Ablation of T cell immunity differentially influences tumor risk in inbred BD rat strains.

Inbred rat strains BDIX and BDIV are constitutionally susceptible and resistant, respectively, to the development of malignant peripheral nerve sheath tumors (MPNST) induced by neonatal exposure to N-ethyl-N-nitrosourea (EtNU). They represent a model system for analysis of molecular and cellular processes underlying differential cancer susceptibility. A point mutation in the Neu/ErbB-2 gene is an early marker of Schwann precursor cells at high risk of malignant conversion and is diagnostic of the resulting MPNST predominantly developing in the trigeminal nerves. Initially considerable amounts of Neu/ErbB-2-mutant cells arise in nerve tissue of both rat strains subsequently disappearing in resistant BDIV rats, but persisting and giving rise to MPNST in susceptible BDIX animals. An almost identical cellular immune response-sequentially involving macrophages, T helper- and cytotoxic T lymphocytes-is mounted in the trigeminal nerves of EtNU-treated rats of both strains. In this study, T cell maturation was prevented by neonatal thymectomy following EtNU-exposure. While resistance against MPNST development significantly decreased in BDIV rats MPNST incidence and survival time remained unaltered in thymectomized BDIX rats. Contrary to euthymic animals a number of both thymectomized BDIV and BDIX rats developed MPNST lacking the Neu/ErbB-2-mutation. This suggests that Schwann cells initiated by other genetic alterations can progress to full malignancy in immune-compromised rats only. T cell-dependent resistance against tumorigenesis originating from non-Neu/ErbB-2-mutant Schwann precursors might thus be shared by both strains while BDIV T lymphocytes additionally prevent the development of Neu/ErbB-2-mutant MPNST. Rat strain-specific differences in the interaction of T lymphocytes with (pre)malignant Neu-mutant cells may thus critically contribute to susceptibility and resistance towards EtNU-induced MPNST development.

The interaction mode of premalignant Schwann and immune effector cells during chemically induced carcinogenesis in the rat peripheral nervous system is strongly influenced by genetic background.

Contrary to rats of the highly sensitive inbred strain BDIX, BDIV rats are resistant to the induction of malignant schwannomas by N-ethyl-N-nitrosourea, arising predominantly in the trigeminal nerves. A point mutation of the neu/erbB-2 gene diagnostic of N-ethyl-N-nitrosourea-induced rat schwannomas is an early marker of Schwann precursor cells at high risk of subsequent malignant transformation. Neu-mutant cells initially arise at a similar frequency in sensitive and resistant animals. However, these cells disappear from the trigeminal nerves of resistant rats while giving rise to highly malignant schwannomas in susceptible animals. The resistance of BDIV rats obviously includes mechanisms to recognize and eliminate premalignant cells. The involvement of a cellular immune response was investigated in trigeminal nerves of both strains at different times after neonatal carcinogen exposure. An inflammatory reaction involving sequentially CD4(+) macrophages and T helper cells, CD8(+) cytotoxic T cells, and ED1(+) and ED2(+) macrophages was detected as a consequence of N-ethyl-N-nitrosourea treatment as early as postnatal day 40, briefly after the emergence of premalignant neu-mutant Schwann cells. It persisted throughout the observation period (40-250 days). However, there were no gross differences in immune cell counts between tumor-susceptible and tumor-resistant rats, except for a moderate increase of ED2(+) macrophages in N-ethyl-N-nitrosourea-treated BDIX rats only. Differential interactions of immune effector cells with premalignant Schwann cells may thus be involved in genetically determined tumor susceptibility or resistance, which could include functional differences of immune effector cells and/or a differential capability of premalignant Schwann cells to escape or counteract the cellular immune response.

Genetics of susceptibility of rats to trigeminal schwannomas induced by neonatal administration of N-ethyl-N-nitrosourea.

A genetic analysis was done on the induction of trigeminal schwannomas by N-ethyl-N-nitrosourea [(ENU) CAS: 759-73-9] in susceptible LE rats, resistant WF rats, and their F1, F2, and reciprocal backcross hybrids. Both sexes of all strains were given a neonatal sc injection of 40 mg ENU/kg body weight and were sacrificed at 6 months after treatment. Many neurogenic tumors were induced in the central nervous system of all strains of rats. However, the incidence of trigeminal schwannomas in LE rats was 93% in males and 86% in females, whereas in WF rats the incidence was 24% in males and 20% in females. F1 and F2 hybrids showed an intermediate susceptibility (62 and 82% in F1 males, 79 and 86% in F2 males, 26 and 38% in F1 females, and 65 and 77% in F2 females). The incidence in hybrids backcrossed to LE was high (90 and 100% in males and 77 and 83% in females), and the incidence in hybrids backcrossed to WF was low (35 and 38% in males and 11 and 7% in females). The findings suggest that susceptibility to the induction of trigeminal schwannomas by ENU does not result from the expression of genes that are simple dominant or recessive genes. A genetic model involving three independently segregating loci may explain the experimental results. In all strains of rats, particularly the F1 hybrids, males were more susceptible than females to the induction of trigeminal schwannomas by ENU.

Reduction of ethylnitrosourea-induced neoplastic proliferation in rat trigeminal nerves by nerve growth factor.

Ethylnitrosourea administered to pregnant rats on the 21st day of gestation at a dose of 50 mg/kg resulted in early neoplastic proliferation or neurinomas of trigeminal nerves in 90% of the offspring at 90 days of age. Treatment with nerve growth factor either postnatally following ethylnitrosourea administration or transplacentally prior to ethylnitrosourea treatment appeared to result in a reduction of neurinomas at 90 days of age.

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.

Suppression of ethylnitrosourea-induced schwannoma development involves elimination of neu/erbB-2 mutant premalignant cells in the resistant BDIV rat strain.

Contrary to the response of rats of the highly sensitive inbred strain BDIX, BDIV rats are resistant to the induction of malignant schwannomas by exposure to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). In BDIX rats, a point mutation at nucleotide 2012 in the transmembrane region of the neu/erbB-2 gene has proved to be a very early marker of initiated Schwann precursor cells with an elevated risk of malignant transformation, and is diagnostic of the resulting schwannomas. To gain insight into the cellular and molecular mechanisms responsible for the resistance of the BDIV strain, comparative quantitative neu mutation analyses combined with histomorphological studies were performed on the trigeminal nerves of EtNU-treated BDIV and BDIX rats as well as on their (BDIX x BDIV) F1 progeny. It was found that neu-mutant Schwann cells are initially present at comparable frequency in the trigeminal nerves of both resistant and sensitive animals. Contrasting with the progressive multiplication of mutant Schwann cells in BDIX trigeminal nerves, however, the numbers of mutant cells began to decrease during the intermediary phase of the carcinogenic process in BDIV animals, and premalignant neu-mutant cells were no longer detectable by the time BDIX rats developed full-blown trigeminal schwannomas. The resistance of BDIV rats thus involves the elimination of initiated neu-mutant Schwann cells during the postinitiation period of EtNU-induced schwannomagenesis via mechanisms that remain to be clarified.

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.

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.

[Synthesis and animal experiments of ethylnitrosourea].

Ethylnitrosourea (ENU) was synthesized with raw materials such as ethylamine and sodium nitrite. The product was a yellowish powder. Its melting point, solubility and infrared absorption spectrum coincided with those reported in the literature. The result form elementary analysis for the product was almost the same as the theoretical value. The product was applied to rats by various methods. It was found that ENU was a cancerogenic compound, especially to the nervous system. More details about the animal experiments will be published in some other papers.

[Carcinogenicity in BD-IX rats of 7 homologues of N-nitroso-N-n-alkylureas in different stages of postnatal development (author's transl)]. / Karzinogene Wirkung von 7 Homologen der N-Nitroso-N-n-Alkyl-Harnstoffe in verschiedenen postnatalen Entwicklungsphasen an BD-IX-Ratten.

Carcinogenic activity of seven N-Nitroso-N-n-alkylureas was investigated in 679 BD-IX rates in different stages of postnatal development after administration of a single s.c. dose of the test compound. The results are compared with a former investigation using N-nitroso-N-ethylurea. The nitrosoureas in a series from the methyl- to the n-octyl derivative showed a pronounced neurotropic carcinogenic effect on treatment during the first ten days after birth. Malignant neurinomas of the heart, subcutaneous sarcomas at the site of injection and malignant tumors in other organs varied distinctly, depending mainly on the time of treatment. All observed effects were similar in all 7 alkylnitrosoureas investigated.

Spinal tumors induced by neonatal administration of N-ethyl-N-nitrosourea in Wistar rats.

The carcinogenic effect of N-ethyl-N-nitrosourea (ENU) administered by single neonatal injection (40 mg/kg) was examined in wistar rats. By 2 months after ENU administration, 30% of the examined animals had spinal cord tumors. After months all rats had neurogenic tumors, and the incidence of spinal tumor was as high as 86%. Spinal cord tumors were observed at all levels of the white matter of the spinal cord without any predilection site, though spinal root tumors were located exclusively on lumbosacral plexuses. Most of the spinal cord tumors were oligodedrogliomas or glioependymomas, whereas all the spinal root tumors were anaplastic schwannomas.

Strain differences of tumorigenic effect of neonatally administered N-ethyl-N-nitrosourea in rats.

The tumorigenicity of neonatally administered N-ethyl-N-nitrosourea (ENU) was studied in four different inbred strain rats, that is Wistar/Furth (WF), Long-Evans (LE), F1 of Wistar/Furth and Long-Evans (F1) and Fischer 344 (F344) rats. All strains developed tumors of the nervous system with high incidence (97-100%) during 6 months of observation. The incidence of tumor of the central nervous system, including the brain (82-88%) and the spinal cord (53-76%), was high in all strains, but that of the peripheral nervous system, including the cranial nerve (21-89%) and the spinal root (13-93%), differed by strain. The peripheral nervous system of WF and F344 rats had a low susceptibility to the tumorigenic effect of ENU, but that of LE rats had a high susceptibility. Many brain tumors were induced in the temporal and frontal cortex and subcortex in all strains of rats. Spinal cord tumors were observed at all levels of the white matter of the spinal cord without any predilection site. Spinal root tumors were located in lumbosacral plexuses in WF and F344 rats, but in LE and F1 rats cervical and thoracic root tumors were also observed. Histological examination revealed that most of the brain and spinal cord tumors were oligodendroglioma, but in F344 rats about half of the brain tumors were mixed glioma. Epidermoid cysts of the lumbar spinal cord were observed only in F344 rats. Tumors of the peripheral nervous system were so-called anaplastic schwannoma.

Ganglion cells and their satellites in experimental trigeminal schwannomas in the rat.

Ten neurinomas of ganglion semilunare nervi trigemini induced in rats with ethylnitrosourea contained numerous neurons entrapped into solid neoplastic tissue. Ganglion cells were mostly well preserved and presented the receptors to the lectin Con A, i.e. the ability for its binding to alpha-glucose and alpha-mannose. Positive GFAP immunoreaction in neuronal satellite cells suggests that the function of satellites corresponds to that of intracerebral astrocytes and that the preservation of ganglion cells depends on their satellites.

[The effect of ethylnitrosourea in the activity of the NAD-linked glycero-3-phosphate dehydrogenase in the Vth nerve of developing rats and its relation to myelination and tumour induction (author's transl)]. / Der Einfluss von Athylnitrosoharnstoff auf die Aktivität der Nad-abhängigen L-alpha-Glyzerin-3-Phosphatdehydrogenase während der Myelinisierung und Tumorinduktion im N. trigeminus der Ratte.

The activity of NAD-linked L-alpha-glycero-3-phosphate dehydrogenase (E.C. 1.1.1.8.) in the Vth nerve from albino rats treated with a single subcutaneous injection of ethylnitrosourea in the first day of life and from controls was determined biochemically in 10 different age groups ranging from 7 to 70 days. The specific activities of the controls rise rapidly from the 7--35 days of life and remain there after almost at the same level. The activity of the ENU treated animals is significantly lower than that of the controls and reaches only at the age of 63 days the values of the 35 days old untreated animals. About 26% of the activity values of the treated animals are below the lower confidence limit of the controls.

Effect of age at treatment on the incidence and location of neurogenic tumors induced in Wistar rats by a single dose of N-ethyl-N-nitrosourea.

The effect of age at treatment on the incidence and location of neurogenic tumors induced by N-ethyl-N-nitrosourea (ENU) was investigated in 232 Wistar rats of both sexes. Rats were given 40 ng/kg of ENU on the 16th day of gestation (group I), on the day of birth (group II), and on the 1st week (group III), 2nd week (group IV), 3rd week (group V) and 4th week (group VI) after birth. Up to 6 months of observation, the brain consistently showed the highest susceptibility ranging from group I (93%) to group VI (36%), followed by the spinal cord (group I, 34%; group II, 64%; group III, 43%). However, the trigeminal nerve was only susceptible in group I (27%) and group II (36%) and the spinal root was susceptible exclusively in group II (46%). Most of the tumors obtained were oligodendrogliomas or mixed gliomas. Glioependymomas of the spinal cord were predominant only in group II. The temporal and paraventricular regions and hippocampus were the preferred sites of brain tumors in group I and II, but in groups III and IV frontal tumors were predominant. Mesenchymal tumors of the kidney were also induced, mainly in groups III (16%) and IV (16%).

Effects of sex difference, gonadectomy and estradiol on N-ethyl-N-nitrosourea-induced trigeminal nerve tumors in rats.

The occurrence of trigeminal nerve tumors (TNTs) induced by neonatal administration of N-ethyl-N-nitrosourea (ENU) in WF x LE F1 (F1) rats was studied with special reference to sex difference, effect of gonadectomy and estradiol (E2) administration. Experimental groups 1-6 were treated with 40 mg ENU/kg of body weight neonatally. They consisted of male, female, castrated male, ovariectomized female, E2 pellet (0.1 mg, s.c.) supplemented and gonadectomized male and female rats respectively. Rats of groups 7-12 served as the respective controls without ENU. All the rats were killed at 8 months of age. Levels of serum E2 and E2 receptor (ER) of the TNTs were also examined. It was noted that the incidence of TNT was higher in males (79%) than in females (48%, P less than 0.05) and did not change by castration in males (91%) but increased in ovariectomized female rats (74%, P less than 0.05). Administration of E2 followed by gonadectomy inhibited the occurrence of TNTs in male rats (59%) but not in female rats (60%). No TNT was observed in any control groups. Kidney tumors were the second most frequent tumors next to nervous system tumors in the present experiment. The incidence of kidney tumors was much higher in females (38%) than in males (4%, P less than 0.05) and decreased by ovariectomy, whereas it increased in male rats by E2 administration. ER levels of TNTs and trigeminal nerve tissue were less than 1 fmol/mg protein. These results suggest that in rats treated with ENU neonatally, E2 has an inhibitory effect on the induction of TNTs but may not be regulated through ER. E2 also shows a promoting effect on kidney tumorigenesis.

The effect of nerve growth factor and antibodies to nerve growth factor on ethylnitrosourea-induced neoplastic proliferation in rat trigeminal nerves.

Sprague-Dawley (CD) rats were injected intravenously with ethylnitrosourea at a dose of 20 mg/kg on day 20 of gestation. This exposure resulted in early neoplastic proliferation or development of a neurinoma of the trigeminal nerve in 58% of the offspring at 90 days of age. Implantation of osmotic microinfusion pumps containing 2.5S nerve growth factor prior to ethylnitrosourea administration significantly reduced the incidence of early neoplastic proliferation. Postnatal implantation of microinfusion pumps containing 2.5S nerve growth factor also resulted in a significant but less pronounced reduction of early neoplastic proliferation. Immunoglobulin G directed against nerve growth factor (anti-nerve growth factor) did not influence the incidence of early neoplastic proliferation when administered via microinfusion pumps implanted on day 15 postnatally. These findings suggest that nerve growth factor has a protective effect on the developing nervous system against ethylnitrosourea-induced carcinogenesis.

Immunohistochemical investigations in experimentally induced tumors of the nervous system.

35 tumors of brain, spinal cord and cranial and peripheral nerves were induced with ENU (ethyl-nitrosourea) in the offspring of treated BD-IX pregnant rats. 36 tumors--35 of the nervous system, one nephroblastoma--were observed in 14 rats. With these results, the number of experimental nervous system tumors of the own collection induced in BD-IX rats and classified next to the rules of human neurooncology, amounts to 2,216. All 35 tumors of the nervous system were treated by a panel of immunohistochemical reactions comprising antibodies against cytoskeleton intermediary filaments such as GFAP (glial fibrillary acid protein), neurofilament proteins, vimentin and cytokeratins and some nervous system antigens such as NSE (neuron specific enolase), MBP (myelin basic protein) and S-100 protein. In central tumors, considered to be malignant gliomas, focal reactivity against vimentin and GFAP was found. Expression of other tested markers was weak or absent. In neurinoma of trigeminal and peripheral nerves, reactivity to S-100 antigen was lacking, whilst there was strong reaction to the vimentin antigen.