TABLE Il
EFFECT OF LINDANE ON RNA AND DNA
OF RAT TISSUES
(mg/em dry wt)
. RNA
Tissues DNA RNA DNA

Control 4.24 797 1.88

Heart
Test 452 14.20 3.14
Control 3.82 25.30 6.62

Liver
Test 352 26.10 TAQ
Control 950 15.20 2.46

Kidney

Test 7.95 12.30 1.55
Control = 4.71 8.45 180°

Brain
Test 4.03 8.70 2.16
Control 40.30 20.80 0.52

Spleen

Test 5830 40.40 0.69

of heart and spleen aud a decrease in brain and
kidney. Little change in DNA was observed in
the liver, The spleen DNA was consistently
high in quantity, and showed a remarkable in-
crease ‘allowing exposure to lindane for even
relatively short periods of time. It is ay jt
from these data that alterations of considerable
magnitude can result from the presence of lin-
fe whlch thse chnoges effec the phyilogy
to wi s the
of the cell or contribute to the symplonatolosy
of toxicity is not kyown. |

Effect of Lindane'on Heart Sarcosome Activ-+
ity. From the general toxicological features of
lindane poisoning on higher animals, a working
theory was formulated that the’ physiological
effects were, perhaps in some part, attributable
to an effect on the active transport of important
metabolites in the tissues. Since this phe-
nomenon is energy dependent, it was reasoned
that a study of the chemical reactions responsi-
ble fog. providing energy in a form suitable for
this function was a logical starting point in an

Two often-studied oxidative enzyme systems
were selected for initial study — eytochrome

oxidase and succinic dehydrogenase, “These are
known to be linked biochemically to the syn-
thesis of ATP.in cells and tissues, and in addi-
tion, have several other relati s and
characteristics which emphasize the fogic of
selecting them for study in the present overall
research plan: Morrison and Brown (6) showed
that the cytochrome oxidase activity of cock-
roach coxal muscle was sensitive to chlorinated
hydrocarbons; both of the systems are. located
predominantly in the particulate fraction of the
cell and in this regard are available for certain
assay purposes in future studies of interface
phenomena within cells; and reliable histo-
chemical techniques are available for the study
of such oxidative reactions,

Table III shows that the cytochrome oxidase
activity of sarcosomes from the heart of the

TABLE III

EFFECT OF LINDANE ON SPECIFIC
ENZYME ACTIVITY OF RAT
HEART SARCOSOMES

(x MO, per mg protein per hour)

we Succinic Cytochrome
Tissue’ Deh Oxidase
Control 36.4 108.0
Test 40.5 180.0

"Females exposed 2 weeks

female rat was markedly increased by lindane,
given aaily for 14 days, at a dosage level of 17
mg,/kg. of body weight. A smaller, but signifi-
cant effect was noted on succinoxidase activity.
Control animals received the oil solvent only.:
The Uptake of S-35 Methionine by Chick .
Tissues, Methionine, along with other amino
acids and non-protein metabolites, is known to
be selectively absorbed by the intestinal mucosa
and the,kidney tubules, and actively trans-
ported reross tissue membranes (7). Critical
experiments involving $-35 labeled methionine,
were carried out to test part of the initial hypo-

investigation of the specific biochemical effects _ thesis concerning the mechanism of chlorinated
of chlorinated hydrocarbons on higher animals, hydrocarbon toxicity, - .
-3-



it is noteworthy that approximately 95% of the
methionine (measureable) taken up by the
heart sarcosomes was, at the time of assay, still
in the form of the free amino acid (chromato-
graphic analysis), Any statement in explana-
tion of this effect on the movement of methio-
nine must await the results of studies on the
more specific biochemical effects of these in-
secticidal agents, Of special importance wili be
those studies devoted to the determination of
the capacity of chlorinated hydrocarbons to
uncouple oxidative phosphorylation, If this
is found to be the case, the increased 0, con-
sumption, increased cytochrome oxidase activ-
ity, decreased rate of methionine uptake and
many of the other observable effects of
chlorinated-hydrocarbon intoxication would be

closer to adequate explanatior,

1

2 4eep

x

s.
10.

ik

REFERENCES

Lowry, O. H., Rosebrough, N. S., Farr, A, L. and
Randall, R. J, J. Bol. Chom. 193:285, 1951.

Dische, Z, Microchemie 8:4, 1930,

|. Mejbaum, W., Z. Physiol. Chem. 258:117, 1939.

Schneider, W. C., J. Biol. Chem, 161:293, 1043.

Schneider, W. C. and Klug, H. L., Cancer Research
6:691, 1948.

Morrison, P. E. and Brown, A.W.A,, J. Econ. Entomol.
41(3):123, 1954.

1. Daugherty, J. W., Symposium, Amer. Soc. Trop. Med.,

‘Amar. J. Trop. Med. and Hyg, 6:464, 1957.

Dounce, A. L., Witter, BR. F., Monty, K. J., Pate, S.
tad Cottne, HLA. J. Blophy: Bockem. Cyfol. 1:13,
Annau, E., Canad, J. Blochem. Phystol, 32:178, 1954.
Brown, G. B. and Roll, P. M., The Nucleic Acids, Il,
Academie Press, N. Y., 1085.

Harvey, G. T. and Brown, A.W.A., Canad. J. Zool,
29:42, 1931.

ACKNOWLEDGMENTS

The authors wish to
tion to Dr, Paul W. Smith
toxicological matters in the investigation and in

their apprecia-
his help in certain

the preparation of the manuscript. We also
iat to thank Dr, Wallace. Frieder for the
use of facilities in the Radiobiology tory.
! ’
, / .
V '
: .
*
-6-

PAA AC 68-3383


_

chemical Research, $-35 labeled methionine
was obtained from Schwarz Bioresearch Inc,
Protein was estimated according to the
method of Lowry et al (1), Nucleic acids were
determined by pentose analysis; DNA by 2-
desoxy-ribose (8) and RNA by ribose (3).
Isolation of the nucleic actds from the tissues
and the removal of lipid materials was accom-
plished according to the methods each
hneider (4) and Schneider and Klug (5).
Nucleic acids so measured were found to com-
pare favorably in amount with those deter-
mined by others using different criteria, Total
nitrogen was determined by standard micro-
Kjeldahl techniques. The heart sarcosomes and
liver mitochondria were isolated by differential
centrifugation in 0.44 M sucrose. Succinoxidase
and cytochrome oxidase activities were meas-
ured manometrically following the usual
ization ures, Radio-activity
Measurements were made with a Sharp Low
Beta counter, Chromatographic analyses were
carried out on Whatman No, 4 filter paper using
ascending principles with phenol and 2, 4-
lutidine as the solvents. Specific procedural
details are described in connection with appro-
priate results below.

RESULTS

The DNA Content of Rat and Chick Tissues.
In reporting results concerning the activities of
enzyme ne systems in tissue preparations from ani-
mals differing in age, sex, nutritional state,
experimentation, etc., it is unt im-
portance that a proper and adequate choice of
units be made for comparing and ting
the various biochemjcal data. Investigators use
many different references for this purpose; wet
weight of tissue, dry weight, total nitrogen con-
tent, in content, etc, Each pf these units
may be adequate and appropriate for
occasions, but in every case copitrol studies are
necessary as a means of demonstrating the re-
liability of the unit for comparison purposes.
These are seldom provided. Too frequently
such well recognized variables as tissue ‘water
content and the gain or loss of non-active con-
nective tissue components are ignored,

Probably the most useful and reliable unit for
the expression of biochemical activities is on the
basis of. the tissue DNA content, This value

allows a reasonably close correlation of activity
measurements with the amount of chemically
active material (cells) included in the tissue
preparations. Comparisons between different
tissues, tissues from different animals and tis
sues experimentally altered can thus be justifi-
ably made, In audition, the increase f the
significance of experimental results is worth
more than the extra effort required to make the
estimate of DNA. The results in Table I, show-
ing the variability in DNA content of compar-
able amounts of different tissues of the rat and

TABLE 1 :
VARIATION IN DNA WITH AGE
(Milligrams per gram wet weight)
Rat Fowl
Organ Age Age

1Wk 3Mo_ 1Yr 1Wk 3Mo &Mo
Heart _...... 3.88 0.67 0.71 156 086 081
Liver .....444 108 2.16 3.39 150 199
Kidney ......5.84 2.02 2.28 1.79
Brain __..... 234 132 122 181 140 128
Spleen .....7.48 7.75 6.22 4.04 4.73 6.09

chick, are evidence of the advisability of mak-
ing this determination. It is apparent that on
the basis of DNA content, considerable differ-
ences obtain between tissues which vary as to
the source and | age.

Effect of Lindane Exposure on DNA and
RNA. In experiments designed to study some
of the biochemical effects of lindane toxicity,
additicns! ovidence was disclosed indicating the
desirability of basing enzyme kinetics data on
concurrently derived DNA values, Table IT
shows the DNA and RNA content of certain
tissues following exposure of female rats to
daily intraperitoneal injections of 17 mg, of
lindane in paraffin oil per kg, of bady weight,
for a’ two-week period, Controls received
paraffin oil only, These results are in agree-
ment with earlier results on rats which were
exposed to lindane for 31 days, In these ani-
mals there was a pronounced increase in DNA


Twelve-week old chicks were given 42 micro-
curles of $-35 labeled L-methionine by the oral
route, At allotted times duplicate 50 mg
samples of various tissues were extirpated,

ed, and placed in stainless steel countin
Blanchets 1 inch in diameter and 5/16 incl
leep. The samples were then wet-ashed with
cone, HNO,, dried with low heat in vacuo, and
their radioactivity measured. Corrections for
decay and self-absorption were made,

Table IV gives the results of this study of the
rate of methionine uptake by the various tissues
of the chick.

TABLE IV

*-METHIONINE UPTAKE BY
CHICK TISSUES

(Counts/min/mgm dey wt)

TISSUE 1Hr
Mucosa .............$

6Hrs 12Hrs 18Hrs
1010 925 = 1013
358. 37 294
739 T18 648
382 525 424
4 311 300
744 = (1027 789
%4 x) 27

3 Ww 2

These data ¢how that methionine is picked
up by the intestinal mucosa, blood, liver, spleen
and lung at such a rapid rate that by the end
of the h the uptake by these tissues i
at a maximum or nearly so, the case of the
mudésa this is ‘confirmation of its high meta-
bolic activity gnd its known high turnover rate
of metabolites,

The maximum uptake of fnethionine by brain
was not observed unti] the twelfth hour, ‘The
reason for this slow rate is a subject for specula-
tion at this time, as is the limited uptake of
methionine by cardiac and skeletal muscle.

gThe Effect of Dieldrin on Methionine Up-
take, The influence of chlorinated hydrocarbons
on this biological activity was then tested, The
chlorinated. terpene, dieldrin, was dissolved in
acetone and introduced in specified amounts

into gelatin capsules, The acetone was allowed
to evaporate and the encapsulated dieldrin was
fed to tweive-wweek old chicks in daily amounts
of 20 mg./kg. for three days. At this time the
animals were given, by mouth, 40 microcuries
of the S-35 labeled L-methionine. At the end
of six hours the hearts and livers were removed
and the heart sarcosomes and liver mitochondria
isolated by procedures adapted from Schneider
(4) and ince et al (8), The six hour inter-
val was selected as this represented the mini-
mum time required for maximum methionine
uptake by cardiac muscle, Special precautions
were taken in washing the late fractions
before they were prep for counting the
radio-activity and in all cases the final wash
was, itself, counted to assure the complete
removal of isotope-labeled methionine from the
exterior of the particles, This final wash never
materially exceeded the back count, The
results are presented in Table V. The decline
in the rate of methionine uptake by liver and
heart tissues was found to be of significance.

TABLE V
EFFECT OF DIELDRIN (2 wks) ON S*
METHIONINE TRANSPORT
6 Hour
Tissue Count
“Control =—-28,000
Heart? Test 26,300
Heart Control 880
Sarcosomes** Test 540
+ Evert pe e400
Liver Control 8820

Mitochondria** Test TT70
*Counis/mia/gm dry wt
**Counte/min/ particulate fraction from 1 ym tisue

DISCUSSION

It 4s generally recognized that of the man
consequences ot chlorinated hydrocarbon in
toxication in higher animals, liver involvement,
as evidenced by cloudy swelling, hydropic de-
generation, jogeniety of hepatic cell
granules, liver enlargement, cell vacuola-

_tion, ete, is among the most consistently

“4+


9 By ASTIA 7

if
wer
- gl,

PROBLEMS IN
AERIAL APPLICATION:

f. SOME BIOCHEMICAL
EFFECTS OF LINDANE AND
DIELDRIN ON VERTEBRATES

62:10

ASTIA

f AUG "3 1962 }
Bwish

TISIA A

FEDERAL AVIATION AGENCY
‘ Aviation. Medical Service
i’ Research Division « -
Civil Aeromedical Research Institute
Branch
Oklahoma City, Oklahoma

MAY, 1962


observed. However, little is known of the
causative factors underlyin the appearance of
these conditions, Ava! able information sug-
gests that alterations in membrane permeability
and active transport mechanisms may be, at
least in part, responsible for the observable
histopathology, Should this suggestion find
sufficient experimental support, the usefulness
of these chlorinated hydrocarbons as basic re-
search tools could surpass the practical ad-
yances that application of this information
would provide in civil aviation and industrial
medicine,

In connection with the studies on en:
requirements for active transport, efforts so far
to demonstrate a specific uncoupling effect by
chlorinated hydrocarbons on phosphate esteri-
fication in the synthesis of high energy phos-

hate bonds (ATP) have met with technical
J iiculties. However, useful and interesting
ancillary information has resulted,

The measurements of the nucleic acid con-
tent of tissues under normal and experimental
conditions have yielded interesting findings,
some new, some confirmatory, concerning ee
assay of biochemical functions and the activities
of tissue components, One cannot fail to rec-
ognize that the actual amounts of chemically
(metabolically) active material in a given
tissue sample may vary markedly with the in-
stantaneous physiological condition of the ani-
mal and of he tissue, In the present studies,
unpublished findings have shown an actual
alteration in the we chlogbat of certain tissues
following exposure to ated hydrocarbons.
After 3] days on lindane, the water content of
the rat heart was’ elevated whereas that of the
liver was decredsed, Annau (9) reported a
rise. in both DNA and RNA of liver parenchyma,
but no, change i the nuclete, wid content of

5 e feeding g chlorina'

hy fon te, to g for from 13 to 25
days This is at somey @ with the results

resent study, jut many o! e experi-
mental Ponditions were different, including the
test animal and the insecticide. ‘In_ spite of
these ay t discrepancies, it is clear that
Ingestigators in the field of toxicology, whose
task includes the determination of cellular bio-
chemical activities, cannot be too demanding
in the proper choice of assay controls or in the
choice of references used to exnress findings.

Evaluation of the significance of observed
alterations in RNA and DNA content of tissues
must await the solution of some of the basic

roblems in nucleic acid metabolism, Brown
and Roll (10) point out that “,. . we cannot
adequately describe the process by which nu-
cleic acids are formed in the living cell”, and,
“many situations have been described in which
the administration of a compound and the in-
duction of a certain physiological state in an
animal, is followed by alterations in the amount
or in the renewal of tissue nucleic acids, ... it
does not follow that they are directly impli-
cated in nucleic acid synthesis”, -

The observations on the effects of lindane on
oxidative enzyme activity are of interest in
relation to the findings of other workers, Mor-
rison and Brown (6) found that 10°M lindane
depressed wome oxidase activity of the
coxal muscle of the cockroach, Periplaneta
americana, but the e to 10°*M lindane
resulted in a transitory increase in activity, In
seeming contradiction, Harvey and Brown (11)
observed a marked increase in 0, consumption
of Blatella following the injection of toxic
amounts of lindane. There is at present neither
reason nor basis for comparing insecticide ef-
fects on with these on vertebrates.
In fact there is to date no acceptable informa-
tion indicating whether or not the various
effects of most insecticides on biochemical
activities are direct effects of the unmetabolized
compound, direct effects of the metabolically
altered compound, or indirect effects, mediated
through undefined channels, Work aimed
toward the solution of this problem is of fore-
most importance in the final description of
insecticide effects.

The most im ressive evidence that chlori-
nated hy fect active transport
Mrechenione is found in the results of experi-
ments in which L-methionine translocations
were followed by the use of the radioactively-
labeled compound, There can be little doubt
that ‘dieldrin, under the conditions of the tests,
not, ofily depresses methionine across
cell membranes, but also affects'its movement
across the heart sarcosome interface. In the
experiments reported, transport into the liver
cells was reduced approximately 6%, whereas
transport into the particulate fraction of the

_cell was reduced 39%, In examining these results

-S-


PROBLEMS IN AERIAL APPLICATION:

1. SOME BIOCHEMICAL EFFECTS OF LINDANE
AND DIELDAIN ON VERTEBRATES

JACK W. DAUCHERTY, ?H. D.°
DANE EUGENE LACEY, B. 3.
PATRICIA KOBTY, B. 5.

. Schl, Cepochenicry Section
Pharmecology-Biachemietry

10

MAY, 1908


PROBLEMS IN AERIAL APPLICATION:

I. SOME BIOCHEMICAL EFFECTS OF LINDANE
AND DIELDRIN ON VERTEBRATES

JACK W. DAUGHERTY, PH. D.°
DANE EUGENE LACEY, B. S.
PATRICIA KORTY, B. S.

INTRODUCTION

Despite the numerous studies that have been
made on insecticide toxicology since the first
use of DDT, the modes‘of action of even the
most thoroughly studied of the newer organic
agents are relatively unknown, Investigators

ve practically ignored ten! it
area of research which has Pplica in in-
dustrial and aviation medicine, ayid animal
husbandry, In addition, contribu to theo-

retical toxicology and bi are likely, A
investigation of the biochemical ef-
ects of organic I agents has been

initiated at the Civil Aeromedical Research
Institute, _It is hoped that from these studies,
preventivd; protective and therapeutic measures
will develop which will decrease the morbidity
and mortality rates in the field of aerial
application,

EXPERIMENTAL

The work in this paper represents
one part of a continuing program. The rats
used in this investigation were of the Sprague-
Dawley strain, as bred and supplied by Holtz-
inan, were obtained when 140-160 grams
in weight and cies laboratory itions
for ent an
mental a: The were White Racks,
obtained locally when one day old, and kept
under laboratory conditions until used, Com-
mercia] (Ralston) animal rations were used

throughout,’ bon
hydrocarbon insecticidés, lin-
dane and dieldrin, were obtained from the
Nutritional Biochemicals Corporation. Adeno-
sine-monophosphate, sdenosine diphosphate,
were

EDTA, c and succinic
supplied by the California Corporation for Bio-

-1-