BER gene polymorphisms associated with key molecular events in bladder cancer.

AIM: Base excision repair (BER) gene polymorphisms are known to play an independent role in predisposition to developing different cancers as well as to be associated with clinicopathological traits of the disease modifying its clinical outcomes. One of the underlying mechanisms is presumed to include interplay between BER gene polymorphisms and key mutational, epigenetic and chromosomal events in tumor tissues. The present study was aimed at elucidating potential gene-gene interaction and assessing their mutual effects in bladder cancer (BC). MATERIALS AND METHODS: The earlier obtained data on genotyping patients with verified diagnosis of BC for OGG1 rs1052133 (Ser326Cys) and XRCC1 rs25487 (Arg399Gln) polymorphisms were used for this study. The tumor tissue samples from the same patients were analyzed for mutations, epigenetic variations and losses of heterozygosity in some key genes involved in divergent pathogenic pathways of BC. RESULTS: It was shown that the OGG1 (326 codon) heterozygous genotype as well as the minor 326Cys allele can intensify a mutational response of the RAS locus in urothelial carcinomas in the total cohort of patients simultaneously decreasing the mutation rates in the PIK3CA locus in smokers. The XRCC1 (399 codon) heterozygous genotype as well as the minor 399Gln allele reduced the frequency of LOH in the PTEN and TNKS genes, but did not affect the mutational variability in any locus tested. Both polymorphisms influenced the methylation status, carriers of OGG1 326Ser/Cys or Ser/Cys+Cys/Cys genotypes demonstrating increased frequency of methylated RUNX3 and ISL1 genes whereas the similar effect of XRCC1 polymorphism concerning methylation of p16 and TIMP3 genes. When dividing the total cohort into groups based on the extent of tumor spread, the observed associations were characteristic of non-muscle invasive BC. CONCLUSION: The BER gene polymorphisms contributed to modification of key molecular events in urothelial carcinomas. Their mutual effects mainly manifested in non-muscle invasive BC. The underlying mechanisms as well as possible clinical outcomes need to be further explored to propose novel prognostic biomarkers for BC.

[The polymorphism of DNA repair genes XPD, XRCC1, OGG1, and ERCC6, life expectancy, and the inclination to smoke].

The polymorphism of excision repair genes XPD Asp312Asn, XRCC1 Arg399Gln, OGG1 Ser326Cys, and ERCC6 Met1097Val was analyzed by PCR-RFLP in 370 representatives of the Belarusian population of average, old, and elderly ages. Correlation analysis showed that the frequencies of wild-type homozygous combinations significantly increase with age in the group of subjects over 70 years old in the case of the interaction of two genes, XPD 312 and XRCC1399, or three genes, XPD312, XRCC1399, and ERCC6 1097. In a subgroup of the long-lived, this relationship is manifested in case of a pairwise interaction of gene XPD 312 with XRCC1 399 or ERCC6 1097, as well as an interaction of three genes, XPD 312, XRCC1 399, and ERCC6 1097. The data suggest that the optimum activity of repair processes may favor longevity. It is shown that the frequency of the Asp/Asp genotype is reduced, and the frequency of the Asn allele of the XPD 312 gene is increased in the subgroup of smokers as compared with nonsmokers, which apparently indicates an association of this gene polymorphism with an inclination to smoke. The problem requires further study.

Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells.

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

[DNA-repair modulation using derivatives of 1,4-dihydroisonicotinic acid as an example].

The influence of two derivatives of 1,4-dihydroisonicotinic acid on DNA-repair involved in chemical mutagenesis in Drosophila germ cells has been investigated. The compounds tested decreased the level of EMS-induced chromosome breakage and point mutations due to stimulation of maternal repair of DNA primary damage induced in spermatozoa as well as due to activation of DNA-repair in larvae and imago premeiotic stages of Drosophila males. Deficiency of DNA-repair systems leads to decrease in female and male germ-cell sensitivity to antimutagen action.

[Biological effect of low doses of alkylating agents in drosophila studies]. / Vliianie malykh doz na biologicheskie éffekty alkiliruiushchikh agentov v issledovaniiakh na drozofile.

The low dose (0.05-0.1 mM) influence of alkylating agents on germ cell survival and male fertility, the level of embryonic and postembryonic lethality as well as the sex-linked recessive lethal (SLRL) frequency induced by high alkylating agent doses was studied in Drosophila melanogaster. The pretreatment of adult males with low doses of methyl and ethyl methanesulfonate (MMS and EMS) did not change or even enhanced EMS cytotoxicity and mutagenicity in both mature sperm and premeiotic cells. On the contrary, the low EMS dose pretreatment of larvae protected them against higher mutagen doses increasing male fertility, decreasing embryonic and postembryonic lethality in F1, and leading to three-fold reduction in the SLRL frequency in F2. The adaptive response was dependent on the Drosophila developmental stage exposed to challenge mutagen doses, since the protection was maximal in larvae and practically absent when the high dose was administered to adult males. The adaptive response observed does not seem to be associated with DNA repair, but it is rather due to other protective mechanisms.

[Effect of the yellow locus on sensitivity of drosophila sex cells to chemical mutagens]. / Vliianie lokusa yellow na chuvstvitel'nost' polovykh kletok drozofily k khimicheskim mutagenam.

The effect of the yellow (y) locus on germ cell sensitivity to the alkylating agent ethyl methanesulfonate (EMS) has been studied in Drosophila. Since DNA repair is one of the most important factors that control cell sensitivity to mutagens, the approaches used in our experiments aimed at evaluating the relationship between germ-cell mutability and activity of DNA repair. Germ-cell mutability and repair activity were assessed using several parameters, the most important of which was the frequency of the recessive sex-linked lethal mutations (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin wild; y; y ct v; y mei-9a) were treated by mutagenic agents and then crossed to Basc females. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that the yellow mutation significantly enhanced the premeiotic cell sensitivity to EMS, presumably, due to the effect on DNA repair. In the second series of experiments, the effect of the maternal DNA repair was studied and, accordingly, mutagen-treated Basc males were crossed to females of various genotypes including y and y mei-9a ones. The crosses involving y females yielded F1 progeny with high spontaneous lethality, whereas in F2, the frequency of spontaneous mutations was twice higher. The germ cell response to EMS depended also on female genotype: the effect of yellow resulted in increased embryonic and postembryonic lethality, whereas the RSLLM frequency decreased insignificantly. The latter result may be explained by elimination of some mutations due to 50% mortality of the progeny. The results obtained using the above two approaches suggest that the yellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.

Anticlastogenicity of two derivatives of 1,4-dihydroisonicotinic acid in mouse micronucleus test.

Effects of two derivatives of 1,4-dihydroisonicotinic acid (1,4-DHINA) against the monofunctional alkylating agent ethyl methanesulfonate (EMS) were studied in the micronucleus test in (CBA x C57Bl/6(j)) mice. Adult males and pregnant females were treated with an antimutagen (i.p.) and 12h later they were exposed to EMS (i.p.). The frequencies of micronucleated (MN) polychromatic erythrocytes (PCEs) in mouse bone marrow and foetal liver were analysed 6, 12, 18, 24, 30, 36, 48 or 24, 48 and 72 h after the mutagen injection. In adults, the maximum number of MNPCEs was observed 36 or 24h after the EMS administration. In foetuses, which were treated in a maternal organism, such peak was found at 24h. Pre-treatment of mice with the antimutagens 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (DHP) and glutapyrone (GP) decreased the yield of MNPCEs in male bone marrow. Having been observed at a peak of MN induction, the anticlastogenic effect of DHP (1/10 LD(50) or 340 mg/kg) reached 30%. DHP at the doses of 0.5-1mM/kg did not affect the EMS-induced frequency of MNPCEs in bone marrow, whereas GP inhibited it at the similar millimolar concentrations. Simultaneously with maternal bone marrow, foetal liver cells were analysed for MNs in the transplacental test. The anticlastogenic effect of DHP (1/10 LD(50)) was found to be more prolonged and higher in females than in males and to average 50%, but this antimutagen was not efficient in foetuses. Both antimutagens did not change the polychromatic/normochromatic erythrocyte (PCE/NCE) ratio as compared with EMS action. Results presented indicate a peak of EMS-induced micronucleated cells in mouse bone marrow 24 or 36 h and in foetal liver 24h after animal treatment. Two 1,4-DHINA derivatives exhibited anticlastogenic activity in adults, but not in foetuses.

[Modification of the repair processes in chemical mutagenesis in Drosophila melanogaster]. / Modifikatsiia protsessov reparatsii pri khimicheskom mutageneze u Drosophila melanogaster.

The effect of 1,4-dihydroisonicotinic acid derivative (1,4-DHINA), glutapyrone, on the frequency of sex-linked recessive lethals (SLRL), which were induced by ethyl methanesulfonate (EMS) in spermatozoa, was studied in Drosophila males and females under different exposure conditions. This test was used to analyze repair processes in EMS mutagenesis. Glutapyrone manifested the protective effect after precultivation of males at the stage of larvae and females with glutapyrone. This preparation was shown to have no effect on the frequency of EMS-induced mutations under other conditions (precultivation and subsequent cultivation of mature males with glutapyrone). These data confirmed a key role of indirect mechanisms responsible for the effect of the examined antimutagen. Glutapyrone may be assumed to improve the synthesis or functioning of enzymes involved in the repair of O6-ethylguanine. After adaptive treatment of Drosophila with low doses of the mutagen, glutapyrone increased the fertility of parents but did not suppress chemical mutagenesis.

[Mechanisms of the inhibitive action of 1,4-dihydroisonicotinic acid derivatives in chemical mutagenesis]. / Mekhanizmy ingibiruiushchego deistviia proizvodnykh 1,4-digidroizonikotinovoi kisloty pri khimicheskom mutageneze.

1.4-Dihydroisonicotinic acid derivatives were used as an example to examine the mechanisms of action of exogenous antimutagens in chemical mutagenesis in eukaryotic organisms. The chemical mutagenesis was stimulated by alkylating agents, mutagens of direct action. The behaviour of alkylating mutagenesis was analysed, which gives an insight into the stages at which antimutagens may be involved in this process. Chemical mutagenesis was demonstrated to be suppressed due to lower molecular doses of the mutagen and by affecting the mechanisms of implementation of chemically induced mutations. Great emphasis was laid on the mechanisms of action of mutagens mediated by the intracellular antimutagenic and reparative systems. A special role is played by the analysis of exogenous antimutagens affecting DNA reparation as the most important step in the process of conversion of primary damages to fixed mutations. The paper presents a review of literature on basic problems of chemical mutagenesis modification.

[1,4-dihydroisonicotinic acid (1,4-DHINA) derivatives, inhibitors of chemical mutagenesis]. / Proizvodnye 1,4-digidroizonikotinovoi kisloty (1,4-DGINK)-- ingibitory khimicheskogo mutageneza.

The paper summarizes the results of studies into the modifying effect of some 1,4-dihydroisonicotinic acid (1,4-DHINA) derivatives during chemical mutagenesis in eukaryotic organisms. The compounds under study are effective in reducing the incidence of point and chromosomal mutations induced by ethylmethane sulfonate (EMS) in Drosophila sex cells, displaying the specificity of the influence depending on some factors. The effects of these antimutagens on the formation and repair of chromosomal breaks were studied during mutagenized sperm storage in Drosophila female receptacula semenis. A relation was found between the female sensitivity to antimutagens and the appropriate modifying effect, on the one hand, and the oocytic genotype, on the other. The protective effect of one of 1,4-DHINA derivatives against alkylating agents (EMS and Thiophosphamide) was confirmed by in vivo experiments in mice and in cultured human lymphocytes. The revealed mechanisms of modified chemical mutagenesis both in the Drosophila sex cells and in the mammalian somatic cells are in favour of those of protective action, which are mediated by the antimutagenic cell system, as well as, perhaps, by repair processes.

[The effect of antioxidants on the formation of ethylmethane sulfonate-induced chromosomal breaks in relation to the maternal repair systems in Drosophila melanogaster]. / Vliianie antioksidantov na formirovanie indutsirovannykh étilmetansul'fonatom razryvov khromosom v zavisimosti ot sistem materinskoi reparatsii u Drosophila melanogaster.

The features of EMS-induced chromosomal breakage formation and the modes of its modification by the Drosophila repair systems were studied. The storage of mutagenized spermatozoa in female receptacula seminis is accompanied by accumulation of chromosomal breaks. Transformation of primary DNA lesions to structural chromosomal mutations was found to be regulated by the maternal repair systems. Treating the females with 1,4-dihydroisonicotinic acid derivatives and potassium salt of phenozane was demonstrated to reduce the frequency of EMS-induced chromosomal breakage during the whole storage period. The effects of antioxidants on maternal repair of EMS-induced endpoints were examined. Their antioxidative action seems to be displayed by inhibiting error-prone pathways of postreplication repair.

A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster.

The 1,4-dihydropyridine derivative 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (1,4-DHP) was studied for antimutagenic effects in the dominant lethal test and in the sex-linked recessive lethal test of Drosophila melanogaster. The observed effects were compared with those of the radioprotectors cysteine and cysteamine and with those of the phenolic antioxidant butylated hydroxytoluene (BHT). In a wide range of concentrations, including low ones, 1,4-DHP reduces the frequency of EMS-induced genetic damage (point mutations and chromosome breakage). A reduction of the mutation rate induced by EMS in adults could be observed independently of the developmental stages (larvae or imago) pretreated with 1,4-DHP. The protective effect of this new antimutagen against the alkylating agent depended on both the 1,4-DHP dose and the level of the EMS-induced mutation rate. The effect of 1,4-DHP was more pronounced than that of the studied radioprotectors. It is concluded that dihydropyridine-type compounds are able to protect eukaryote germs cells from genetic damage produced by direct-acting mutagens such as EMS.

[Sensitivity of individual Drosophila to the mutagenic action of ethyl methanesulfonate]. / Izuchenie chuvstvitel'nosti otdel'nykh osobei Drozofily k mutagennomi deistviiu étilmetansul'fonata.

The genetic effect of some factors is generally evaluated as an average response of all individuals, without taking into account their potential differences. The presence of individual sensitivity in separate Drosophila organisms to the mutagenic influence of ethyl methanesulfonate was shown when analysing recessive sex-linked lethal mutations in germ cells of males. Different sensitivity of separate individuals to mutagens reflects the existence of cryptic genetic variability in Drosophila strains on a large scale. It is advisable to take into account individual sensitivity of organisms to mutagenic factors, when conducting mutation research and studying genetic consequences of biosphere pollution.

[Cytogenetic effects of 1,4-dihydropyridine in various test systems]. / Tsitogeneticheskii éffekt 1,4-digidropiridina v razlichnykh test-sistemakh.

Cytogenetic effect of 1,4-dihydropyridine was studied in different test-systems. The preparation is shown to decrease the level of complete sex-chromosome losses in Drosophila and chromosome aberration frequency in Allium fistulosum seedlings. The preparation does not affect spontaneous mutability of bone marrow cells in mice, high doses of the preparation have no mutagenic potential. Thus, 1,4-dihydropyridine shows antimutagenic activity reducing the chromosome mutation level in sex and somatic cells of eucaryotic organisms. Absence of the effect on mice chromosomes may testify to the specificity of 1,4-dihydropyridine action.