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This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCR on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/beta-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.

The most prominent neoplastic lesions in mice in the 2-year studies of o-nitrotoluene and riddelliine were hemangiosarcomas. Fifteen o-nitrotoluene-induced hemangiosarcomas of the skeletal muscle, subcutaneous tissue, and mesentery; 12 riddelliine-induced hemangiosarcomas of the liver; and 15 spontaneous subcutaneous hemangiosarcomas were examined for genetic alterations in ras, p53, and beta-catenin genes. Mutations in at least one of these genes were identified in 13 of 15 (87%) of the o-nitrotoluene-induced hemangiosarcomas with missense mutations in p53 exons 5-8 detected in 11 of 15 (73%) of these neoplasms. Seven of 15 (47%) hemangiosarcomas from mice exposed to o-nitrotoluene had deletions at exon 2 splice sites or smaller deletions in the beta-catenin gene. K-ras mutation was detected in only 1 of the 15 (7%) o-nitrotoluene-induced hemangiosarcomas. In contrast to the o-nitrotoluene study, 7/12 (58%) riddelliine-induced hemangiosarcomas had K-ras codon 12 GTT mutations and, when screened by immunohistochemistry, 9/12 (75%) had strong staining for the p53 protein in malignant endothelial cells, the cells of origin of hemangiosarcomas. Riddelliine-induced hemangiosarcomas were negative for the beta-catenin protein. Spontaneous hemangiosarcomas from control mice lacked both p53 and beta-catenin protein expression and ras mutations. Our data indicated that p53 and beta-catenin mutations in the o-nitrotoluene-induced hemangiosarcomas and K-ras mutations and p53 protein expression in riddelliine-induced hemangiosarcomas most likely occurred as a result of the genotoxic effects of these chemicals. It also suggests that these mutations play a role in the pathogenesis of the respective hemangiosarcomas in B6C3F1(1) mice.

In this study we used liver neoplasms induced by several chemical carcinogens to investigate potential nuclear targets associated with beta-catenin/Wnt signaling and potential membrane-associated beta-catenin binding partners. Strong expression of cyclin D1, in a pattern similar to that observed previously for beta-catenin, was observed by Western analysis for all five hepatoblastomas examined regardless of treatment. Increased expression of cyclin D1 was also detected in 12 of 35 (34%) hepatocellular neoplasms. Ten of 15 tumors (67%) that had mutations in the Catnb gene had upregulation of cyclin D1, while only 2 of 20 tumors (10%) without Catnb mutations had increased cyclin D1 expression. Immunohistochemical analysis confirmed strong expression of cyclin D1 in most nuclei of hepatoblastomas and scattered nuclear staining in hepatocellular tumors that had Catnb mutations. Increased c-Jun expression was observed in 19 of 30 (63%) hepatocellular tumors and all hepatoblastomas, although upregulation was not completely correlated with Catnb mutation. C-Myc expression was not increased in the tumors. Reduced expression of E-cadherin, which interacts with beta-catenin at the membrane, was observed in some tumors, but this did not correlate with Catnb mutation. Expression of the epidermal growth factor receptor, which may have a role in beta-catenin tyrosine phosphorylation, was lower in some tumors than in normal tissue depending on chemical treatment. The results provide evidence that increased expression of cyclin D1 and c-Jun may provide an advantage during tumor progression and in the transition from hepatocellular neoplasms to hepatoblastomas. Moreover, it is likely increased cyclin D1 expression results at least in part from Catnb mutation, beta-catenin accumulation, and increased Wnt signaling.

Four putative quantitative trait loci (QTLs) that influence susceptibility to the induction of lung adenomas by urethane in an F2 cross between A/J and BALB/cOlaHsd have been mapped. Following microsatellite typing of mice with resistant and susceptible phenotypes at 97 microsatellite marker loci, a major locus was identified on chromosome 18 with a lod score of 15. This was responsible for an 8- to 10-fold increase in tumor multiplicity in males and females, respectively, having the AA and CC genotypes at the D18Mit188 marker locus. It mapped close to Dcc (deleted in colorectal cancer). A locus on chromosome 4 (lod score 6.5) had the resistant allele in strain A/J and the susceptible allele in BALB/c, with a 14-fold difference in tumor multiplicity between mice of the AA and CC genotypes. This mapped close to the Cdkn2a (cyclin-dependent kinase inhibitor 2A) locus, which is commonly deleted in mouse lung tumors. Two loci with smaller effects (lod scores 3.03 and 3.25) were identified on chromosomes 1 and 11. There was also significant sexual dimorphism in tumor multiplicity both among 151 F2 hybrids and among 52 mice resulting from a backcross to strain A/J, with males having higher tumor counts than females., (Copyright 1998 Academic Press.)