Suppression of sulfoconjugation reduces the protective effect of ortho-aminoazotoluene on hepatocarcinogenesis induced by diethylnitrosamine in mice.

The effects of ortho-aminoazotoluene on carcinogenic activity of diethylnitrosamine were studied in CBA and ICR mice. Injection of ortho-aminoazotoluene before and after diethylnitrosamine led to a significant reduction of its anticarcinogenic effect, judging from significantly lower level of liver tumors. Pentachlorophenol, inhibitor of sulfotransferase (catalyzing the terminal stage of ortho-aminoazotoluene metabolic activity), stimulated its carcinogenic effect on mouse liver. On the other hand, pentachlorophenol reduced the protective effect of ortho-aminoazotoluene on diethylnitrosamine-induced hepatocarcinogenesis in mice. Presumably, the carcinogenic and anticarcinogenic effects of ortho-aminoazotoluene were realized by its initial form or intermediate (non-sulfated) metabolites.

[Inhibitory effect of ortho-aminoazotoluene on diethylnirtosamine hepatocarcinogenesis in suckling mice. Phenomenon and possible mechanism].

The modifying effect of the one compound on carcinogenicity of the other in the 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 metabolic level, because OAT has no effect on metabolism of DENA in mouse liver. Our findings can be unequivocally explained by the 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.

[Mutagenic activation and carcinogenicity of aminoazo dyes of 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.

OAT and 3'MeDAB azo compounds similarly cause liver tumors in GR mice, but differently modify activities of FoxA transcription factors.

Transcription factors of the FoxA family (forkhead box A) regulate cell metabolism and differentiation and maintain specificity of liver cell proteome and phenotype of mature hepatocytes. The relationship between hepatocarcinogenicity of azo compounds o-aminoazotoluene (OAT) and 3'-methyl-4-dimethylaminobenzene (3'MeDAB) for GR mice and one of the early events, modulation of the DNA-binding activity of FoxA transcription factor, was studied. Single injection of 3'MeDAB to 12-day-old mice caused liver tumors in 100% males and females similarly as OAT, a well-known mouse hepatocarcinogene. The DNA-binding activity of FoxA in the liver decreased 2.5-3 times by OAT, this resulting in a 40% reduction of glucocorticoid induction of tyrosine aminotransferase (liver-specific gene). In contrast to these, 3'MeDAB did not modify FoxA protein activities or the degree of glucocorticoid induction of tyrosine aminotransferase.

[O-aminoazotoluene inhibits DENA-induced hepatocarcinogenesis when used together in mice].

When used separately, diethylnitrosamine (DENA) initiates 4-6 times more neoplastic lesions in the liver of suckling mice than ortho-aminoazotoluene (OAT) lower. However, after combined treatment with either carcinogen the total number of hepatic lesions was significantly than that in mice injected with DENA only. Similar inhibition of DENA-induced hepatocarcinogenesis was observed when OAT was administered 8-12 hrs after DENA. Our findings cannot be adequately explained except by competition of the carcinogens for supposedly target molecules of protein origin, presumably, transcription factors, which contribute to generation of genetic information. They have different affinities for different compounds and, therefore, suffer different damage from the latter functioning.

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.

Detection of target genes of FOXA transcription factors involved in proliferation control.

To reveal the mechanism of tumor-suppressing activity of FOXA proteins in liver, a search for potential target genes of these transcription factors involved in proliferation control was carried out. In the first step, we have used data from the literature concerning gene expression in mouse liver (high content of FOXA proteins) and kidney (FOXA expression is absent) obtained by hybridization on microchips. A search for FOXA binding sites in regulatory regions of forty differentially expressing genes involved in proliferation control was carried out using the computer method SITECON. Eleven genes containing clusters of potential FOXA sites incorporating 3-6-fold repeats of TTTG were revealed. The FOXA-specific interaction with such microsatellite sites was confirmed by gel-retardation technique using the GST-fused protein containing the DNA-binding domain of FOXA2. Six genes containing clusters of confirmed binding sites--Cul2, Cdc73, Ptk, Pdcd, Creb, and Ppp2r5d--were selected. The effect of hepatocarcinogen orthoaminoazotoluene (OAT), which lowers the FOXA activity, on expression of these genes was studied by the real-time PCR. OAT was shown to increase sharply the level of mRNA of the Cul2 and Cdc73 genes.

Promoting effect of o-aminoazotoluene on hepatocarcinogenesis is accompanied by the increase in inflammatory and proliferative processes in liver tissue and decrease in the concentration of free thyroxin in the blood.

o-Aminoazotoluene in noncarcinogenic doses promoted the development of liver tumors in female ICR mice induced by administration of diethylnitrosamine during early ontogeny. Severe inflammatory infiltration and proliferation of oval cells were found in liver tissue of these animals. The concentration of free thyroxin decreased in the blood. Our results supplement published data that promoters of hepatocarcinogenesis inhibit thyroid function.

o-Aminoazotoluene does induce the enzymes of its own mutagenic activation in mouse liver.

The objective of this study was to investigate the CYP1A1 and CYP1A2 mRNAs and enzyme activities in mouse liver during induction with o-aminoazotoluene (OAT) as well as the capability of the hepatic S9-fraction from OAT-treated mice to induce its own activation to mutagens in the Ames test using S. typhymurium strain TA98. The data obtained indicate that when used at appropriate doses, OAT is a PAH-type inducer of mouse hepatic microsomal monooxygenases, which activity is not less than that of the known inducer 3,4-benzo[alpha]pyrene. In the absence of S9-fraction enzymes no OAT-mediated mutagenicity was observed in the Ames test. In the presence of the S9-fraction from OAT-pretreated mice, OAT induced as high revertant numbers, as it did in the presence of the S9 fraction from the liver of Aroclor 1254-treated mice. Thus, OAT does induce the enzymes of its own mutagenic activation in mouse liver.

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.

[mRNA level and cytochrome P450 1A activity in the liver of C57BL mice induced by various xenobiotics]. / Uroven' mRNK i aktivnost' tsitokhromov P450 1A v pecheni myshei linii C57BL pri induktsii razlichnymi ksenobiotikami.

The rate of hepatic cytochrome P450 Cypla1 and Cyp1a2 induction was investigated in C57BL male mice during induction with o-aminoazotoluene (OAT), benzo[a]pyrene (BP) and 1,4-dihydroxyanthraquinone (AQ). The Cypla1 mPNA level determined by quantitative RT-competitive PCR increased more than three orders of magnitude during induction with OAT and BP compared with untreated animals and remained unchanged during induction with AQ. The Cypla2 mRNA level was only 8.5, 18.7 and 1.9 times higher during induction with OAT, BP and AQ respectively than in untreated mice. At the same time 7-Ethoxyresorufin-O-deethylase (EROD) and 7-Methoxyresorufin-O-demethylase (MROD) activities of Cypla were also investigated in liver. The increase of Cypla1 mRNA level correlated with the increase of EROD activity. This suggests involvement of the transcriptional mechanism of the inducibility of this enzyme. In the case of Cypla2 there was insignificant increase of its mRNA level but high catalytic activity registered in liver in response to injection of the inductor of MR metabolism. This can imply the posttranscriptional mechanism of Cypla2 regulation. During induction with AQ the Cypla1 mRNA level remained unchanged, but the EROD activity increased almost 20-fold. The latter suggests insufficient specificity of this substrate for Cypla1. Thus, on the basis of the data obtained, the mRNA level can be considered as more accurate estimation of Cypla1 and Cypla2 inducibility, than the determination of the enzyme activity.

Lack of effect of furfural on unscheduled DNA synthesis in the in vivo rat and mouse hepatocyte DNA repair assays and in precision-cut human liver slices.

The ability of furfural to induce unscheduled DNA synthesis (UDS) in hepatocytes of male and female B6C3F(1) mice and male F344 rats after in vivo administration and in vitro in precision-cut human liver slices has been studied. Preliminary toxicity studies established the maximum tolerated dose (MTD) of furfural to be 320 and 50 mg/kg in the mouse and rat, respectively. Furfural was dosed by gavage at levels of 0 (control), 50, 175 and 320 mg/kg to male and female mice and 0, 5, 16.7 and 50 mg/kg to male rats. Hepatocytes were isolated by liver perfusion either 2-4 h or 12-16 h after treatment, cultured in medium containing [3H]thymidine for 4 h and assessed for UDS by grain counting of autoradiographs. Furfural treatment did not produce any statistically significant increase or any dose-related effects on UDS in mouse and rat hepatocytes either 2-4 h or 12-16 h after dosing. In contrast, UDS was markedly induced in mice and rats 2-4 h after treatment with 20 mg/kg dimethylnitrosamine and 12-16 h after treatment of mice and rats with 200 mg/kg o-aminoazotoluene and 50 mg/kg 2-acetylaminofluorene (2-AAF), respectively. Precision-cut human liver slices from four donors were cultured for 24 h in medium containing [3H]thymidine and 0-10 mM furfural. Small increases in the net grain count (i.e. nuclear grain count less mean cytoplasmic grain count) observed with 2-10 mM furfural were not due to any increase in the nuclear grain count. Rather, it was the result of concentration-dependent decreases in the mean cytoplasmic grain counts and to a lesser extent in nuclear grain counts, due to furfural-induced cytotoxicity. In contrast, marked increases in UDS (both net grain and nuclear grain counts) were observed in human liver slices treated with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM aflatoxin B(1) and 0.005 and 0.05 mM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. This study demonstrates that furfural does not induce UDS in the hepatocytes of male and female B6C3F(1) mice and male F344 rats after oral treatment at doses up to the MTDs. Moreover, human liver slice studies suggest that furfural is also not a genotoxic agent in human liver.

[Effect of the hepatocarcinogen ortho-aminoazotoluene on induction of tyrosine-aminotransferase by glucocorticoids in mouse liver]. / Vliianie gepatokantserogena orto-aminoazotoluola na induktsiiu tirozin-aminotransferazy gliukokortikoidami v pecheni myshei.

o-Aminoazotoluene (OAT) suppressed more than twofold the glucocorticoid induction of tyrosine aminotransferase (TAT) 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 (GR) were not affected in either line. 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.

Involvement of transcription factor HNF3gamma in the effect of o-aminoazotoluene on glucocorticoid induction of tyrosine aminotransferase in mice sensitive to its hepatocarcinogenic action.

In the rodent liver, hepatocarcinogens inhibit the glucocorticoid induction of several liver-specific genes, including tyrosine aminotransferase (TAT). A distinct positive correlation exists in mice between the extent of inhibition of TAT induction after acute administration of o-aminoazotoluene (OAT) and the frequency of liver tumors after chronic exposure to the carcinogen. To elucidate the mechanism of the carcinogenic action, the effects of OAT on the DNA-binding activity of several transcription factors participating in the glucocorticoid regulation of TAT gene expression were studied. The experimental inbred male mice were sensitive (A/He and SWR/J, tumor induction frequency of 75-100%, TAT induction inhibition of 35-50%) and resistant (CC57BR/Mv and AKR/J, 0-6% and 10-15%, respectively) to OAT. Gel retardation experiments showed that hepatocyte nuclear factor 3 (HNF3)gamma DNA-binding activity was strongly reduced in nuclear extracts from the livers of OAT-treated A/He and SWR/J mice but only slightly reduced in CC57Br/Mv and AKR/J mice. The DNA-binding activities of Ets, AP1 family members, and GME binding proteins were unaffected. HNF3gamma DNA-binding activity was reduced by 1 h after OAT administration and remained low for 1 mo, as did inhibition of TAT induction in the liver. These results suggested that the inhibitory effect of OAT on the glucocorticoid induction of TAT is mediated by reduced HNF3gamma DNA-binding activity.

Expression of CYP1A in liver of A/Sn and CC57BR mice differing in sensitivity to hepatocarcinogenesis induced by o-aminoazotoluene.

The induction of P450 cytochromes Cyp1a1 and Cyp1a2 in the liver of male mice differing in sensitivity to the carcinogenic effect of o-aminoazotoluene (OAT) has been studied. The level of Cyp1a1 and Cyp1a2 mRNA was assayed by quantitative competitive polymerase chain reaction (PCR). In both OAT-treated strains, the level of Cyp1a1 mRNA increased more than 1000-fold, while the amount of Cyp1a2 mRNA increased only two- or threefold. Interstrain differences in the Cyp1a mRNA level were revealed. The level of Cyp1a1 mRNA in liver of OAT-induced A/Sn mice was three times higher than in CC57BR mice. The amount of Cyp1a2 mRNA in control and OAT-treated mice was 7 and 13 times higher, respectively, than in CC57BR mice. The enzyme activities of cytochromes P450 1a were assayed. An increase in Cyp1a1 mRNA level in OAT-treated mice correlated with an enhancement of the benzo[a]pyrene hydrolase and 7-ethoxyresofurin o-deethylase activities; the correlation between the level of Cyp1a2 mRNA and 7-ethoxyresofurin o-demethylase activity was much less pronounced. Interstrain variation in Cyp1a1 and Cyp1a2 activities was also shown at the enzyme activity level. We suppose that quantitative differences in the Cyp1a2 mRNA level play a key role in OAT-induced hepatocarcinogenesis.

[Cytochrome P-450 induction and the subsequent induction of an immune response in rats during the chronic administration of xenobiotics]. / Induktsiia tsitokhroma P-450 i posleduiushchaia induktsiia immunnogo otveta u krys pri khronicheskom vvedenii ksenobiotikov.

The long-term administration of xenobiotics carcinogens o-aminoazotoluene (o-AAT) and benz(a)pyrene (BP) to rats was found to cause induction of the liver cytochrome P-450 system which gradually decreases in spite of continued administration of the agents. Induction of microsomal oxygenases under these conditions is followed by induction of the immune response to o-AAT and BP. The data obtained correspond to the conception of the immunochemical functional system of homeostasis implying that the cytochrome-450 system and the immunity system are functionally linked and are elements of the common functional adaptive system of the organism.