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Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice. The induction of skin tumors in the hairless p53+/- mice was accomplished by daily exposure to two different UV-doses of approximately 450 J/m2 and 900 J/m2 from F40 lamps corresponding to a fraction of about 0.4 and 0.8 of the minimal edemal dose. Marked differences in skin carcinogenesis were observed between the p53+/- mice and their wild type littermates. Firstly, at 900 J/m2, tumors developed significantly faster in the heterozygotes than in wild types, whereas at 450 J/m2 there was hardly any difference, suggesting that only at higher damage levels loss of one functional p53 allele is important. Secondly, a large portion (25%) of skin tumors in the heterozygotes were of a more malignant, poorly differentiated variety of squamous cell carcinomas, i.e. spindle cell carcinomas, a tumor type that was rarely observed in daily UV exposed wild type hairless mice. Thirdly, the p53 mutation spectrum in skin tumors in heterozygotes is quite different from that in wild types. Together these results support the notion that a point mutation in the p53 gene impacts skin carcinogenesis quite differently than allelic loss: the former is generally selected for in early stages of skin tumors in wild type mice, whereas the latter enhances tumor development only at high exposure levels (where apoptosis becomes more prevalent) and appears to increase progression (to a higher grade of malignancy) of skin tumors.

The nucleotide excision repair (NER) pathway comprises two sub-pathways, transcription coupled repair (TCR) and global genome repair (GGR). To establish the importance of these separate sub-pathways in tumor suppression, we exposed mice deficient for either TCR (Csb), GGR (Xpc) or both (Xpa) to 300 ppm 2-acetylaminofluorene (in feed, ad libitum) in a unique comparative exposure experiment. We found that cancer proneness was directly linked to a defect in the GGR pathway of NER as both Xpa and Xpc mice developed significantly more liver tumors upon 2-AAF exposure than wild type or Csb mice. In contrast, a defect in TCR appeared to act tumor suppressive, leading to a lower hepatocellular tumor response in Xpa mice (tumor incidence of 25%) as compared to Xpc mice (53% tumor-bearing mice). The link between deficient GGR and tumor proneness was most pronounced in the liver, but this phenomenon was also found in the urinary bladder. As tumor induction by 2-AAF appeared almost exclusively dependent on a defect in GGR, we examined whether gene mutation induction in the non-transcribed lacZ locus could reliably predict tumor risk. Interestingly, however, short-term 2-AAF exposure induced lacZ mutant levels in Csb mice almost as high as those found in Xpa or Xpc mice. This indicates that lacZ mutant frequencies are not correlated with a specific DNA repair defect and eventual tumor outcome, at least not in the experimental design presented here.

Phytoestrogens such as isoflavonoids and lignans have been postulated as breast cancer protective constituents in soy and whole-grain cereals. We investigated the ability of isoflavones (IFs) and flaxseed to modulate spontaneous mammary tumor development in female heterozygous Tg.NK (MMTV/c-neu) mice. Two different exposure protocols were applied, either from 4 wk of age onward (postweaning) or during gestation and lactation (perinatal). In the postweaning exposure study, mice were fed IFs or flaxseed in a high-fat diet. In addition, flaxseed in a low-fat diet was tested. Postweaning exposure to IFs and flaxseed tended to accelerate the onset of mammary adenocarcinoma development, although tumor burden at necropsy was not changed significantly. Perinatal IF exposure resulted in enhanced mammary gland differentiation, but palpable mammary tumor onset was not affected. However, tumor burden at necropsy in the perinatal exposure study was significantly increased in the medium- and high-IF dose groups. Comparison of both exposure scenarios revealed a strongly accelerated onset of tumor growth after perinatal high-fat diet exposure compared with the low-fat diet. This study shows that breast cancer-modulating effects of phytoestrogens are dependent both on the background diet and on the timing of exposure in the life cycle.

Phytoestrogens, like isoflavonoids and lignans, have been postulated as possible colorectal cancer protective constituents. To investigate this hypothesis, two high-fiber sources rich in lignan precursors, i.e., rye bran and flaxseed, were tested for their ability to modulate intestinal tumor development in ApcMin mice. Test diets consisted of a control diet (a Western-style diet, adjusted for fiber and/or phytate content) supplemented with 5% flaxseed or 30% rye bran. Chemical analysis of diets and blood samples confirmed the enhanced systemic exposure of mice fed the test diets to the major lignan precursors, i.e., secoisolariciresinol and matairesinol. No statistically significant difference was observed in the incidence and multiplicity of small intestinal and colon tumors at terminal sacrifice between mice fed the control diet or the diet supplemented with 5% flaxseed. With the rye bran diet a statistically significant enhancement of the number of small intestinal tumors in female mice was observed. The number of colon tumors, however, was comparable between the control and rye bran-fed mice of either sex. Furthermore, no activating point mutations in the K-ras oncogene nor positive immunohistochemical staining for the p53 gene were observed in a set of 48 colon tumors. In conclusion, our results demonstrate that increased intake of lignan precursors from flaxseed or rye bran, administered in a Western-style diet, does not protect against intestinal tumor development in an appropriate animal model for intestinal neoplasia such as the ApcMin mice.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in which repair of ultraviolet (UV)-induced DNA damage is impaired or is totally absent due to mutations in genes controlling the DNA repair pathway known as nucleotide excision repair (NER). XP is characterized, in part, by extreme sensitivity of the skin to sunlight, and XP patients have a more than 1000-fold increased risk of developing cancer at sun-exposed areas of the skin. To study the role of NER in chemical-induced tumorigenesis in more detail, the authors developed Xpa-/- homozygous knockout mice with a complete defect in NER (designated as Xpa mice or XPA model). Xpa mice develop skin tumors at high frequency when exposed to UV light, and as such, they mimic the phenotype of human XP. Moreover, the Xpa mice also appear to be susceptible to genotoxic carcinogens given orally. Based on these phenotypic characteristics, the Xpa mice were considered to be an attractive candidate mouse model for use in identifying human carcinogens. In an attempt to further increase both the sensitivity and specifi city of the XPA model in carcinogenicity testing, the authors crossed Xpa mice with mice having a heterozygous defect in the tumor suppressor gene p53. Xpa/p53+/- double knockout mice develop tumors earlier and with higher incidences upon exposure to carcinogens as compared to their single knockout counterparts. Here the authors describe the development and features of the Xpa mouse and present some examples of the Xpa and Xpa/p53+/- mouse models' sensitivity towards genotoxic carcinogens. It appeared that the Xpa/p53 +/- double knockout mouse model is favorable over both the Xpa and p53+/- single knockout models in short-term carcinogenicity testing. In addition to the fact that the double knockout mice respond more robustly to carcinogens, they also appear to respond in a very discriminative way. All compounds identified thus far are true (human) carcinogens, and, therefore, the authors believe that the Xpa/p53+/- mouse model is an excellent candidate for a future replacement of the chronic mouse bioassay, at least for certain classes of chemicals.

XPA-deficient mice have a complete deficiency in nucleotide excision repair, and as such they display a cancer predisposition after exposure to several carcinogens. Besides being sensitive to genotoxic agents applied to the skin, they are also susceptible to human carcinogens given orally, like benzo[a]pyrene (B[a]P). To study the role of the tumor suppressor gene p53 in DNA repair, gene mutation, and tumor induction, we crossed XPA-deficient mice with p53 knockout mice and lacZ (pUR288) gene marker mice. When treated orally (by gavage) with B[a]P, the XPA−/−/p53+/− double transgenic mice developed tumors much earlier and with higher frequency compared to their single transgenic counterparts. The major tumor type found in all genotypes was generalized lymphoma mainly residing in the spleen; several sarcomas were observed in p53+/− and XPA−/−/p53+/− mice. Next, we determined lacZ mutation frequencies in several (non)target tissues. It appeared that in the spleen (the major tumor target tissue) of XPA−/− and XPA−/−/p53+/− mice the lacZ mutation frequency was significantly elevated (80–100 × 10−5), and was two times higher as found in spleens of B[a]P-treated WT and p53+/− mice (P = 0.003). In nontumor target tissues like liver and lung, we found a moderate increase in the lacZ gene mutation frequency (30–40 × 10−5), which was independent of the genotype. The results obtained with the DNA-repair deficient XPA mice indicate that a significantly increased lacZ mutation frequency in a particular organ/tissue is an early marker for tumor development at later stages at the same site. However, the synergistic effect of a XPA−/−- and a p53+/−-deficiency in tumor development is not reflected by an absolute increase in the lacZ mutation frequency in the major tumor target tissue of XPA−/−/p53+/− or p53+/− mice compared to that of XPA−/− and WT mice, respectively. Environ. Mol. Mutagen. 34:124–130, 1999 © 1999 Wiley-Liss, Inc.

Data from epidemiological studies suggest that isoflavones in soy may have a protective effect on the development of colon cancer in humans. Therefore, we have investigated whether soy isoflavones will inhibit intestinal tumour development in Apc Min mice. The mice were fed a Western-type high risk diet (high fat, low fibre and calcium) containing two different isolates of soy protein as a protein source. For the control and test groups this resulted in the administration of about 16 and 475 mg of total isoflavones per kg diet, respectively. As a positive control, a third group of mice was administered a low isoflavone diet supplemented with 300 ppm sulindac. No significant differences in the incidence, multiplicity, size and distribution of intestinal tumours were observed between Min mice fed low and high isoflavone-containing diets. However, a clear reduction in the number of small intestinal tumours was observed for the sulindac diet. Thus, in contrast to epidemiological studies, our results demonstrate that high amounts of soy isoflavones present in a Western-type high risk diet do not protect against intestinal tumour development in a relevant animal model such as the Min mice.

Defects in the xeroderma pigmentosum complementation group A-correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer-prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet-B and 7,12-dimethylbenz[a] anthracene-induced skin tumors. Here we report that XPA-deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA-deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P-treated wild-type and heterozygous mice. Our results show for the first time that XPA-deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin.

The usefulness of transgenic E(mu)-pim-1 mice over-expressing the pim-1 oncogene in lymphoid tissues, as sensitive test organisms was studied in a short-term carcinogenicity study. The mice were fed standard diet Altromin 1314 supplemented either with 0.03% 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) for 7 months or with 0.03% 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for 6 months. PhIP and IQ are heterocyclic amines formed during cooking of meat and fish and are mutagenic to bacteria and cultured mammalian cells. PhIP is a potent mouse lymphomagen, while IQ is a liver carcinogen and also causes lung tumors and tumors of the forestomach in mice. We found that transgenic E(mu)-pim-1 mice are highly susceptible to PhIP induced lymphomagenesis but do not respond to the IQ treatment. PhIP feeding of E(mu)-pim-1 mice not only increased the total number of T-cell lymphomas but also decreased the latency time compared to either transgenic or wild-type controls. The effect was most pronounced in the treated female E(mu)-pim-1 mice, which showed a higher incidence of PhIP induced T-cell lymphomas than transgenic males and a strongly reduced latency period after PhIP treatment compared to non-transgenic mice. Our results suggest that the transgenic E(mu)-pim-1 mouse may be a useful model for short-term carcinogenicity screening of potential genotoxic carcinogens having the lymphoid system as target tissue. The carcinogen IQ which does not have the lymphoid system as a target was not recognized in this model.

Chronic use of phenacetin-containing analgesics has been associated with the development of renal cancer. To establish genotoxicity as a possible cause for the carcinogenic effect of phenacetin, we exposed wild type and DNA repair deficient Xpa-/- and Xpa-/-/Trp53+/- mice (further referred as Xpa and Xpa/p53 mice, respectively), carrying a reporter lacZ gene, to 0.75% (w/w) phenacetin mixed in feed. Xpa mice completely lack the nucleotide excision repair pathway, and as such they are sensitive to some classes of genotoxic compounds. Phenacetin exposure induced a significant increase of lacZ mutations in the kidney of both Xpa and Xpa/p53 mice. A minor response was found in liver, whereas no lacZ mutation induction was observed in the spleen of these animals. Interestingly, the observed phenacetin-induced mutant frequencies were higher in male than those found in female mice. This gender difference is probably due to a difference in metabolic rate. Phenacetin-induced mutations mainly consisted of point mutations rather than deletions. The mutational spectra in the kidney of treated WT and Xpa mice were quite similar. Taken together, these results demonstrate that the human carcinogen phenacetin acts as a weak genotoxic agent in an in vivo mouse model system.

As part of the international evaluation program coordinated by ILSI/HESI, the potential of DNA repair deficient Xpa- /- mice and the double knockout Xpa- /- .p53+ /- mice for short term carcinogenicity assays was evaluated. For comparison also wild-type C57BL/6 mice (WT) were included in these studies. Four test compounds were administered to groups of 15 male and 15 female Xpa- /- mice, Xpa- /- .p53+ /- mice and WT mice for 39 weeks. The model compounds investigated were haloperidol, reserpine (nongenotoxic rodent carcinogens, putative human noncarcinogens), phenacetin (genotoxic rodent carcinogen, suspected human carcinogen), and D-mannitol (noncarcinogen in rodents and humans). The test compounds were administered as admixture to rodent diet at levels up to 25 mg/kg diet for haloperidol, 7.5 mg/kg diet for reserpine, 0.75% for phenacetin, and 10% for D-mannitol. These levels included the maximum tolerable dose (MTD). Survival was not affected with any of the test compounds. Haloperidol, reserpine and D-mannitol were negative in the carcinogenicity assay with Xpa- /- and Xpa- /- .p53+ /- mice, showing low and comparable tumor incidences in controls and high-dose animals. The results obtained with phenacetin may be designated equivocal in Xpa- /- .p53+ /- mice, based on the occurrence of a single rare tumor in the target organ (kidney) accompanied by a low incidence of hyperplastic renal lesions and a high incidence of karyomegaly. These results are in agreement with the currently known carcinogenic potential of the 4 test compounds in humans.

The usefulness of transgenic Eµ-pim-1 mice bearing in their genome the pim-1 oncogene supplemented with an upstream immunoglobulin enhancer and a downstream murine leukaemia virus long terminal repeat, as sensitive test organisms was studied in two short-term carcinogenicity studies. The mice were fed standard diet Altromin 1314 supplemented either with 0.03% 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) for 7 months or with 0.03% 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for 6 months. PhIP and IQ are heterocyclic amines formed during cooking of meat and fish and are mutagenic to bacteria and cultured mammalian cells. PhIP is a potent mouse lymphomagen, while IQ is a liver, lung and forestomach carcinogen in mice. We found that transgenic Eµ-pim-1 mice are highly susceptible to PhIP induced lymphomagenesis but do not respond to IQ treatment. PhIP feeding of Eµ-pim-1 mice not only increased the total number of T-cell lymphomas but also decreased the latency time compared to either transgenic or wild-type controls. The effect was most pronounced in the treated female Eµ-pim-1 mice, which showed a higher incidence of PhIP induced T-cell lymphomas than transgenic males and a strongly reduced latency period after PhIP treatment compared to non-transgenic mice. Our results suggest that the transgenic Ep-pzm-i mouse may be a useful model for short-term carcinogenicity screening of potential genotoxic carcinogens having the lymphoid system as target tissue. Carcinogens that do not target this tissue, like IQ, however will not be recognised.

The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) catalyses the first and rate-limiting step in polyamine biosynthesis. Its activity is markedly increased in rapidly growing or regenerating tissue and is subject to regulation by a variety of trophic and mitogenic stimuli. ODC is therefore believed to play an essential role in the onset of cellular proliferation. In a molecular-biological approach to investigate ODC regulation upon induction by tumor promoters in rat liver we isolated an almost full-length rat ODC cDNA clone of 2.4 kb (designated pODC.ElO) from a cDNA library of testosterone-induced rat kidney poly(A)+ RNA. Characterization by restriction-endonuclease mapping and sequence analysis showed strong homology to mouse ODC cDNA sequences previously published [Gupta and Coffino, J. Biol. Chem. 260 (1985) 2941–2944; Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673–1677; Hickok et al., Proc. Natl. Acad. Sci. USA 83 (1986) 594–598]. This homology is most pronounced in the 461-aa-spanning coding region, amounting to 94% and 97% at the DNA and protein levels, respectively. In the 423-nt 5′ leader the rat-mouse homology (approx. 75%) is most pronounced in a region of about 175 nt directly upstream from the translational start site. The leader sequence also contains a perfect inverted repeat of 54 nt and ten additional upstream ATG triplets, which are all followed by nonsense codons before the initiating ATG. In the 633-nt 3′ trailer region of pODC.ElO an additional polyadenylation signal is observed more than 300 nt upstream from the 3′ end. Rat-mouse homology is about 80% up to this first polyadenylation signal and is considerably less thereafter. The presence of two alternate polyadenylation sites most likely accounts for the 3′ size heterogeneity observed in the two ODC mRNAs of 2.1 and 2.6 kb, respectively. In rat liver both mRNAs are coordinately induced by different tumor promoters. Finally, Southern blot analysis of normal rat liver and rat hepatoma DNA revealed that rat ODC, as in other rodents, belongs to a multigene family.