Role of apoptosis for mouse liver growth regulation and tumor promotion: comparative analysis of mice with high (C3H/He) and low (C57Bl/6J) cancer susceptibility.

Apoptosis constitutes one of the organisms defense lines against cancer. We investigated whether failure of apoptosis may be concurrently causative for the high cancer susceptibility in C3H/He as compared to C57BL/6J mice (low cancer susceptibility). First, in short-term in vivo experiments (7-21 days), mouse liver growth (C3H/He, C57BL/6J) was induced by administration of phenobarbital (PB; 2 days 500 ppm + 5 days 750 ppm via the food) or nafenopin (NAF; 7 days 500 ppm via the food), cessation of PB or NAF treatment served to initiate liver involution. Liver weight, DNA content, hepatocyte ploidy and apoptotic activity were studied as endpoints. Secondly, in a long-term study liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w.) to 5-weeks-old C57Bl/6J and C3H/He mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w.) for up to 90 weeks. Cell proliferation and apoptosis in normal liver tissue and (pre)neoplastic tissue was quantitatively analysed by histological means. The short term studies revealed that PB and NAF-induced mouse liver growth is essentially due to cell enlargement (hypertrophy). A moderate increase of liver DNA content was brought about by hepatocellular polyploidization; C3H/He mice exhibited the most pronounced ploidy shift, corresponding to their high cancer susceptibility. Upon cessation of PB or NAF treatment, regression of liver mass was neither associated with a loss of DNA nor an increase in apoptoses in the liver of C3H/He and C57Bl/6J mice; food restriction did not enforce the occurrence of apoptosis. Thus, the mouse strains did not differ with respect to the occurrence of apoptosis. In the long-term study, PB promoted liver tumor formation in all strains, exhibiting quantitative differences in growth kinetics of preneoplasia rather than a specific biological quality. Quantitative analysis of apoptosis in normal and (pre)neoplastic liver tissue of C3H/He and C57BL/6J mice revealed no clue to explain their different cancer susceptibility. Rather, cell proliferation seems to be the prevailing determinant of tumor promotion in the liver of both mouse strains.

Intestinal microflora plays a crucial role in the genotoxicity of the cooked food mutagen 2-amino-3-methylimidazo [4,5-f]quinoline.

We investigated the impact of the intestinal microflora on the genotoxicity of 2-amino-3-methylimidazo[4,5-f] quinoline (IQ), a mutagenic/carcinogenic heterocyclic amine commonly found in fried meats and fish. In parallel, we also examined the effect of the microflora on the protective effect of glucotropaeolin (GT), a glucosinolate contained in cruciferous vegetables, towards IQ-induced genotoxic effect. Conventional (NF), human flora associated (HFA) and germ free (GF) rats were treated either with 90 mg/kg IQ alone, 150 mg/kg GT alone or a combination of the two by gavage and DNA damage was determined in liver and colon cells using the alkaline single cell gel electrophoresis (SCGE) or comet assay. IQ caused a significant effect in both organs of all groups. However, DNA damage was most pronounced in NF animals. In colon cells, DNA migration was 6-fold more in IQ-exposed rats as compared with untreated controls. The effect measured with liver cells was similar. In comparison to NF rats, in HFA rats, tail length of the comets was 22 and 53% lower in liver and colon cells, respectively. Significantly weaker effects were seen in GF animals (66 and 75% lower damage in hepatocytes and colonocytes, respectively, than in NF animals). Pretreatment with GT led to a complete reduction of IQ-induced DNA damage regardless of the microbial status of the animals. In addition, a moderate decrease in spontaneous DNA damage was seen in animals that received GT alone. Our results show that the microflora has a strong impact on the genotoxic effects of IQ. We conclude that the alkaline SCGE assay with rats harbouring different flora opens new possibilities to investigate the role of intestinal bacteria on health risks caused by dietary carcinogens.

Effects of cruciferous vegetables and their constituents on drug metabolizing enzymes involved in the bioactivation of DNA-reactive dietary carcinogens.

Epidemiological studies give evidence that cruciferous vegetables (CF) protect humans against cancer, and also results from animal experiments show that they reduce chemically induced tumor formation. These properties have been attributed to alterations in the metabolism of carcinogens by breakdown products of glucosinolates, which are constituents of CF. The present article gives an overview on the present state of knowledge on the impact of CF and their constituents on enzymes that are involved in the metabolism of DNA-reactive carcinogens. The development of in vitro models with metabolically competent cell lines led to the detection of potent enzyme inducers contained in CF such as sulforaphane. Recently, we showed that Brassica juices induce glutathione-S-transferases (GST) and cytochrome P-450 1A2 in human hepatoma cells (HepG2) and protect against the genotoxic effects of B(a)P and other carcinogens. Earlier in vivo experiments with rodents indicated that indoles and isothiocyanates, two major groups of glucosinolate breakdown products, attenuate the effects of polycyclic aromatic hydrocarbons (PAHs) and nitrosamines via induction of GST and inhibition of cytochrome-P450 isoenzymes, respectively. Our own investigations showed that CF are also protective towards heterocyclic amines (HAs): Brussels sprouts- and garden cress juices attenuated IQ-induced DNA-damage and preneoplastic lesions in colon and liver of rats. These effects were paralleled by induction of uridine-di-phospho-glucuronosyl transferase (UDPGT) which is very probably the mechanism of protection against HAs by cruciferous vegetables. There is also evidence that consumption of CF might protect humans against cancer. In matched control intervention studies with these vegetables, it was shown that they induce GST-activities in humans but overall, results were inconclusive. Recently, we carried out crossover intervention studies and found pronounced GST-induction upon consumption of Brussels sprouts and red cabbage, whereas no effects were seen with white cabbage and broccoli. Furthermore, we found that the isoenzyme induced was GST-pi which plays an important role in protection against breast, bladder, colon and testicular cancer. No induction of the GST-alpha isoform could be detected. Urinary mutagenicity experiments gave further evidence that CF affect drug metabolism in humans. Consumption of red cabbage led to changes in the pattern of meat-derived urinary mutagenicity. Overall, CF are among the most promising chemopreventive dietary constituents and further elucidation of their protective mechanisms and the identification of active constituents may contribute to the development of highly protective Brassica varieties.

Nuclear Ras: unexpected subcellular distribution of oncogenic forms.

The Harvey-ras gene encodes small guanine nucleotide binding proteins, mutant forms of which are associated with a number of human malignancies. Based on studies with truncated forms of the protein it is known that correct post-translational processing of Ras is essential for cytoplasmic membrane localization and function. Surprisingly, immunofluorescence analysis provided evidence that in addition to its cytosolic localization, activated H-Ras(Val 12) was also localized in the nuclei of transformed cells both in vitro and in vivo. Immunoblot analysis of nuclear fractions was consistent with results found by immunohistochemistry. Moreover, inhibition of protein farnesylation prevented the nuclear targeting of activated H-Ras(Val 12) and NFkappaB. Alterations in subcellular distribution pattern and phosphorylation of the cell cycle inhibitor p27, which is involved in Ras driven tumor growth, coincided with nuclear localization of H-Ras(Val 12). Proteins are often not functional until they are transported to their final destination. Indeed, Ras was found to complex with NTF2 a factor involved in nuclear protein import and export. Therefore it is suggested that NTF2 is the actual carrier for oncogenic Ras. In view of these observations the question arises whether the nuclear localization of H-Ras(Val 12) in tumors is important in oncogenic activation or whether it is a response to apoptosis. J. Cell. Biochem. Suppl. 36: 1-11, 2001.