Genetic basis of sex-specific resistance to neuro-oncogenesis in (BDIX x BDIV) F(2) rats.

The identification of cancer susceptibility- and resistance-mediating genes is an essential prerequisite for prevention and early diagnosis of malignant tumors. Model organisms are helpful to identify variant alleles involved in pathways affecting individual cancer risk. BDIX and BDIV rats of both sexes are highly susceptible and resistant, respectively, to the development of N-ethyl-N-nitrosourea (ENU)-induced malignant peripheral nerve sheath tumors (MPNST), predominantly in the trigeminal nerves. Nevertheless, female (BDIV x BDIX) F(2) intercross rats have a lower MPNST incidence and a longer latency time than males. Six of seven autosomal gene loci (Mss1-Mss7) controlling genetic susceptibility and resistance in (BDIV x BDIX) F(2) hybrids exert allele- and sex-specific effects on tumor incidence and/or latency time of variable strength. Homozygous BDIV alleles at Mss4 or Mss7 located on rat chromosomes 6 and 10, respectively, are sufficient to cause almost complete resistance to ENU-induced MPNST development in female F(2) rats regardless of the genotype of the other locus. Both loci display only weak effects on male cancer risk. Survival curves of ENU-treated F(2) females depleted of animals with homozygous BDIV alleles at Mss4 and Mss7 are not significantly different from those of males, suggesting that these loci account mainly for the excess tumor resistance observed in female F(2) rats. By haplotype analysis Mss4 and Mss7 could be narrowed down to 20 and 12 Mb, respectively, providing a basis for the positional identification of candidate genes.

Differential oncogenic effects of methylnitrosourea.

We investigated differences in the oncogenic effects of methylnitrosourea (MNU) which were induced by varying dose schedules and changing administration routes. The nervous system represented the target organ when MNU was given intravenously to rats. Carcinogen by other routes resulted in decreased numbers of neurogenic tumors and the appearance of neoplasms at the injection site. Increased oral doses of MNU caused shorter survival times, a decreased incidence of neuroglial tumors, and increased numbers of thymic lymphomas and mesenchymal tumors of the nervous system. The results suggest that many tissues are susceptible to the oncogenic effects of MNU, but the degree of exposure necessary for neoplastic transformation varies.

Inhibitory effect of sodium ascorbate on ethylurea and sodium nitrite carcinogensis and negative findings in progeny after intestinal inoculation of precursors into pregnant hamsters.

To assess their carcinogenic effects, the ethylnitrosourea (ENU) precursors, ethylurea and sodium nitrite, [were administered to pregnant hamsters as a single intragastic] dose on day 15 of gestation, or introduced into the cecum on day 14. Since sodium ascorbate (NaASC) inhibits the biosynthesis of nitrosamides, identical doses of the precursors were given concomitantly with NaASC. Progeny of mothers treater intragastrically developed significant incidences of neurogenic tumors of the peripheral nervous system, with a predominance in females. The concurrent administration of NaASC with ENU precursors prevented carcinogenic effects in the progency, whereas the simultaneous inoculation of the precursors into the cecum produced no carcinogenic effects in the offspring.

Desmosterol in rat central and peripheral nervous systems during normal and neoplastic growth.

Desmosterol (5, 24-cholestadien-3beta-ol; delta 24-cholesterol; 24-dehydrocholesterol), an immediate precursor of brain cholesterol, increased in malignant intracranial tumors induced in rats by nitrosourea derivatives. The average increase in desmosterol was higher in intracerebral gliomas than in neurinomas of the trigeminal nerve. Similarly, desmosterol increased only slightly in developing normal trigeminal nerve compared to the high levels observed in developing cerebrum. The differences may have been partly related to the predominantly growing cell type, i.e., glial (central nervous system) or Schwann (peripheral nervous system) cells seen at the time of study.

Morphologic and biochemical characteristics of transplantable neurogenic tumors induced by N-ethyl-N-nitrosourea in inbred BD IX rats.

Brain and nerve tumors were induced transplacentally in inbred BD IX rats by systemic application of N-ethyl-N-nitrosourea. Because primary gliomas and neurinomas produced in this way are composed of heterogeneous cell populations, changes in tumor morphology were expected to occur during serial transplantation in syngeneic hosts. In this study such changes in morphology were correlated with the expression of two biochemical nervous system markers, S-100 protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Several changes were observed during serial transplantation, including increased growth rate (even after one passage), preferential growth of anaplastic versus differentiated glial and Schwann's cells, varying degrees of fibrosarcomatous changes after prolonged serial transplantation, and reduced levels of S-100 protein. In contrast, tumors derived from biochemically differentiated clonal cell lines retained their morphologic and biochemical characteristics to a much greater extent, even after prolonged periods of sc transplantation.

Oncogenic response of rats with x-ray-induced microcephaly to transplacental ethylnitrosourea.

The relation of congenital malformations to tumor development was examined. Pregnant Sprague-Dawley rats were given 200 rads of X-rays on the 15th or 16th day of gestation and injections of 10 mg ethylnitrosourea (ENU)/kg 1-4 days later, or they were irradiated or injected only. Surviving weanlings that had been irradiated had micrencephaly and other malformations. Offspring exposed to ENU only had no external deformities. By 15 months of age 16.7% of the offspring exposed to X-rays and ENU prenatally had developed neurogenic tumors, whereas 62.2% of those exposed to ENU alone had developed tumors. Those only irradiated had no tumors. Both of the former groups developed oligodendrogliomas, mixed gliomas, ependymomas, and schwannomas, but the first manifestations of tumors occurred later in the group receiving the combined treatment. This delay persisted furing the subsequent period of the study.

Reduction of N-nitroso-N-ethylurea-induced neurogenic tumors by X-radiation: a life-span study in rats.

Whole-body X-irradiation after neonatal injection with N-nitroso-N-ethylurea (ENU) significantly reduced the incidence of induced neurogenic tumors in inbred HMT rats kept for their complete life-span. After administration of 10 mg ENU/kg and 1.25 Gy X-radiation, the incidence of schwannomas but not of gliomas was reduced as compared to the incidence in rats given 10 mg ENU/kg only. In contrast, after administration of 4 mg ENU/kg, 1.25 Gy reduced the incidence of gliomas but not of schwannomas. Administration of 1.25 Gy alone induced a remarkably high incidence of rats with neurogenic tumors (20%). Latency of tumor detection was not significantly affected by radiation. Among the most frequently occurring nonneurogenic tumors, squamous cell carcinomas were reduced in incidence by treatment with ENU, 1.25 Gy X-radiation, or both combined. No treatment affected the incidence of pituitary or mammary tumors. There was a preponderance of ovarian tumors in rats given 4 mg ENU/kg + 1.25 Gy. An incidental finding was the occurrence of granular cell tumors in 7 rats from different treatment groups.

Effect of nerve growth factor on the transplacental induction of neurinomas by ethylnitrosourea in Sprague-Dawley rats.

Administration of nerve growth factor (NGF) to the offspring of Sprague-Dawley rats transplacentally exposed to 50 mg/kg ethylnitrosourea on the 20th day of gestation resulted in a significant reduction of trigeminal and peripheral nerve neurinomas. Forty, 60, and 80 micrograms of NGF was administered in five s.c. doses, one dose on each of days 12-16, 90-94, and 210-214 postnatally. Of the 34 rats in the NGF-treated group, 11 animals were affected with trigeminal nerve neurinomas as compared to 18/34 in the NGF-untreated group (P less than 0.05). In the peripheral nerves (spinal cord nerve roots) there were five and 11 neurinomas, respectively, in each group of 34 rats. When the total numbers of neurinomas (trigeminal and peripheral nerves) between these groups were compared (16/34 versus 29/34), the significance of neurinoma reduction was P less than 0.01. Five trigeminal and two peripheral neurinomas in the NGF-untreated group were shown by immunohistochemical staining to contain nerve growth factor receptor protein, whereas none of the neurinomas in the NGF-treated group were positive for the receptor protein. The results obtained from this experiment lend support to the hypothesis that NGF has the capability to reduce the oncogenic consequences of ethylnitrosourea exposure perhaps by the process of maturation and/or differentiation of the transformed cells, and that this effect may depend upon the presence of receptor binding sites.

The effect of gonadal ablation on transplacentally induced neurogenic tumors in hamsters.

The effects of gonadectomy on tumors induced transplancentally by the ethylnitrosourea precursors, ethylurea and sodium nitrite, were investigated in hamsters. The pregnant hamsters were exposed to four daily doses of ethylurea (100 mg/kg) and sodium nitrite (50 mg/kg) administered from Day 12 to 15 of pregnancy. Weaned offspring were gonadectomized when they reached the age of 5 weeks. Orchiectomized male progeny showed a multiplicity and greater frequency of peripheral nervous system tumors and of any other tumor types than did intact males or their ovariectomized and intact female siblings. The possible inhibitory effects of endogenous androgens on the development and growth of neurogenic tumors in the peripheral nervous system and the influence of an induced endocrinal imbalance on prenatally induced neoplasms are discussed.

Prenatal multicarcinogenesis by ethylnitrosourea in mice.

The Role of prenatal age, sex, and the maternal background upon the incidence, multiplicity, and spectrum of tumors induced by ethylnitrosourea has been studied in the offspring of reciprocal hybrids of the same genotype. The first generation (F1) offspring of C57BL/6J X C3HeB/FeJ and the reciprocal hybrids were observed throughout their life-span for tumor development following single i.p. injections of ethylnitrosourea (60 microng/g) given to pregnant mothers at 12, 14, 16, or 18 days of gestation. Animals exposed to ethylnitrosourea survived on the average for only 90 weeks. They developed by that age tumors in lungs, livers, ovaries, nervous system, and forestomach. Control mice killed at 90 weeks were essentially free of tumors. The fetal age at the time of administration of the carcinogen was one of the most significant modulators of tumor development in lung, liver, ovaries, and nervous system. The sex of the animals influenced the rate of development of liver tumors, whereas maternal background affected the multiplicity of lung tumors.

Neurogenic tumors in rats induced by ethylnitrosourea.

Ethylnitrosourea (ENU) was injected intravenously into Sprague-Dawley rats on day 15 of gestation at doses of 0, 2.50, 6.25 and 10.00 mg/kg. The resulting 1980 progeny were observed for up to 24 months in a life-time study (900 rats) or for periods of 171-325 days in a serial sacrifice study (1080 rats). The rats in both studies were randomized into three groups, one exposed to a radiofrequency, one sham-exposed and one cage control. Since no effects of the radiofrequency were observed on the ENU-induced tumors, the exposure groups were combined to facilitate study of the tumors by dose rate over time. All rats were necropsied and major organs were examined histologically including the brain, entire spinal cord, trigeminal nerves and all tumors. A total of 48 spinal cord tumors (SCT), 251 spinal nerve tumors, 264 cranial nerve tumors and 1058 brain tumors were studied. The tumors were characterized by incidence, histologic type, volume, malignancy and multiplicity. Ethylnitrosouria, as given in this study, was determined to be an effective carcinogen reliably inducing (in order of frequency) brain, cranial nerve, spinal nerve and SCT. Dose of ENU correlated positively with the frequency, multiplicity, volume, malignancy, and negatively with latency of brain tumors and to a lesser extent with nerve tumors.

Induction of malignant peripheral nerve sheath tumors in European hamsters with 1,1-dimethylhydrazine (UDMH).

A rate of up to 43% of malignant peripheral nerve sheath tumors (PNST) was induced in European hamsters (EH) after weekly s.c. administration of 1,1-dimethylhydrazine (UDMH). The overall neoplastic response in the treated EH was also elevated as compared to the untreated controls. Histologically, the malignant PNST were neurofibrosarcomas and melanotic as well as unpigmented schwannomas. The occurrence of melanotic schwannomas is briefly discussed with regard to the histogenesis of this rare tumor type.

Treatment with field bean protease inhibitor can effectively repress ethylnitrosourea (ENU)-induced neoplasms of the nervous system in Sprague-Dawley rats.

The ability of field bean protease inhibitor (FBPI) to inhibit ethylnitrosourea (ENU)-induced tumours of the nervous system of Sprague-Dawley rats was investigated. Groups of 1-day-old rats were injected intraperitoneally (i.p.) with neurocarcinogenic amounts of ENU and a few hours later, one group was treated i.p. with 80 mg of FBPI per kg body weight. This treatment was carried out three times a week for the first month and five times a week for the next month. Animals were killed when they were neurologically ill and their neural tissues were assessed for lesions. Those FBPI-treated rats which showed no illness were also killed to terminate the experiment about 8 weeks after the last rat of the control group was affected with paralysis. The neural tumours induced in all groups were predominantly large tumours found in the cerebrum of the rats. ENU-treated rats showed a 100% incidence of nervous system tumours with a mean time of manifestation of neurological symptoms of 282 days, which was significantly shorter in comparison to that noted in the FBPI-treated group. The latter group showed an incidence of 58.3%, i.e. a significant reduction of 41% in the incidence of neural tumours, as well as a lower mean value for the number of tumours per rat. All these aspects indicated that FBPI is a potential neurooncopreventive agent. A neural tumour incidence of 100% in the rats treated with heat-inactivated FBPI confirmed that the tumour suppressive activity of FBPI is related to its protease inhibitory activity.

neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence but low allelic representation.

Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T --> A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

The effects of 860 MHz radiofrequency radiation on the induction or promotion of brain tumors and other neoplasms in rats.

Sprague-Dawley rats were irradiated with a continuous- wave (CW) or a pulsed-wave (P) radiofrequency (RF) for 6 h/day, 5 days/week from 2 up to 24 months of age. The RFs emanated from dipole antennas (1 W average output) 2.0 +/- 0.5 cm from the tip of each rat's nose. The RFs had an 860 MHz frequency, and the specific absorption rate was 1.0 W/ kg averaged over the brain. Fifteen groups of 60 rats (900 total) were formed from offspring of females injected i.v. with 0 (groups 1, 2, 9, 10, 13), 2.5 (groups 5, 6, 7, 8, 11, 12, 14) or 10 mg/kg (groups 3, 4, 15) ethylnitrosourea (ENU) to induce brain tumors. Groups 1, 3, 5 and 7 received the PRF, and groups 9 and 11 the CWRF; groups 2, 4, 6, 8, 10 and 12 were sham-irradiated, and groups 13-15 were cage controls. All rats but 2, totaling 898, were necropsied, and major tissues were studied histopathologically. There was no statistically significant evidence that the PRF or CWRF induced neoplasia in any tissues. Additionally, there was no significant evidence of promotion of cranial or spinal nerve or spinal cord tumors. The PRF or CWRF had no statistically significant effect on the number, volume, location, multiplicity, histological type, malignancy or fatality of brain tumors. There was a trend for the group that received a high dose of ENU and was exposed to the PRF to develop fatal brain tumors at a higher rate than its sham group; however, the result was not significant using the log-rank test (P = 0.14, 2-tailed). No statistically significant differences were related to the PRF or CWRF compared to controls in the low- or zero-dose groups regarding tumors of any kind.

[Deoxyribonucleases in spontaneous and experimental tumors of the nervous system]. / Desoxyribonucleasen in spontanen und experimentellen Tumoren des Nervensystems

DNA- and RNA-concentrations, as well as in vitro activities of DNase I (EC 3.1.4.5), DNase II (EC 3.1.4.6), and DNase I inhibitor, have been determined in 63 spontaneous (man) and 22 experimentally induced (rat) nervous system blastomas of various types and of different degrees of malignancy. Generally, a distinct elevation of DNA concentrations and of the ratio (Q) of DNase II- to DNase I-activities has been observed when compared with control values. A statistically significant relationship could be demonstrated between increase of DNA concentrations and Q in experimentally induced neurinomas of rats as well as in human astrocytomas and glioblastomas. Whereas the increase of Q may be a biochemical expression of elevated DNA synthesis of tumour cells, no conclusions can be drawn as to the role of DNases in the process of malignant transformation.

S-100 protein and glial fibrillary acidic protein (GFAP) in experimentally induced and spontaneous tumours of peripheral nerves in BDVI rats. Light microscopic and immunohistochemical study.

Twenty-five ethylnitrosourea (ENU) induced and 24 spontaneous tumours of peripheral nerves as well as 28 spontaneous mesenchymal tumours in BDVI rats were studied by light microscopy and immunohistochemically for the presence of S-100 protein (S-100). Early ENU-induced schwannomas*) representing the thickenings of nerves showed weak or negative S-100 immunoreactivity. S-100 positivity (both in the cytoplasm and nucleus) was observed in all large ENU-induced tumours found in animals dying or killed at advanced age. Immunostaining was present in both cystic and solid areas of schwannomas. S-100 positivity was found in 20 of 24 spontaneous schwannomas: 14 of 20 positive tumours contained cysts. Twenty-seven schwannomas (12 ENU-induced and 15 spontaneous) were studied for the presence of glial fibrillar acid protein (GFAP) and 13 were positive (7 had cystic areas). GFAP-positivity was relatively high in 4 tumours (3 ENU-induced and 1 spontaneous); these tumours also showed intense S-100 reactivity. Immunoreactivity for S-100 occurred more frequently and was much more intense than that for GFAP. The incidence of spontaneous peripheral nerve tumours in BDVI males reached 4%, cystic schwannomas being the most frequent type. All spontaneous mesenchymal tumours except lipoma (S-100 positive) were negative for S-100 protein and for GFAP.

[Pathological study of malignant peripheral nerve sheath tumor in rat].

Sequential morphological studies of rat malignant peripheral nerve sheath tumors induced with ethylnitrosourea were performed by light microscopy, transmission electron microscopy, scanning electron microscopy, immunohistochemistry and nude mice transplantation. The result indicated that rat malignant peripheral nerve sheath tumor was actually a sarcoma arising from Schwann cell within the peripheral nerve, which possesses some basic morphological characteristics as those of human malignant peripheral nerve sheath tumor, and is considered worthy to be used as a model for further studies.

[Glial fibrillar acidic protein in peripheral nerve tumors in rats]. / Glial'nyi fibrilliarnyi kislyi protein v opukholiakh perifericheskikh nervov krys.

Twenty-seven peripheral nerve tumours of BD VI rats from the histological archives of the International Agency for Research on Cancer were immunohistochemically studied for GFAP reactivity. Twelve tumours were induced by nitrosoethylurea (NEU) and 13 tumours were spontaneous. Thirteen tumours (7 of them NEU-induced and 6 spontaneous) revealed GFAP-reactivity which was relatively high (large areas of GFAP-positive cells) in 4 neoplasms (3 induced and 1 spontaneous). GFAP-positive tumours were not histologically distinguishable from GFAP-negative tumours. GFAP-positive tumours were also positive for S-100 protein although the incidence and the intensity of GFAP-reactivity were much lower than that of S-100 reactivity. Early NEU-induced schwannomas were negative for GFAP and S-100; all GFAP-and S-100-positive tumours were large neoplasms with an invasive growth. GFAP-positivity was observed in both solid and cystic schwannomas. The results of the study represent the additional data on the observation of GFAP outside of the central nervous system.