S. I. Ilnitskaya

Species‐specific effects of the hepatocarcinogens 3′‐methyl‐4‐dimethyl‐aminoazobenzene and ortho‐aminoazotoluene in mouse and rat liver

The effects of rat‐specific hepatocarcinogen 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB), mouse‐specific hepatocarcinogen ortho‐aminoazotoluene (OAT), non‐species‐specific hepatocarcinogen diethylnitrosamine (DENA), and non‐carcinogenic 4′‐methyl‐4‐dimethylaminoazobenzene (4′‐MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA‐binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen‐susceptible and ‐resistant animals. Species‐specific hepatocarcinogens 3′‐MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non‐carcinogenic 4′‐MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA‐binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species‐specific effects of OAT and of 3′‐MeDAB on HNF3 DNA‐binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32–0.47g/mL interval of ammonium sulfate concentration. In contrast, non‐specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.

Correlation between hepatocarcinogenic effect of estragole and its influence on glucocorticoid induction of liver-specific enzymes and activities of FOXA and HNF4 transcription factors in mouse and rat liver

It is known that the carcinogenic effect of estragole, a component of essential oils of many spicy plants, is characterized by species, tissue, and sex specificity. It causes mainly liver tumors in female mice but is not carcinogenic for male mice and for rats. In this work, the estragole hepatocarcinogenicity was shown for female mice of previously not studied ICR line. The strict correlation between estragole hepatocarcinogenicity and its ability to decrease the level of glucocorticoid induction of liver-specific enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) was found. Inhibition of TAT and TO inducibility by estragole takes place only in female mice but not in male mice and in rats. Studying the estragole effect on DNA-binding activity of transcription factors, present mainly in liver and regulating expression of genes encoding liver-specific proteins, has shown that estragole decreases FOXA and HNF4 activities but not activities of C/EBP and HNF1, and this happens only in female mice, for which this substance is hepatocarcinogen, but not in male mice and in rats. Pentachlorophenol, preventing hepatocarcinogenic effect of estragole, abolishes inhibitory influence of the latter on the TAT and TO glucocorticoid induction and restores DNA-binding activity of FOXA and HNF4. Thus, a correlation was revealed between the estragole hepatocarcinogenic effect and decrease in DNA-binding activity of transcription factors FOXA and HNF4, which might be indicative of the role of these factors in tumor suppression mechanisms in liver.

Effects of o-aminoazotoluene on liver regeneration and p53 activation in mice susceptible and resistant to hepatocarcinogenesis

The susceptibility to hepatocellular carcinoma (HCC) varies greatly within human populations in response to environmental risk agents. The mechanisms underlying differential susceptibility are still largely unknown and need to be clarified to improve HCC chemoprevention and therapeutic treatment. Inbred rodent strains with established predispositions for hepatocarcinogenesis offer the opportunity to identify intrinsic susceptibility and resistance factors. Previously, we have characterized mouse strains showing differential susceptibility to o-aminoazotoluene (OAT) and established that susceptibility does not result from OAT metabolism or genotoxicity in the livers of resistant and susceptible mice. In this study we have found that OAT differently affects hepatocyte proliferation in mice after partial hepatectomy (PH). OAT inhibited hepatocyte proliferation by 60-80% in the livers of susceptible mice, whereas resistant mice showed less than 15% inhibition. The inhibition resulted in significant delay of hepatic mass recovery in susceptible mice. OAT induced p53 stabilization and transcriptional activation in response to carcinogen treatment to the same degree in both, susceptible and resistant mice. Taken together, our data support inhibition of hepatocyte proliferation as a major cause for increased mouse susceptibility to hepatocarcinogenesis, and acceleration of functional liver recovery may offer a way to increase resistance to hepatic neoplasms. These results may have relevance to clinical observations of HCCs and implications for HCC chemoprevention and treatment.

Mutagenic activation and carcinogenicity of aminoazo dyes ortho-aminoazotoluene and 3′-methyl-4-dimethylaminoazobenzene in experiments on suckling mice

It is found that after administration of 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB,) which was hepatocarcinogenic to rats, in suckling mice, the number of neoplastic lesions in the liver of mice was 3 times higher than after analogous administration of equimolar dose of ortho-aminoazotoluene (OAT)). However, in the Ames test (TA-98 strain of Salmonella typhimurium) with activation by hepatic enzymes (S-9 fraction) of both intact and Aroclor-1254-induced mice and rats OAT contributed by an order of magnitude to revertant colonies compared to 3′-Me-DAB. In vivo inhibition of sulfotransferase activity, the enzyme which catalyzes the final stage of the mutagenic activation of aminoazo dyes, had no effect on carcinogenicity of 3′-Me-DAB but more than 4 times elevated that of OAT. It was concluded that the mechanism of carcinogenic action of aminoazo dyes studied is not genotoxic and that the carcinogenic potential of OAT is lost in the process of mutagenic activation.

Relationship between high sensitivity of suckling mice to hepatocarcinogenic and antiglucocorticoid effects of o-aminoazotoluene and diethylnitrosamine.

Suckling mice were more sensitive to the hepatocarcinogenic effect of various carcinogens compared to adult animals. After treatment with o-aminoazotoluene and diethylnitrosamine HNF3-DNA-binding capacity and glucocorticoid-induced liver tyrosine aminotransferase activity in suckling mice decreased more significantly than in adult animals.

Inhibitory effect of ortho-aminoazotoluene on diethylnitrosamine-induced hepatocarcinogenesis in suckling mice. Phenomenon and possible mechanism

The modifying effect of one compound on the carcinogenicity of another in combined application is attributed usually to some changes in the carcinogen metabolism, i.e. its activation or inactivation. In this paper, when used separately, diethylnitrosamine (DENA) induced 4–6 times more neoplastic lesions in the liver of suckling mice than ortho-aminoazotoluene (OAT) did. However, after combined treatment with both carcinogens the total number of hepatic lesions was significantly lower than that in mice treated with DENA only. Similar effect was observed when OAT was administered 3 days before or 3 days after DENA injection. The observed protective effect is not mediated at a metabolic level, because OAT has no effect on metabolism of DENA in mouse liver. Our findings can be unequivocally explained by competition of the carcinogens for target protein molecules, presumably transcription factors, participating in hepatocyte differentiation, which differently interact with and are diversely impaired by different compounds.

Potentiation of Antitumor Effects of Cisplatin by Tumor Necrosis Factor-β

Recombinant tumor necrosis factor-β potentiated the inhibitory effect of cisplatin on intramuscularly transplanted GA-1 tumor and liver metastasis in mice. The antitumor effect was related to cytotoxic and cytostatic activities of the test preparations rather than to initiation of apoptosis.

Effect of estragole on liver tumors glucocorticoid-mediated induction of liver-specific enzymes, and the activity of transcription factors FOXA and HNF4 in mouse and rat liver

The carcinogenic effects of estragole in mice of an earlier unexplored strain ICR have been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and tryptophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice or in rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and resistant animals. Estragole decreases the HNF4 and FOXA DNA-binding activities only in susceptible female mice, but not in insusceptible male mice or in rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and tryptophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.

The Effect of Hepatocarcinogen o-Aminoazotoluene on Induction of Mouse Liver Tyrosine Aminotransferase by Glucocorticoids

Abstracto-Aminoazotoluene (OAT) suppressed more than twofold the glucocorticoid induction of tyrosine aminotransferase in the liver of SWR mice, which are sensitive to the hepatocarcinogenic effect of OAT, but not in resistant AKR mice. The hormone- and DNA-binding activities of the glucocorticoid receptor were not affected in either strain. The OAT-dependent suppression proved to be associated with a decrease in the DNA-binding activity of HNF3 in liver cell extracts. The content of the HNF3 mRNA did not change, suggesting a posttranscriptional effect of OAT.

The effect of changes in CYP2E1 activity in the liver on toxicity and carcinogenicity of diethylnitrosamine in mice

In this work, the biological effects of diethylnitrosamine (DENA) have been studied under controlled conditions of its metabolism in mice of different ages. The results indicate that the general toxic and hepatocarcinogenic effects of DENA are mostly due to the parent compound, whereas the alkylating metabolites cause hepatic cell damage. Our findings cast doubt on the conventional understanding of the exclusive role of mutagenic activation in the carcinogenic action of chemicals.