BDIV Rats Research Articles

Abstract 4807: Braf mutations initiate the development of rat gliomas induced by postnatal exposure to N-ethyl-N-nitrosourea (ENU)

Abstract Purpose A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T -> A mutations in the transmembrane domain of the neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas was unknown. The objectives of this study were to identify driver mutations in ENU-induced rat gliomas. Methods We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one. Results T:A->A:T and T:A->C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41-55% of all somatic single nucleotide mutations). T->A mutations were detected in all 5 rat gliomas at Braf codon 545 (V545E), which corresponds to the human BRAF V600E. Additional screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V545E mutation, while peritumoral brain tissue of either strain (n=16) had the wild-type sequence. The gliomas were immunoreactive to BRAF V600E antibody. Conclusions Braf mutation is a frequent early event in the development of rat gliomas caused by a single dose of ENU. Citation Format: Kaishi Satomi, Qi Wang, Ji Eun Oh, Barbara Hutter, Benedikt Brors, Nicolle Diessl, Hai-Kun Liu, Stephan Wolf, Otmar Wiestler, Paul Kleihues, Bernd Koelsch, Andrea Kindler-Rohrborn, Hiroko Ohgaki. Braf mutations initiate the development of rat gliomas induced by postnatal exposure to N-ethyl-N-nitrosourea (ENU) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4807. doi:10.1158/1538-7445.AM2017-4807

Braf Mutations Initiate the Development of Rat Gliomas Induced by Postnatal Exposure to N-Ethyl-N-Nitrosourea

A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T->A mutations in the transmembrane domain of the Neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas remains enigmatic. We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one. T:A->A:T and T:A->C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41% to 55% of all somatic single nucleotide mutations). T->A mutations were identified in all five rat gliomas at Braf codon 545 (V545E), which corresponds to the human BRAF V600E. Additional screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V545E mutation, whereas peritumoral brain tissue of either strain had the wild-type sequence. The gliomas were immunoreactive to BRAF V600E antibody. These results indicate that Braf mutation is a frequent early event in the development of rat gliomas caused by a single dose of ENU.

Genetic basis of sex-specific resistance to neuro-oncogenesis in (BDIX × BDIV) F2 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.

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.

Development of a new method for the analysis of sphinganine and sphingosine in urine and tissues.

The fumonisins are inhibitors of de novo sphingolipid biosynthesis in vitro and in vivo and thus possibly interfere with the regulation of cell growth, differentiation, and neoplastic transformation. In addition, the ratio of free sphinganine (Sa) to free sphingosine (So) has been proposed as a marker of exposure for animals or humans consuming feed or food contaminated by these toxins. A method to analyze these sphingolipid bases has been proposed [Merrill et al., 1988: Anal Biochem 171:373-381; Riley et al., 1994a: JAOAC 77:533-540] but involves numerous steps and consequently is not ideally suited to the analysis of large numbers of samples, as is often required in epidemiological studies. A new method was therefore developed for the analysis of the Sa/So ratio in tissues as well as human and rat urine. Briefly, the method involves isolation of exfoliated cells from as little as 0.5 ml of rat urine or 2 ml of human urine followed by a rapid and efficient extraction of sphingolipid bases in ethyl acetate, an optimized derivatization step with o-phthaldialdehyde and a high-pressure liquid chromatography separation on a 250 mm x 4.6 mm. 5 microns Kromasil C18 column, with a 4-step phosphate buffer/methanol gradient. Fluorescence was monitored at 340 nm excitation, 455 nm emission, and retention times for So, Sa, and C-20 Sa were about 11, 14, and 22 min, respectively. The method was adapted to tissue analysis by partially digesting approximately 30 mg tissue with trypsin to permit isolation of a cell pellet before extraction of the sphingolipids as described above. The method was applied to the analysis of So and Sa in urines and tissues of fumonisin B1 (FB1) treated and untreated male BDIV rats. The Sa/So ratio in urine of untreated rats varied from 0.1 to 0.7, and for treated rats (between 1-5 mg FB1/kg body weight daily by gavage), the ratio was between 1.2-10. In kidney, the ratio was 0.1 in control rats and varied from 4 to 10.3 in treated rats. In human urine, measurements could easily be made in 2 ml of urine in females, but in males much larger volumes were required due to the low levels of sphingolipid bases.

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.

Gender‐specific polygenic control of ethylnitrosourea‐induced oncogenesis in the rat peripheral nervous system

The inbred BD rat strains constitute a model system for analysis of the genetic basis of susceptibility or resistance to the development of neural tumors, as they exhibit distinct strain-specific differences regarding the sensitivity to tumor induction by the alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Among the different BD strains, BDIX and BDIV rats, respectively, are either highly susceptible or entirely resistant to the development of EtNU-induced malignant schwannomas of the peripheral nervous system (PNS), predominantly of the trigeminal nerves. We have previously mapped one locus associated with susceptibility/resistance to schwannoma induction to the telomeric third of chromosome 10 (Mss1) in segregating (BDIX x BDIV) crosses. We report on the genetic mapping of 6 further loci controlling tumor incidence or survival time on chromosomes 1 (Mss2), 3 (Mss3), 6 (Mss4), 13 (Mss5) and 15 (Mss6) as well as on chromosome 10 (Mss7) close to the centromere. Interestingly, most of these loci mediate gender-specific effects of variable strength ranging from minor influences on tumor development to complete tumor resistance. The gender specificity is reflected by the fact that male (BDIX x BDIV) F2 rats exhibit a 2-fold higher incidence of EtNU-induced schwannomas than females as well as a shorter survival time. A number of human nervous system tumors too arise with a marked gender bias. Genes mediating gender-specific predisposition of developing malignant schwannomas in the rat may be relevant for the human individual risk of developing nervous system tumors.

The induction and persistence of altered sphingolipid biosynthesis in rats treated with fumonisin B1

The fumonisins produced by Fusarium spp. are carcinogenic in rodents and possibly carcinogenic to humans. One action of fumonisins is to inhibit the enzyme ceramide synthase and as a consequence disrupt de novo sphingolipid biosynthesis. Measurement of the ratio of sphinganine (Sa) to sphingosine (So) in urine, serum and tissues has thus been made in various animal species as a marker of exposure to this mycotoxin. In this study in male BDIV rats, the effect of increasing daily gavage doses (0.01–5mg/kg b.w.) of fumonisin B1 (FB1) on the Sa/So ratio in various tissues and urine was examined as was the persistence of the effects after cessation of treatment. The lowest dose of FB1 which induced an alteration of Sa/So in any tissue was 1mg/ kg b.w. The tissue most affected was the kidney, with the liver affected to a much lesser extent and the lung and oesophagus not at all. Only a modest indication of an alteration in Sa/So ratio was obtained in the urine. The large interindividual variations in urinary Sa and So in this strain of rat, even in the absence of FB1 treatment, made interpretation of these data difficult. After cessation of treatment, the Sa/So ratio returned to normal in kidney after a period of 1–3 weeks. These data suggest that altered Sa/So ratios reflect recent exposure to FB1 in the rat.

Seventh Japanese–German workshop on molecular and cellular aspects of carcinogenesis

Seventh Japanese–German workshop on molecular and cellular aspects of carcinogenesis

Seventh Japanese-German workshop on molecular and cellular aspects of carcinogenesis

Seventh Japanese-German workshop on molecular and cellular aspects of carcinogenesis

Allele-specific losses of heterozygosity on chromosomes 1 and 17 revealed by whole genome scan of ethylnitrosourea-induced BDIX x BDIV hybrid rat gliomas.

The induction of neural tumors by N-ethyl-N-nitrosourea (EtNU) in inbred strains of rats has evolved as a valuable model system of developmental stage- and cell type-dependent oncogenesis. Tumor yield and latency times are strongly influenced by genetic background. Compared with BDIX rats, BDIV rats are relatively resistant to the induction of brain tumors by EtNU, with a lower tumor incidence and latency periods prolonged by a factor of 3. To characterize genetic abnormalities associated with impaired tumor suppressor gene function in neuro-oncogenesis, losses of heterozygosity (LOHs) and microsatellite instability (MI) were investigated in brain tumors induced by EtNU in (BDIV x BDIX) F(1) and F(2) rats. The polymerase chain reaction was used to amplify 55 polymorphic microsatellite markers spanning the entire rat genome. The tumors displayed different histologies and grades of malignancy, corresponding to part of the spectrum of human gliomas. MI was not observed in any of the tumors. LOH of rat chromosome 1q was predominantly detected in oligodendrogliomas and mixed gliomas, with a 30% incidence in informative cases. 11p15.5, the human genome region syntenic to the consensus region of LOHs observed on rat chromosome 1, has been shown to be involved in the formation of gliomas in humans. Furthermore, rat brain tumors of different histologies often showed allelic imbalances on chromosome 17p. In both cases of LOH, there was a clear bias in favor of the parental BDIV allele, suggesting the involvement of tumor suppressor genes functionally polymorphic between the two rat strains.

LINKAGE OF NEUTROPHIL RESPONSE TO A MUTAGEN GENE (Nrm‐1) TO THE AGOUTI LOCUS IN THE RAT

The ACI or BDIV rats responded with decreased or increased neutrophil levels in blood after the N-methyl-N-nitrosourea (MNU) administration. The F1 hybrids had decreased neutrophil counts, and the BDIV x (BDIV x ACI)F1 backcross offspring showed two phenotypes. The sex of the rats and the neutrophil response to MNU assorted independently. The results indicated that the neutrophil response to MNU was regulated by autosomal gene Nrm-1 with two alleles. The Nrm-1d regulates the decrease and the Nrm-1i regulates the increase of neutrophils in blood after the MNU administration. The results were confirmed by the SKUMIX computer program. We found that the Nrm-1 gene was linked to the agouti locus (chi-square = 10.3, P less than 0.001). The map distance between two genes was 33 +/- 5 cM. The Nrm-1 gene thus resides on the linkage group IV of the rat.

Identification in rats of N-nitrosonipecotic acid as a major urinary metabolite of the areca-nut alkaloid-derived nitrosamines, N-nitrosoguvacoline and N-nitrosoguvacine

N-Nitrosamines derived from areca-nut alkaloids have been implicated in cancer of the oral cavity and esophagus caused by betel quid chewing in India and other Asian countries. A major urinary metabolite of N-nitrosoguvacoline and N-nitrosoguvacine, both present in saliva of betel quid chewers of ppb levels, was isolated from rat urine and identified as N-nitrosonipecotic acid by comparison with the authentic compound. When a dose of 50 or 500 μg/rat of either compound was administered orally to BDIV rats, 66–85% of the dose was excreted in the urine as N-nitrosonipecotic acid and 2–8% as N-nitrosoguvacine. These N-nitrosamino acids could be analysed in the urine of betel quid chewers as a marker of exposure to areca-nut specific nitrosamines.

Dietary Restriction of Tyrosine and Phenylalanine: Inhibition of Metastasis of Three Rodent Tumors<xref ref-type="fn" rid="FN2">2</xref>

The effects of a combined dietary restriction of tyrosine and phenylalanine on metastasis were investigated with the use of 3 rodent tumor cell lines: B16-bladder 6 (BL6) melanoma inoculated into (C57BL/6 X DBA/2)F1 mice, Lewis lung (3LL) carcinoma inoculated into C57BL/6 mice, and RT7-4bs hepatocarcinoma inoculated into BD-IV rats. When examined for effects on spontaneous metastasis, dietary restriction of tyrosine and phenylalanine had no effect on metastasis to draining lymph nodes in either BL6 or 3LL tumors. However, the restriction did reduce metastasis of RT7-4bs cells to draining lymph nodes by 60%. In all tumor systems, the dietary restriction effectively inhibited the subsequent growth of lymph node tumors. The most marked effect of the dietary restriction was on spontaneous hematogenous metastasis, which was almost totally blocked for BL6 cells and was reduced by 85% for RT7-4bs cells. Tumor-associated splenomegaly also was completely inhibited in 3LL tumor-bearing mice. The selective dietary amino acid restriction failed to reduce initial lung colonization in the experimental metastasis assay but clearly inhibited subsequent tumor outgrowth in the lungs. These findings demonstrate that modification of host nutritional status by restriction of the dietary intake of tyrosine and phenylalanine exerts a dramatic antimetastatic effect directed particularly on spontaneous hematogenous metastasis. Although the preliminary data suggest a primary modulating effect on tumor cell populations growing in diet-restricted animals to reduce inherent metastatic ability, the actual mechanisms remain to be defined.

A carcinogenicity study of styrene-7,8-oxide in rats

Styrene-7,8-oxide (200 mg/kg body wt) was given orally to female BDIV rats on 17th day of pregnancy. Their offspring received 96 weekly doses of styrene oxide (100–150 mg/kg body wt). Following the continuous administration of styrene oxide, and increased incidence, statistically significant, of forestomach tumours was observed in rats of both sexes. The incidence of tumours occurring at other sites was similar in treated and control animals. The present results show that styrene oxide is a direct-acting carcinogen producing benign and malignant tumours of the forestomach.

Benzo[a]pyrene metabolites: formation in rat liver cell-culture lines, binding to macromolecules, and mutagenesis in V79 hamster cells.

Benzo(a]pyrene was metabolized in liver cell lines derived from BC-IV and BD-IV rats which included several chemically-transformed lines (IAR-6-1; IAR-19; IAR-28), one spontaneous transformant (IAR-27) as well as one non-malignant line (IAR-20). Cultures were treated with tritiated benzo[a]pyrene over a 5-day period. The cells and medium were extracted with ethyl acetate and the distribution between organic-soluble and water-soluble metabolites determined. Organic-soluble metabolites consisting of dihydrodiols, phenols and quinones were determined by high-pressure liquid chromatography, and macromolecular binding of BP to each cell line was measured over a 24-h period. Comparisons between binding and overall metabolism were not directly proportional in these liver cell lines. However, there was a positive correlation for benzo[a]pyrene mutagenesis in the V-79 hamster cell assay with 8-azaguanine as a marker when the cell lines with the highest (IAR-20) and lowest (IAR-27) metabolic competence were used as activating cell layers.

Carcinogenicity of dapsone of mice and rats.

Dapsone (4,4'-diamino-diphenyl sulfone) has been tested for possible carcinogenicity in long-term animal experiments. BDIV rats and C₇ Bl mice received a 3.5% aqueous suspension of dapsone by intragastric intubation. Treatment was started in pregnant females during the last part of pregnancy, continued during lactation, then given to the offspring after weaning, five times a week for 104 weeks. The dose administered was 100 mg/kg to both rats and mice; total doses ranged from 10-16 g per rat and 1.2-1.4 g per mouse. Separate groups of animals received a combined treatment of dapsone with urethane or benzo (alpha) pyrene, to investigate the possible additive or synergistic action of dapsone with known carcinogens, as well as the possible inhibiting effect of dapsone on carcinogenesis. Unusual tumors, viz. spleen sarcomas (related to severe fibrosis of the spleen) were detected in male rats, and higher morbidity from C-cell thyroid carcinomas was observed in treated rats of both sexes than in control rats. There was no evidence that dapsone can modify the action of other chemical carcinogens. It was noted that: (1) although the increase in the incidence of tumours in dapsone-treated animals over that observed in untreated controls is statistically significant, the increase is relatively low; (2) the tumours appeared after lifetime treatment with maximum tolerated doses; (3) in rats, spleen sarcomas were observed mostly in males; this may indicate a possible hormone-dependence of the observed carcinogenic effect. The present results therefore provide only limited evidence of carcinogenicity of dapsone in rats.

Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine.

1. BD-IV rats were given labelled dimethylnitrosamine (2 mg/kg) by stomach tube on weekdays (Monday to Friday) for up to 24 weeks. The rats killed after 2, 4, 8, 16 and 24 weeks of treatment (72 h after the final dimethylnitrosamine gavage) and DNA was isolated from the pooled livers, kidneys and lungs. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. Throughout the experiment, the content of 7-methylguanine in liver DNA was approx. 16 times that in kidney and lung. The amount of this product increased in the DNA of all three tissues up to 16 weeks, but by 24 weeks had decreased by 20% in the liver and 46% in the other tissues. 3. O6-Methylguanine was not detected in liver DNA, but was easily measured in kidney and lung DNA after 4 weeks of dimethylnitrosamine administration. The amount of O6-methylguanine in kidney and lung DNA increased relative to that of 7-methylguanine, and by 24 weeks was 60% of the 7-methylguanine content in both tissues. 4. Incorporation of radioactive C1 breakdown products of dimethylnitrosamine into normal purines in DNA increased continuously in all three tissues. 5. The results are discussed with respect to the specific hepatocarcinogenic effect of chronic administration of dimethylnitrosamine and the possible contribution of increased DNA repair and DNA synthesis.