I. Introduction
606. The new human and mammalian data reviewed in
the preceding sections pennit a revision of some of theestimates of hazards arrived at by the Committee in its
1972 report (589). Ltming (295) has drawn attention tosome of the main problems in risk assessment that still
exist, and Liining and Eiche (303), Oftedal (383),
Newcombe (360), Sankaranarayanan (494,495), Schalet
and Sankaranarayanan (562), Searle (508, 509, 510),
and Selby and Selby (528) have considered the extent towhich some of the new data bear relevance to humanhazard evaluations. In addition, a task group of the
ICRP is currently engaged in preparing a document on
the impact of a radiation-induced increase in mutation
rate on the frequency of serious genetic disease in man(381). In what follows, an attempt will be made first to
summarize the pertinent new information and theninquire whether (or not) and how it can be used to makegenetic risk estimates. As in the 1972 report, such
evaluations will be made using both the direct and the
doubling dose methods.

2. Direct method(a) Recessive mutations
607. It may be recalled that from a reanalysis of
dose-rate data (gamma irradiation) for the induction of
specific-locus mutations in · mouse spermatogonia(paras. 276-280), Lyon et al. (318) suggested that at very
low dose rates, the risk of induction of these mutational
events may be higher than hitherto assumed and that
this might be due to very low or no spermatogonial cellkilling (relative to that which occurs at rates such as
0.009 R/min or higher). However, the available data also fit
the concept of no change in mutation rate at exposure rates
below 0.8 R/min, and besides, there are now two newlines of evidence. Firstly, the data currently available
from the continuing work of W. L. Russell and Kelly(486, 487) (paras. 282-286) show that when a 300-R
gamma-ray exposure is delivered to mouse sperma·
togonia at rates of 0.0056 R/min and 0.0007 R/min, themutation frequencies are practically identical but
significantly above controls. Secondly, in parallel
experiments, also at 300 R, Oakberg and Palatinus (378){paras. 285, 400-402) have demonstrated that thesurvival of stem-cell sperrnatogonia under x irradiation isunaffected at a rate of 0.0007 R/min, reduced to 37 percent of controls at a rate of 0.0056 R/min, and to I 6 percent of controls after 94 R/min. Yet, the mutation rates
obtained at the two low rates are identical and highest
after high exposure-rate x-irradiation, showing nocorrelation between sensitivity to cell killing and to
mutation induction. It therefore does not seem justified
to alter the earlier premise used in hazard evaluations,
namely that at low doses and dose rates, the rates of
induction of point mutations in spermatogonia is about
one third of that observed at high doses and dose rates.
608. As was fully discussed in the 1972 report, the two
major uncertainties in the estimate by the multiplicative
method of the rate of induction per genome of recessive
point mutations (i.e., specific-locus rate multiplied by
the assumed number of loci that make up the genome)
relate to (a) whether the average specific-locus rate basedon the 12 loci is representative of the mutational
sensitivity of the genome in general and {b) the numberof loci in the genome capable of mutating to recessive
visibles. For spontaneous recessive mutations, it is
known that the forward mutation rate at unselected lociis at least an order of magnitude lower than that at
specific loci. The situation with respect to theradiation-induced rate for recessive visibles at unselectedloci is not entirely known, although such data on
dominant visibles indicate a very much lower rate
relative to that for recessive specific loci (506, 589).
Thus, the problem of selected versus unselected loci andthe differences in their mutational potentialities will
always remain. Since there is no easy way to get a
precise estimate of the number of loci in the genome,
the reliability of the genome estimate arrived at by themultiplicative method will continue to remain questionable.
609. Autosomal recessive lethals which represent
mutations at unselected loci have been found to be
induced at a much lower rate per gamete than would
have been expected on the basis of the specific-locus
507

experiments. If for the sake of argument; one divides the
per gamete induction rate for recessive lethals(0.9 10-4 rad- 1 ) by the per-locus specific-locus rate
(1.7 10- 7 rad- 1 ), one gets an improbably low figure ofabout 500 as the total number of loci capable ofmutating to recessive lethals.
610. In view of these considerations, it would appear
preferable to use the direct estimate based on recessive
lethals in hazard evaluations. The value of thespecific-locus data in this context lies in the information
they provide on the effects of various physical andbiological variables on mutation induction. If this basic
conceptual change is accepted as a more reliable way toexpress risks, then the rate of induction of recessive
lethals can be used as a meaningful starting point.
611. As was pointed out earlier, the estimated rate of
0.9 10- 4 rad- 1 per gamete (high dose, high dose-rateirradiation) is for autosomal recessive lethals acting inutero. The unpublished observations of W. L. Russell
referred to in the 1972 report have shown that the
proportion of pre-natal lethals averaged over the loci (atspecific loci) is less than one half of the total number ofmutations. If true in general, then the rate of inductionof recessive mutations is likely to be at least
2 X 0.9 10- 4 = 1.8 10-4 rad -i per gamete. Dividing thisby 3 (to correct for expected effects at low dose-rate,
low-dose conditions applicable to man) one gets a figureof 0.6 10-4 rad- 1 per gamete for spermatogonial
irradiation.
612. In making these calculations, it has been assumed
that the rate of induction of recessive lethals reflects the
genome rate for the induction of point mutations ingeneral. This assumption derives support from the factthat recessive lethals represent events as unselected lociand from the fact that a majority of radiation-induced
specific-locus mutations studied in the mice behave as
recessive lethals in the homozygous condition.
613. There are no data on the induction of recessive
lethals in mouse females. Since, under radiation
conditions applicable to man, maturing mouse oocytes
show a very low level of mutational sensitivity andimmature oocytes are virtually insensitive to the
induction of specific-locus mutations, it may bepresumed that the risk in females will be low, but noquantitative estimates can be given.

(b) Dominant mutations
614. There are two sets of data in mice which allow arough estimate to be made , of the risk of inducing
mutations with dominant effects. These data refer to(a) dominant visible mutations and (b) skeletal mutations appearing in the F 1 generation after x or gamma
irradiation. For chronic gamma irradiation, the mean
rate of induction of dominant visible mutations is
1.0 10-7 rad- 1 per gamete (506). This can be converted
into a per-locus rate by dividing it by the number of lociin the mouse (approximately 75) at which dominantvisible mutations have occurred. Assuming an equal
mutation rate in man, this figure (1.3 10- 9 ) can then be
multiplied by the assumed number ofloci (1000) which
determine dominant traits in man to get an overall rate508

for dominant mutations. This procedure was used in the1972 report. The Committee was. however, aware of theuncertainties and limitations of this approach and havenot used it in the present document.
615. As far as skeletal mutations are concerned, there
are new data on heritability and rate of induction. The
new data of Selby and Selby (528) (paras. 335-339)
confirm that abnormalities classified as presumed
mutations on the basis of Ehling's criteria(paras. 332-333) are probably mostly heritable. The dataof Selby and Selby, when appropriately corrected for
dose-fractionation and dose-rate effect, 28 give the samerate of induction as that which could be derived from
Ehling's data (namely, a rate of 4 10-6 rad- 1 per
gamete). In both the investigations, the authors
recognized that this may be an underestimate of the true
rate (paras. 336, 338).
(c) The relationship between rates of induction ofrecessive and dominant mutations and the effectsof these mutations in the progeny
616. In its 1972 report, the Committee arrived at
estimates of the effects of recessive and dominant
mutations in the generation following paternal irradiation (see table 47) in the following way: for recessives,
the induction rate (1500 per 10 6 gametes per rad or 36
per 10 6 gametes per rad) was multiplied by 2-5 per cent( this range of figures being based on the averagesemi-dominant effects of recessive lethals in Drosophila)
and the resultant figures (30-75 per 10 6 or 1-2 per 106 )were considered as representing the '·expression" ofmutations in the F 1 generation. In fact, what thesefigures imply is that the mean Darwinian fitness ofheterozygotes for these mutations will be reduced by
2-5 per cent relative to those not carrying the mutations
and that, on the average, these mutant genes will persist
in the population for 20 (5% semi-dominance) or 50 (2%semi-dominance) generations subsequent to their induction. In other words, the impact of natural selection on
mutant genes is through their heterozygous effects andthe duration of persistence of the mutant genes will
vary, depending on, among other things, theirsemi-dominant effect in heterozygotes, i.e .. the smaller
their semi-dominant effects, the longer will they persist
in the population.
617. For dominant visibles, the procedure was simple:
the induction rate and the expression in the F 1 wereconsidered the same (2 per 10 6 ). Based on the limited
information 1hen available, mutations causing dominantskeletal defects were considered together with recessive
mutations, since it was thought that they might
represent heterozygous manifestations of recessive
mutations.
618. In the light of new data on mutations causing
dominant skeletal defects in the mouse, the finding that
11 The frequency of 1.4 10 · 2 is divided br 600 (dose inrad) and further divided by 1.9 (to correct for the enhancementin mutation frequency due to the fractionation regime
employed, based on findings in specific-locus work) and by 3 (toget a rate at low dose-rates, again based on specific-locus work)(1.4 10 • 2 )/(1.9) (3) (600) ""4 10 ·•.

four out of four such mutations tested thus far (525)behaved as recessive lethals when made homozygous,
other evidence (para. 625), and the premise that
dominance and recessivity are attributes of thephenotype and not of genes. the Committee feels that it
is now possible to improve the estimates given in 1972
along the following Jines: (a) by considering dominant
and recessive mutations together to estimate effects in
the F 1 and (b) by attempting to express these effects insocially meaningful terms, i.e., in terms of handicaps or
disabilities that are expected.
619. An overall estimate of the risk from the inductionof mutations causing dominant effects in the progeny(and this includes recessives with semi-dominant effects
as well as dominants) can be derived using the data on
the rate of induction of skeletal abnormalities in themouse. The main argument for this rests on the premisethat many of the skeletal abnormalities recovered in themouse experiments are similar to rare dominants andrare irregularly inherited dominant conditions in man,
which together constitute a sizeable portion of human
genetic diseases. However, to convert the rate ofinduction of mutations causing skeletal abnormalities in
the mouse to an overall rate for all dominant mutations
with serious effects in man, one needs information on(a) the proportion of dominant conditions in man whose
main effect is in the skeleton and (b) the proportion ofskeletal abnormalities studied in the mouse which, at thehuman level, is likely to cause a serious handicap.
620. Firstly, a perusal of McKusick's latest tabulationof monogenic disorders in man (328) will reveal that outof the 583 "proved" autosomal dominants, 328 areclinically important; in the latter group, 74 ( or roughly
20 per cent) involve one or more parts of the skeleton toa v:uying extent. However, this figure is undoubtedly areflection of the ease of diagnosis of such abnormalities
by phenotypic inspection and/or radiography. The true
figure is therefore likely to be lower and, in the opinionof Carter (82) and McKusick (328a), is of the order of
10 per cent.
621. Secondly, not all of the skeletal abnormalities ofmutational origin studied in the mouse will impose aserious handicap in humans. Selby (525) suggests that
this proportion of abnormalities (leading to serious
handicaps) may range from 25 to 75 per cent; in Carter's
opinion, a figure of 50 per cent may be accepted as atentative estimate and this has been confirmed in a
detailed discussion of the mutants by Selby (525) and
McKusick (328a).
622. It follows that, if the estimated rate of inductionof mutations causing skeletal abnormalities in the mouse( 4 10-6 rad - 1 per gamete under low-LET, low-dose or
low dose-rate irradiation; spermatogonia) is multipliedby 10 and then divided by 2, the resultant rate,
20 10- 6 rad -i per gamete, is one of induction of
mutation having dominant effects on any of the bodily
systems in man. In other words, following I rad ofpaternal (spermatogonial) irradiation, 20 out of every
million progeny will be expected to carry mutations
which cause one or another kind of dominant genetic
disease, in the first generation.

623. It is well known in both man and mouse that
dominant mutations may show pleiotropism and thus
may affect both the skeleton and non-skeletal systems(such as the spleen and stomach in the case of mousedominant hemimelia). Similar skeletal anomalies may
also tum up in different genetic syndromes in both man
and mouse. However, in making the calculations referred
to above, allowance was made for such pleiotropism.
624. Since in man skeletal abnormalities are frequently
observed in association with chromosomal aberrations,such as complete trisomies and partial monosomies andtrisomies (208a), it is important to establish to what
extent the skeletal conditions described by the Selbys
could have been chromosomal rather than genie inorigin. Nine mutant lines (mainly involving sterile andsemi-sterile individuals)have been tested cytologically andthree contained translocation heterozygotes. Two of thesethree seemed to be associated with the skeletal effects. It
should be noted that (a) no unbalanced forms, such as
trisomies, were found; (b) the possibility that some rarehuman apparent dominants are associated with chromosome anomalies cannot be ruled out; and (c) effects ofbalanced translocations have not been included in any
other category in the risk assessments. For these reasonsand those given in paragraph 340 the amount of overlap
with the chromosomal aberration risk category is likely
to be very small, although it cannot be estimated in theabsence of detailed refined chromosome analysis.
625. On the other hand, a substantial fraction of these
dominant conditions ar~ likely to be lethal in thehomozygote and can therefore also be placed in therecessive lethals category. Of 164 dominant mutations in
the mouse (apart from those concerned with enzyme
polymorphisms and immunological traits) at least 45, or
27 per cent, appear to be lethal in the homozygote. It isquite clear, however, that the dominant manifestation is
the important one from the point of view of risk
·estimation. Homozygotes for human deleterious dominants have rarely been observed, but if so, are lethal.
626. Although the many, uncertainties involved in
extrapolating from mutations causing dominant skeletal
effects in the mouse to the overall number of harmful
dominants in man are realized, it is felt that theinduction rate of 20 per progeny per rad of paternal
irradiation ( to spermatogonia), as calculated in paragraph 622, is the best estimate which can be obtained in
our present state of knowledge. It is therefore the rate
given in table 49.

(d) Reciprocal translocations
627. For the induction of reciprocal translocations,more information, including some limited human data, is
now available (paras. 131. 184-187). The combined
human and marmoset spermatogonial data (at doses of
100 rad and below, acute x-irradiation) indicate atranslocation rate of 6.94 I 0-4 rad -l per cell, on theassumption of linearity (para. 185). A much lower rate
could be estimated from the two sets of data now
available for the rhesus monkey (paras. 182. 183), but
for reasons discussed earlier (paras. 186, 187), it has
been decided to base estimates of risk on the human andmarmoset data in this report.
509

628. For estimating the rate at low doses or after
chronic exposures, the following lines of evidence frommouse work are pertinent. When a total dose of 300 radof x rays is administere_d in 30 or 60 equal fractions, thefrequencies of translocations observed in the spermatocytes are reduced by a factor of 4 relative to acutedoses (315). At the lowest x-ray dose rate of
0.09 rad/min (300-rad level), the reduction in frequency
is by a factor of 2 (512). The efficiency of chronic
gamma irradiation at 0.02 rad/min (600-rad level) is
lower by a factor of about 10 relative to acuteirradiation (517); at a lower dose rate of 0.007 rad/min,over a range of up to 1200 rad, the dose-effectrelationship is linear and here too, there is a dose-rate
reduction factor of at least 10 (Pomerantzeva et al.;refs. 415 and 417). Finally, in the experiments of Searle
et al. (516) with a gamma-ray dose of 1128 rad delivered
over a period of 28 weeks at a rate of 0.004 rad/min. therate of induction was 1.40 10- 5 rad -i per cell, which islower by a factor of about 20 relative to that after acuteirradiation. (In the work of Pomerantzeva et al (417),
however. a lowering of the rate from 0.007 to
0.003 rad/min did not lead to any sizeable reduction in
the efficiency of gamma irradiation.)
629. If these results are considered applicable totranslocation induction in human spermatogonia, the useof reduction factors of, respectively 4, 2 and 10 (tocorrect the rate of 6.94 10-4 rad- 1 ) for low-dose x rays,
low dose-rate x rays, and chronic gamma irradiation,
would appear to be reasonable. The corresponding
figures then are (rad- 1 per cell): 1.74 10-4 (low-dose
x rays), 3.47 10-4 (low dose-rate x rays), and
0.694 10-4 (chronic gamma irradiation).
630. The induction rate of importance for hazard
evaluations is the one that relates to recoverable
translocations in the F 1 progeny. Although in its 1972
report the Committee used a reduction factor of 8 to get
the above from spermatocyte data (based on the work of
Ford et al (170)), it would appear prudent (on the basisof new evidence reported in paragraphs 131 and 132) toassume that the rate of recovery in the F 1 will be one
fourth of that in spermatocytes. This means the rates
will be (rad- 1 per gamete): 0.44 10-4 (low-dose x rays),
0.87 10-4 (low dose-rate x rays), and 0.174 10-4 (chronic
gamma irradiation).
631. It follows that if males are exposed to low-dose
x rays, low dose-rate x rays, or chronic gamma
irradiation, the expected number of balanced andunbalanced translocation-carrying zygotes in the F 1 willbe, respectively, 44 and 88, 87 and 174, or 17 and34 per million conceptions per rad of exposure.
Assuming further (as the Committee did in its 1972
report) that only about 6 per cent of the unbalanced
zygotes will result in children with multiple congenital
anomalies, there will be about 5, 10 or 2 such children
per million conceptions after one rad of paternal(spermatogonial) exposure. One third of the remaining
unbalanced zygotes (83 X 1/3= 28, 164 X 1/3= 55,
32 X 1/3 = 11) would fall into the recognized abortion
category, and the remaining two thirds would die soearly as to go undetected.
632. It bears reiterating here that the rates oftranslocation induction used above are based on human510

and marmoset cytogenetic data with corrections fortransmission and expected effects at low doses and dose
rates being based on mouse data. Should it turn out thatthe rate of induction in human spermatogonia is moresimilar to that in the rhesus monkey. the estimates mayneed revision, and consequently the quantitative figuresarrived at must be considered provisional at present. In
the 1972 report. the rate used for human spermatogonia
was based on that in the mouse (~.3 10-4 rad- 1 per
gamete, acute x irradiation, semi-sterility data), multiplied by 2 to take into account the two-fold difference
between man and mouse in effective chromosome arm
number and divided by factors of 4 or 9 to get the rates
under low dose or chronic exposure conditions,
respectively. The change in approach was dictated inpart by the lack of validity of the arm number
hypothesis (paras. 450-465) for interspecific extrapolations and in part by the availability of new data inman, the marmoset and the mouse on the transmissionof translocations to the F 1 progeny.
633. The rate of translocation induction in human
females is not known. The data for mouse females
discussed earlier (para. 166) show that in maturingoocytes exposed to acute x irradiation, the rate is
0.16 10-4 rad- 1 per gamete (semi-sterility data).
Although there is no direct evidence on the response ofimmature oocyte stages to the induction of translocations at low doses and dose rates, the data on
specific locus mutations and on X-chromosome losses(see paras. 230-231 for the latter) strongly support theview that the rate for translocations is also likely to be
low, bi.it no quantitative estimate can be given.

(e) Other structural aberrations
634. It seems likely that the risk from the induction of
structural aberrations other than reciprocal translocations will be yery small. In the mouse, "dominantlethals, due to · isochromatid exchange etc., withresultant chromosomal breakage, can be transmitted
after oocyte irradiation, though apparently not after
spermatogonial irradiation because of germinal selection.
It seems likely that most of them act too early toconstitute a real hazard. Deficiencies are also induced atcomparatively high frequency after oocyte irradiation;
however not enough is known yet about their probableoverall rate of induction or deleterious effects ( especiallyin the heterozygote) to make any estimate of the genetic
damage they may cause'' (508).

(f) Sex-chromosome losses
635. The complete data on the x-ray induction of
sex-chromosome losses in male mice {para. 229) support
the conclusion reached by the Committee in 1972,
namely that the risk from this class of genetic damage
after spermatogonial irradiation is very low or nil. The
new data for female mice (table 32 and para. 231) showthat the frequencies of sex-chromosome losses are low
and not significantly above the controls both after acutex irradiation with 50 R and after chronic gamma ray
exposures ( 400 R) delivered at a rate of 0.006 R/min.
These findings therefore suggest that if the humanoocytes respond like those of the mouse, the risk isprobably very low. but no quantitative estimates can begiven.
636. The risk from the induction of other chromosomal
aberrations cannot be reliably estimated at present. As
far as chromosome gains are concerned, there is no clear
evidence in the mouse, for their induction after
spermatogonial irradiation (450, 506). In oocytes
however, Yamamoto et al (627) obtained somepresumptive evidence for the x-ray induction of
non-disjunction in older mice (para. 237), but these data
do not lend themselves to risk estimation because ofsome statistical problems (para. 238). The work of
Uchida and Lee (582) (paras. 239-241) also provides
evidence for the induction of non-disjunction in oocytes,
but the total number of non-disjunctional events is too
scanty to define the form of the dose-effect relationship.
The results in humans are conflicting (paras. 33-37) andfurthermore, there is a large maternal-age component. In
view of these, it seems judicious to refrain frompredicting the overall extra risk of trisomy in theoffspring of women exposed to irradiation until somemore information becomes available.

3. Doubling dose method
637. Turning now to the method of expressing risks inrelation to the natural incidence of genetic diseases inman (i.e., the doubling dose method), it should be
stressed that this method has certain advantages as wellas limitations: these were dealt with in the 1972 reportof the Committee. Suffice to point out that its chiefadvantage lies in the fact that it expresses risks in morecomprehensible terms than the direct method, although
one of the main assumptions, namely, that there is
proportionality between spontaneous and induced rates,
still remains an unproved one. To employ the doublingdose method, one needs, in addition to otherinformation, the following kinds of data: (a) the natural
incidence of the different classes of genetic disease inman; (b) the relationship between incidence andmutation rate; (c) the doubling dose; (d) the radiationdose to which the population is exposed.
638. The data of Trimble and Doughty (576). with thesubstantial modifications suggested for regular dominantdiseases (para. 11) and those summarized by Nielsen andSillesen (363) on the incidence of chromosome
anomalies (see last column in table 9), provide therequisite information on point (a). Of the totalfrequency of 0.6 per cent chromosomal anomalies
recorded in new-born surveys (table 11), roughly two
thirds (0.4 per cent) is constituted by numericalsex-chromosomal and autosomal anomalies plus
aneuploid structural abnormalities. This 0.4 per cent canbe considered as the rate of incidence of chromosomal
diseases, although this figure (which does not include
balanced reciprocal translocations) may be an underestimate since there is evidence that heterozygosity forbalanced reciprocal translocations in some cases,
especially in those involving the sex-chromosomes, canresult in a handicap to carriers (143, 144). The evidenceof Jacobs et al. (250) from new-born surveys thataneuploid structural rearrangements of the chromosomes
detectable with non-banding techniques may be a rarecause of congenital malformations needs to be viewed
with some caution since such malfonnations may still berecognized later in life.
639. As far as point (b) is concerned, it is safe toassume that the incidence of autosomal dominants andX-linked diseases is essentially proportional to themutation rate, i.e., their incidence will increase afterradiation exposures. The incidence of recessive diseases
is only very indirectly related to mutation rate. The
incidence of numerical chromosomal disorders andunbalanced structural anomalies will be directly
proportional to mutation rate.
640. For diseases of complex aetiology, such as
irregularly inherited dominant diseases, multifactorialdiseases and congenital malformations (together consisting about 9 per cent in the British Columbia Survey),
as was mentioned earlier (para. 15), we shall assume thatthe mutational component (i.e., the proportion of these
diseases that will respond to radiation exposures in amanner similar to that of simple monogenic dominantdiseases) is of the order of 5 per cent (para. 15). In termsof absolute effects expected at equilibrium, this isequivalent to assuming that 0.45 per cent diseases ofcomplex aetiology (i.e., 5 per cent of 9 per cent) areirregularly-inherited dominants, and these will behave
like simple dominants in their response to irradiation.
The reason for not lumping these two classes is that
under the radiation conditions assumed for theestimation of risks (see below) the expected effects inthe first generation are different for these two classes.
641. Coming now to the doubling dose, as was
discussed earlier, the BEIR report used a range of
20-200 rad; the 1972 UNSCEAR report used the figureof 100 rad for males. The examination of available
evidence in the mouse suggests that the use of a I 00-rad
doubling dose (for both sexes) will not underestimate
the risk (495, 510). The ICRP Task Group (381) has also
used this figure in its calculations. Further justification
for the use of this figure derives from the recent
Hiroshima and Nagasaki data (para. 39), which suggestthat for normal radiation conditions applicable to thehuman population (low LET, low dose or low dose rate),
the minimal doubling dose for the type of damage
resulting in death during the first 17 years of life is atleast 138 rad for males and over 1000 rad for females.
We shall therefore use 100 rad as the doubling dose inthis report.
642. To facilitate comparisons with the estimates given
in table 49, we shall assume that the population is
exposed at a rate of 1 rad per generation (low-LET. low
dose rate). The resultant estimates are given in table 50(top half). It can be seen that (a) the expected incrementin the first generation is estimated to be one fifth of thatat equilibrium for simple dominant and X-linked diseases
and one tenth of that at equilibrium for diseases ofcomplex aetiology (based on the assumptions used in the
BEIR report); (b) for chromosomal diseases, the first
generation incidence is nearly the same as at equilibrium;and (c) the total increment expected in the first511

generation is 63 new cases of genetic disease per 10 6
(0.06 per cent of current incidence) and 185 cases per
10 6 at equilibrium (0.17 per cent of current incidence).

there was no correlation between cell killing and themutation rate. In view of this, the reduction factor of 3
to estimate effects at low dose rates has been retained.

643. A comparison of the present estimates with thoseof the BEIR report (bottom half of table 50) will showthat there are some discrepancies between the two setsof estimates. The differences in certain numericalestimates derive from the use of (a) different current
incidence figures (10. 7 per cent in the present report.
6 per cent in the BEIR report); (b) a single doublingdose of 100 rad rather than a range of doubling doses
from 20 to 200 rad; (c) a single figure instead of a rangeof figures to quantify the mutational component indiseases of complex aetiology (5 per cent instead of
5-50 per cent); and (d) a numerical estimate for theeffects of radiation on the increment in chromosomal
diseases (based on data from new-born surveys)· in thepresent report.

648. Since there are no data on the induction ofrecessive lethals in female mice, no estimates are possible
for this kind of damage in human females. However, theevidence from specific-locus experiments strongly
support the view that the risk in females is low.

644. It is worth pointing out here that the firstgeneration effects for simple dominant diseases(20 per 10 6 ) and for irregularly inherited dominants(5 per 10 6 -a total of 25 per 10 6 live-born-is quite
similar to the figure of 20 per 10 6 arrived at using thedirect method.

4. Summary and conclusions
645. This chapter has been devoted to (a) a comparisonof the main conclusions reached by UNSCEAR and
BEIR in their 1972 reports; (b) summarizing the major
new findings that are pertinent for the evaluation ofgenetic radiation hazards (details of which werediscussed in the different chapters of this annex); and(c) presenting revised estimates of risk on the basis of
the information currently available. The estimatesarrived at are given in table 49 ( direct method) and in
table 50 (doubling dose method); these effects are those
expected following low-LET irradiation of the germ-cellstages most at risk (spermatogonia in males and· oocytes
in females) and are for conditions of radiation exposure
most relevant, namely, low doses and chronic exposures.
646. The rate of induction of recessive mutations has
been estimated to be 0.6 10-4 rad -i per gamefo. This
figure is derived from the rate of induction of autosomal
recessive lethals in male mice following acute x irradiation of spermatogonia (0.9 10-4 rad- 1 per gamete),corrected for (a) low dose-rate irradiation conditions(i.e., the use of a reduction factor of 3 which gives
0.9 10-4 + 3= 0 3'10-4 ) and (b) assuming that the
proportion of recessive lethals scored by the pre-natal
method represents about one half of all recessive
mutations (i.e., multiplying 0.3 10-4 by 2, we obtain

0.6 10-4 ).
647. The Committee examined the possibility that, atvery low dose rates, the mutation-rate reduction factorof 3 (correction (a) above) may not be applicablebecause of the lack of cell killing in the context of(a) new data on specific locus mutations at low dose
rates and (b) new data on the survival of stem-cell
spermatogonia at different dose rates and concluded
that, at the dose rates studied (one high and two low),
512649. The overall dominant effects from the inductionof mutations in man (including recessives withsemi-dominant effects and dominants), estimated as
20 10-6 rad- 1 per gamete, has been arrived at using the
expected rate of induction of mutations affecting theskeleton in mice (following spermatogonial irradiation at
low dose rates) as the starting point ( 4 10 - 6 rad -i per
gamete). This figure was multiplied by 10 to take into
account the fact that approximately 10 per cent of
dominant mutations recorded in man are associated withskeletal abnormalities and divided by 2 to take into
account the possibility that about 50 per cent of theskeletal mutations found in the mouse, if induced inhumans, could cause serious handicap.
650. There are no data on the induction of skeletal
mutations in female mice, and consequently no attempt
has been made to make a quantitative estimate of risk
for the induction of dominant mutations in human
females. The risk is expected to be low. however, if theresults of specific-locus tests in female mice areconsidered applicable to human females.
651. The rate of induction of reciprocal translocations
have been estimated to be 0.44 10-4 , 0.87 10 - 4 , and
0.174 10-4 rad- 1 per gamete for low-dose x rays, lowdose-rate x rays, and chronic gamma irradiation,
respectively. With these rates of induction, theconsequences following I rad of paternal exposure are
2-10 congenitally malformed children, 11-5 5 recognizedabortions and 22-109 early embyronic losses per 10 6
conceptions. The rates of induction have been derived
using the limited human and marmoset cytogenetic data
(spermatogonial irradiation) obtained in experiments
involving acute x irradiation and making corrections for
expected effects at low doses and dose rates and fortransmission to the F 1 progeny using mouse results as a
guide. The Committee wishes to stress that the risk fromthe induction of translocations as given above is subject
to uncertainty in view of the limited nature of the
primary data and the fact that within primates there isconsiderable heterogeneity; in fact, the data from
Rhesus monkeys suggest that the rates may be much
lower than in both the marmoset and the mouse, andshould this suggestion be confirmed in subsequent
experiments with primates, especially the monkey
and/or man, there may be grounds for revision of theestimates.
652. The reason for using human and marmoset datafor hazard evaluation (and not the rate of induction inmice with a multiplication factor of 2 to apply to man,as was done in the 1972 report) is that the basic premisefor the use of the multiplication factor of 2 (namely. thehypothesis of linear relationship between effective

l

r
I

chromosome arm number and the relative sensitivity to
the induction of dicentrics or translocations) now
appears invalid.

paragraph) in the first generation and is about the sameas that estimated using the direct method (20 per million
live-born; see paragraph 649).

653. There are no data which will permit an evaluationof the risk from the induction of translocations inhuman females. Again, on the basis of mouse results withanother end-point of genetic damage, namely the loss of
X-chromosomes, it would appear that the risks are low,
but no quantitative estimates can be given.

VII. SUGGESTIONS FOR FUTURE
RESEARCH

654. The risk from the induction of other kinds ofstructural aberrations is likely to be small by analogy
with the mouse, but no estimate can be made at present.
655. For the induction of sex-chromosome losses. theavailable mouse data suggest that in both males andfemales (at low doses or after chronic gamma
irradiation) the risk is low or negligible. The lack of asignificant difference in X-loss frequencies (between
controls and the irradiated groups of mice), viewed inconjunction with the fact that in humans, a predominantmajority of the XO conceptions are inviable, would
suggest that in humans, too, the risk is likely to be quite
low.
656. Although in mouse experiments, presumptive
evidence for the induction of non-disjunction has beenobtained at low doses, in view of some uncertainties(lack of information on dose-effect relationship, statistical problems and complications due to maternal-age
effects), it is not possible to make a realistic quantitative
estimate for this kind of damage.
657. Estimates of doubling doses arrived at in themouse for various end-points of genetic damage, as wellas the data on the mortality of children born to A-bomb
survivors in Hiroshima and Nagasaki, suggest that the
doubling dose in man {both sexes) is unlikely to belower than 100 rad.
658. The doubling dose of JOO rad has been used inconjunction with the frequencies of spontaneous
incidence of genetic disease in man to compute, theexpected increments. The spontaneous incidence figures
used are (per cent): dominant and X-linked diseases, 1;
recessive diseases, 0.1; chromosomal diseases, 0.4;
diseases with complex aetiology, .9.0. Under theassumption that the population is exposed to low-LET,
low dose-rate irradiation at the rate of I rad per
generation, it was estimated that in the first generation
there will be, per million live-born, 20 additional cases
suffering from dominant and X-linked diseases, 38 fromchromosomal diseases, and 5 cases from diseases ofcomplex aetiology. The total, 63, represents 0.06 percent of the current incidence (10.5 per cent). At
equilibrium {which will be reached after different
numbers of generations depending on the kinds ofdisease), this total increment will be 185 cases per
million live-born, or 0.17 per cent of current incidence.
659. The overall increment due to the induction of
dominant mutations (simple dominants plus those withirregular penetrance), estimated by the doubling dose
method is 25 cases per million (20 + 5; see preceding

660. In this annex, the progress that has been made in
mammalian and human genetics, cytogenetics, molecularradiobiology and somatic cell genetics since thepublication of the 1972 report has been reviewed, andrevised estimates of genetic risks to man from exposure
to ionizing radiation have been presented. Theseestimates reflect our current state of knowledge; as will
be evident from the Committee's consecutive reports on
this subject, the process of risk assessment is acontinuous one: new advances help us to view old
problems with a new perspective, to examine the validityof assumptions and extrapolation procedures used, andto identify areas of research which are likely tocontribute to a better understanding of the genetic
radiosensitivity of human beings. The Committee feels
that, in order to increase our precision in risk
assessment, more research effort along the following
main lines will be useful (the order in which these arelisted do not reflect the order of importance):(a) Additional surveys on the prevalence ofhereditary defects and disease in human populations(similar to that carried out in British Columbia).
including an ascertainment of chromosomal diseases anddiseases of late onset;(b) Mutation monitoring in man by biochemical
techniques:(c) Continuation of studies on non-disjunction
and the effects of radiation thereupon;(d) Continuation of genetic and cytogenetic
studies on Hiroshima and Nagasaki populations;( e) Continuation of comparative
studies in different mammalian species;

radiation(f)Further studies on the relationship between
the frequencies of reciprocal translocations scoredcytogenetically in spermatocytes and those observed in
genetic tests, especially at low doses and after low
dose-rate irradiation;(g) Collection of more cytogenetic data on the
induction of reciprocal translocations in primate species
using when possible, human testes which happen to havebeen irradiated for medical reasons;(h) Continuation of studies on the radiationinduction of structural chromosomal aberrations (including translocations) in mammalian oocytes:(i)
Further studies on the role of factors
influencing the induction of structural aberrations insomatic as well as germinal cells, such as the amount anddistribution of heterochromatin in the chromosomes,
the basis for differential sensitivity to radiation-induced
breakage observed between chromosomes or chromosome regions;(j)Further studies on the repair of radiationinduced chromosome breaks;

513

Continuation of studies aimed at thecharacterization of the radiation-induced mutational
events at biochemically-defined loci in mammals andother organisms such as Drosophila;(k)Continuation of studies on the nature ofradiation-induced specific-locus and dominant mutations;(!)(m) Studies on the induction of autosomal
recessive lethals in female mice;Continuation of studies on the induction
and characterization of mutations affecting the skeleton
and other apropriate bodily systems in the mouse;(n)( o) Studies on the induction of mutations ingerm cells and somatic cells at very low doses and dose
rates and the development of techniques to facilitatesuch studies;(p) Continuation of genetic and cytogenetic
studies with mice on the effects of incorporated
radioisotopes;(q) Continuation of studies on the factors thatmodify radiation-induced genetic damage:(r)
Development and exploitation of moremutational assay systems in somatic cells in vitro and in

viva;
(s)
Studies on the nature of radiation-induced
damage at the DNA level and its relationship tomutations and chromosome aberrations;(t)Validation of the assumptions involved in
the use of the doubling dose approach for hazard
evaluation;(u) Continuation of studies on the interaction ofradiation and chemical mutagens.

TABLE 1. SINGLE GENE DOMINANT DISORDERS BY ICDA CODE
Number of
personsa{fectedb

ICDA
code"

282.0
286.3
312.9
313.9
314.9
330.0
330.4
330.9
331.0
350.0
354.0
358.0
377.9
389.9
520.5
733.2
743.4
744.2
744.3
744.5
744.7
744.8
752.2
753.1
755.0
755.2
755.3
755.5
755.8
756.0
756.6
756.6
756.7
757.0
757.2
757.9
759.6 '
759.8

17
4
1
1
1
6
4

3
2
1
2
2
4

3
3
3
29

1
27
8
3
24
2

1
21
2
1
5
2

16
57
30
1
l

15
3
12

34

ICDA titlec

Familial acholuric jaundice
Vascular haemophilia
Moderate idiopathic mental retardation
Severe idiopathic mental retardation
Profound idiopathic mental retardation
Neuropathic muscular atrophy
Myotonia atrophica
Unspecified neuromuscular disorder
Hereditary chorea
Facial paralysis
Polyneuritis and polyradiculitis
Homer's syndrome
Unspecified disease of retina and optic nerve
Impairment of hearing, one 01 both ears
Hereditary disturbances in tooth structure
Myositis ossificans
Neurofibromatosis
Buphthalmos
Congenital cataract
Aniridia
Congenital blepharoptosis
Anomalies of eye
Hypospadias
Cystic kidney disease
Polydactyly
Reduction deformity of upper limb
Reduction deformity of lower limb
Anomaly of upper limb (including shoulder girdle)
Generalized flexion contracture of limb joints
Anomalies of skull and face bones
Chondrodystrophy
Osteogenesis imperfecta

Generalized anomalies of skeleton
Hereditary oedema of legs
Anomalies of skin.
Unspecified anomalies of skin, hair and nails
Tuberous sclerosis
Congenital syndrome, affecting multiple systems

Total 352
Source: Based on the British Columbia Survey (576).
= International Classification of Diseases Adapted, 8th revision

0 ICDA(244).
bsince ascertainment for many conditions, especially congenital
malformations, Is not routinely good for all birth cohorts, and since thesenumerators are not broken down by birth period, valid estimates of rates ofindividual conditions cannot be calculated from these numbers.
"These titles do not describe single disease entities but rather the nature of
the groups of conditions to which the ICDA rubrics apply. Detailed diacnostic
lists for each ICDA code are given in the ICDA, 8th revision (244 ),

514

i

TABLE 2. SINGLE GENE RECESSIVE DISORDERS BY ICDA CODE

ICDA
code

243.0
253.1
253.9
270.0
270,7a
270.8
271.0
271.l
271.2
272.2
273.0
273.3
273.4
273.6
273.8
275.0
275.1
279.0
282.4
284.0
311.9
315.9
330.0
330.1
330.2
330.3
332.0
333.0
333.1
333.9
377.9
389.0
389.9
583.0
742.0
743.1
743.8
744.1
744.2
744.3
747.2
747.9
751.5
752.7
753.1
756.5
756.7
757.2
759.8

Number of
persons
affected
3

1
1
41
28
11

5
8

5
4
151

1
6

22
16
7

5
5
10

2
1
1
3
19
24
28

5
10
4

2
1
13

20

1
1
6

1
2
16
1
1

1
1
5
19
3

3
17
9

ICDA title

Cretinism of congenital origin
Anterior pituitary hypofunction
Unspecified disease of pituitary gland
Phenylketonuria
Albinism
Congenital disorders of amino acid metabolism
Von Gierke's disease
Unspecified glycogen storage disease
Galactosaemia
Lipid storage disorders
Cystic fibrosis
Hepatolenticular degeneration
Disorders involving metabolism of minerals
Disorders of steroid metabolism
Congenital disorders of metabolism
Agammaglobulinaemia
Hypogamrnaglobulinaemia
Unspecified metabolic diseases
Mediterranean anaemia
Aplastic anaemia
Mil_d idiopathic mental retardation
Unspecified idiopathic mental retardation
Neuropathic muscular atrophy
Familial progressive spinal muscular atrophy
Amyotonia congenita
Progressive muscular dystrophy
Hereditary spinal ataxia
Amaurotic family idiocy
Progressive cerebra! leukodystrophy
Unspecified hereditary diseases of nervous system
Unspecified diseases of retina and optic nerve
Deafness, both ears
Impairment of hearing, one or both ears .
Nephritis, unqualified
Congenital hydrocephalus
Microcephalus
Congenital anomalies of nervous system
Micropthalmos
Buphthalmos
Congenital cataract
Anomaly of aorta
Unspecified anomaly of circulatory system
Atresia of biliary ducts
Pseudohermaphroditism
Cystic kidney disease
Chondrodystrophy
Generalized anomalies of skeleton
Anomalies of skin
Syndromes affecting multiple systems

Total 550
Source: Based on the British Columbia Survey (576).
Note: See notes to table 1.

aSpecial registry code.
TABLE 3. SINGLE GENE X-LINKED DISORDERS BY lCDA CODE
ICDA
code

257.9
265.1
270.7a
273.4
273.8
275.0
275.1
282.2
282.9
285.0
286.0
286.1

Number of
persons
affected

1

2
4
6

7
11
20
1

1
1
40
11

ICDA title

Unspecified ovarian dysfunction
Rickets, late effectAlbinism
Disorders involving metabolism of minerals
Congenital disorders of metabolism
Agammaglobulinaemia
Hypogammaglobulinaemia
G-6-PD deficiency anaemia
Hereditary haemolytic anaemia
Hypochromic anaemia with iron loading
Haemophilia A
Haemophilia B

515

TABLE 3 (continued)

ICDA
code

Number of
persons
affectedICDA tir/e

286.9
312.9
330.3
330.4
377.3
377.9
742.0
752.7
756.7
752.2
757.9
759.8

1
1
52
1
24
1
1
4
2
3
I
2

Coagulation defectModerate idiopathic mental retardation
Progressive muscular dystrophy
Myotonia atrophica
Colour blindness
Unspecified disease of return and optic nerve
Congenital hydrocephalus
Pseudohermaphroditism
Generalized anomalies of skeleton
Anomalies of skinUnspecified anomaly of skin, hair or nails
Syndromes affecting multiple systems

Total

198

Source: Based on the British Columbia Survey {576).
Note: See notes to table I.
aspecial reaistry code.

TABLE 4. DISEASESDUE TO SINGLE GENESDOMINANq: BY ICDA CODE

ICDA
code

Number of
persons
affectedICDA title

252.1
282.4
282.5
286.9
744.6

1
1
2
3
5

Hypoparathyroidism
Mediterranean anaemia
Haemoglobinopathies
Coagulation defects
Retinitis pigmentosa

Total

OF

UNCERTAIN

12

Source: Based on the British Columbia Survey (S76).
Note: See notes to table I.

TABLE 5. AUTOSOMAL CHROMOSOME 1NOMALIES BY ICDA CODE

Number of
persons

ICDA
code

affected

759.3
759.4

972
44
Total

1 016

ICDA title

Down's syndrome
Syndromes due to autosomal abnormality except
trisomy 21

Source: Based on the British Columbia Survey (576).
Note: See notes to table I.
TABLE 6. SEX CHROMOSOME ANOMALIES BY ICDA CODE

ICDA
code

Number of
persons
affectedICDA title

44

Syndrome due to sex chromosome anomaly \l,ith
indeterminate sex
Syndrome due to sex chromosome anomaly withhypospadias
Syndrome due to sex chromosome anomaly withanomalies of ribs and sternum
Syndromes due to sex chromosome anomalies

752.0
752.2
756.4
759.5
Total

47

Source: Based on the British Columbia Survey (576).
Note: See notes to table I.

516

TABLE 7. CONGENITAL MALFORMATIONS BY ICDA CODE

Number of
ICDA

persons

code

affected

740.0
741.0
741.9
742.0
743.0
743.1
743.2
743.3
743.8
743.9
744.0
744.1
744.2
744.3
744.4
744.7
744.8
744.9
745.0
745.1
745.2
745.3
745.4
145.5
745.8
745.9
746.0
746.1
746.2
746.3
746.4
746.S
746.6
746.7
746.8
746.9
747.0
747.1
747.2
747.3
747.4
747.S
747.6
747.8
748.0
748.1
748.2
748.3
748.4
748.S
748.6
749.0
749.1
749.2
750.0
750.1
750.2
750.3
750.8
750.9
751.0
751.1
751.2
751.3
751.4
751.S
751.6
751.7
751.8
751.9
752.0
752.1
752.2

123
259
317
372
20
83

85
2
6
27

9

32
18
198
20
106
196
14

65
179
SS
46
93
2
8
3

22
72

163
1 014

499
10
219
41

101

477
347
91

22
102
8
13
12
8

23
15
4

38

8
4

21
460
291
571

SS
746
42
27
32
11
44

24
73
123
107
16

18
4

5
2
11

612
692

JCDA title

Anencephalus
Spina bifida v,ith hydrocephalus
Spina bifida without mention of hydrocephalus
Congenital hydrocephalus
Encephalocele
Microcephalus
Anomalies of brain
Anomalies of spinal cord
Anomalies of nervous system
Unspecified anomalies of brain, spinal cord. andnervous system
Anopthalmos
Mier op thalmos
Buphthalmos
Congenital cataract
ColobomaCongenital blepharoptosis
Anomalies of eye
Unspecified anomalies of eye
Anomalies of ear causing impairment of hearing
Accessory auricle
Anomalies of ear
Unspecified anomalies of ear
Branchial cleft, cyst or fistula; preauricular sinus
Webbing of neckAnomalies of face and neck
Unspecified anomalies of face and neck
Common truncus
Transposition of great vessels
Tetralogy of Fallot
Ventricular septa! defectAtrial septa! defectOstium atrioventriculare commune
Anomalies of heart valves
Fibroelastosis cordis
Anomalies of heart
Unspecified anomalies of heart
Patent ductus arteriousus
Coarctation of aorta
Anomalies of aorta
Stenosis or atresia of pulmonary artery
Anomalies of great veins
Absence or hypoplasia of umbilical artery
Anomalies of peripheral vascular system
Anomalies of circulatory system
Choanal atresia
Anomalies of nose
Web of larynx
Anomalies of larynx, trachea and bronchus
Congenital cystic lung
Agenesis of lung
Anomalies of lung
Qeft palate
Qeft lip
Qeft palate with cleft lip
Anomalies of tongue
Pyloric stenosis
Trachea-oesophageal fistula
Oesophageal atresia and stenosis
Anomalies of upper alimentary tract
Unspecified anomalies of upper alimentary tract
Meckel's diverticulum
Anomalies of intestinal fixation
Hirschsprung's disease
Atresia and stenosis of rectum and anal canal
Anomalies of intestine
Atresia of biliary ducts
Anomalies of gallbladder, bile ducts, and liver
Anomalies of pancreas
Anomalies of digestive system
Unspecified anomaly of digestive system
Indeterminate sex
Undescended testicle
Hypospadias

517

TABLE 7 (continued}

ICDA

code
752.3
152.5
752.6
752.7
752.8
752.9
753.0
753.1
753.2
753.3
753.4
153.5
753.6
753.8
753.9
754.0
755.0
155.1
755.2
155.3
755.5
155.6
155.1
155.8
155.9
756.0
756.1
756.2
756.3
156.4
156.5
756.6
756.7
756.8
756.9
757.0
757.1
757.2
757.3
757.9
758.0
758.1
758.2
758.8
158.9
759.0
759.2
159.8
159.9
Total

Number of
persons
affected
13

I
32
12
50
5
66
35
123
79
46
13
257
21
10
2 205
279
252
182
62

86
1 076

238

38
2
147
11
63

2
125
8
3
16
151
51
4

123
40
4
8
4
3

43
18
I
9
14

39
14

ICDA title

Epispadias
Anomaly of ovary, fallopian tube or uterus
Anomalies of vagina and external female genitalia
Pseudohermaphroditism
Anomalies of genital organs
Unspecified anomalies of genital organs
Renal agenesis
Cystic kidney disease
Obstructive defects of urinary tract
Anomalies of kidney
Anomalies of ureter
Ex trophy of urinary bladder
Atresia and stenosis of urethra and bladder neckAnomalies of bladder and urethra
Unspecified anomalies of urinary system
Talipes cavus
Polydactyly
Syndactyly
Reduction deformity of upper limb
Reduction deformity of lower limb
Anomaly of upper limb (including shoulder girdle)
Congenital dislocation of hip
Anomaly of lower limb (including pelvic girdle)
Generalized flexion contracture of limb joints
Unspecified anomaly of unspecified limb
Anomalies of skull and face bones
Anomalies of lumbosacral joint
Anomalies of spine
Cervical rib
Anomalies of ribs and sternum
Chondrodystrophy
Osteogenesis imperfecta
Generalized anomalies of skeleton
Anomalies of muscle, tendon and fascia·
Anomalies of musculoskeletal system
Hereditary oedema of legs
Pigmented nevus
Anomalies of skinAnomalies of hairAnomaly of skin, hair, or nails
Anomalies of spleen
Anomaly of adrenal glandAnomalies of thyroid gland
Congenital anomalies
Unspecified congenital anomaly
Situs inversus
Forms of monster
Specified syndromes
Unspecified multiple congenital anomalies

15 728Source: Based on the British Columbia Survey (!:76).
Note: See notes to table 1.

518

TTABLE 8. OTHER MULTIFACTORIAL DISORDERS BY !CDA CODE

ICDA
code

242.0
250.0
269.0
281.0
295.2
295.8
295.9
310.9
311.9
312.9
313.9
314.9
315.9
345.0
345.1
345.2
345.3
345.4
345.5
345.9
370.0
370.1
370.3
370.9
373.0
373.9
493.0
722.1

Number of
persons
affecred

2
401
105
1
2
51
6
542
327
311
126
129
321
202
292
1
19
3
29
842
40
2
2
9
3 517
6
203
260
Total 7 751

JCDA title

Toxic diffuse goitre
Diabetes mellitus with mention of acidosis or coma
Sprue and steatorrhea
Pernicious anaemia
Schizophrenia, catatonic type
Schizophrenia
Unspecified type of schizophrenia
Borderline idiopathic mental retardation
Mild idiopathic mental retardation
Moderate idiopathic mental retardation
Severe idiopathic mental retardation
Profound idiopathic mental retardation
Unspecified idiopathic mental retardation
Generalized non-convulsive epilepsy
Generalized convulsive epilepsy
Status epilepticus
Partial temporal lobe or psychomotor type epilepsy
Partial motor type epilepsy
Unspecified partial type epilepsy
Unspecified type of epilepsy
MyopiaHyperopia
Astigmatism
Unspecified refractive errors
Strabismus, convergent
Unspecified strabismus
AsthmaJuvenile osteochondrosis of hip

Source: Based on the British Columbia Survey (576).
Note: See notes to table 1.

TABLE 9. ESTIMATES OF THE FREQUENCY OF GENETIC DISORDERS IN A BIRTH COHORT
Cases per 100 live-born
British Columbia Survey(576)

Stevenson

UNSCEAR

Disease category(566)(586, 587)

MinimalAdjusted

Dominant
Recessive
X-linked
Chromosomal
Congenital malformations
Other multifactorial
Unknown

3.32a
0.21
0.04
b
1.41
1.48

0.95
0.21
0.04
0.42c
2.50
1.50

0.06
0.09
0.03
0.16
3.58 }
1.58(0.60)1

0.08
0.11
0.04
o.2Qd
4.28
4.73
(2.70)1'

5.50

9.44

Totald

}

b

b

6.46

5.62

}

UNSCEARpresent
estimate

i.oe
0.1

f

0.4

} 9.0
10.sh

0 lncludin1 both trivial and serious anomalies.

bNo information.
clncluding Down's syndrome (0, I 5), other autosomal trisomies (0.05), Klinefelter's syndrome(0. I 7), Turner's syndrome (0,03) and Crldu·chat syndrome (0.02) but excludes XXX females (0.12)
and individuals with translocations (0,50).
d All but about 3 per cent are due to Down's syndrome.
elncluding X-llnked disorden.
/included in the dominant category.
IFrequency of unknown diseases is not included in the tout.
hFor the evaluation of radiation hazards the figures used are slightly different; see table 50 andparagraphs 12 and 19.

519

TABLE 10. ESTIMATES OF MUTATION RATES FOR HUMAN GENES

Trait

Population examined

Achondroplasia

Denmark
Northern Ireland, UK
Munster, Germany, Fed. Rep. of

Aniridia

Denmark
Michigan, USA

Dystrophia myotonica

Northern lreland, UK
Switzerland

Retinoblastoma

England, UK; Michigan, USA
Switzerland; Germany, Fed. Rep. of
Hungary
Netherlands
Japan
FranceMutation rate

Numberof mutants
per 10 6
gametesA. AUTOSOMAL MUTATIONS

Acrocephalosyndactyly(Apert's syndrome)

England, UK
Munster, Germany, Fed. Rep. of

Osteogenesis imperfecta

SwedenMunster, Germany, Fed. Rep. of

Tuberous sclerosis(epiloia)

I 10" 5
1.3 10-s
6-9 10-•

10
13
6-9

2.9-5 10-•
2.6 10·•
8 10-•
1.1 10" 5

2.9-5
2.6
8
11

6-7 10-•6-7

6 10-•
1.23 10-•8 10·•
5 10·•

6
12.3
8

3 10-•

3
4

4 10-'

5

Oxford, England, UK
China

0.7-1.3 10-•
1.0 10-•I.OS 10-•
6 10-•

10.S
6

Ncurofibromatosis

Michigan, USA
Moscow, USSR

1 10-•
4.4-4.9 10-s

100
44-49

Polyposis intestini

Michigan, USA

1.3 10-•Marfan's syndrome

Northern Ireland, UK

Polycystic diseaseof the kidneys

7-13
10

13

Denmark

4.2-5.8 10-•
6.5-12 10-•65-120Diaphyseal aclasis(multiple exostoses)

Munster, Germany, Fed. Rep. of

6.3-9. l 10-•

6.3-9.1

von Rippel-Lindau syndrome

Germany, Fed. Rep. of

4.2-5.8

1.8 10-,0.18

B. SEX-LINKED RECESSIVE MUTATIONS
Haemophilia

Denmark
Switzerland
Munster, Germany, Fed. Rep. of

3.2 10-•2.2 10-•2.3 10-s

32
22
23

Haemophilia A

Hamburg, Germa:iy, Fed. Rep. of
Finland57
32

Haemophilia B

Hamburg, Germany, Fed. Rep. of
Finland5.7 10·•3.2 10-•
3 10-•
2 10-•

Duchenne-typemuscular dystrophy

Utah, USA
Northumberland and Durham, UK
Siidbaden, Germany, Fed. Rep. ofNorthern Ireland, UK
Leeds, England, UK
Wisconsin, USA
Bern, Switzerland
Fukuoko, Japan
North-east England, UK
Warsaw, Poland

9.5 10-•4.3 10-•
4.8 10·•6.0 10-s
4.7 10-•9.2 10-•
7.3 10-•
6.5 10·•10.S 10-•
4.6 10-•

Incontinenta pigmenti(Bloch-Sulzberger)

Munster, Germany. Fed. Rep. of

0.6-2.0 10-•

Oculofaciodigital
syndrome (OFD)

Milnstcr, Germany, Fed. Rep. of

5 10-•

Source: Reference 599.

520

3
2

95
43
48
60
47
92
73

65
105
46
6-20
5

------------ - - - - - - - - - - - - - - - - - - - - - - -TABLE 11. lNCIDENCE OF CHROMOSOMAL ABNORMALITIES IN NEW-BORN INFANTS
Autosomal abnormalities

Sex-chromosome anomalies
Males
Number of infants
47,

47,

XYY XXY

Females\,s"

Survey ce11tre

Male

FemaleTotal

Hamilton, Canada
London, Canada
Winnipeg, Canada
Arhus, Denmark
Edinburgh, UK
Boston, USA
New Haven, USA
Moscow, USSR

493
I 066
7 176
5 751
7 849
9 048
2 176
1 303

437
I 015
6 763
5 387
3 831
2 177
1 197

2
93oe
2 081
4
13 9394
11 1483
10
11 680
9 048
3
4 353
3
2 50od

1
I
6
6
9
6
4
1

6

34 87220 80755 679

34

30

Total

29

C

.

Numerical {trlsomics)

-s"

,s"

.

I:!

45,

47,

X

XXX

5

2
9
5
8

7
5

+D

2
3
2

+E

3
I
2

3

1

2

C

I:!

20

122 (0.22%)

7

3

7

+G

C

2
14
16
17
7
3
4

1

63

3

76 (0.14%)

Structural (euploid)
Robertsonian

D/D
I
12
15
6
5
2

2
41

D/G

Structural (aneuploid)
Reciprocal
Q
Tand
ReelSuper- I:!
inserb1ver- Robert-pro- b1ver- Dele- 1u'1ner.. ,stionalsions sonian cal T sions lions ary
C

.

I
2
4
2
1
1

11
15
10
5
3

11

48

I
I
2

I

2
6c

6b

1

2

2

4

5

11

2

3

107 (0.19%)

7

2

7

4

Freque11cy
Total {%)

5
10
65
93
78
44
22
19

0.54
0.48
0.47
0.83
0.67
0.49
0.51
0.76

336

IQ.60

30 (0.05%)

Sources: References 38,212, 2SO, 289,363.
aMost of these are mosaics.
bun balanced Y-autosome translocations.
cSupernumerary small metacentric chromosomes including two mosaics.
dThese 2500 infants urc II random sample of 10 237 infants born during the study period (1969-1972) and included IS lwin-polrs, 29 new-born infants with various congenital malformations, s stillborn and 13 delivered prematurely (38).
elncluding I twin-pair and I triplet,TABLE 12. CHROMOSOMAL ANOMALIES IN SPONTANEOUS ABORTIONSa

Karyotype

CanadaCa"

UnitedKingdomCreasyFranceet al.
Boue

Monosomy X

1268

Autosomal

8

31

18
l (A)

l (G)

Trisomy A
B
C
D
E
F
G

l
1
2
6
9
l
6

Double trisomy

1

Triploidy XXX

6
2
1

XXY
XYY

11

2
17
21
60
3
29
18
18
1

Not karyotyped
Mosaics 46/49

xxxx
XXYY

Not karyotyped

2
3
7

6
11
12

6

16

2

10
2

57
92
7
27
2

5

2

Japan
Kaji
et al.

1

12

39

2
1
7
11
20

1
2
11
16
27

9

6

2
1
1
14
2

2

2
7

3

4

3
2
2
1
3

Denmark
Therkelsson

2

s

3
3
l

8

1
l

4

3

s

9
5

2

1

2

3
I

7
5

33
20

2sc

43

3

s

17

50

287

919

35

94

63

2
l

6
4

2

5d

10

20

82

138

2
l
1

2

Other anomalies
Total

12
6
86
109
172
7
87

5

USSR
Kuliev2bHypertriploids
Tetraploidy

140

Germany,
Fed. Rep.
of
UnitedUnited States
Degen•
Kingdom
hardt
Dhadial Arakaki Szulma11 Thiede

1524

Sources: References 45, 403.
a Results from 11 series in 9 different countries.
bone had an extra C-group chromosome (70, XXY, +C), while the other was tetrasomic for an £-group chromosome (70, XXY, +E).
clncluding 1O structural anomalies, the remainder being mixoploids or other anomalies.
drncluding 3 structural anomalies and 2 mosaics.

TABLE 13. COMPARISON OF THE FREQUENCIES OF INCIDENCE OF
DIFFERENT KINDS
OF CHROMOSOMAL ANOMALIES INSPONTANEOUS ABORTIONSKind of anomaly

Boue and Boue (45)

Orher a.Jthors

Frequency
Number ('fo)

Frequency
Number (%)

43

15.2
0.1
52.1
1.7
20.1
6.0
4.7

199
2
393
8
101
36
69

24.6
0.2
48.6
1.0
12.5
4.S
8.5

919

100

808

100

Monosomy·X
Monosomy (autosomal)
TrisomyDouble trisomy
Triploidy
Tetraploidy
Others

140
l
479
16
185

Total

SS

Source: Compiled from the data in table 12.

522

~-

'

TABLE 14. RELATIVE FREQUENCYAUTOSOMALTYPESOFSPONTANEOUS ABORTIONSOF DIFFERENTIN
TRISOMIESGroup of 392
trisomiesa
Extraautosome
involved

Number

A
B
C
D
E
F
G

20
6
50
73
160
7
76
392

Total

Group o/479
trisomiesb

Frequency

Frequency
Number (%)

(%)

5.1
12.8
18.6
40.8
1.8
19.4

12
6
86
109
172
7
87

2.5
1.3
17.9
22.8
35.9
1.5
18.2

100

479

100

1.5

aThe series of table 12, that of Boue excepted.
b References 45, 2 S 3.

TABLE 15. MATERNALAGE
INRELATIONTO
DIFFERENT TYPES OF CHRmmSOMAL ANOMALIES
Number of cases
observed

Karyotype

Monosomy X
TrisomyTriploidy
Tetraploidy
Translocation
NormalMean maternalage (years)

134
448
167
53
26
509

27.57 ± 0.88
31.25 ± 0.60
27.38 ± 0.79
26.79 ± 1.40
26.96 ± 2.32
27.48 ± 0.45

13
7
72
92
157
8
78
14

29.62 ± 2.18
33.43 t 7.13
30.93 ± 1.67
32.49 ± 1.33
29.58 ± 0.88
30.13 ± 5.32
33.17 ± 1.40
35.00 ± 6.09

Autosomal trisomy
47,A+
47,B+
47,C+
47,D+
47,E+
47,F+
47,G+
48

Source: Reference 45.

TABLE 16. X·RAY-INDUCED DOMINANT LETHALS IN MALE MICE AT DIFFERENT POST-IRRADIATION INTERVALS
Ma.ting ratio, 1 male to 3 females per week; 30 irradiated and 10 control males
Irradiation level 200 R

WeekofmatingGroup

Control
Irradiated

1
2
3
4
5
6
7
8
9

Numberoffemalespaired

31
15
16
14
14
15
13

14
13
5

ProNumber portion
of
of
total
femalespregnant (%)

28
15
15
12
12
14
8
1
11
5

90
100
94
86
86
93
62
7
85
100

Corporalutea

Implants

259
134
118
110
102
121
70
8
126
54

239
111
103
83
71

92
47
2
93
39

Averagenumberof liveembryosperpregnantLiveembryos female

221
89
78
55
57
69
36
2
84
34

7.9
5.9
5.2
4.6
4.8
4.9
4.5
2.0
7.6
6.8

PreImp/anration
loss

Postimplan•
ration
loss

(%)

(%)

Aa

sb

7.7
17.2
12.7
24.5
30.4
24.0
32.9
75.0
26.2
27.8

7.5
19.8
24.3
33.7
19.7
25.0
23.4

25
34
42
39
38
43

13

18
28
13
19
17

9.7
12.8

4
14

6

Induced dominantlethality (%)

2

Source: Reference 133.
a Index A for dominant lethality is based on the number of live embryos per pregnant female.

blndex B for dominant lethality is based on the number of live embryos per total implantation.

523

TABLE 17. FREQUENCY OF DOMINANT LETHALITY IN LITTERS CONCENED IN THE FIRST AND THIRD WEEKS AFTERX IRRADIATION OF FEMALE MICE

Numberoffemales

Dose
{rad}

0

Corpora
/urea

Pre-

Implants

Liveembryos
Total
implants

Inducedpostimplanrationb
loss(%)

Corpora
lutea

Toral
implants

Live

per

per

embryos

femalefemaleimplanrationa
loss{%)

22

222

201

194

10.1

9.14

9.5

0.965

18
16
15
14

199
154
164
165

162
125
138
141

151
110
121
116

11.1
9.6
10.9
11.8

9.00
7.81
9.20
10.07

18.6
18.8
15.9
14.5

0.932
0.880
0.877
0.823

9.1
14.7

15
19
18
18

151
201
181
187

121
154
94
107

116
122
67
67

10.1
10.6
10.1
10.4

8.07
8.11
5.22
5.94

19.9
23.4
48.1
42.8

0.959
0.792
0.713
0.626

0.7
17.9
26.1
35.1

First week
100
200
300
400

3.4

8.8

Third week
100
200
300
400

Source: Reference S 13.
acaJculated as-Number of implants/Number of corpora Jutea) X 100b· Number of embryos/Number of implants in the irradiation series
calculated as ( I ) x I oo
Number of embryos/Number of implants in the controls

TABLE 18. DOMINANT LETHALITY AFI'ER FISSION-NEUTRON IRRADIATION OF FEMALESFirst-week mating
Induced lethality
Dose
(rad)

Pregnant
females

Corporalutea
per female

0

16
15
18
16
16

10.0
12.7
11.3
9.6
9.9

50
100
150
200

Implants
per femaleLiveembryos
per female

Live embryos
Corpora lutea

Live embryos
Tora/ implants

Postimplantation

Total

9.6
11.4
9.7
8.7
7.8

8.6
10.2
8.4
6.0
5.0

0.856
0.805
0.749
0.627
0.503

0.890
0.895
0.869
0.691
0.645

-0.6
2.4
22.4
27.5

6.0
12.5
26.8
41.2

Source: Reference 51 3.

TABLE 19. FERTILITY AND DOMINANT LETHALITY IN FEMALE GUINEA-PIGS AND GOLDEN HAMSTERS TREATED WITH
XRAYS

Dose
(rad)

Total
number Numberofnotfemales pregnant

Corporalutea

Implants

LiveembryosperfemaleDeadAb·normalCorpora Im·
plants
/utea
per
per
femalefemale

2
3
7
9

0
0
1
2

0
0
0
0

3.6
4.0
4.4
4.8

3.1
3.4
3.3
2.9

3.0
3.3
2.9
2.3

1
0
1
3

0
0
3
0

1
0
0
0

3.7
3.5

3.4
3.3
3.5
2.8

3.3
3.3
3.3
2.6

65

4
1

72
58

6

1
0
1

9

I

0
0
l
0

3.3
3.1
3.4
3.2

3.1
3.1
3.1
2.8

Live
embryos

Moles

GUINEA-PIG
Mating at first oestrus

62
85
69
60

60
82
61
49

0
100
200
400

20
25
21
26

5

101
93
94

0
100
200
400

20
21
20
20

0
1
0
0

74
69
78
,73

68
65
69

66
65
65

SS

52

0
100
200
400

23
21
24
21

2
0
1
0

79
74
92
77

70
66
79

0
0
0

72

Second oestrus

3.9
3.7

Three months

68

65

3.8
3.5
4.0
3.7

GOLDEN HAMSTER
Mating at first oestrus

0
100
200
400

524

11
23
21
23

l
2
2
4

178
270
245
278

165
245
192
180

138
196
147
112

19

38
36
65

3
6
3
1

s
s

6
2

12.7
12.9
12.9
14.6

11.8
11. 7
10.1

9.5

9.8
9.3
7.7
5.9

Dose
(rad)

Total
number Numberofnotfemales pregnant

Corporalutea

Implants

Liveembryos

Moles

Dead

Ab·
normalCorporalutea
perfemaleIm.
plantsperfemaleLive
embryosperfemaleGOLDEN HA11STER (continued)
Second oestrus
0
100
200
400

20
20
20

2
2
2
3

143
226
231
280

133
204
190
211

104
161
135
142

0
100
200
400

20
25
22
31

1
5
2
12

230
252
254
275

207
223
214
219

171
175
178
175

0
400

12
13

2
10

126
34

111
28

13

21
32
40
47

6
5
12
11

2
6
3
11

13.0
12.6
12.8
16.5

12.0
11.3
10.6
12.4

9.5
8.9
7.5
8.4

34
40
27
33

1

5
4
2

1
3
5
9

12.1
12.6
12.7
14.5

9.5
11.2
10.7
11.5

8.5
8.8
8.9
9.2

14
5

3
2

3
1

12.6
11.3

11.0
8.5

9.1
6.7

One monthThree months

91
20

Source: Reference 121.

TABLE 20. FREQUENCY OF DOJl.fiNANT LETHALS IN FEMALE GUINEA-PIGS AND GOLDEN HAMSTERS TREATED WITH
X RAYS
Fraction of dominant lethals (%)
Dose
(rad)

Implants

Live embryos

Live embryos

Corpora lutea

Implants

Corpora lutea

Preimp/antation

Post•
imp/antation

Total

2.2
13.2
25.8

0.4
8.6
15.6

2.6 ± 6.9
21.3 ± 7.2
37.5 ± 7.1

3.7
18.0

2.9
2.6

6.6 ± 6.0
20.1 ± 6.7

1.8
0.3

3.1
8.1

4.9 ± 7.2
8.5 ± 7.6

2.1
15.5
30.1

4.4
8.5
25.6

6.4 ± 5.1
22.6 ± 5.1
48.1 :!: 4.3

2.9
11.6
18.2

9.1
14.9

2.1 ± 6.5
19.6 ± 9.1
30.3 ± 6.5

1.7
6.4
11.6

8.2
2.7
6.5

9.8 ± 7.8
8.9±8.1
17.3±7.8

6.5

13.0

18.6

GUINEA-PIG
Mating at first oestrus
0
100
200
400

0.861 ± 0.041
0.842 ± 0.036
0.742 ± 0.045
0.638 ± 0.050

0.986 :t 0.022
0.965 ± 0.020
0.884 ± 0.039
0.817 ± 0.050

0
100
200
400

0.919 ± 0.032
0.942 ± 0.028
0.885 ± 0.036
0.753 ± 0.050

0.971 ± 0.021
1.00 ± 0.030
0.942 ± 0.028
0.946 ± 0.031

0
100
200
400

0.886 ± 0.036
0.892 ± 0.036
0.859 ± 0.036
0.883 ± 0.037

0.929 ± 0.031
0.985 ± 0.015
0.911 ± 0.032
0.853 ± 0.043

0.833 ± 0.044
0.812 ± 0.039
0.656 ± 0.049
0.521 ± 0.052
Second oestrus

0.892 ± 0.036
0.942 ± 0.028
0.833 ± 0.042
0.712 ± 0.053
Three months

0.823 ± 0.043
0.878 ± 0.038
0.783 ± 0.043
0.753 ± 0.049
GOLDEN HAMSTER
Mating at Just oestrus

0
100
200
400

0.927 ± 0.020
0.907 ± 0.018
0.784 ± 0.048
0.648 ± 0.033

0.836 ± 0.029
0.800 ± 0.026
0.766 ± 0.031
0.622 ± 0.036

0. 775 ± 0.031
0. 726 ± 0.027
Q.600 ± 0.031
0.403 ± 0.029
Second oestrus

0
100
200
400

0.930 ± 0.021
0.903 ± 0.020
0.823 ± 0.047
0.754 ± 0.033

0.782 ± 0.036
0.789 ± 0.028
0. 711 ± 0.055
0.673 ± 0.047

0
100
200
400

0.900 ± 0.031
0.885 ± 0.020
0.843 ± 0.039
0.796 ± 0.041

0.855 ± 0.025
o. 785 ± 0.041
0.832 ± 0.026
0.799 ± 0.027

0400

0.881 ± 0.029
0.824 ± 0.065

0.820 ± 0.036
0.714 ± 0.085

0. 727 ± 0.03 7
0.712 ± 0.030
0.584 ± 0.059
0.507 ± 0.039
One month

0. 770 ± 0.051
0.694 ± 0.038
0.701 ± 0.042
0.636 ± 0.043
Three months

o. 722 ± 0.040
0.588 ± 0.022

Source: Reference 121.

525

TABLE 21. DOSE-EFFECT DATA FOR THE INDUCTION OF
LOCATIONS IN MOUSE SPERMATOGONIA

RECIPROCAL

TRANS-

Single exposures at a high exposure rate

Exposure
(R)

0
50
100
150
200
300
400
500
600
700
800
1200

Time intervala(days)

Number ofcells scored

Number ofanimals

600
600
600
600
600
600
800
800
800
800
900
600

6
6
6
6
6
6
8
8
8
8
9
6

27
28
32
35
48
54
62
68
91
121
182

Proportion ofabnormal cells

Proportion oftranslocations

(%)

(%)

0.0
0.7 ± 0.3
1.5 ± 0.5
2.5 ± 0.6
4.0 ± 0.8
7.8 ± 1.1
12.1 ± 1.2
14.8 ± 1.4
16.0 ± 1.4
11.6 ± 1.2
8.7 ± 1.0
3.8 ± 0.8o.o
0.7 ± 0.3
1.5 ± 0.5
2.5 ± 0.6
4.3 ± 0.8
7.8 ± 1.1
13.5 ± 1.3
17.0 ± 1.5
19.5 ± 1.6
13.5 ± 1.3
10.2 ± 1.1
14.5 ± 0.9

Source: Reference 419.
aBetween irradiation and sampling.

TABLE 22. YIELD OF RECIPROCAL TRANSLOCATIONSIN SPERMATOCYTES FOLLOWING X IRRADlA TION
OF MOUSE SPERMATOGONIA.
Repeated 400-R fractions separated by 8-week intervals

Total exposure
(R)

0
400
800
I 200
1 600

2 OOO

2400
2 800

Number of cells
scoredYield oftranslocations
(%)

1 OOO

0

2 150
1 450
900
600
600
600
600

13.8 ± 0.8
19.7 ± 1.2
31.1 ± 1.9
47.8 ± 2.8
60.2 ± 3.2
64.5 ± 3.3
80.7 ± 3.7

Source: Reference 420.

TABLE 23. FREQUENCY OF CYTOLOGICALLY SCORED
AND GENETICALLY RECOVERED RECIPROCAL
TRANSLOCATIONS FOLLOWING X IRRADIATION
OF MOUSE SPERMA TOGO NIA.

Minimum
Frequency of Number frequency of
Ratio
translocationtrans/ocations of F 1
hettrozygotesb recovered/
Exposure scoreda
males
scored{%)(R){%)
rested

0
150
300
600
1 200

0.0
2.5 (2.5)
7.8 (5.7)
17.2 (16.2)
4.5 (3.8)

2 633
993
1 018
1 075
1 038

o.o
0.6
0.88
1.90
0.50

0.25
0.12
0.11
0.13

Source: Reference 56.
avalues in parentheses are proportions of cells with asingle translocation.
bCytologically confirmed semisteriles.

526

•
I

-r
I

TABLE 24. FREQUENCY OF CHROMATID ABERRATIONSIN MI OOCYTES AT VARIOUS TIMES AFTER AN
ACUTE X-RAY EXPOSURE OF 200 R

Interval(days}

Number ofcells scored

I

100
100

3

125

5
7

100
125
100

14
21

Frequency ofdeletions

Frequency ofinterchanges

('lo}

{%)

1.0 ± 1.0
4.0 ± 2.0
4.8 ± 2.0
6.0 ± 2.5
10.4 ± 2.9
6.0 ± 2.5

1.0 ± 1.0
1.0 ± 1.0
0.8 ± 0.8
6.0 ± 2.5
12.8 ± 3.2
6.0 ± 2.5

Source: Reference S8.

TABLE 25. FREQUENCY OF CHROMATID ABERRATIONSIN MI OOCYTES 14 DAYS AFTER VARIOUS X-RAY
EXPOSURES

Exposure(R)

Number ofcells scored

0
50
100
200
300
400

200
150
150
125
150
100

Frequency ofdeletions

Frequency ofinterchanges

{%)

(%)

1.3 ± 0.9
4.0 ± 1.6
10.4 ± 2.9
16.0 ± 3.3
37.0 ± 6.1

1.3 ± 0.9
4. 7 ± 1.8
12.8 ± 3.2
23.3 ± 3.9
56.0 ± 1.5

Source: Reference S8.

TABLE 26. YIELD OF TRANSLOCATIONS IN SPERMATOCYTES FOLLOWING SPERMATOGONIAL IRRADIATION IN
DIFFERENT MAMMALIAN SPECIES(a) Data of Lyon and Cox (309).

RabbitDose orexposure(rad or R)
0
100
200
300
400

SOO
600

Animals Cells
3
3
3
4
3
3
3

l 117
1 285
l 008
1 923
967
l 248
l 055

Guinea-pig

Golden hamster

Yield oftranslocations (%)

Yield oftranslocations ('fo)

Yield oftranslocations (%)

Definiteandpossible Definite

Definiteandpossible Definite

Definiteandpossible Definite

0.2
1.8
3.1
4.9
1.0
1.1

0.1
1.6
2.6
4.2
0.8
0.6
0.3

0.5

Animals Cells

s
5
6

s
s
s

5

1 013
1 408
1 633
1 518
1 24S
1 307
I 051

0.1
1.2
1.7
2.0
1.4
1.6
1.0

0.2
1.6
2.3
2.2

1.8
2.1
1.6

Animals Cells

s
s
s

4
4
4

5

1 406
1 273
1 504
1 873
l 127
968
l 268

0.3
1.1
2.3
3.6
1.3

o.s

0.1
0.7
1.7
3.0
1.2
0.3

0.8

0.5

( b) Data of Brewen and Preston (54).

Do.re orexposure(rad or R)
0

so

100
200
300

Yield of translocations (%)
Guinea-pig

Chinese hamster

0
1.4 :t 0.6
2.7 :1: 0.9
6.1 :1: 1.1
8.0 :1: 2.001.0 :1: 0.6
2.0 :1: 0.8
4.3 ± 1.2

527

TABLE 27. TRANSLOCA TION YIELDS AFTER VARIOUS X-RAY DOSES TO MONKEY
SPERMATOGONIA

Monkey No.

Dose (rad)Interval between
irradiation andexaminatfon(months)

Translocation yield
Number ofcells analysed

Definite andpossible

Definite

English monkeys (ref. 314)a
O+S
300+ SS + 200
200+ S
100
100
100
100

lL
R
2L
R
3L
R
4L
R

124
186
517
82
391
253
248
146

8
8
11

9
9
6.5
9
6.5

0.8
2.7
1.0
4.9
4.1
2.0
0.4
1.4

0.8
2.7
0.8
4.9
1.8
1.2
0.4
1.4

0.0
0.0
0.0
1.6
0.3
0.0

0.0
0.0
0.0

1.4
1.5
1.0

0.8
1.2
1.0

Dutch monkeys (ref. 314)
300
300
200
200
100
0

l
2
3
4
5
Control

291
1
36
495
777
30

8
8
8
8
8

1.2

0.3

o.o

Combined results
1 815
1 130
478

100
200
300

Dutch monkeys (ref. 70)
1-3

0

4
5
6
7
8
9

100
100
200
200
300
300

100 + 100 +
300
1 700
350
850
550
400
175

8
8
8
8
8
8

0.0
0.06
1.1

2.0
1.1

0.0
1.1

Combined results
0.2
1.6
0.3

2 050
1400
575

100
200
300

aEnglish monkeys No. 1 and No. 2 were unilaterally irradiated; all others were bilaterally
irradiated. L = left testis; R right testis; S shielded.

==

TABLE 28. YIELD OF RECIPROCAL TRANSLOCATIONS IN SPERMATOCYTES FOLLOWINGSPERMATOGONIAL IRRADIATION IN MARMOSET AND MAN COMPARED WITH
MOUSE DATA

Marmoset

Man
Dose Number of(rad) individuals Cells
0
25
50
78
100
200
300
600

Yield(%)

2

200

0

2

371

4.0: 1.0

2300

7.0 ± 1.3

2180

6.1 ± 1.8

Source: References 57,419.

528

Number ofanimals Cells

Yield(%)

2
2
2

600
600
600

0
2.8 ± 0.7
3.3 ± 0.8

2
1
2

600
200
600

7.8 ± 1.1
7 .5 : 1.9
7.0 ± 1.1

Mouse
Number ofanimals
Cells

Yield(%)

6

600

0

6

600

0.7 ± 0.3

6
6
6
8

600
600
600
800

1.5 ± 0.5
4.3 ± 0.8
7.8 ± 1.1
19.5 ± 1.6

·•T

TABLE 29. YIELDOF
TRANSLOCATIONSINSPERMATOCYTESFOLLOWING
SPERMATOGONIAL IRRADIATION WITH SINGLE OR FRACTIONATED EXPOSURES IN
THE GOLDEN HAMSTER AND GUINEA-PIG

Golden hamster

Guinea-pig

Yield(%)
Total
dose Radiation(rad) procedure

oa

ob

oc

od
200
400
400
400
400
300
600
600
600
600

singlesingle2 X 200
2 X 200
8 X 50singlesingle2 X 300
2 X 300
12 X 50

Yield(%)

Interval Number Number Definite
Number Number Definite
between of ani- of cells andof ani- of cells andfractions mals
scored possible Definite mals
scored possible Definite

5
5
5

1 013
1118
906

0.2
0.2
0.1

0.1
0.1
0.0

6

7

1 633
1 245
1 607
I 350
1 800

2.3
1.8
4.5
3.5
2.7

1.7
1.4
3.9
3.1
1.9

5

1 051

1.6

1.0

5
5

2.5

4
4
3

5
24 h
8 weeks
1 week6524 h
8 weeks
1 week

6

1446

3.1

5
4
3
4

5
4
4
4

1 406
778
604
765
1 504
1 127
881
1 039

4.8
3.2

0.1
0.0
0.0
0.0
1.7
1.2
4.1
2.5

1 873
1 268
763
1 042
1 193

3.6
0.8
7.6
3.1
5.0

3.0
0.5
7.0
2.0
4.1

0.3
0.1
0.2
0.3
2.3

1.3

Source: Reference 308.
acontrol for single-dose experiments.
bcontrol for 2 X 200 rad (24 h), 2 X 200 rad (8 week) experiments in golden hamster and for2 X 200 rad (24 h) and 2 X 300 rad (24 h) experiments in guinea-pig.
ccontrol for 12 X SO rad (1 week), 8 X SO rad (1 week) experiments in golden hamster for
12 X SO rad (I week) experiments in guinea-pig.
dcontrol for 2 X 300 rad (8 week) and 2 X 200 rad (8 week) experiments in guinea-pig.

TABLE 30. EMBRYONIC MORTALITY lN PROGENY OF MALES WITH OR WITHOUTDETECTABLE TYPES OF CHROMOSOME ABERRATION

Category ofmale

Number of
pregnant
femalesWith 1 RT 0
With 2RT
With insertion
Normal

120
8
2
849

Embryonic
mortality
Total
Live
Dead
implants embryos implants {%)
806

58
14
6 743

369
18
7
6 161

437
40

7
582

54.2
69.0
50.0
8.63

Implants
per female

Live embryos
per female

6.72 ± 0.20 3.08 ± 0.15
7.25
2.25
7.00
3.50
7.94
7.26

Source: Reference S20.
aRT = Reciprocal translocation.

TABLE 31. EMBRYONIC MORTALITY IN PROGENY OF MALES WITH RECIPROCAL TRANSLOCATIONS (RT)

Total
Male
Number number of
Number of RT
femalesNumberCorpora
Live
Dead
not
pregnant lutea
Implants embryos embryos Moles

Embryonic
mortality(%)

Implants
per female

Live embryos
per female

Golden hamster
1
2
3
4
6
8
12
13

2
1

I
1
1
1
1
1
Total (lRT)aControlb

5

85

11
39
36
31
47
69
14
40

1
3
3
4
8
4
0
2

33
57
37
43
38
33
19
43

570

276

24

270

9
8
9
8
10
3
9

0
0
0
0
0
0
0
0

71
125
104
107
108
112
34
105

45
99
76
78
93
106
33

56

0

695

43

542

478

393

12

9.0
11.0

2.2
4.3
4.5
3.4

75.6
60.6
52.6
60.3
49.5
34.9
57.6
52.9

8.7
11.6
10.6
11.0
9.4

Average

52.6

10.3

4.87

53

13.6

11.1

9.14

9.5

5.9
6.9
4.7
4.4

529

TABLE 31 (continued)

Total
Male
Number number of
Number of RT
femalesNumbernor
pregnant

Embryonic
Deadmortality
CorporaLive
Implants embryos embryos Moles (%)
lutea

Implants
per female

Live embryos
per female

Guinea-pig
2
4
6
9
11
15
17

l
l
l

5

I
1
l
1

10
10
8
12
6
4
7

l
l
l
3
0
4

22
42
34
52
13
21
9

Total (lRT)a

57

15

193

Controlb

7
25
15
30
9
1

0
0
0
1
0
1
0

141

92

2

-.

146

40

14
31
30
37
8
13
8

130

5

50.0
19.4
50.0
18.9
37.5
30.8
87.5

2.8
3.4
4.3
3.4
2.7
3.3
2.7

Average

42.0

3.2

1.9

4

3.1

3.3

3.2

7
6
15
6
3
3
7
47

0

126

Source: Reference 123.
al RT = one reciprocal transloca tion.
bcontrol data taken from Cox and Lyon (121 ).

TABLE 32. FREQUENCY OF INDUCED X-CHROMOSOME ANOMALIES FROMIRRADIATION OF FEMALE GERM-CELL STAGES IN MICE

Cell stage
sampledInterval,
irradiation
to mating(days)

Frequen~y of OXP
Exposure Number
Induceda
Total
Exposure rateof
(10-s R-1)
R
(R/min) females (%)

Preleptotene
through
diplotene

>42

221

l-42c
1-42
>42c
1-42

400
50
50
400

Control
Dictyate
Dictyate
Dictyate
Dictyate

Control

66-78
66-78
66-78
0.6

15 006
12 256
10 17210 900

391

0

0-1e
8-421
>42K

400
400
400

0.006
0.006
0.006

0

Control
Dictyate
Dictyate

1 221

0Control
Dictyate
Dictyate
Dictyateea. 80

1-49
1-49

400
65-70
8 x soh 65-70
00.08-o.33b

Reference

0.13-l.24b452,456

1.38-1. 78
odod
0.76-0.88

468,489,490

0.52
od
od

468,484,485

1.80-2.04
1.02-1.14

76,46800.61-0.77
0.02-0.08
0.09
0.36-0.40

11 528

0.06

1 906
1 779
6 644

0.26
0-0.060.08

12 1630.04-0.05

5 170
6 099

0.77-0.87
0.46-0.51

5 154

0.04

Source: Reference 450.
acalculated as treated minus overall spontaneous control frequency. Data for the contemporary
control are shown.grouped with the treated sets.
bThe lower number {or the single number, where only one is given) represents only genetically
and/or cytologically verified cases; the higher number represents cases observed in original phenotypic
scoring. All are derived from XX mothers only.
cconsiderable differences in frequency are observed within this interval. Readers wishing to
make more precise quantitative comparisons between these results and those from lower dose rates arereferred to the full data.
dFrequency of treated group not significantly higher than contemporary control.
eMatings made during this interval utilized oocytes that received almost the entire 400-R dose
during the last six weeks of their development.
fMost matings made during this intervai utilized oocytes that received only a small part of thedose during the last six weeks of their development.
KMatings made during this interval utilized oocytes irradiated prior to their last six weeks ofdevelopment.
hwith 75-minute intervals between the fractions.

530

1.4
2.8
2.1
2.7
1.7
2.3
0.3

TABLE 33. SPECIFIC-LOCUS MUTATION FREQUENCIESRESULTING FROM X IRRADIATION OF MALE MICEAT VARIOUS AGES

X rays, 300 R
Age atilladiation(days)

Number ofoffspringscreened

Numberofmurations

New-born
2
4
6
8
10
14
21
28
35
AdultdSS 456

16a
2
3
4
7
3
9
5c
6
4
40

8 127
8 784
7 559
8 463
8 580
8 397
8 920
10 0099 056
65 548

95%confi·
dence limits

Frequency
per locus
rur• R· 11

Lower Upper

13.7
11.7
16.3
25.2
39.4
16.6
51.0
26.7
28.5
21.0
29.1

7.1 b 22.6b
2.1 39.2
4.4 43.9
8.6 60.5
18.5 77.5
4.5 45.0
25.3 95.1
7.3b 72.lb
12.4 61.0
7.2 50.S
21.0 39.0

Source: References 526, 527.
a Including three clusters of two mutations each.

blncluding correction for clusters using Russell's approach(480).

clncluding two clusters of two mutations each.
dData of W. L. Russell (474).

TABLE 34. ARBITRARY GROUPING OF SPECIFIC-LOCUS
MUTATION RATE DATA FOR YOUNG MALE MICETO ILLUSTRATE THE LIKELY TIME FOR
TRANSITION IN MUTATIONAL RESPONSE
Age at the timeof irradiation(days)

Mutation frequency
per locus

95% confidence limits(JO-• R" 1)

Lower

Upper

13.7
17.5
30.6
29.I

7.1 b
8.7
20.4b
21.0

22.6b
32.6
42.3b
39.0

New-born
2-6
8-35
AdultaSource: Reference 527.
a Data of W. L. Russell (474).

blncluding correction for clusters using Russell's approach(480).

TABLE 35. A COMPARISON OF THE fREQUENCIES OF
SPECIFIC-LOCUS MUTATION AFTER IRRADIATION
OF MOUSE SPERMATOGONIA GIVEN IN ONE DOSE
OR IN 1WO EQUAL FRACTIONS SEPARATED BY
VARIOUS TIME INTERVALS

X rays, 1000 R

Interval
betweenfractions

Total
offspring

None '(single
exposure a
44 6492 ha
148 791
11 16424 h"
4 daysb
7 168
7 daysb
8 271> 15 weeksa. c
4 904

Mutation
Number frequency
per locus
ofmutations (JO"')

29
12
39
11
11

10

8.5
11.52
49.91
21.92
19.00
29.13

95%confidence limits
Lower Upper

6.21
5.95
35.45
10.94
10.94
13.98

13.33
20.12
68.22
39.22
39.22
53.57

a Data of W. L. Russell (472).

boata of Cattanach and Moseley (92).
c600 R + 400 R.

531

TABLE 36. YIELD OF SPECIFIC-LOCUS MUTATIONS OBTAINED AFTER IRRADIATION OF MOUSE FEMALES WITH ACUTE
AND FRACTIONATED DOSES OF X RAYS

Number ofmutants
Group

Dose

Total
progeny

A+Bb
C+ Dc

1 X 200
20 X 1021578
20 398Mutations
per locus
95% conFuience(JO"' rad"') limits

Up to 20 days 0

7
1

2.32
0.35

Number ofmutants

Lower

Upper

Total
progeny

0.93
0.0088

4.78
1.95

34 81339 887

Mutations
per locus
95% confidence(JO"' rad·'; limits

Up to 7 weeksa

9
1

Lower

Upper

0.85
0.0045

3.51
1.00

1.85
0.18

Source: Reference 312.
Note: The mutation rates given above are not corrected for controls.
a Duration from end of radiation treatment to conception.
bln group A, mating was soon after irradiation; in group B, it was one week later.
c!n group C, 4 fractions each of I O rad were administered at 2-h intervals in one day: radiation was given over a 5-<lay period(4 X I O rad X S); mating: 3 days after the last dose. In group D, 20 fractions each of 10 rad were administered over 5 days in a week

(separated by 24-h intervals) for 4 weeks; mating: soon after completion of the last treatment.

AND
OF
OFFSPRINGTABLE 37. DISTRIBUTION
MUTATIONS AT SEVEN SPECIFIC LOCI IN
CONCEPTIONS OCCURRING DURING SUCCESSIVEWEEKS AFTER IRRADIATION OF FEMALE MICEWITH VARIOUS EXPOSURES AND EXPOSURE
RATES
Xrays, 200R
at 90 R/min
Week

2
3
4
5

6
7
8
9

X rays, SOR
at 90 R/min

Gamma rays,
400 R at 0.8 R/min

Off·springMutationsOff·springMutationsOff·springMutations

28 547
3 604
454
1138
8 395
3 327
328
17

13
2
1
0
12

84 614
8 016
2 904
9 587
45 17916 30413 8689 092
11 0967 433
50 570

6
0
0
1
3
3
0
0
0
0
0

49 039
7 928
737
844
7 342
4 990
170
2
4

14
3
1
4
6
2
0
0
0

5

5

1

10

11 on

0
0
0
0

Source: Reference 483a.

TABLE 38. NUMBER OF MUTATIONS AT SEVEN SPECIFIC LOCI IN VARIOUS LOW-LEVELRADIATION AND FRACTIONATION EXPERIMENTS ON FEMALE MICE

Exposure ordose given(R or rad}

0
215
258
215
· 400
400
615

Exposure rate ordose rate(Rfmin or
rad/min)

Weighted meaneffective exposure or dose a Number ofoffspring(R or rad}

51.
51.6b

0.009
51.6c
0.009
0.009
0.05

172
207
212
283
284
615

Number ofmutations

Mutationfrequency
per locus
r10·•1

Reference

3 or 8II
02Il2.1 or 5.6
9.7
18.6
0.0
20.8
9.9
14.0

483a
312
471
312
471d
471
84a

204 639
14 671
7 692
21 20413 74214 40210 1770 oose delivered to oocytes in the 6 weeks prior to evaluation.
boose delivered over 4 weeks in 20 fractions of 10.7 rad.
cDose delivered over S days in 2 0 fractions of I 0. 7 rad.
d Animals irradiated were old, previously bred females.

532

TABLE 39. INDUCTION OF DOMINANT LETHALS BY 2 3 9 Pu IN THE GERM CELLS OF MALE
MICE

Experlmental
series

E4
ES

E3
E4

ES

E3

Group

Week
after
matingTotal
numberofimplants
N

Dead
Dead
implants
implants(earlyPre(late
Fredeaths) quencya deaths) quencyb
(%)
(%)
"ED
"LD

Control
0.05 µCi
Control
0.05 µCi
Control
0.1 µCiControl
0.25 µCi
Control
0.25 µCi
Control
0.5 µCi

1-13
1-13
1-18
1-18
1-24
1-24
1-13
1-13
1-18
1-18
1-24
1-23

2 502
2 962
3 971
3 663
5 492
5 182
2 502
2 436
3 971
3 773
5 492
2 467

221
298
331
368
384

8.8
10.1
B.3
10.0

564

10.9
8.8
10.2
8.3
11.2
7.0
ll.6

221
248
331
424
384
287

1.0

45
71
40
113
SS
161
45
42
40
112
55

44

2.0
2.7
I.I
3.4
1.1

3.5
2.0
1.9
I.I
3.3
1.1

2.0

Total
dead
Freimpla(ltS quencyc
(%)
"T

266
369
371
481
439
725
266
290
371
536
439
331

10.6
12.5
9.3
13.1
8.0
14.0
10.6
11.9
9.3
14.2
8.0
13.4

Source: Reference 304.
a100 nEo/N.
b I 00 nLo/(N-11£D).
clOO nT/N.

TABLE 40. CHROMOSOME ABERRATIONS IN METAPHASE-1 SPERMATOCYTES OF MICE AT DIFFERENT TIMES AFTERINJECTION OF 2 ' 9 Pu OR AFTER CHRONIC GAMMA-RAY EXPOSURES

Weeks ofexposure

Series

I: >39Pu

II: 239 Pu

I

'°Co

0
6
12
18

Testis mass(mg)

Estimated
testis dosea
(raJ)

00
14 (lO)e30 (25)e
44

Right

Left

NumberNumberd withofspermaQuadrltocytes Fragvalentsc
scoredb ments

Ring1

ChainsTota/sdReference

300
400
400
400

1 (0.3)4 (1.3)8 (2.0) 4 (1.0)
9 (2.2) 16 (4.0)
12 (3.0) 9 (2.3)

0
3
1st
2

0
1
4
3

0
4 (1.0)19 (4.8)
5 (1.3)

)

600
700
800
800

2 (0.3). 2 (0.3)
11 (2.6)' 5 (0.7)
12 (1.5) 8 (1.0)
12 (1.9)~ 4 (0.S)K(1.9Y,
2 (1.0)/39 (4.l)i 16 (l.6)h

0
4
6
1

2
1
2

2 (0.3)5 (0.7)8 (1.0)4 (0.5)- (0.74)

125 ± 291 ± 688 ± 273 ± 80119.2 ± 3.7
021
13 ± 1111.4 ± 6.4 103.4 ± 6.4
96.3 ± 8.0
28
18 ± 2102.7 ± 5.3
18 ± 134
109.0 ± 5.8 111.0 ± 3.5
Meanh
17 ± 1; IS.Se 107.7 ± 3.1 103.0 :t 3.4
0118.9 ± 6.1
01128: 10sse 42.2 ± 1.3
28
Mean controls(' 39 Pu and ••coof series m

300
1 OOO

Reciprocal rranslocan·ons

3K

10'

0

6

33

516

17 (1.7)
0.22

aEstimated from left testis.
bscored in the ri1ht testis.
cResulting from reciprocal translocations arisin1 in spermatogonia (see next columns) or chromatld interchanges.
dPercentage In parentheses.
eEstimated spermatogonial dose, not taking into account the lnhomogenous distribution of 2 39 Pu in the testis in the plutonium
series.

,.

/Three cells each had 2 rinp of 4.
K1ncludin1 a chain of 3 + univalent and 2 chains of 4.
h1ncluding I ring of 6,
1Including I ring of 6, 9 rings of 4 and 6 chains of 4.
iFragments per cell,kone cell recorded as having both single and double fragments.

533

OF
FERTILITY
TABLE 41. RECOVERYOF
X-IRRADIATED MALE MICE FOLLOWING SINGLEOR FRACTIONATED (24-H INTERVAL) EXPOSURES

Age (days)

Total number
Number of of females
testedreplicates

Overall median
day of returnto fertility(a) Various ages at 300 R single

10
10
10

1
1
1

5
10
Adult

42
51
51

(b) Various ages at 500 R single

10
11
10

1
1
1

5
10
Adult

44
53
56

(c) Various ages at 1 OOO R single

3
5

1
1

10
Adult

2

10
10
10
20

1

79
84
74
102°

(d) Adults at iarious exposures

Exposure (R)300
400
100 + 300
300 + 100
500
600
100 + 500
500 + 100
300 + 300
700
800
100 + 700
700+ 100
300 + 500
500 + 300
900
1000
700 + 300
100+ 900
900 + 100
500+ 500

20
20
7
10
47

2
2
1
1
5
5
2
3
1
2
4
2
2
1
2
2
2
1
1
1
2

52
53
53
51
56
67
62
62
57
67
71
81
73
82
81
88
102
111
135
108
102

so

20
29
10
20
40
20
20
10
20
20
20
10
10
10
20

Sources: References 90, 94.
Same data as for the adult at I OOO R in part (d)0

TABLE 42. COMPARISON OF RADIATION-INDUCED MUTATION RATES WITH FREQUENCYOF LABELLED SPERMATOGONIAL STEM CELLS IN THE MOUSE 207 AND 414 HOURSAFTER X IRRADIATION
Frequency of labelled cells (o/o)

Exposure
(R)

0
100.
300
500
600
1000
soo + soOd

Labelling 6 x 12.5 µCl
of'H-TdR
24 h before
x irradiation
Tfme after irradiation

Labelling 3 X 12.5 µCi
of'H-TdR
24 h before
x irradiation
1Yme after irradiation(lo-s,

207h

414 h

207h

414 h

(o/o)

0.75

7.8
15.6

0.7
1.6

16.3

1. 7

2.4
39.1

0.2
3.1

29.2
29.0
24.3
27.5
26.3
10.2c
47.2b

22.8
33.8b
33.3b
39_9b
36.Sb
1S.3b
35.5b

26.8
11.2
9.3
3.2
2.3

Mutations
per locus"

8.72
13.29
9.28
49.91

Sources: References 367a, 657.
aFrom W. L. Russell (471).
bSignificantly above control.
cSignificantly below control.
d Fractions given 24 h apart.

534

Cell survival
comparedwith control

TABLE43. COMPARISON OF THE YIELDS OF DICENTRICS (IN LYMPHOCYTES) AND
RECIPROCAL TRANSLOCATIONS (IN SPERMATOCYTES, AFTER SPER.\iATOGONIAL X
IRRADIATION) IN MAN, MARMOSET, RABBIT ANDTIIE RHESUS MO!\'KEY

Translocarion yield(%)
Dicentric yield
Dose
(rad)

Dlcentric yield(%)

Definiteand possible

0
78
200
600

8.0
35.2
250.0

DefiniteTranslocation yield0
4.0 ± 1.0
7.0± 1.3
6.1 ± 1.8

542

2Marmoset"
0
25
50
100

0
2.8 ± 0.7
3.3 ± 0.8
7.8 ± 1.1
7.5 ± 1.9
7.0 ± 1.1

1
2
2
6
12

20.5

1.84 ± 0.3&d
2.5 (estimated)
3.08 ± 0.54

1.56 ± 0.34d
1.8 (estimated)
2.59 ± 0.50

4-5
5.7
-7-8

33.6

4.94 ± 0.85

4.20 ± 0.77

-7-12

3.0
6.7
16.3
44.6
84.9

200
300

Rabbit
100
153
200
203
300
305

6.3c

8.0b
13.0
23.0
50.0

Rhesus monkeye
0

100
200
300

(600){
4.9 (600)
13.9 (600)26.6 (600)

0- (500)/
0.2 (1 950)
1.6 (1 400)
0.3(515)25

8
89

aData given in the paper of Brewen and Preston (57).
bEstimatcd from the data of Scott and Bigger (502); x irradiation at 100 rad/min.
coata of Bajerska and Liniecki ((20) and personal communication); x irradiation at 18 rad/min.
doata of Lyon and Cox {309), x irradiation at 88 rad/min.
eData of van Buul (70).
!Number of cells in parentheses.

TABLE 44. COMPARISON OF INTRA-LOCUS FORWARD MUTATION RATES PER HAPLOID GENOME IN DIFFERENTORGANISMS a
Low-LET radiation

Test system

Haploid DNA content(pg)

Loci and cell stages where
mutations were studied

Hordeum vulgare(barley)

6.7(10.0, 11.S)b

44 eceriferum loci59 eceriferum loci
26 erectoides loci
86 chloroplast loci130 chloroplast loci
1 OOO chloroplast loci(estimated)
1 locus (DDT resistant)

Zea mays(maize)5.5

Mus musculus(mouse)3.0(2.26)b

Arabidopsis thaliana(cruciferous plant)

0.27

1 locus (bronze)
Spermatogonia
7 locl7 loci minimum
6 lociMaturing oocytes
7 loci, 50 R
200 R
200 R
400 R
Immature oocystes
7 loci, SOR
4 thiamine loci

Mutation rate
per locus(rad- 1)
3.0 10·'2.2 10·'1.4 10-•1.3-1.7 10-•0.85-1.2 10-s
1.1-1.5 10·'

ReferencesDNA
content

Mutationrate

440,441

395,442443
395
444,445443,444443

440,441447

448

195,419
see text
449

1.7 10·•<2.3 10·•

2.2 10-1c
8.1 10-1d
7.8 10· 1

444,446

1.78 10 -,e3.95 10·'
1.78 10· 1
5.50 10· 1

195,419
195,419
211
195,419
195,419•0
1.1-1.~ 10

440

450

535

TABLE44 (continued)
References
Loci and cell stages where
mutations were studied

Mutation rate
per locus(rad- 1)

0.14
0.18

Sperma togonia
8 cluomosome III loci1.28 10-•(0.17, 0.22)b

10 oogonia
I O X-chromosome lociPost-meiotic sperm
8 chromosome III loci

Haploid DNA content(pg)

Test system
Drosophila melanogaster(fruit fly)DNAcontent

Mutationrate

433,448451

405

0.5-1.54 10- 1

4 X-chromosome loci
5 loci5 loci2 adenine-3 loci(ad·3A, 3B)
1 aza-3 locus
All 3 loci considered
All 3 loci considered452

4.7 10-•3.0 10-1
2.9 10-•1.4 10-•1.2 10·•3.0 10-•
5-6 10·•2.0 10-•8.7 10-•1.0 10-a

Neurospora crassa(mold)

0.038(0.042)b

Saccharomyces
cerevisiae (yeast)

0.008
0.014. 0.015(0.024)b

6 early adenine loci1 can-I locus
1 can-I locus

6.0 10-•1.6 10-•1.0 10-1 0(maximum)

Schizosaccaromyces pombe(yeast)0.012. 0.015(1C)
0.023, 0.029(2C)

5 early adenine loci5 early adenine loci

0.8-1.1 10-•} f
0.4-0.8 10-•405
453,454
455
456
457

458
459
410
410,458
410,459

425,460

416
358
411

461,462414
415

Source: Reference S62.
aThe intra-locus rates given here must be viewed in conjunction with the differences between systems in their resolving power lo
detect intragenic change.
bDNA values given by Abrahumson et al. (4).
cThis rate is not intragenic.
dFor the mouse, except for the rate 8.1 10-•, the rates given may not all represent intra-locus rates.
eThe rates obtained in the radiation experiments were corrected using the spontaneous rate of 1.4 10-• per locus (589).
/The two ranges of mutation rate estimates were obtained by taking the rates given by the respective authors and dividing by 2 toallow for the fact that cells with 2C DNA amounts were treated.

TABLE 45. INTRA-LOCUS MUTATION RATE ESTIMATES FOR TiiE INDUCTION OF SPECIFIC LOCUS
MUTATIONS IN POST-MEIOTIC MALE GERM CELLS OF DROSOPHILA MELANOGASTER, CYTOLOGICALPOSITION OF THE LOCI AND ESTIMATES FOR TiiE SIZE OF THE BANDS IN THE SALIVARY
CHROMOSOME
Locufl
g
f
rb
y
w
crd
sn
V

cm
pn
m
car

ras
ac
SC

Mutation rate per locus (R • •)5.0 10-•(between that ofg and w)

2.5 10-•(between that ofw andpn)
9.0 10-•(between that ofpn and car)3.3 10-•(2.0 10-• yJ
1.6 10-•(6.8 10-•)d. 10-•-1.6

Salivary band positionb

Estimated relative size of tile
bands from 1 = small to 5 = large

12B6-7±15Fl-2
4C6-8
lBl
3C2 or 3Cl.5
7B3 or 4
701-2
lOAl
6E6
2D6-2EllOEl or 2

1
4
2
2c
1c
1
4

18Dl-24

5
1?
3 or 4

9E4-7
1B2
1B3-4

2c

5

Sources: References 27S, 593.
aThe loci are ranked in descending order of mutation rate.
bsalivary band positions are from Lefevre (275), who in a personal communication makes the following
amendment: rb, cm and car are not exactly where they are supposed to be.
c According to Lefene (274 ), there appears to be more than a single complementation group associated -...ith at least
some of the bands in 1 B. In addition, Lefevre (personal communication) points out that y and ac are the sole occupants of
I 81·2 and that cytogenetic analysis in collaboration with Garcla-Bellldo suggests that sc is a duplicated gene which
occupies I 83-4. The possibility that w is associated with a very faint band between 3Cl and 3C2 has been discussed by
Sorsa, Green and Beerman (546).
dln consideration of the arguments of Muller (351) that at er, car and ras most of the mutations unaccompanied bychromosome aberrations are male lethal (and may therefore be considered as true gene mutations giving a lethal effect), theintra•locus rates given in parentheses Include these mutations as well; for the et locus the rate is 3.3 10· 1 •

536

-~

TABLE 46. FACTORS WHICH POSSIBLY DISTURB THE EXACT DETERMINATIONOF MUTATION FREQUENCIES IN CULTURED MAM~IALIAN CELLS
Factors as such relevant for selectionof HG-PRT-deflcient mutants in

Factors possibly disturbing the exact determinationof mutation frequencies

Phenomena
involved

Human
diploidskin {fibroblastic
cells)

Chinesehamster(epithelioid
cells)

LS178Ymouse
lymphoma(cells insuspension)

+

+

+

+,

+

+

V79

A. Recovery ofmlllants

GrowthOccurrence of spontaneous mutants in course of experiment
Differential growth rate of wild-type cells and fully expresse<l mutants
Differential growth rate of wild-type cells and newly induced mutants

Cloning
efficiency

Differential cloning efficiency of wild-type cells and fully expressed mutants
Differential cloning efficiency of wild-type cells and newly induced mutants
Absolute cloning efficiency of wild-type cells

+
+

+

+
+

Metabolicco-operation

Humoral metabolic co-operation
Intraclone metabolic co-operation
Interclone metabolic co-operation

+
+

+

+

B.

±

Determination of expression time

Rate of decreaseof wild-tn,egene products

Dilution of normal gene products by cell division
Rate of turnover of normal gene products

+
+

+
+

+
+

Treatmentof cells

Dose-dependent reduction in growth rate
Delay in mutation induction

+

+

+

Source: Reference 538.

TABLE 47. RISK OF INDUCTION OF VARIOUS KINDS OF GENETIC DAMAGE IN MAN PER l RAD
AT LOW DOSES OR AFTER CHRONIC EXPOSURES
Estimated in 1972
Expected rate of induction per million
End-point

Spermatogonia

1. Recessive point mutations

OocytesExpression in F 1 per million conceptions after spermatogonia/ i"adiation

Very low

30-75
(1-2)

2. Dominant visibles

2C

3. Skeletal mutations

4. Reciprocal translocationsd

Very low

5. X-chromosome losses

Very low

6. Other chromosome anomalies

Very low

Total genetic damage

I 5211

Total genetic darnage 1300

82 congenitally malformed children, 19 unrecognized
early embryonic losses and 9 recognized abortions!
8 early embryonic losses and/or abortion~k
Very low(51)hSource: Reference 589.
Note: Dashes indicate that inadequate or no information is available.
0 Estimate based on mouse specific-locus data.
bEstimate based on the per genome rate for recessive lethals induced in mouse spermatogonia.
clncluded under end-point I above (see paragraph 594 or Annex E of reference 589).
dFigures apply to low-dose x Irradiation. Estimates for chronic 11amma Irradiation are SO per cent ·lower,e Balanced products.
/For low dose x irradiation; for chronic pmma irradiation, figures should be halved (see paragraph 621 or Annex E of reference589).
Kobtaine<l by addins I SOO+ 2 + 4 + 1 sin the column.
hobtained by adding 36 + 2 + 4 + Is in the column.
1Relative to spontaneous incidence of genetic diseases among live-born, based on an estimated "doubling dose" of I 00 rad.
frn terms or incidence of genetic disease among live-born.
k After oocyte Irradiation.

537

~I
'

TABLE 48. ESTIMATED EFFECT OF 5 RAD PER GENERATION ON A
POPULATION OF ONE MILLION, INCLUDING CONDITIONS FOR WHICH
THERE IS SOME EVIDENCE OF A GENETIC COMPONENT

Effect of 5 rad per generation
Disease classification

incidence

generation

Equilibrium

Dominant diseases
Chromosomal and recessive diseases

10 OOO
10 OOO

50-500Relatively
slight250-2 500
Very slow
increase

Congenital anomalies
Anomalies expressed later
Constitutional and degenerative
diseases

15 OOO
10 OOO

5-500

50-5 OOO

60-1 OOO

300-7 500

Total

15 OOO
60 OOO

Source: Reference 34.

TABLE 49. RISK OF INDUCTION OF VARIOUS KINDS OF GENETIC DAMAGE IN MAN PER
1 RAD AT LOW DOSE RATES OR LOW DOSES OF LOW-LET IRRADIATION

Expected rate of induction
per million gametes
resulting from
irradiation of
End-poinr
1. Autosomal mutations'2
2. Dominant visiblesb
3. Skeletal mutationsc
4. Balanced reciprocal translocationr
5. Unbalanced products of end-point 4
above
6. X-chromosome lossh
7. Other chromosome anomalies

Spermatogonia

OocytesExpression
in rustgeneration
per ml1lion births

2Qd

60
Very low

4
17-87

Low

Lewi

34-174
Very low

Low

Low

2-lOg

Note: Dashes indicate that inadequate or no information is available.
aPresumed to include small deficiencies. Based on rate of induction of mutations in mice thatare lethal in the homoznous condition, which is doubled to give the overall rate (para. 611 ).
baased on those scored in the course of specific-locus experiments in mice.
cDetected in mice by dominant effects.
dOverall rate of dominant effects, based on skeletal mutations and presumably including
dominant visibles and heterozygous effects of autosomal mutations.
eoerived from human and marmoset cytogenetic data under the assumption that the frequencyof translocations,in the F 1 progeny is one fourth of that observed in spermatocytes.
/Effects such as those given for end-point S In the next footnote will become manifest in
generations following the first.
l'Expressed as congenital malformations. In addition, there would be 11 ·S 5 recognizedabortions and 22-109 early embryonic losses.
hoetected in mice by X-chromosomal markers.

538

I

.1

TABLE 50. ESTIMATED EFFECT OF l RAD PER GENERATION OF LOW-DOSE, LOW
DOSE-RATE, LOW-LET IRRADlATION ON A POPULATION OF ONE MILLION LIVEBORN IND1VIDUALSAssumed doubling dose, 100 rad
fffect of 1 rad per generation
Disease classifica tiona
Autosomal dominant and X-linked
diseases
Recessive diseases
Chromosomal diseases
Congenital anomalies
Anomalies expressed later
Constitutional and degenerative
diseases
Total

Cu"enr incidenceb)

First generationC

Equilibrium

10 ooQd
1 100
4 oooe20

100

Relatively slight

Very slow increase

38/

40

90 OOOK5h

45h

105 200

63

185

0.06

Percentage of current incidence

0.17

Recalculated BEIR assessments
Autosomal dominant and X-linked
diseases
Recessive and chromosomal diseases
Congenital anomalies
Anomalies expressed later
Constitutional and degenerative
diseases
Total)

Percentage of current incidence

10 OOO
10 OOO

20

100

Relatively slight

Very slow increase

40 OOO

2-20;

20-200;60 OOO25-4oi

I 25-30oi

100

0.04-0.07

0.21-0.50"Follows that given in the BEIR Report (34).
bBased on the results of the British Columbia Survey with certain modifications; see table 9.
cThe first generation incidence is assumed to be about one fifth of the equilibrium incidence
for autosomal dominant and X-linked diseases; for those included under the heading "congenital
anomalies etc." it is one tenth of the equilibrium incidence. For rationale see reference (34).
dsee table 9.
eBased on the pooled values cited in Nielsen and Sillesen {363); includes mosaics but excludes
balanced translocations.
/The first generation incidence is assumed to include all the numerical anomalies and threefifths of the unbalanced translocations (the remaining two fifths being derived from a balanced
translocation in one parent),
KJncludes an unknown proportion of numerical (other than Down's syndrome) and structural
chromosomal anomalies.
hsased on the assumption of a S per cent mutational component.
;The range reflects the assumption of S and SO per cent mutational components; see text for
explanation.
i Rounded-off figures.

539

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