O. A. Timofeeva

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.

Synthesis and evaluation of oligo-1,3-thiazolecarboxamide derivatives as HIV-1 reverse transcriptase inhibitors

A set of oligo-1,3-thiazolecarboxamide derivatives able to interact with the minor groove of nucleic acids was synthesized. These oligopeptides contained different numbers of thiazole units presenting dimethylaminopropyl or EDTA moieties on the C-terminus, and aminohexanoyl or EDTA moieties on the N-terminus. The inhibition of such compounds on HIV-1 reverse transcriptase activity was evaluated using different model template primer duplexes: DNA x DNA, RNA x DNA, DNA x RNA and RNA x RNA. The biological properties of the thiazolecarboxamide derivatives were compared to those of distamycin, another minor groove binder which contains three pyrrole rings. Similar to distamycin, the thiazole containing oligopeptides were good inhibitors of the reverse transcription reaction in the presence of DNA x DNA. But in contrast to distamycin, the oligothiazolide derivatives were able to inhibit reverse transcription in the presence of RNA x DNA or DNA x RNA template primers. Both distamycin and oligothiazolecarboxamides had low affinity for RNA x RNA duplexes. The inhibition obtained with the newly synthesized thiazolecarboxamides showed that these compounds were more powerful and versatile inhibitors of the RT-dependent polymerization than the natural minor groove binder distamycin.

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.

[Pulmonary carcinogenesis susceptibility-associated single-nucleotide polymorphisms in K-ras intron 2 affect the binding of factor Gata-6 but not gene expression].

The sequence of K-ras intron 2, which has been associated with lung tumor susceptibility in inbred mouse strains, was analyzed in susceptible strain GR and in resistant strains PT and UT. In the latter case, the intron had a tandem repeat of a 37-bp sequence with variant GC of its two single-nucleotide polymorphisms (SNPs), as earlier reported for resistant strains AKR, C57BL/c, and C3H/A. Strain GR did not differ in intron structure from susceptible strains A/He and ICR, having one copy of the 37-bp sequence with SNP variant CA. By gel retardation assay, a DNA probe corresponding to “susceptible” allele CA of K-ras region 278–307 formed an additional complex with nuclear proteins extracted from lungs, as compared with probes corresponding to the “resistant” GC and “intermediate” CC alleles. With specific antibodies, the protein binding to the susceptible allele was identified as transcription factor GATA-6. Reverse transcription with subsequent multiplex PCR did not reveal any significant difference in K-ras expression for susceptible and resistant strains. The results suggest that SNPs of K-ras intron 2 do not affect the level of K-ras expression but do control the binding of GATA-6, which plays an important role in lung differentiation.

The mechanisms of endogenous control of hepatocyte proliferation are different in mice of two inbred strains (BALB/c and AKR).

Experiments on the fusion of stimulated fibroblasts of NIH 3T3 mice with hepatocytes obtained from an intact liver of BALB/c mice showed that the hepatocytes only slightly, if at all, suppress the proliferative activity of the fibroblast nuclei in the heterokaryons [1]. Conversely, hepatocytes obtained from a partially recovered liver of mice of the same strain (15 days after partial hepatectomy) suppress the proliferative activity of the fibroblast nuclei in the heterokaryons. A shortterm (for 2 h) preincubation of these hepatocytes with a protein-synthesis inhibitor, cycloheximide, completely eliminated their ability to suppress DNA synthesis in the fibroblast nuclei in the heterokaryons [2]. Based on these results, we assumed that the mechanism of suppression of the proliferative activity of the fibroblasts in a regenerating liver may be connected with the appearance of intracellular inhibitors of proliferation in these cells at the final stages of regeneration. In view of this, the endogenous inhibitors of hepatocyte proliferation should be identified. To solve this problem, we studied the mechanisms of hepatocarcinogenesis and attempted to determine the mechanisms of resistance of mice of different inbred strains to some chemical carcinogens. The basis of these experiments was as follows.

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.

Mutations in codon 61 of H-ras gene in mouse liver tumors induced by potent and weak carcinogens

PCR with subsequent sequencing showed that codon 61 of H-ras gene is presented by mutant variants in 7 of 13 liver tumors induced by neonatal injection of potent hepatocarcinogens o-aminoazotoluene or nitrosoethylurea in CBA mice, while in ICR mice tumors induced by these carcinogens carried only wild-type allele. Only mutant alleles of the studied codon were detected in 8 tumors induced with a weakly carcinogenous mutagen ethylmethansulfonate in ICR mice. In 7 of 15 cases these alleles were presented by two variants: AAA and CTA simultaneously (normal codon CAA) in CBA mice injected with o-aminoazotoluene and by CGA and CTA in ICR mice receiving ethylmethansulfonate.