Ethylnitrosourea Treatment Research Articles

Exposure to Ethylnitrosourea Before Implantation Induces Congenital Malformations in Mouse Fetuses

Abstract The effects of ethylnitrosourea (ENU) on the development of preimplantation mouse embryos were investigated. ICR mice were treated intraperitoneally with single doses of 25, 50 or 100 mg ENU/kg body weight on day 0, 1, 2 or 3 of gestation, or with single doses of 25, 50 or 75 mg ENU/kg on day 8 of gestation. The uterine contents were examined on day 18 of gestation, and viable fetuses were inspected for external and skeletal malformations. No significant differences were observed in the number of implants between the ENU‐treated groups on day 1, 2 or 3 of gestation and controls, while the number of implants in all of the groups treated with ENU on day 0 of gestation was significantly decreased compared to that in the control group. The frequencies of early postimplantation deaths were significantly increased in all of the groups treated with ENU on each gestational day before implantation, compared to the control frequencies. ENU treatment before implantation caused dose‐dependent increases in the incidence of externally or skeletally malformed fetuses. Cleft palate, exencephaly and umbilical hernia were the most common types of external malformations in the groups treated with ENU before implantation and in the control group. The skeletal malformations seen in the ENU‐treated groups were malformed vertebrae, malformed ribs, and bending of appendicular skeleton. Fused ribs was the most common skeletal malformation seen in the control fetuses. The type distributions of external and skeletal malformations induced by the treatment with ENU before implantation is quite different from those of fetal malformations induced by the treatment with ENU at the organogenesis stage. The results in the present study demonstrate that embryos before implantation in the uterus are susceptible with regard to the induction of congenital malformations by chemicals, and I propose that a large portion of the external malformations in fetuses treated at the preimplantation stages is the result of increased yields of spontaneously‐occurring malformations.

Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice.

Transgenic B6C3F1 mice carrying a lacI target gene were exposed to acute and multiple doses of ethylnitrosourea (ENU), and germ cells from the seminiferous tubules were assayed for mutation 3 and 90 days after treatment. Relative to untreated controls, the mutation frequency increased 3.2- and 19.9-fold at 3 and 90 days after treatment, respectively. Mutant lacI genes recovered from untreated and treated groups were sequenced, and the spectra of mutations were determined. Eighty-five percent (11/13) of the spontaneous mutations resulted in G.C-->A.T transitions, all of which occurred at CpG dinucleotides. Fifteen of 22 sites (68%) found mutated 3 days after ENU treatment occurred at G.C base pairs, although some of these are expected to be spontaneous mutations. Ninety days after treatment, 13 of 19 sites (68%) found mutated occurred at A.T base pairs. The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in vivo spectra seen in ENU studies by using transmissibility assays and the hprt gene. These findings represent significant progress toward defining the in vivo spectra of ENU mutagenesis in mammalian germ cells.

Congenital malformations in the offspring of male mice treated with ethylnitrosourea at the embryonic stage.

Ethylnitrosourea (ENU) was i.p. injected into ICR female mice at 25 or 50 mg/kg on day 10 of gestation, and male newborns treated with ENU at the embryonic stage were obtained. The treated males were aged for 10 weeks and then mated to untreated females of the same strain. The male germ cells used in the copulation correspond to primordial germ cells (PGC) at the time of ENU treatment. The F1 offspring were sampled as fetuses on day 18 of gestation and inspected for external and skeletal malformations. Evidence of F1 teratogenesis due to the mutagenized PGC was obtained when the fetuses from the males treated with 25 mg ENU/kg were inspected the frequencies of skeletally malformed fetuses in the ENU-treated series and the control being, respectively, 1.0% (14/1,360) and 0.3% (3/1,017). The fetuses from 50 mg/kg-treated males did not show a significant increase in malformations, most probably reflecting a high vulnerability of PGC to the killing effect of ENU. The findings in this study suggest that germ cell stage at PGC is at risk for the induction of congenital malformations by environmental chemicals.

Effect of Lung Development on the Histological Pattern of Lung Tumors Induced by Ethylnitrosourea in the C3HeB/FeJ Mouse

The number, size, and histological pattern of lung tumors collected 6 months after being induced in C3HeB/FeJ mice by ethylnitrosourea (ENU) treatment on one of days 13-19 of gestation, or days 5, 15, or 35 after birth, or as 9-month-old adults was investigated. The number of lung tumors induced increased when treatment occurred between days 13-16 and then decreased when treatment occurred on days 17-19 of gestation. There was a marked increase in lung tumor numbers induced on day 5 after birth, which decreased to much lower levels on days 15 and 35 after birth and in the treated adult mice. The size of lung tumors was greater in males than females in all groups and decreased as the age at the time of ENU treatment increased. A greater proportion (60-65%) of papillary tumors were induced following treatment on days 13-16 whereas, with one exception, alveolar lining tumors were the predominant histological type when treatment occurred after day 16. When treatment occurred on day 5 after birth, papillary tumors were once again the predominant type (66%) induced. The changes in tumor numbers induced and in the histological type of tumor induced occur at periods when major shifts in the histological development of the lung occur. The results indicate that the histological type of tumor induced by ENU treatment is developmentally regulated and suggests that alveolar lining and papillary tumors behave as two separate populations of tumors.

Induction of dominant cataract mutations in golden hamster by ethylnitrosourea

In in vivo experiments using the spot test with mice, diethylhexylphthalate (DEHP) was co-recombinogenic and anti-mutagenic. In two independent experiments, DEHP was able to increase in combination with ethylnitrosourea (ENU) the frequency of animals with spots of genetic relevance from about 12% (ENU alone) to about 15% (ENU + DEHP). This enhancement can be exclusively attributed to an enhancement of recombination. In historical as well as in current experiments, with astonishing accuracy, about 8% of all spots induced with ENU alone are black, near-white or twin spots, i.e., presumed products of reciprocal recombination. Under the influence of DEHP, about 20% of all colored spots were black, near-white or twin spots. These co-recombinogenic effects are very strong, considering that the low frequency of twin spots with 0.3% (historical ENU control) or 0.6% (current ENU control) after ENU treatment alone has been enhanced up to a frequency of 3%. While ENU alone induces about 14% light brown colored spots, depending exclusively on gene mutations, under the influence of DEHP the frequency was only 7%, i.e., DEHP was anti-mutagenic. Pentachlorophenol was ineffective in enhancing or reducing recombination or mutations. There was no difference between ENU and ENU + PCP treatment. Since a number of ‘non-genotoxic’ carcinogens with tumor-promoting activities like d-limonene, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) have shown similar effects in the spot test, it is suggested that DEHP may have tumor-promoting activity in carcinogenicity tests.

Tumour incidence in the progeny of male rats exposed to ethylnitrosourea before mating

BDVI male rats were given a single i.p. dose of 80 mg/kg b.w. ethylnitrosourea (ENU), and each rat was then mated at weeks 1, 2, 3, 4 and 5 after treatment with 3 untreated females. A decrease in the fecundity of the treated males was observed, particularly when they were mated 5 weeks after ENU treatment. The average litter size was lower in the treated group, especially for females mated in week 4. No significant differences in pre- or post-weaning mortality were noted between control and treated groups. A slight, non-significant increase in the incidence of brain tumours was observed in the progeny of treated males compared with the controls. The incidence of thyroid tumours was significantly higher in controls but this difference disappeared when adjustment was made for litter effect and intralitter dependence.

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics in F 1 mice derived from post-spermatogonial treatment with ethylnitrosourea

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics was determined in F 1 mice derived from post-spermatogonial germ-cell stage treatment with 2 × 80, 160 or 250 mg/kg ethylnitrosourea. A total of 5 dominant cataract mutations, 3 dominant cataract mutation mosaics, 1 specific-locus mutation and 9 specific-locus mutation mosaics were recovered in 15,542 screened F 1 offspring. Results indicate that ethylnitrosourea treatment increases the mutation rate of dominant cataract and recessive specific-locus alleles in post-spermatogonial germ-cell stages of the mouse and that the mutations occur mainly as mosaics. Genetic confirmation of newly induced mutations occurring as mosaics is more problemtical for induced recessive alleles than for induced dominant alleles and should be considered when evaluating such mutagenicity results.

Diaplacental induction by ethylnitrosourea of tumours at the pial border of the central nervous system in (T × HT)F1 mice

Ethylnitrosourea (ENU) was diaplacentally applied to (T X HT)F1 mice at a dose of 40 mg/kg on different gestation days during organogenesis and early fetal stages by i.p. injection to the dams. The animals were particularly sensitive to induction of tumours at the central nervous system (CNS)-skull-vertebra interface (30 and 20% in ENU-treated male and female offspring respectively, compared with 1% in controls). ENU treatment during the late organogenesis stage (gestation days 8-11) proved to be less efficient in tumour induction than during the subsequent early fetal period (gestation days 12-14). Ninety-two per cent of the CNS tumours were located at the interface between the CNS and the osseous surrounding. The distribution of these tumours at the pial border was inhomogeneous: 69% were found at the brain-skull border, 6% of the tumours occurred within the cervico-thoracal districts and 25% within the lumbo-sacral districts of the spinal cord-vertebra interface. Histological classification revealed a preferential occurrence of neuroepithelial tumours in male offspring (approximately 20%) and only approximately 7% Schwann cell tumours and approximately 3% tumours of meningomesenchymal origin. In female offspring neuroepithelial tumours and Schwann cell tumours were observed at about an equal rate (9-10%), in contrast to meningo-mesenchymal tumours (1%). Nearly 98% of these tumours were situated at the basal districts of the space between the CNS-skull and spinal cord-vertebra. This indicates a particular sensitivity of the basal neurothelium, a derivative of neural crest cells, for ENU-induced carcinogenesis. The pluripotency of these cells during the mid-gestation period apparently enables growth of different histopathological tumour types, which arise independently from each other.

Simultaneous induction of mutagenic and cancerogenic effects in T × HT mice with transplacental ethylnitrosourea treatment

The relationship between mutagenesis and carcinogenesis was investigated in T x HT crossbred mice using diaplacental application of ethylnitrosourea (ENU) at different stages of embryonal development. Mutagenesis was detected by induction of coat color spots, and the carcinogenic response was investigated in a long-term follow-up study of the F1-generation. The animals were particularly sensitive to induction of tumors at the central nervous system (CNS)-skull/vertebra interface (30% and 20% in ENU-treated male and female offspring, respectively, compared with less than 1% in controls). There was a correlation between the appearance of these tumors and the presence of color spots. This correlation was low but statistically significant in female offspring. Three other types of tumors showed a correlation with the presence of coat color spots. Liver tumors were significantly increased in color spot-positive females but unchanged in males. Lung tumors were reduced in color spot-positive males and appeared earlier in color spot-positive females. There was a lower incidence of lymphoma/leukemia in all spot-positive mice. The reduction in tumor incidence beyond the spontaneous rate in spot-positive animals might be caused by a high cytolethal response to ENU in the relevant organs and tissues.

Administration of Ethylnitrosourea to Neonate Hamsters Increases Growth and Frequency of SV40-Induced Fibrosarcomas

The in vivo interaction between the chemical carcinogen ethylnitrosourea (ENU) and the oncogenic simian virus 40 (SV40) was studied. Inbred newborn Syrian golden hamsters were injected subcutaneously with SV40 (5 x 10(6) plaque-forming units), ENU (0.5% solution, 125 or 25 mg/kg body wt), or equal mixtures of the two. Animals that received SV40 and ENU developed more tumors (100% vs 52%) within a shorter latent period (10 weeks vs 18 weeks) than animals that received SV40 alone. Animals given SV40 and ENU showed increased mortality and increased metastatic tumors (54.2% vs 30.8%) compared with those given SV40 alone. The SV40 and ENU group also exhibited multiple (greater than 10 nodules) pulmonary metastases (33.3% vs 7.7%) and metastases in multiple organs (12.5% vs 0%) compared with animals injected with SV40 alone. No difference in primary tumor size, histology, and SV40 T-antigen content was detected between SV40- and SV40/ENU-induced tumors. Four weeks after SV40 or SV40 plus ENU treatment, animals were challenged intradermally with 2.7 x 10(6) SV40-transformed hamster cells. Five weeks after challenge, 89.5% of the animals treated with SV40 and ENU and 45.4% of animals treated with SV40 developed tumors at the challenge site. Newborn animals given SV40 and ENU developed larger tumors at the challenge site (P less than 0.002) than newborns treated with SV40 alone. Thus, administration of ENU to hamsters during the neonatal stage of development produced a long-lasting systemic effect that enhanced tumor development by transplanted SV40-transformed hamster cells.

The effect of dose fractionation on the frequency of ethylnitrosourea-induced dominant cataract and recessive specific locus mutations in germ cells of the mouse

A combined dominant cataract-recessive specific locus mutation experiment for fractionated exposure to ethylnitrosourea (2 × 80 mg/kg, 24-h fractionation interval) was designed to determine if lower doses of ethylnitrosourea are more effective in inducing dominant cataract mutations as suggested by previous results. This observation was not confirmed by the present experiment. The extensive, statistically more reliable specific locus results indicate an additive effect of fractionated ethylnitrosourea treatment. A saturable repair system for ethylnitrosourea-induced DNA damage has been previously documented (Karran et al., 1979; Sega et al., 1986; Van Zeeland et al., 1985). Two parameters inherent to a saturable system, the minimal time required for the saturated system to recover and the minimal dose to saturate the system are important, and results of experiments employing a fractionation exposure protocol must be interpreted relative to these two parameters. Longer fractionation intervals or smaller doses result in a reduced mutagenic effect. Due to the inherently lower experimental variability of the specific locus mutation assay as compared to the dominant cataract assay, the specific locus assay is the test of choice to determine factors affecting the mammalian germ cell mutation rate. The dominant cataract test requires a larger investment of experimental resources to achieve a comparable degree of accuracy. The dominant cataract mutation test is important in assessing the mutation rate to dominant alleles in germ cells of mammals. Due to the immediate expression of the mutant phenotype in newly occurring dominant mutations, a dominant mutation assay screens a genetically relevant endpoint in an assessment of the mutagenic hazard for man in mouse experiments. A multi-endpoint design screening specific locus, dominant cataract, and biochemical mutational endpoints (Ehling et al., 1985) allows a systematic comparison of mutagenic results for different classes of mutations in the same animals.

Nonrandom distribution of structural mutants in ethylnitrosourea-treated cultured human lymphoblastoid cells.

Two-dimensional polyacrylamide gel electrophoresis has been used to detect somatic cell gene mutations altering protein structure, following ethylnitrosourea treatment of cultured human lymphoblastoid cells. A total of 267 polypeptides encoded by 263 loci were scored in a series of 1143 lymphoblastoid clones. Sixty-five electrophoretic mutants were detected at a total of 49 loci. Sixteen of the 65 mutations were phenotypically repeat mutations, occurring at 11 loci. Furthermore, structural mutations occurred more frequently at loci known to be polymorphic. These results provide evidence that the mutations that are detectable at the protein level by two-dimensional polyacrylamide gel electrophoresis do not occur at random and that their frequency is greater among polymorphic loci.

Ethylnitrosourea treatment increases lectin binding to mouse germ cells

Germ cell toxicity was assessed by investigating the binding of FITC-labeled lectins to mouse testis cells before and 18 days after treatment with ethylnitrosourea (ENU). Flow cytometry of testis cells dual-labeled with FITC-lectin plus the DNA stain, propidium iodide, allowed analysis of haploid (1C), diploid (2C), and dividing (4C) cell populations. Soybean agglutinin, wheat germ agglutinin, concanavalin A and Limax flavus agglutinin bound to normal mouse testis cells containing 1C, 2C or 4C DNA. Asparagus pea lectin and Bandeireae simplicifolia I isolectin B 4 did not. ENU treatment reduced the number of testis cells and increased lectin binding, particularly of those lectins which bound to untreated cells.

Postnatal development and neoplastic disease pattern in NMRI mice after combined treatment with ethylnitrosourea and X-irradiation on different days of the fetal period.

Mice were X-irradiated on day 14, 15 or 16 of gestation with 1.0 Gy. This did not result in an increased tumour frequency in the offspring until 12 months. Mice treated with ethylnitrosourea (ENU) (45 mg/kg) on these gestation days developed a significantly increased tumour frequency in the lungs and liver, and in the ovaries after treatment on day 15 of gestation. Additionally this experiment was the first to show that ENU treatment on gestation day 14, 15 or 16 results in haemangiosarcomas of the subcutis at a low incidence (2.0, 2.4, 2.6 per cent). After combined treatment with these two agents in the sequence X+ENU and an interval of 4 h, a significantly increased incidence rate of animals with tumours was observed in the offspring treated on gestation day 14 or 16. Moreover, the treatment on gestation day 16 exhibited the highest tumour frequency per examined animal (5.7) of all treatment groups. This result is due to a relatively uniform increase of all tumor types. Within this pattern, the frequency of liver tumours was most marked. The diagnosed liver tumours were significantly augmented after X+ENU treatment on day 16. In the reverse sequence (ENU+X), the total tumour outcome was not significantly altered compared with the effects of ENU alone. However, detailed analysis also showed a significant augmentation of the liver tumour frequency with treatment on day 15.

Nerve growth factor modification of the ethylnitrosourea model for multiple schwannomas.

The administration of ethylnitrosourea (ENU) to pregnant rats late in gestation or to neonatal rats results in the induction of Schwann cell tumors in a high percentage of perinatally exposed animals. Exogenous administration of nerve growth factor (NGF) significantly reduces the number of Schwann cell tumors and other neurogenic tumors developing in ENU-treated rats. Administration of antibodies directed against NGF prior to neonatal ENU exposure results in a substantial increase in the incidence of Schwann cell tumors, particularly in the trigeminal nerves of both rats and mice. Transplacental ENU treatment causes early neoplastic proliferation (ENP) at 90 days of age in the Schwann cell population of trigeminal nerves in nearly all exposed rats. A variety of NGF treatment protocols (single or multiple inoculations or microinfusion prior to or following ENU exposure) resulted in a significant reduction in ENU-induced ENP in trigeminal nerves. These results indicate that NGF may convey protection either directly or indirectly, by an unknown mechanism, to Schwann cells and other supportive neural cells by reducing their sensitivity to ENU-induced neoplastic transformation.

The frequency of dominant cataract and recessive specific-locus mutations in mice derived from 80 or 160 mg ethylnitrosourea per kg body weight treated spermatogonia

A systematic comparison of the frequency of dominant cataract and recessive specific-locus mutations in mice has been extended to include results for 80 and 160 mg ethylnitrosourea per kg body weight spermatogonial treatment. The frequency of confirmed dominant cataract mutations in the historical control, 80 and 160 mg/kg ethylnitrosourea treatment groups was 1/22 594, 8/5090 and 14/6435, respectively. The frequency of recessive specific-locus mutations in the same dose groups was, respectively, 19/227 805, 20/13 274 and 35/8658. These present results confirm previous results, which indicate that ethylnitrosourea is effective in inducing both recessive specific-locus and dominant cataract mutations although the per locus mutation rate to recessive alleles was observed to be approximately 6 times greater than the per locus mutation rate to dominant alleles. The exclusion of certain classes of lens opacity variant phenotypes, previously demonstrated not to be due to a dominant mutation, from the group of suspected dominant cataract mutations subjected to a genetic confirmation test has greatly improved the efficiency of the test. A total of 23 dominant cataract mutations were confirmed from a group of 67 phenotypic variants. Of the 23 confirmed dominant cataract mutations, 8 were shown to have reduced transmission to the following generation of offspring expressing the mutant phenotype. These results are also consistent with previous results for ethylnitrosourea or radiation treatment in which it was shown that approximately one-third of the recovered mutations have reduced penetrance. One group of dominant cataract mutations, with phenotypic effects on the polar, sub-capsular or corneal regions, is overlay represented in the group of recovered mutations with a reduced transmission of offspring expressing the mutant phenotype. Two hypotheses are suggested for this observation, both dependent on the fact that the regions affected indicate that the mutations are expressed later in the development of the eye. Either all carrier individuals have not expressed to phenotype at the time of examination and classification, or later acting mutations are more subject to environmental interactions resulting in more variable expression. Finally, it is argued that a dominant cataract mutation test represents a most practicable protocol to screen for induced dominant mutations in germ cells of the mouse. The imposition of the criterion that suspected variants be subjected to a genetic confirmation test has at least two advantages beside the fact that results represent unambiguous mutational events. First, variants which are sterile can be identified, and the penetrance and fertility of confirmed mutations can be estimated. Second, a group of recovered mutations controlling a phenotype system is required to analyse genetically the loci controlling the system.

Induction of gene mutations in mice: The multiple endpoint approach

The multiple endpoint mammalian mutagenesis approach developed in our institute screens in the same animal for recessive specific-locus alleles at 7 loci, approximately 30 loci coding for dominant-cataract mutations, 23 loci controlling protein-charge changes and 12 loci for enzyme-activity alterations. Experiments to screen for the approximately 70 loci in the same offspring of treated male mice were performed with ethylnitrosourea (ENU), procarbazine and X-ray exposure. Mutations were recovered for each genetic endpoint in all treatment groups where a sufficient number of offspring was scored. ENU treatment is highly effective in inducing mutations to all genetic endpoints. The mutations were confirmed by breeding tests. The mutation rates to specific-locus and enzyme-activity alleles were both higher than the mutation rates to either dominant-cataract or protein-charge alleles. The advantages and possibilities of the multiple endpoint approach are discussed in detail.

Comparison of methods for detecting mitomycin C- and ethyl nitrosourea-induced germ cell damage in mice: sperm enzyme activities, sperm motility, and testis weight.

Testes weights, sperm motility and enzyme activities in single sperm were compared with respect to their ability to detect either developmental or mutational damage to germ cells. Male mice were injected i.p. with 2.5 mg/kg mitomycin C (MC) or 50 or 100 mg/kg ethylnitrosourea (ENU) or saline and were then killed at times such that sperm derived from treated vas sperm (SZ), spermatids (ST), preleptotene-late-spermatogonial cells (PLSG), spermatogonial cells (SG), or spermatogonial stem cells (SGS) could be evaluated. Testis weights decreased significantly as early as 1 wk after treatment, with the greatest decrease reached 3-4 wk after treatment, followed by recovery to normal levels 10-15 wk after treatment. We conclude that testis weight, which is easily obtained, is a sensitive indicator of germ cell damage by these agents. Sperm from each animal were evaluated for sperm motility, acrosin activity, succinic dehydrogenase (SDH) activity with or without the competitive inhibitor malonate or after exposure to 60 degrees C for 10 min. The latter two assays were to detect sperm enzymes resistant to the inhibitor or heat. The presence of the acrosin protein was also detected immunologically. Sperm motility decreased most from treatment of PLSG and SG. After MC or ENU treatment, the greatest loss of acrosin activity and of the acrosin protein was also noted in sperm derived from treated PLSG and SG. MC and ENU failed to induce SDH activity in single sperm resistant to 60 degrees C heat inhibition or to inhibition by malonate. Of the sperm assays, acrosin activity proved to be the most sensitive indicator of germ cell damage and was the simplest to measure.

Role of nerve growth factor in ethylnitrosourea-induced neural carcinogenesis.

The sensitivity of the rat nervous system to malignant transformation by ethylnitrosourea (ENU) is a function of age at treatment. From late gestation, nervous structures decrease in sensitivity with age as non-neural structures increase in susceptibility. There is a decrease in the proportion of neural tumors induced by ENU and an increase in survival time when nerve growth factor (NGF) levels are elevated in the fetal or neonatal stage. If antibodies directed against mouse beta-NGF (anti-NGF) are administered prior to neonatal ENU treatment, neural tumors appear earlier, although in the same proportion as with treatment by ENU alone. This effect is not observed if the ENU is administered first. This phenomenon seems to be attributed to an increased number of trigeminal nerve neurinomas, which have a shorter latent period than other nervous system tumors. The induced neurological tumors in rats treated neonatally with anti-NGF prior to ENU seem to be almost exclusively neurinomas in the peripheral nervous system. Fetal anti-NGF treatment leads to an increased number of intracerebral gliomas and a longer survival time, which corresponds to the longer latent period of these tumors. The role of NGF in the sensitivity of the rat nervous system to carcinogenesis by ENU and its possible implications in the development of the nervous system discussed.

Influence of five different postnatal lifelong treatments on the transplacental carcinogenicity of ethylnitrosourea in sprague-dawley rats

Sprague-Dawley rats received in 10 mg/kg body weight (group I) or 30 mg/kg body weight (group II) ethylnitrosourea (ENU) orally on the 19th day of pregnancy. Their offspring were treated with Bacille Calmette-Guerin, human albumin, hydrocortisone, cyclophosphamide or nicotine starting on the 6th day of life. The ENU treatment significantly reduced the life expectancy of all offspring. Treatment of the offspring did not influence tumor frequency, induction time or localization of neurogenic malignant tumors. Cyclophosphamide treatment of group II offspring increased the number of females bearing mammary carcinomas.