N-nitrosoethylurea Research Articles

Intestinal Formation of N-Nitroso Compounds in the Pig Cecum Model

N-Nitroso compounds (NOC) are a group of compounds including N-nitrosamines and N-nitrosamides, which are well-known for their carcinogenic, mutagenic, and teratogenic properties. Humans can be exposed to NOC through the diet and environmentally, or NOC can be formed endogenously in the stomach and intestine. In the intestine, the formation of NOC is supposed to be afforded by the gut microbiota. In this study, the formation of the N-nitrosamines, N-nitrosomorpholine (NMOR) and N-nitrosopyrrolidine (NPYR), and the N-nitrosamides, N-nitrosomethylurea (NMU) and N-nitrosoethylurea (NEU), was investigated in the pig cecum model after the incubation of the corresponding precursor amine or amide with nitrite or nitrate. Following the incubation with nitrate, the formation of NMOR, NPYR, NMU, and NEU was detectable with the microbiota being responsible for the reduction of nitrate to nitrite. After the incubation of nitrite a chemical formation of NOC was shown.

Effect of light/dark regimen on N-nitrosoethylurea-induced transplacental carcinogenesis in rats

Pregnant females were randomly subdivided into three groups (24 rates per group) and kept at the 12:12 h light/dark regimen (group 1), at the constant light illumination (24 h a day, group 2) or at the continuous darkness (group 3). N-nitrosoethylurea (NEU) has been injected into the tail vein of all rats (80 mg/kg) on the 18–19th day of the pregnancy. After the delivery the lacting dams and their progeny during the lactation period (1 month after delivery) were kept also at the three different light/dark regimens. Then all offspring from each group was kept at the 12:12 h light/dark regimen, males and females separately, and were observed until natural death.. The exposure to constant light significantly promoted the transplacental carcinogenesis whereas the exposure to constant darkness inhibited it. The incidence of total tumors, tumors of both a peripheral nervous system and kidney was 2.6; 2.5 and 8.5 times higher, and survival significantly shorter, correspondingly, in rats from the group 2 exposed to the constant light regimen as compared to the group 1 (12:12 h light/dark regimen) ( P<0.05). On the other hand, the exposure to the continuous darkness during the pregnancy and the lactation period significantly inhibited the transplacental carcinogenesis in the offspring of rats treated with NEU. The incidence of total tumors, tumors of a peripheral nervous system was by 2.4 and 2.7 times less, and survival longer, respectively, in exposed to the darkness rats from the group 3 as compared to the group 1 (12:12 h light/dark regimen) ( P<0.05). Thus, our data firstly have shown the modifying effect of light-dark regimen on the realization of the transplacental carcinogenesis induced by NEU in rats.

Age- and dose-dependent transplacental carcinogenesis by N-nitrosoethylurea in Syrian golden hamsters.

Syrian golden hamsters have a very short period (15 days) of gestation. The implantation of the blastocyst occurs on day 5, embryogenesis proceeds very rapidly thereafter and neural tube closure is completed by day 9. In the present study the effects of two different doses of N-nitrosoethylurea (NEU) administered at various stages of gestation were quantitatively evaluated in Syrian golden hamsters. NEU at either 0.2 or 0.5 mmol/kg was administered transplacentally as a single i.p. injection to pregnant hamsters on gestation days 7, 8, 9, 10, 11, 12, 13, or 14. The incidence, latency period and multiplicity of tumors varied with the dose of NEU and the stage of development at the time of NEU administration. Although tumors of the peripheral nervous system predominated, a variety of other tumors, including melanomas and visceral tumors of epithelial and mesenchymal origin, were also observed in hamster offspring exposed transplacentally to NEU. Sensitivity to transplacental carcinogenesis was maximal during late gestation and very low before day 9.

Effect of stage of development on frequency and pathogenesis of kidney tumors induced in Noble (Nb) rats exposed prenatally or neonatally to N-nitrosoethylurea.

Wilms' tumor of kidney, a common human childhood neoplasm, is modeled by nephroblastomas induced by prenatal exposure of some rodents to alkylating agents. Noble (Nb) rats are especially susceptible. We studied the ontogeny of susceptibility by treatment with N-nitrosoethylurea (NEU) on gestation day 10, 12, 14, 16 or 18 or neonatal day 1, 3, 5, 7, or 10. No nephroblastomas were observed in offspring exposed to NEU on day 10 or 12 of gestation. In contrast, nephroblastomas commonly occurred in rats exposed on gestation day 14, 16 or 18 of gestation, with the highest incidence (48%) after treatment on day 18. Nephroblastomas were rare ( < 10%), but renal mesenchymal tumors were common (25-30%) in rats exposed to NEU on day 1 or 3 after birth. In rats exposed to NEU on day 7 or 10 only renal mesenchymal tumors were seen. Thus our results suggest that the stage of differentiation of fetal and neonatal kidneys at the time of NEU administration determines the frequency and type of kidney tumors induced in Nb rats. Since NEU induces both nephroblastomas and mesenchymal tumors in this strain, this experimental model may prove useful for the study of molecular mechanisms involved in the development of these two histogenetically different types of kidney tumors.

Effect of anoxia on changes in phosphoinositide content and single unit activity in the cat cerebral cortex

Preneoplastic changes in organ cultures of mouse embryonic kidney (OCEK) under transplacental N-nitrosoethyl urea (ENU) did not increase under the influence of Ado-Cbl. The frequency of focal proliferation in OCEK under transplacental influence of Ado-C bl and ENU was 2.2 times less than the combination of carcinogen with MeCbl (32.2% and 71.9%, respectively, P less than 0.01). The number of DNA-synthesising epithelial cells in OCEK were less under transplacental influence of Ado-Cbl (8.6 +/- 1.4%) than in MeCbl or FA (19.4 +/- 1.8% and 16.2 +/- 1.3% respectively, P less than 0.01).

Ki‐ras mutations in spontaneous and chemically induced renal tumors of the rat

A high frequency of point mutations at codon 12 of the Ki-ras gene has previously been reported for rat kidney mesenchymal tumors induced by methylating N-nitroso compounds. In this study, we analyzed renal tumors with divergent histogenesis, i.e., mesenchymal tumors (sarcomas), cortical epithelial tumors (carcinomas), and embryonal tumors (nephroblastomas). Renal mesenchymal tumors and carcinomas were induced in juvenile or young adult Wistar rats by a single dose of N-nitrosodimethylamine (NDMA) while nephroblastomas were induced in Nb hooded rats by a single transplacental dose of N-nitrosoethylurea (NEU). Nephroblastomas developing spontaneously in WAB/Not rats were also examined. Amplification of Ki-ras sequences from formalin-fixed, paraffin-embedded tissue by the polymerase chain reaction was followed by direct DNA sequencing. GGT----GAT point mutations at codon 12 of the Ki-ras gene were found in 9 of 12 (75%) renal mesenchymal tumors and in 9 of 12 (75%) cortical epithelial tumors induced by NDMA. Even higher incidences were observed in nephroblastomas (8/8; 100%) induced by NEU and in spontaneous nephroblastomas (10/11; 91%). These results indicate that Ki-ras mutations are frequent events during the development of kidney tumors irrespective of their histogenesis and suggest that they may play an important role in renal carcinogenesis in rats. These data further indicate that mutational activation of Ki-ras proto-oncogenes in carcinogen-induced rat kidney tumors occurs in a tissue-specific, rather than cell-specific, manner.

Renal tubular cell or hepatocyte hyperplasia is not associated with tumor promotion by di(2-ethylhexyl)phthalate in B6C3F1 mice after transplacental initiation with N-nitrosoethylurea

B6C3F1 mice of both sexes that had been exposed transplacentally on day 18 of gestation to 0.5 mmole N-nitrosoethylurea (NEU) were fed either normal diets or diets containing di(2-ethylhexyl)phthalate (DEHP) at 6,000 ppm beginning at 6 wk of age and continuing to 78 wk of age. At 52 and 78 wk of age, 6-26 mice from each group received a single injection of 5-bromo-2'-deoxyuridine (Brdu) at 200 mg/kg i.p. and were sacrificed 1 h later for determination of the levels of renal and hepatic DNA synthesis by the Brdu immunohistochemical technique. No differences occurred in incidences of gross or microscopic renal tubular cell tumors between the NEU (males 15%, females 21%) and NEU-DEHP groups (males 10%, females 15%) at 78 wk. The labelling index (LI) of renal cortical tubular cells was significantly increased at 78 wk (22.3 +/- 3.7/mm2 for males, 21.8 +/- 1.2 for females) in mice given NEU and DEHP as compared with NEU alone (9.7 +/- 1.0 for males, 6.9 +/- 0.7 for females). The number and sizes of focal hepatocellular proliferative lesions (FHPL), including hyperplastic foci, hepatocellular adenomas and carcinomas, were quantified by image analysis and stereology. DEHP significantly enhanced the mean volume and volume % of FHPL, including liver tumors, but not numbers of FHPL/liver. Hepatocyte LI was also not affected, at least as detected by the technique used, while FHPL had significantly increased LI (14.5-48.3) as compared with normal hepatocytes (0.5-2.4). This study provides some evidence that enhanced chronic cell replication in the kidney may not always be associated with renal carcinogenesis of tumor promotion, while tumor promotion in liver may be a consequence of increased DNA synthesis in initiated or focus cells rather than in nonproliferative parenchymal hepatocytes, which may not be target cells of some tumor promoters.

Transplacental initiation of liver, lung, neurogenic, and connective tissue tumors by N-nitroso compounds in mice

Epidemiological studies have implicated nitroso compounds as possible causative agents for human childhood cancers, including those of neurogenic orgin. Published evidence from animal models, which is reviewed in this report, indicates that capacity for metabolic activation of nitrosamines is limited in rodent fetuses and that nitrosamines are correspondingly weak transplacental carcinogens. The C3H mouse fetus, however, has both moderate capability for activation of N-nitrosodimethylamine (NDMA) and proven susceptibility to transplacental causation of neurogenic tumors by a nitrosourea. We tested whether NDMA could act as a transplacental carcinogen in the C3H mouse, and whether it or N-nitrosodiethylamine (NDEA) would initiate neurogenic tumors. N-Nitrosoethylurea (NEU) served as positive control. C3H HeNCr MTV − pregnant mice were treated ip on Gestation Day 16 or 19 with NDMA (0.1 mmol, 7.4 mg/kg, maximum nonfetotoxic dose), NDEA (0.5 mmol, 51 mg/kg), or NEU (0.4 mmol, 47 mg/kg). NDMA had significant transplacental carcinogenic effects, resulting in an increase in percentage female offspring with hepatocellular carcinomas and in average number of liver tumors after treatment on either gestational day, compared with controls. In the males there was a significant increase in numbers of liver tumors and carcinomas following Day 19 exposure. An increase in incidence of histiocytic and undifferentiated sarcomas was also of statistical significance. There was no change in number of pulmonary tumors. One intracranial schwannoma resulted. NDEA had no effect when given on Gestation Day 16, but caused a significant increase in liver and lung tumor numbers in both sexes when treatment was on Day 19. NEU induced the expected high incidence of lung tumors, significantly increased liver tumor incidence in females (Day 19 exposure), and produced schwannomas in 14 and 35% of the offspring after Days 16 or 19 treatment, respectively. The results show that NDMA at even a low dose had significant transplacental carcinogenic effects, including one schwannoma, which was most unlikely to have occurred spontaneously. However, this single neurogenic tumor contrasts with the absence of similar neoplasms in mice exposed transplacentally to NDEA, in view of the generally greater efficiency of ethylating agents as carcinogens for the nervous system in rodents. These data thus neither conclusively support nor refute the hypothesis that nitrosamines may initiate neurogenic tumors in fetuses.

Transplacental Initiation of Liver, Lung, Neurogenic, and Connective Tissue Tumors by N-Nitroso Compounds in Mice

Transplacental Initiation of Liver, Lung, Neurogenic, and Connective Tissue Tumors by N-Nitroso Compounds in Mice. ANDERSON, L. M., HAGIWARA, A., KOVATCH, R. M., REHM, S., AND RICE, J. M. (1989). Fundam. Appl Toxicol. 12, 604–620. Epidemiological studies have implicated nitroso compounds as possible causative agents for human childhood cancers, including those of neurogenic origin. Published evidence from animal models, which is reviewed in this report, indicates that capacity for metabolic activation of nitrosamines is limited in rodent fetuses and that nitrosamines are correspondingly weak transplacental carcinogens. The C3H mouse fetus, however, has both moderate capability for activation of N-nitrosodimethylamine (NDMA) and proven susceptibility to transplacental causation of neurogenic tumors by a nitrosourea. We tested whether NDMA could act as a transplacental carcinogen in the C3H mouse, and whether it or N-nitrosodiethylamine (NDEA) would initiate neurogenic tumors. N-Nitrosoethylurea (NEU) served as positive control. C3H/HeNCr MTV− pregnant mice were treated ip on Gestation Day 16 or 19 with NDMA (0.1 mmol, 7.4 mg/kg, maximum nonfetotoxic dose), NDEA (0.5 mmol, 51 mg/kg), or NEU (0.4 mmol, 47 mg/kg). NDMA had significant transplacental carcinogenic effects, resulting in an increase in percentage female offspring with hepatocellular carcinomas and in average number of liver tumors after treatment on either gestational day, compared with controls. In the males there was a significant increase in numbers of liver tumors and carcinomas following Day 19 exposure. An increase in incidence of histiocytic and undifferentiated sarcomas was also of statistical significance. There was no change in num ber of pulmonary tumors. One intracranial schwannoma resulted. NDEA had no effect when given on Gestation Day 16, but caused a significant increase in liver and lung tumor numbers in both sexes when treatment was on Day 19. NELl induced the expected high incidence of lung tumors, significantly increased liver tumor incidence in females (Day 19 exposure), and produced schwannomas in 14 and 35% of the offspring after Days 16 or 19 treatment, respectively. The results show that NDMA at even a low dose had significant transplacental carcino genic effects, including one schwannoma, which was most unlikely to have occurred spontaneously. However, this single neurogenic tumor contrasts with the absence of similar neoplasms in mice exposed transplacentally to NDEA, in view of the generally greater efficiency of ethylat ing agents as carcinogens for the nervous system in rodents. These data thus neither conclusively support nor refute the hypothesis that nitrosamines may initiate neurogenic tumors in fetuses.

Carcinogenic and Co-carcinogenic Effects of Radiation in Rat Mammary Carcinogenesis and Mouse T-cell Lymphomagenesis: A Review

The importance of the promotion stage and of the physiological condition of target cells at the time of initiation is illustrated in both the rat mammary carcinogenesis and the mouse T-cell lymphomagenesis. In the former, prolactin was shown to be a powerful promoter regardless of the initiating agent. Prolactin was also found to be useful in detecting the carcinogenicity of the small doses of carcinogens; a high r.b.e. of 2.0 MeV fission spectrum neutrons was demonstrated by the application of prolactin to radiation-initiated mammary carcinogenesis in rats. In the latter, total-body irradiation involving both bone marrow and thymus facilitates chemically-initiated T-cell lymphomagenesis in mice. This could be attributed to the amplification of the cell population susceptible to a chemical carcinogen in the target tissue during the recovery phase after irradiation. The dual effect of a carcinogen, acting in the different phases of carcinogenesis was suggested by the split administration of N-nitrosoethylurea (NEU) in the induction of T-cell lymphomas. It is emphasized, through these findings, that besides the initiation by a genotoxic agent, the availability of a promoter or an inhibitor determines the fate of initiated cells, and that a modifier of target cells also plays a crucial role in the efficient induction of a tumour.

Synergistic effect of radiation and N-nitrosoethylurea in the induction of lymphoma in mice: cellular kinetics and carcinogenesis.

The effect of a combined treatment with radiation and N-nitrosoethylurea (NEU), or a split administration of NEU in inducing lymphoma was studied in female C57BL/6N mice. A single intragastric administration of 5 mg of NEU was only slightly lymphomagenic, inducing thymic lymphomas in 20% of mice, while the incidence was elevated to 92% if the NEU treatment was preceded (by 5 days) by 400 rad of total-body X-irradiation, which alone is seldom lymphomagenic. A high yield of lymphoma (84-93%) was also obtained if 5 mg of NEU was delivered in two split doses of 4 mg and 1 mg with a 4 day interval. Drastic injury to both the thymus and bone marrow caused by either 400 rad total-body X-irradiation or the first dose of NEU (4 mg) was followed by a vigorous regeneration within a few days. The maximum induction rate of lymphoma was obtained when the subsequent dose of NEU (1 mg) was given at the peak of DNA synthesis in the bone marrow and thymus following the first treatment. The data indicate that the principal effect of the irradiation or the first dose of NEU was to provide a susceptible cell population, and that a high yield of lymphoma was brought about through the action of the subsequent dose of NEU on a sufficient number of target cells engaged in heightened DNA synthesis.

Transplacental carcinogenic effect of N-nitrosoethylurea in dogs

During the late period of pregnancy (on the 50th and 53rd days post coitum) 2 dogs were injected intraperitoneally with 100 mg/kg (a single administration) of nitrosoethylurea. Nine puppies were born. During the 9 year period of observation no tumours were detected in mother-dogs. Three of 9 offspring developed the following tumours: brain haemangioblastoma (on the 526th day after administration), nephroblastoma and spleen angioleiomyoma (on the 845th day after administration) and adenoma of thyroid gland (after 7 years). Among other lesions 3 offspring-dogs developed polycystic kidneys. The revealed tumours of brain, kidney and spleen could be connected with transplacental carcinogenic effect of N-nitro-soethylurea (NEU), while neoplasms of these localizations and types rarely occur in dogs spontaneously.