Carcinogenesis vol.8 no. 11 pp. 1635-1643, 1987

Combination experiments with very low doses of three genotoxic

iV-nitrosamines with similar organotropic carcinogenidty in rats

M.R.Berger, D.SchmahJ and H.Zerfoan1

Institute of Toxicology and Chemotherapy and 'institute of Experimental

Pathology, German Cancer Research Center, Im Neuenheimer Feld 280,

D-6900 Heidelberg, FRG

The study was designed to assess the syncarclnogenk activity of very low doses of A'-nitrosodiethylamine (NDEA),

A'-iutrosopyrrolidine (NPYR) and A'-nitrosodiethanolamine

(NDE1A) in the liver of 1800 male Sprague-Dawky rats. The

iV-nitrosamines were administered throughout the rats' lives

individually and in combination at three logarithmically

spaced dose levels contained in drinking water. The dose levels

in the individual dose—response experiments ranged from the

lowest concentrations of previous experiments (NDEA, 0.1

mg/kg; NPYR, 0.4 mg/kg; NDE1A, 2.0 mg/kg) to dosages

10 times lower and comprised a high, medium and low dose

(escalation factor: 3.16). The high dose of the combination contained the three nitrosamine concentrations used as the medium doses of the individual nitrosamines. The medium

combination dose resulted from the combined administration

of the three lowest dosages, and the low combination dose

consisted of three nitrosamine dosages which amounted to

one-third of the low dosages respectively. Administration of

these dosages was associated with a dose-dependent incidence

of liver cancer: NDEA induced 45, 3.8 and 2.5%; NPYR

caused 21.3, 5 and 1.3%; NDE1A generated 7.5, 1.3 and

2.5%; and the combinations induced 16, 4.2 and 1.7% respectively. Untreated controls showed 0.6% liver cancer incidence. Besides the liver, the gastrointestinal tract, the

neurogenk tissue, the urinary tract and the hematopoietk and

lymphatic tissue were affected by tumor incidences increased over that of controls. There was, however, no well-defined

dose dependency as with the liver tumors. These results indicate dose dependency of liver tumor formation even at very

low exposure levels of the individual agents. The carcinogenic

effects of the hepatotropic A'-nitrosamines summed up in combination. The observed additivity was linear. Dose levels,

which alone would presumably not have been carcinogenic,

effected a significant cancer risk in combination.

Introduction

The number of conceivable experimental designs for investigating

combination effects in chemical carcinogenesis is almost infinite.

Thus, for two or more compounds used in combination, differences may exist with regard to the compounds' physiochemical

properties, type of activity (local or resorptive), supposed mechanism of action (genotoxic or epigenetic), potency, mode of

exposure (oral, by inhalation, etc.), organotropism, and last but

not least, with regard to the respective exposed organism (animal

•Abbreviations: NDEA, W-nitrosodiethylamine; NDE1A, N-nitrosodiethanolamine; NPYR, iV-nitrosopyrrolidine; H/M/LD, high/medium/low dose; H/M/

LCD, high/medium/low combination dose; PAS reaction, periodic acid-Schiff

reaction; PAHs, polycyclic aromatic hydrocarbons.

species, condition of target organ, pharmacokinetic characteristics of the individual laboratory animal, etc.). In view of the

complexity of the number of variables it is important to elaborate

essential characteristics to find an experimental design that is

suited for the investigation of combination effects and also applicable to other individual compounds and classes of compounds.

Everybody familiar with the subject matter will realize, however,

that despite meticulous experimental design some uncertainty will

remain when results obtained in one experimental model are

extrapolated to a second model. Thus, every experimental design

will only be an approach to the problem of combination effects

and only very clear-cut results will give generally applicable conclusions.

The present study was designed to investigate the syncarcinogenic effect of three hepatocarcinogenic A'-nitrosamines and, in

particular, the possible summation of carcinogenic effects of very

low doses. In previous related experiments of our (1,2) and of

other groups (3,4), it was found that carcinogenic effects summed

up when chemical compounds with identical organotropism were

administered. The previous experiments were of a more qualitative and at best semiquantitative nature, whereas the present

study was based on strictly quantitative modalities. A disadvantage of such experiments is that the supposed effects occur only

at an advanced age of the animals, because administration of low

doses implies long induction times of tumors. For die same reason

the initial number of animals had to be comparatively large to

ensure a sufficient number of animals at an advanced age, i.e.

when tumors were expected to be seen.

The three A'-nitroso compounds chosen had been individually

tested in dose—response studies. Based on these experiments it

was possible to estimate doses which would induce < 50% tumors

in their target organ when administered throughout the life of

the animal. The three nitrosamines selected were A'-nitrosodiethylamine (NDEA*) (5), A'-nitrosdiethanolamine (NDE1A) (6-8)

and A'-nitrosopyrrolidine (NPYR) (9). It is well known that results

obtained in earlier dose-response studies under different conditions can be extrapolated to other experiments only with limitations. It was dierefore decided to repeat the dose-response

studies with separate administration of the three A'-nitroso compounds. The doses, however, were selected in a range which

covered the lowest dosages of the previous experiments as well

as dosages three and 10 times lower. The results of these experiments then formed the basis of the quantitative calculation in

the combination experiment.

Generally, many benign or malignant spontaneous tumors

occur in older animals. To permit relevant evaluation of the

results obtained in die combination experiment, an especially large

untreated control group was used. Since the previous experiments

essentially showed hepatotropic carcinogenic activity of all three

A'-nitroso compounds, the combination effects were evaluated

with special regard to hepatocarcinogenesis, i.e. the incidence

of hepatocellular carcinomas; yet the whole spectrum of tumors

observed is presented and discussed.

© IRL Press Limited, Oxford, England 1635

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M.R.Berger, D.Schmfihi and H.Zerban

Materials and methods

Chemicals

NDEA, NPYR and NDE1A were synthesized and supplied by Dr R.Preussmann

(Institute of Toxicology and Chemotherapy, German Cancer Research Center,

Heidelberg, FRG). The identity and purity (> 99 %) of the three test compounds

was verified by chromatographic methods using detection both by thermal energy

analysis and mass spectrometry.

Animals and treatment

Eighteen hundred male outbred Sprague - Dawley rats (Charles River Breeding,

Sulzfeld, FRG), 100 ± 10 days old at the start of the experiment, were kept

under conventional conditions (two animals per Makrolon III cage; temperature

23 ± 2°C; relative humidity 55 ± 10%; illuminated period 12 h/day). All animals

received a specific nitrosamine-poor diet which contained <2 p.p.b. of /V-nitrosodimethylamine and of NPYR (1320 N, ahromin, Lage, FRG). This diet was offered ad libitum during the early stages of the experiment. After 4 months the

daily ration was restricted to 21 g per animal, corresponding to 65 kcal, in order

to maintain a mean body wt of 420 g. The rats were randomly stratified into

13 experimental groups, as shown in Table I.

The different group sizes were based on estimated tumor incidences. The Nnitroso compounds were administered in drinking water according to the dosage

schedule shown in Table I for a maximum period of 170 weeks. The highest

dosages of the respective single A'-nhrosamines were selected to correspond to

the lowest dosage of the previous experiments. They are termed 'HD' (high dose).

Two logarithmically spaced lower dosages termed 'MD' (medium dose) and 'LD'

(low dose) were chosen to amount to one-third and one-tenth of the HD dosage.

The highest combination dose (HCD) contained the three nitrosamine concentrations used as the medium doses of the individual Af-nitroso compounds. The

medium combination dose (MCD) resulted from the combined administration of

the three LD dosages and the low combination dose (LCD) consisted of three

nitrosamine dosages which amounted to one-third of the LD dosages respectively.

Fresh solutions of the necessary concentrations were prepared daily five times

a week. The concentration of these solutions was based on a daily consumption

of 7 ml water per 100 g body wt. Over the weekend all rats received plain tap

water ad libitum, except for those animals that had not emptied their carcinogen

solution. Untreated controls received tap water only.

All rats were inspected twice daily and weighed once a month. They were

observed for their entire lifespan and killed only when moribund. Dead animals

were carefully dissected. All macroscoptcally detectable lesions and tumors were

registered. The liver, brain, adrenal and thyroid glands and all macroscopically

visible abnormalities of each animal were fixed in formalin and examined histologically by Dr D.Komitowski (Institute of Experimental Pathology, German

Cancer Research Center, Heidelberg, FRG).

Putative preneoplastic foci of altered hepatocytes were determined in all animals

killed during the first 30 months of the experiment. Liver tissue was taken from

lobes free of macroscopically visible tumors and fixed in Carney's solution. Paraffin

sections were stained with hematoxylin and eosin. Glycogen was demonstrated

in parallel sections by the periodic acid-Schiff reaction (PAS). Foci of altered

hepatocytes were classified according to Bannasch et al. (10). Animals with foci

of clear and acidophilic or mixed and basophibc cells were counted.

Statistical analysis

The tumor rates were analyzed statistically by comparing age-adjusted expected

tumor incidences at different sites with observed incidences (11). Differences

between observed and age-adjusted expected tumor frequencies were compared,

using the x

2

 test. The reported /"-values are two-tailed except for those for which

the use of a trend test is indicated. Differences in survival times between control

and A/-nitrosamine-treated groups were assessed, using the two-tailed rank sum

test according to Wilcoxon (12). The mode of additivity in the combination groups

(linear additivity versus synergistic or antagonistic additivity) was assessed by

analysis of variance methods applied to the frequency data (13).

Results

The essential results are presented in Tables I — VII and Figures

1—3. Untreated control animals had a high mean life-expectancy

exceeding 1000 days (group 1, Table n, Figure 1) due to the

intensive care and the good general condition of the animals.

Malignancies were diagnosed in 29% of the control animals,

while the number of animals bearing benign tumors at dissection

was 2.5-fold higher (72%). The mean tumor rate in tumorbearing animals was one malignant or two benign tumors (group

1, Table H). Three animals (0.6%) developed 'spontaneous'

Table I. Experimental design of administering W-nitrosodiethylamine

(NDEA), JV-nitrosopyrrolidine (NPYR) and W-nitrosodiethanolamine

(NDE1A) to male SD rats

Group No. of Single dose administered/day*b

no.

Median total dose*b

animal s

 NDEA NPYR NDE1A Character NDEA NPYR NDE1A

of dosing

500--- - __ _

2a

2b

2c

3a

3b

3c

4a

4b

4c

5a

5b

5c

80

80

80

80

80

80

80

80

80

100

240

240

0.1

0.032

0.01

-

-

-

-

-

-

0.032

0.01

0.0032

—

-

-

0.4

0.133

0.04

_

-

-

0.13

0.04

0.013

—

-

-

-

-

-

2.0

0.63

0.20

0.63

0.2

0.063

HD

MD

LD

HD

MD

LD

HD

MD

LD

HCD

MCD

LCD

61.1

20.3

6.5

-

-

-

-

-

-

21.0

6.7

2.1

—

-

-

272

85.2

26.4

_

-

-

85

27

8.7

—

-

-

-

-

-

1327

420.8

130.9

409

135

42.2

•mg/kg body wt.

"W-nitrosamines were administered via the drinking water 5 x/week.

TID, high dose; MD, medium dose; LD, low dose; HCD, high

combination dose; MCD, medium combination dose; LCD, low combination

dose.

hepatic tumors, i.e. one hepatocellular adenoma, one hemangioma and one fibrosarcoma (group 1, Table IV). The exceptionally

long mean life-expectancy of the untreated rats accounts for the

fact that these tumors were diagnosed, because they were detected

at a mean lifetime of 1200 days.

All treated animals tolerated the administration of the three

N-nitroso compounds without any symptoms of toxic damage.

The median life-expectancy of treated animals in the individual

groups was not reduced in comparison with the control (Table

II, Figures 1 and 2) except for groups 2a and 2b (high and

medium dose of NDEA, Table IT), in which the median lifeexpectancy was slightly but significantly reduced by 77 and 52

days respectively (P < 0.003).

Regular administration of 0.1 mg NDEA/kg body wt induced

an overall malignancy incidence more than twice as high as in

the control group (65%, group 2a, Table II). Reduction of this

dose to one-third or one-tenth yielded 34 and 29% malignant

tumors respectively (groups 2b, 2c, Table II). In contrast to this

dose—response relationship observed with malignant tumors, an

even distribution of benign tumors was found, not significantly

different from that of controls.

As expected, neoplastic changes were most frequently found

in the liver. For instance, 45% of the animals treated with NDEA

HD (group 2a, Table HI) had liver tumors. The two lower dose

schedules (33 and 10% of NDEA HD) yielded tumors in only

4 and 2.5% respectively (NDEA MD and LD, groups 2b, 2c,

Table HI). The incidence of hepatic tumors was significantly

increased in all three NDEA-treated groups (P < 0.05). According to the histological examination of hepatic tumors, administration of 0.1 mg/kg NDEA (HD) induced more hepatocellular

tumors (30%) than tumors of mesenchymal origin (19%) (group

2a, Table IV). A different distribution was seen in the groups

with lower incidences of hepatic tumors, probably because of

1636

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A combination experiment with three genotoxk /V-nitrosamines

Table

NPYR

Group

no.

1

2a

2b

2c

3a

3b

3c

4a

4b

4c

5a

5b

5c

11. Median survival time, incidences of malignant and benign

, NDE1A or their combination

Mode of

treatment

Control

NDEA HD

NDEA MD

NDEA LD

NPYR HD

NPYR MD

NPYR LD

NDE1A HD

NDE1A MD

NDE1A LD

HCD

MCD

LCD

Median survival time*b

(95% confidence limits)

931 (910-945)

854 (816-892)d

879(812-939)c

914(879-1017)

951 (854-1001)

897 (875-948)

926 (907-989)

928 (897-985)

934 (908-990)

916 (889-968)

914 (875-983)

944 (908-983)

937(903-965)

tumors and tumor numbers per tumor-bearing

Percentage of animals with

tumors

Malignant

(no.)

29 (144)

65 (52)

34(27)

29(23)

44(35)

35(28)

29(23)

35(28)

36(29)

36(29)

48(48)

33(78)

31(75)

Benign

(no.)

72(362)

66(53)

68(54)

73 (58)

69(55)

74 (59)

79(63)

80(64)

75(60)

76 (61)

76(76)

75(181)

74 (178)

animal in male SD rats

No. of tumors per

rat0

Malignant

1.08 ± 0.29

.2 ± 0.46

.0 ± 0.75

.1 ± 0.63

.06 ± 0.24

1.1 ± 0.31

.0 ± 0.0

L.07 ± 0.38

.2 ± 0.47

L.06 ± 0.26

1.1 ± 0.31

1.08 ± 0.27

1.09 ± 0.29

exposed to NDEA,

tumor-bearing

Benign

1.7 ± 0.77

1.6 ± 0.60

1.6 ± 0.69

1.7 ± 0.82

1.8 ± 0.90

1.7 ± 0.69

1.5 ± 0.72

1.5 ± 0.62

1.8 ± 0.92

1.7 ± 0.82

1.6 ± 0.79

1.6 ± 0.77

1.5 ± 0.71

"Days from start of the experiment.

b

Standard significance versus control according to the method of Wilcoxon (12).

cMean ± SD.

d

/" < 0.001 versus control.

CP < 0.003 versus control.

too-I

75

50

25

in

o

E

I °

c too

Untr«at«d control

» NDEA 0.032 mgAfl

• NDEA

B

1/1

50

25- *

 N?T?.0

.-4_'!*gAg.

« NPYR 0.133 mgAfl

• NDELA 2.0

« NOEL* 0.

• NDELA 0.2 mgAg

200 400 600 800 1000 1200 0 200

Time (days)

400 600 800 1000 1200

Fig. 1. Survival curves of male SD rats in per cent of initial group size following peroral administration of (B) NDEA, (C) NPYR and (D) NDELA in

comparison with untreated controls (A). Solid symbols represent liver tumors.

1637

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M.R.Berger, D.Schmahl and H.Zerban

the small numbers (groups 2b, 2c, Table IV). The gastrointestinal

tract ranked second among the organs affected by NDEA: 31,

9 and 11 % of all rats were found to bear tumors in the digestive

tract after administration of the three NDEA regimens respectively (groups 2a—c, Table HI), compared with 5% in the control

group. The differences were significant (P < 0.01). Tumors in

the oral cavity and — in the HD group — in the esophagus

accounted for the increase in tumor incidence in the gastrointestinal tract (groups 2a—c, Table V). Renal tumor incidences were

significantly raised in the three NDEA groups, although the

observed frequencies were very low (1.3, 1.3 and 2.5% in groups

2a, b, c respectively versus 0.2% in the control group: P <

0.03). The test for dose-related trend was significant as well (P

= 0.005). All the other organ-related tumor frequencies did not

differ significantly from the control group.

The overall percentage (44%) of all malignant tumors induced

100

c

o

5 0 -

25-

N

? E* J3.O32 +_NP\R O.j j + NDF.LA 0.6JjVng/kg)

NDEA 0.01 + NPYR 0.04 + NDELA 0.2(mg/kg)

• NDEA 0.0032 + NPYR 0.013 + NDELA 0.063(mflAg)

200 400 600 800

Time (days)

1000 1200

Fig. 2. Survival curves of male SD rats in per cent of initial group size

following combined peroral administration of NDEA, NPYR and NDEIA.

Solid symbols represent liver tumors.

by NPYR HD was lower than in the corresponding NDEA group

but still 1.5-fold higher than in the control group (group 3a, Table

II). The two lower dose schedules of NPYR (NPYR MD and

NPYR LD) induced tumor rates which corresponded to those

recorded in the respective NDEA groups (groups 3b, 3c, Table

II).

The tumor incidence in the liver as the primary target organ

was 21 % after administration of NPYR HD and 5% after NPYR

MD. Both incidences were significantly different from that of

the controls (P :£ 0.002, groups 3a, 3b, Table HI). No significantly increased liver tumor incidence was observed following

NPYR LD (1%, group 3c, Table III). According to the histological classification, the majority of NPYR-induced liver tumors

were of hepatocellular origin (groups 3a-c , Table IV). NPYR

administration did not increase the number of tumors in other

organs, except for the MD in the gastrointestinal tract and the

LD in the urinary tract. The significant increase in gastrointestinal

tumors following NPYR MD was due to a slight increase in

tumors of the oral cavity and the forestomach (group 3b, Table

V), that following NPYR LD was due to a raised frequency of

renal tumors (group 3c, Table HI).

In the case of NDEIA all three dose regimens produced a 1.2-

fold increase in malignant tumors compared with the control

group (groups 4a-c , Table H). Hence no dose —response relationship in terms of the overall malignancy rate was observed.

Administration of NDEIA, however, resulted in significantly different organ-specific tumor incidences compared with the untreated controls. The HD of NDEIA induced 8% liver tumors

(group 4a, Table HI; P < 0.00005), the MD and LD induced

only 1% (non-significant) and 3% (P < 0.04) respectively

(groups 4b, 4c, Table HI). Tumors of hepatocellular origin

prevailed according to the histological examination (groups 4a—c,

Table IV).

The increase in gastrointestinal tumors observed after administration of the LD of NDEIA was significant, though only near

the limit of significance. It was due to the slightly higher rate

of tumors in the oral cavity and in the forestomach (group 4c,

Table m.

Group no.

1

2a

2b

2c

3a

3b

3c

4a

4b

4c

5a

5b

5c

Tumor distribution in different

Mode of treatment

Control

NDEA HD

NDEA MD

NDEA LD

NPYR HD

NPYR MD

NPYR LD

NDEIA HD

NDEIA MD

NDEIA LD

HCD

MCD

LCD

target organs*

Percentage

Liver

0.6 (3)

45 <36f

3.8 (3)c

2.5 (2)e

21.3 (17)c

5 (4f

1.3(1)

7.5 (6)c

1.3(1)

2.5(2?

16 (16)c

4.2 (10)c

1.7 (4/

,b

of animals (no. of animals) with

Gastrointestinal tract

5.2 (26)

31.3 (25)c

8.8 (7y

11.3 (9f

7.5(6)

8.8(7)'

7.5 (6)

7.5 (6)

3.8 (3)

8.8 (7)c

9 (9f

7.9 (19f

5.8 (14)

tumors of the

Neurogenic tisue

10.8 (54)

5 (4)

13.8(11/

12.5 (10)

12.5 (10)

11.3(9)

6.3 (5)

13.8(11)

20 (16)'

10 (8)

17 (17)'

10 (24)

12.1 (29)

Hematopoietk and

lymphatic system

4.6 (23)

5 (4)

5 (4)

2.5 (2)

5 (4)

6.3 (5)

7.5 (6)

8.8 (7)f

6.3 (5)

10 (Sf

3 (3)

4.2 (10)

7.9 (19)e

Urinary tract

0.2 (1)

1.3 (1)'

1.3(1)'

2.5 (2)d

0 (0)

1.3(1)

2.5 (2)d

1.3(1)

1.3(1)

0 (0)

3 (3)d

0.8(2)

0.4 (1)

•Definition as target organs was based on significant differences from group 1 according to footnote b.

''Pooled, age-adjusted analysis for heterogeneity (two-tailed) between observed and expected tumor incidences according to Peto et al. (11).

CP < 0.001 versus control.

dP < 0.01 versus control.

eP < 0.05 versus control.

{P < 0.1 versus control.

1638

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A combination experiment with three genotoxk /V-nitrosamines

Table V). Additionally, the rate of neurogenic tumors was significantly raised following the administration of the MD of

NDE1A (P < 0.03; group 4b, Table IE), and the number of

tumors of the hematopoietic- lymphatic system was significantly

increased by the LD of NDE1A (P < 0.02; group 4c, Table HI).

Combined administration of the MDs of NDEA, NPYR and

NDE1A, i.e. the highest combination dosage, was associated with

a 1.7-fold increase in malignant tumors (group 5a, Table E). This

parameter was still slightly elevated after administration of the

MCDs and LCDs (1.14- and 1.07-fold increase over the controls

respectively; groups 5b, 5c, Table II). In the liver as the main

target organ of all three nitrosamines, the tumor rates were significantly raised to 16, 4 and 2% for the HCDs, MCDs and LCDs

respectively (groups 5a-c, Table IE). Apart from one fibrosarcoma and one cholangioma, all the other hepatic tumors (93%)

were of hepatocellular origin (Table IV). Administration of the

HCDs and the MCDs resulted in significantly increased rates of

gastrointestinal tumors (P < 0.02 for both) due to the high

number of papillomas of the forestomach and of squamous cell

carcinomas of the oral cavity (groups 5a, 5b, Table HI). The

HCD additionally led to a significant increase in neurogenic

tumors and tumors of the urinary tract (group 5a, Table HI; P

Table IV. Classification of primary liver tumors according to histologic type

Group Mode of

no. treatment

Animals with

hepatocellular tumors

Carcinomas Adenomas

% (no.) % (no.)

Animals with mesenchymal tumors

Malignant Benign

hemangioendothcliomas hemangioendotheliomas

% (no.) % (no.)

Animals with

Cholangiomas Fibrosarcomas

% (no.) % (DO.)

1

2a

2b

2c

3a

3b

3c

4a

4b

4c

5a

5b

5c

Control

NDEA HD

NDEA MD

NDEA LD

NPYR HD

NPYR MD

NPYR LD

NDE1A HD

NDE1A MD

NDE1ALD

HCD

MCD

LCD

0

26.3

0

1.3

15

0

1.3

3.8

1.3

0

13

2.1

0.8

(2Db

(0)

(1)

(12)

(0)

(1)

(3)

(1)

(0)

(13)

(5)c

(2)

0.2

3.8

1.3

1.3

6.3

2.5

0

1.3

0

0

3

1.7

0.8

(1)

(3)

(1)

(1)

(5)

(2)

(0)

(1)

(0)

(0)

(3)

(4)

Q)

—

<0.001

0.012

0.005

<0.001

0.003

0.097

<0.001

>0.1

>0.1

<0.001

<0.001

0.012

0

18.

2.

0

0

1.

0

0

0

0

0

0

0

(0)

8(15)b

5(2)

(0)

(0)

3(1)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

0.2 (1)

0 (0)

0 (0)

0 (0)

0

0

0

2.5

0

1.3

0

0

0

(0)

(0)

(0)

(2)

(0)

(1)

(0)

(0)

(0)

0 (0) 0.2 (1)

<0.001

0.002

>0.1

>0.1

0.076

>0.1

0.007

>0.1

>0.1

>0.1

>0.1

>0.1

0

0

0

0

0

0

0

0

1.3

0

0.4

0

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(1)

(0)

(if

(0)

0

0

0

0

1.

0

0

0

0

0

(0)

(0)

(0)

(0)

5(1)

(0)

(0)

(0)

(0)

(0)

0.4 (1)

0 (0)

•Pooled, age-adjusted analysis for heterogeneity between observed and expected tumor incidences according to Peto et al. (11). Reported P-values are twotailed.

bThree animals showed both a hepatocellular carcinoma and a malignant hemangioendothelioma.

cOne animal showed both a hepatocellular carcinoma and a cholangioma.

Tabk V.

Group

no.

1

2a

2b

2c

3a

3b

3c

4a

4b

4c

5a

5b

5c

Incidence and organ

Mode of

treatment

Control

NDEA HD

NDEA MD

NDEA LD

NPYRHD

NPYR MD

NPYR LD

NDE1A HD

NDHA MD

NDHA LD

HCD

MCD

LCD

distribution of gastrointestinal

Percentage of animals (no.

Oral cavity*

Malignant

1 (5)

3.8(3)

6.3(5)

2.5 a)

1.3(1)

3.8 (3)

2.5 a)

2.5 a)

0 (0)

3.8 (3)

1 (1)

2.1 (5)

1.7(4)

Benign

0

0

0

1.;

0

0

0

0

0

0

1

0

0

(0)

(0)

(0)

1(1)

(0)

(0)

(0)

(0)

(0)

(0)

(1)

(0)

(0)

tract tumors

of animals) with

Esophagusb

Malignant

0.2

5

0

0

0

0

0

0

0

0

0

0.4

0

(1)

(4)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(1)

(0)

tumors of the

Benign

0

21.:

0

0

0

0

0

0

0

0

0

0

0

(0)

»(17)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(0)

Stomach0

Malignant

1.8(9)

3.8 (3)

1.3(1)

5 (4)

2.5 a)

0 (0)

1.3(1)

1.3 (1)

1.3(1)

1.3(1)

2 a)

1.7(4)

1.3(3)

Benign

1.8(9)

0 (0)

0 (0)

2.5 a)

0 (0)

3.8 (3)

2.5 a)

2.5 (2)

1.3(1)

2.5 a)

4 (4)

0.8 a)

2.5 (6)

Intestine''

Malignant

0.8 (4)

0 (0)

2.5 a)

0 (0)

2.5 (2)

1.3(1)

2.5 a)

1.3(1)

1.3(1)

1-3(1)

1 (1)

1.3(3)

0.8 a)

Benign

0.2

0

0

0

1.3

1.3

0

0

0

0

0

0.8

0

(1)

(0)

(0)

(0)

(1)

(1)

(0)

(0)

(0)

(0)

(0)

a)

(0)

No. of histologic diagnoses (main group no.)

•Squamous cell carcinoma: 4(1), 1(1), 4(3), 2(4), 7(5); fibrosarcoma: 1(1), ia), 3(4), 1(5); adenocarcinoma: 2Q), 2(3), 2(5); papilloma: ia), 1(5).

b

Squamous cell carcinoma: 1(1), 4(2), 1(5); papilloma; 17a).

c

Squamous cell carcinoma: 7(1), 6^2), 2(3), 1(4); fibrosarcoma: 1(1), ia). 1(3), 1(4); adenocarcinoma: 1(1), ia), 1(4); papilloma: 9{1), 2a), 5(3), 5(4).

d

Squamous cell carcinoma: 1(1); fibrosarcoma: 2(1), 2a), 1(3), 1(4); adenocarcinoma: 1(1), 3(3), 2(4); hemangioma: 1(1), 1(3); polypus: 2(3), 1(5).

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M.R.Berger, D.Schraahl and H.Zerban

Table Via. Tumor distribution in non-target organs*

Group

no.

Mode of

treatment

Control

Percentage of animals (no. of animals) with tumors of the

Mammary gland Pancras Adrenal gland Pituitary gland Thyroid gland Reticulo/histiocytic system Integumental system

8.4 (42) 13.4 (67) 53.6 (268) 17.4 (87) 23.8(119) 5 (25) 5.2 (26)

2a

2b

2c

3a

3b

3c

3a

4b

4c

5a

5b

5c

NDEA

NDEA

NDEA

NPYR

NPYR

NPYR

NDE1A

NDE1A

NDE1A

HCD

MCD

LCD

HD

MD

LD

HD

MD

LD

HD

MD

LD

3.8

6.3

8.8

6.3

13.8

8.8

8.8

8.8

13.8

9

11.3

5.4

(3)

(5)

(7)

(5)

(11)

(7)

(7)

(7)

(11)

(9)

(27)

(13)

3.8 (3)

11.3(9)

6.3(5)

15 (12)

8.8(7)

13.8 (11)

11.3(9)

15 (12)

12.5 (10)

11 (11)

13.3 (32)

8.8 (21)

48.8

45

52.5

50

58.8

57.5

57.5

57.5

51.3

53

48.3

50

(39)

(36)

(42)

(40)

(47)

(46)

(46)

(46)

(41)

(53)

(116)

(120)

8.8

18.8

18.8

15

16.3

16.3

13.8

18.8

21.3

19

22.1

17.5

(7)

(15)

(15)

(12)

(13)

(13)

(11)

(15)

(17)

(19)

(53)

(42)

15

8.8

17.5

21.3

16.3

16.3

18.8

31.3

27.5

15

19.2

25.4

(12)

(15)

(14)

(7)

(13)

(13)

(15)

(25)

(22)

(15)

(46)

(61)

2.5

1.3

3.8

1.3

1.3

1.3

6.3

1.3

5

5

3.8

2.1

(2)

(1)

(3)

(1)

(1)

(1)

(5)

(1)

(4)

(5)

(9)

(5)

1.3

1.3

(1)

(11)

2.6(2)

5

6.3

2.6

1.3

5

5

3

4.2

2.5

(4)

(5)

(2)

(1)

(4)

(4)

(3)

(10)

(6)

"Definition as non-target organ was based on non-significant differences from group 1 according to a pooled,

observed and expected tumor incidences as described by Peto a al. (11).

Table VIb.

Group

no.

, Tumor distribution

Mode of

treatment

in non-target organs"

Percentage of animals (no.

Skeleton Heart

of animals) with tumors of the

External auditory canal Peritoneal cavity

age-adjusted analysis

Testes

for heterogeneity between

Lungs Other organs

Control 1.6(8) 0.8 (4) 0.8 (4) 0.4(2) 0.8 (4) 0.4(2) 0.2 (1)

2a

2b

2c

3a

3b

3c

4a

4b

4c

5a

5b

5c

NDEA HD

NDEA MD

NDEA LD

NPYR HD

NPYR MD

NPYR LD

NDE1A HD

NDE1A MD

NDHA LD

HCD

MCD

LCD

1.3

0

1.3

1.3

1.3

0

0

0

0

3

1.3

1.3

(1)

(0)

(1)

(1)

(1)

(0)

(0)

(0)

(0)

(3)

(3)

(3)

1.3(1)

0 (0)

1.3(1)

0 (0)

1.3(1)

0 (0)

1.3(1)

1.3(1)

1.3(1)

1 (1)

0.4(1)

1.3(3)

0 (0)

1.3(1)

0

1.

1.

0

0

0

0

1

0.

1.

(0)

3(1)

3(1)

(0)

(0)

(0)

(0)

(1)

8(1)

.3(3)

1.3(1)

0 (0)

0 (0)

1.3(1)

0 (0)

0 (0)

0 (0)

1-3(1)

0 (0)

2 (2)

0.8(2)

1.3(3)

0

0

2.

0

0

0

(0)

(0)

5(2)

(0)

(0)

(0)

1.3(1)

1.

0

0

0

0.

3(1)

(0)

(0)

(0)

.8(2)

0

0

1.3

0

0

0

1.3

0

0

0

0.4

0.8

(0)

(0)

(1)

(0)

(0)

(0)

(1)

(0)

(0)

(0)

(1)

(2)

13(1)

0 (0)

1.3(1)

1.3(1)

0 (0)

2.5(2)

1.3(1)

0 (0)

0 (0)

0 (0)

0.8(2)

0.8(2)

•Definition as non-target organ was based on non-significant differences from group 1 according to a pooled, age-adjusted analysis for heterogeneity between

observed and expected tumor incidences as described by Peto et al. (11).

< 0.03 and P < 0.002 respectively) and the LCD caused a

significant increase in tumors of the hematopoietic and lymphatic

system (P<0.04).

The dose —response curves of all malignant liver tumors following the administration of NDEA, NDE1A, NPYR and their

combinations are shown Figure 3. Interestingly, a less steep

increase of malignancies was found in the lower dose range.

To estimate the mode of additivity, i.e. a possible deviation

from simple additivity in an antagonistic or synergistic way, the

sums of the tumor incidences following the MDs and LDs of

the individual Af-nitrosamines were compared with the tumor

incidence following the simultaneous administration of these

dosages, i.e. the HCD and MCD respectively. A difference of

6% was found between the sum of liver tumor incidences observed after the medium doses (10%, groups 2b + 3b + 4b,

Table III) and the liver tumor incidence following the combination

HCD (16%, group 5a, Table HI). This difference, however, was

not significant. When the sum of liver tumor incidences observed

after the MDs is corrected for the background of normally occurring liver tumors by subtracting twice the incidence of the

control, i.e. group 5a is equated to groups 2b + 3b + 4b -

(2 x group 1), the difference is more accentuated (7.2 instead

of 6%) but still insignificant. Only when comparing the respective

incidences of hepatocellular tumors (5 versus 16%, groups 2b

+ 3b + 4b versus group 5a, Table IV) was significance evident

in the x

2

 test (P < 0.02). The importance of this finding, which

would indicate overadditive synergism in carcinogenesis, is

diminished by the fact that the incidence of tumors of hepatocellular origin was merely additive when the sum of tumor rates

induced by the LDs of the individual nitrosamines (3.7%, groups

2c + 3c + 4c, Table IV) was compared with the tumor rate

induced by the combination MCD (3.8%, group 5b, Table IV).

With regard to all liver tumors, the ratio was 6.3:4.2% (groups

2c + 3c + 4c versus group 5b, Table HI) or 5:4.2% when

corrected as described above and thus reversed. Comparison of

both dose levels suggests a linear additivity of /V-nitrosamine1640

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A combination experiment with three genotoxk /V-nitrosamines

Table VII. Distribution of foci of altered hepatocytes in relation to time

Group

DO.

1

2a

2b

2c

3a

3b

3c

4a

4b

4c

5a

5b

5c

Mode of

treatment

Control

NDEA HD

NDEA HD

NDEA LD

NPYR HD

NPYR MD

NPYR LD

NDE1A HD

NDE1A MD

NDE1A LD

HCD

MCD

LCD

% sample size

w

22 (108)

50(40)

33(26)

25(20)

25(20)

26(21)

25(20)

39(31)

24 (19)

21(17)

31 (31)

28(66)

27(64)

12-24 months

No. of animals

within period

56

12

13

12

14

10

6

17

6

6

16

23

27

% animals with

clear/acidophilic

cell focib

11

42

31

17

57

40

17

29

0

0

44

30

37

% animals with

mixcd/basophilic

cell foci"

0

33

0

8

7

0

0

0

0

0

0

0

0

25-30 months

No. of animals

within period

52

28

13

8

6

11

14

14

13

11

15

43

37

% animals with

clear/acidophilic

cell foci"

37

71

31

38

67

46

50

64

62

46

80

61

57

% animals with

mixed/basophilic

cell focib

8

32

8

13

50

9

0

7

7

18

7

14

16

'Animals (per cent of total group size) which were examined for foci of altered hepatocytes.

b

Percentage of animals of investigated sample.

induced syncarcinogenicity. In a further approach the statistical

model

(A) % incidence = a + b\X\ + b^x2 + ^3*3

(x, = dose of NDEA, x2 = dose of NPYR, x3 = dose of

NDE1A) was formally fitted to the frequency data obtained in

the respective groups. Its goodness of fit was compared with that

of this model plus an interaction term of all three carcinogens

which accounts for non-additive influences:

(B) % incidence = a + b^xx + £2*2 + ^3*3 + *i,2,3*1*2*3

The goodness of fit following model A was 0.927 (if statistic)

and that following model B 0.946. The small increase found

indicates that the frequency data observed in the combination can

mostly be explained by the assumption of linear additivity.

Tables Via and VIb present the occurrence of all other tumor

types, their organ relation and incidence. None of these tumors

was treatment related so that they can be classified as 'spontaneous'.

The incidence of putative preneoplastic foci of altered hepatocytes is summarized for all experimental groups and controls in

Table VH. With a few exceptions, the number of animals with

foci of clear and acidophilic glycogen storage cells appearing early

during hepatocarcinogenesis showed a dose-dependence which

was similar to that of hepatic adenomas and carcinomas (see Table

IV) developing later. The higher the single doses of the carcinogens administered the more foci of altered hepatocytes emerged.

This holds true for both the administration of the different nitrosamines given alone and the combined administration of these

compounds. Another interesting result is that the number of

animals with clear and acidophilic cell foci increased with time

and total dose of the carcinogen(s) applied. The mixed and basophilic foci which are considered later phenotypic changes in

hepatocarcinogenesis were only rarely seen in the second year

of treatment but are found in nearly all experimental groups in

the third year. A well-defined dose-dependence of these focal

cell populations is not evident. It should also be mentioned that

70-

0.01

Dose • response relationships for all malignant

tumors of the liver following administration of

NDEA, NPYR, NDELA or their combination

NDEA

NDELA

0.1 1 10

Single dose of N • nitrosamines (mg / kg)

Fig. 3. Log-probit scale on the dose-dependent incidence of all malignant

tumors of the liver following administration of NDEA, NPYR, NDELA or

their combination.

the number of treated animals bearing foci of altered hepatocytes

is not increased over the background of the controls in some

experimental groups. This is not surprising, however, since all

qualitative data of individual animals were collected, which do

not reflect the number and size of foci in these animals.

Discussion

The present study gives a highly accurate account of the doseresponse relationships of very low doses of NDEA, NDELA and

NPYR. The main target organs of these nitrosamines (NDEA:

liver, upper gastrointestinal tract and kidneys; NPYR: liver;

NDELA: liver and nasal cavity; for review see 14,15) could be

confirmed in this study except when significantly increased tumor

1641

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M.R.Berger, D.SchmShl and H.Zerfoan

incidences were found at higher dosages only, which were not

included in our experiment. For example, nasal cavity tumors

following NDE1A were not observed because they have been

described to occur following dosages exceeding 6 mg/kg only

(8). It was demonstrated in rats that the hepatotropic carcinogenicity of these genotoxic N-nitrosamines is additive when they are

administered in combination. This was also true for nitrosamine

doses which alone were not (NDE1A, NPYR) or presumably

(NDEA) not carcinogenic within the lifespan of animals under

the bioassay conditions applied (LCD, group 5c, Tables in and

TV). Thus this experiment confirms and extends previous studies

on the syncarcinogenicity of these agents (1,2).

The importance of these findings becomes more evident when

the dose range used in this study is compared with nitrosamine

amounts resulting from certain life-style habits. According to

Hoffmann et al. (16; for review see 15), for instance, humans

smoking 20 cigarettes/day inhale 1 -9 0 /tg NDEA, 30-330 /xg

NPYR and 0.3-0.4 /tg NDElA/m2 of body surface. Except for

NDE1A, these amounts correspond well to the dosages used per

m

2

 of body surface in the rat experiment (1— 42 /tg NDEA,

6-168 fig NPYR and 27-840 fig NDE1A), which were found

to be carcinogenic in combination.

The assessment of the carcinogenic hazard inherent in very

low doses of /V-nitrosamines was made possible by the long survival of all animals and by the low incidence of 'spontaneous'

liver tumors in control rats. Since no reduction of life-expectancy

was found in treated groups, except following the two high dosages of NDEA (groups 2a and 2b), only the causes of death were

changed by the treatment-related increases in liver cancer incidence. To achieve a sufficient survival time, male rats were used

to exclude reduced life-expectancy in the animal population due

to tumor deaths found more frequently in female rats, e.g. mammary gland neoplasias. Syncarcinogenesis is likely to be affected

irrespective of the sex of the animals. The observed low incidence

of 'spontaneous' liver tumors (0.6%) permitted a smaller number

of animals to be used (17) and yet allowed discrimination of small

increases in liver tumor incidence due to the treatment. Consequently, such low concentrations as 0.14 p.p.m. NDEA, 1.9

p.p.m. NPYR or 2.9 p.p.m. NDE1A, which were ingested via

the drinking water, were significantly carcinogenic in Sprague —

Dawley rats at sufficiently long exposure.

Other authors reported a 6—8% spontaneous liver tumor incidence in Col worth rats (18) and 5-20% in Fischer F-344 rats

(19,20). It was thus considerably more difficult in these experiments to identify carcinogenic effects of very low doses of

nitrosamines.

Under normal environmental conditions, humans — unlike

laboratory animals — are exposed concomitantly or sequentially

to several carcinogens. Syncarcinogenesis is therefore likely to

be an important problem. We tried to approach this aspect by

simultaneously administering genotoxic compounds with identical

organotropism. The characteristic organotropism of the individual

compounds was not changed in the chosen combinations, and

the cytotropism within the liver was almost limited to liver cells

only. Besides the liver, other organs revealed a significant increase in tumor incidence only when at least two of the three

compounds administered in combination effected a treatmentrelated increase in tumor incidences (gastrointestinal tract, neurogenic tissue, urinary tract) over indivual administration. In line

with this observation a significant dose—response relationship

was observed only for liver tumors; this contrasts with all significantly increased incidences of non-liver tumors which were

found following the combinations of NDEA, NPYR and NDE1A.

The decrease from the high to the medium doses and from

the medium to the low doses of the individual compounds and

of the combinations indicates that the dose —response curves

clearly are less steep in the lower dose range, i.e. more tumors

were formed in the low-dose range than the steep decrease from

the high to the medium doses would have suggested (Figure 3).

The resulting graphs show a hockey stick profile which was

described to be indicative of repair (21). Repair phenomena are

supposed to occur following low dosages of certain nitrosamines

such as N-nitrosodimethylamine and NDEA (22,23). Biochemically less C^-ethylguanine can be found in the liver of NDEAtreated rats when the animals are pretreated with NDEA for 5 -10

weeks as compared with unpretreated controls (24,25). The

C^-ethylguanine adduct is considered to be promutagenic and

appears to be removed by an alkyl transferase. The percentage

of removed ethylguanine, however, is lower when higher doses

of the carcinogen are administered. This saturation type behavior

of the enzyme is claimed to be responsible for the observed

hockey stick shape of the curve (21). On the other hand, the

observed profile disputes the efficiency of the repair system at

very low dosages since liver tumor formation was still detectable

at the lowest dosages used.

Unlike the dose-response curves established for liver tumors,

the incidence of esophageal tumors induced by NDEA fell from

26% following administration of 0.1 mg/kg (NDEA HD) to not

detectable levels following 0.032 mg/kg (NDEA MD) and 0.01

mg/kg (NDEA LD), i.e. the dose—response curve of esophageal

tumors was much steeper. The reasons for this discrepancy between liver and esophageal tumors induced by NDEA remain

unclear. The result is, however, in accordance with our previous

experiments on NDEA-caused carcinogenic effects (5), when

esophageal tumors occurred at but not below dosages corresponding to 0.1 mg/kg (NDEA HD). It differs from results reported

by Peto et al. (18), who found measurable tumor incidences of

the esophagus in Colworth rats at a dose range < 0.1 mg/kg/day.

It also differs from the results reported by Lijinski et al. (19)

who observed increased frequencies of esophageal tumors in

Fischer rats after weekly dosages of 0.045 mg/animal. This dose

is in between the two lower dosages of NDEA used in this experiment, which did not cause tumors of the esophagus. The

response of Colworth and Fischer rats is obviously greater than

that of SD rats with regard to the induction of esophageal tumors.

Syncarcinogenesis by polycyclic aromatic hydrocarbons

(PAHs) applied by skin painting to NMRI mice was demonstrated

previously. Similar linear additivity of the carcinogenic effects

of both PAHs (26,27) and N-nitroso compounds was observed.

Thus, compounds which belong to the same class of agents and

have a common target organ, i.e. the skin or liver, show linear

additive effects. Since in both classes of compounds similar or

identical mechanisms of activation lead to the ultimate carcinogen

(28,29), this might explain the observed linear additivity. When,

however, compounds with the same target organ but different

metabolic pathways, such as N-methylnitrosourea and 7,12-dimethylbenzanthracene, were administered concomitantly to 50-

day-old female Sprague—Dawley rats, the observed tumorigenicity was less than additive (30).

The observations on foci of altered hepatocytes preceding the

development of hepatic adenomas and carcinomas are of considerable interest in view of the controversially discussed usefulness of these lesions as endpoints in carcinogenicity testing (31).

The results presented support the concept that there is not only

a qualitative but also a quantitative precursor—product relationship between foci of altered hepatocytes and hepatic tumors

1642

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A combination experiment with three genotaxk JV-nitrosamines

(32-36). In addition, the observations are in line with the proposed sequence of phenotypic cellular changes during hepatocarcinogenesis leading from clear and acidophilic cell foci through

mixed and basophilic cell foci to hepatic adenomas and carcinomas (10, M.Enzmann and P.Bannasch, in preparation).

The few exceptions to a well-defined dose-dependence of the

focal prenoeplastic lesions observed may be explained by the fact

that only animals with foci but not the number and size of foci

in the individual animal were determined in this study. This point

requires a detailed morphometric analysis based on a more systematic selection of liver tissue to be examined. Since single small

foci of altered hepatocytes frequently also appear in untreated

old animals an unequivocal prognosis of tumor development will

only be possible when the volume density of the foci is defined

in both treated animals and untreated controls. With this reservation in mind, our results suggest that the foci of altered hepatocytes may well be taken as endpoints in carcinogenicity testing.

A review by D.Schmahl on combination effects in carcinogenesis is in preparation. Future experiments on syncarcinogenic

effects of genotoxic compounds with different metabolic activation and different target organs will supplement this study and

elucidate the stated theoretically possible interactions in carcinogenesis in defined biological models.

Acknowledgements

We thank Dr L.Edler, Institute of Epidemiology and Biometry, German Cancer

Research Center, for valuable biostatistical consultation. The skilful technical help

of Mrs. A.Weninger, D.Theis and A.Danisman is acknowledged.

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Received on March 30, 1987; accepted on August 10, 1987

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