[Studies of mutagenic activity of positively charged alkyl glycerolipids in Ames test].

Mutagenic activity of non-phosphorous cationic alkyl glycerolipid rac-N-{4-[(2-Methoxy-3-octadecyloxy)propyl]oxycarbonylbutyl}-N,N-dimethyl-N-(2-hydroxyethyl) ammonium iodide was evaluated. According performed Ames assay results indicated on non-mutagenic properties tested compound. Resulting data open the possibility to carry out biological study in vivo for class of ether cationic glycerolipids and further applications as a potential agent of anticancer therapy.

[Analysis of blastomogenic activity of mammal carcinogens in Drosophila using the wts(P4) allele and RNA interference-induced p53 silencing].

The test for somatic mutagenesis and recombination in Drosophila is one of the widely used approaches for determination of possible carcinogenic effects of chemical compounds. The use of heterozygotes for mutant tumor suppressor gene wts enables more direct evaluation of the blastomogenic effects of chemical compounds, by tumor formation in the adult flies. This study presents evaluation of the SMART effectiveness upon the use of Drosophila heterozygotes for the wts(P4) gene, first included into the test system. The increase of the test resolution capacity compared to the literature data for the wts(P2) allele was observed. Using wts(P4) heterozygotes, a total of 20 carcinogenic compounds, and their slightly carcinogenic and noncarcinogenic analogs were tested. Specificity of the method was about 100%, and sensitivity depended on the type of the agent tested. The latter was absolute for the direct action carcinogens, with respect to carcinogens, requiring the metabolic activation. The sensitivity was elective and was limited by the presence of the enzymes capable of activating of these compounds. To increase the test sensitivity, the RNA interference-mediated silencing of the Drosophila p53 functional activity was successfully applied. Moreover, the frequency of wts tumor induction considerably increased both in spontaneous and induced mutagenesis conditions.

[Complementary oligonucleotide invasion into (CA/TG)31-repetitive region of linear and circular DNA duplexes].

(CA/TG)n-repeats belong to microsatellite DNA and are the most widespread among dinucleotide repeats in mammalian genomes, occupying 0.25% of the genome. These repeats are known to be recombination "hot spots", however the molecular mechanisms of this effect are not known. We proposed that the high frequency of recombination in the repetitive regions may be due to duplex conformational characteristics resulting from a special geometry of base-stacking contacts, which permits the initiation of an invasion process of single-stranded DNA into the duplex homologous region. Here we show for the first time a DNA-DNA interaction of oligonucleotides d(CA)10 and d(TG)10 with linear and circular duplexes containing (CA/TG)31-repeats, upon incubation at 37 degrees C in the absence of proteins. Using radioactively labeled oligonucleotides, we showed that duplex-oligonucleotide interaction intensities depend on their molar ratio at a duplex concentration 30 nM. Decreasing the duplex concentration to 3 nM did not influence the intensity of oligonucleotide invasion. It was shown that more than 1%, but much less than 10% of the duplexes participate in the interaction with oligonucleotides, assuming that one molecule of the duplex interacts with one molecule of the oligonucleotide. Analysis of the kinetics of the process reveals invasion of d(CA)10 at the first minute of its incubation with the duplex, while d(TG)10 interacts with the duplex at an even higher rate. We discuss the role of DNA conformation plasticity of (CA/TG)n-repeats in the phenomenon observed, as well as its biological significance, in particular the role of CA-microsatellites in the initiation of homologous recombination.

[Chemical cleavage of DNA with single base mismatches for detection of mutations of unknown localization].

The most promising approach for detection of random point mutations relies upon the DNA chemical cleavage near associated mismatching base pairs. In our study, the series of heteroduplexes with all types of mismatches and extrahelical nucleotide residues surrounded by both A x T and G x C pairs were performed via hybridization of 50-mer synthetic oligonucleotides differing in only one nucleotide at the central position. The chemical cleavage of DNA duplexes immobilized on magnetic beads by means of biotin-streptavidin interaction was carried out with chemicals, which able to attack only nucleobases flipped out of the base stack: potassium permanganate and hydroxylamine reacting to T and C respectively. The chemical reactivity of different mismatches was shown to correlate clearly with the target local structure in a particular sequence context. This work makes up for a deficiency in systematic study of DNA cleavage near mismatches in dependence on their type, orientation and flanking nucleotides. The model system elaborated may be applied to estimate the sensitivity of the methodology and to control the possibility of false-positive and false-negative result appearance, when different protocols for detection of DNA mutations are used. The modification of heteroduplex mixtures by potassium permanganate and hydroxylamine allows to reveal any non-canonical base pair and suggest its type and neighboring nucleotides from the nature of chemical as well as its localization from the length of cleavage products.

[Carcinogenic components of smokeless tobacco and tobacco-free cigarettes].

The investigation deals with an assessment of carcinogenicity and mutagenicity of samples of smokeless tobacco now on the Russian market as well as ash from alternative cigarettes made of aromatic herbs. Our data showed that the levels of polycyclic aromatic hydrocarbons, volatile and tobacco-specific N-nitrosoamines complied with the standards in the producer-countries. Smokeless tobacco extracts failed to show (Ames) any mutagenic effects such as the "read-out frame shift" or "base-pair replacement" patterns. No tobacco-specific N-nitrosoamines were identified in herbal cigarettes. However, polycyclic aromatic hydrocarbons and volatile N-nitrosoamines content appeared to be identical to that of tobacco. Herbal cigarette smoke extracts mutagenicity induced by side-effects of carcinogenic substances was of similar magnitude as well.

[Specific oligonucleotide invasion into the end of DNA duplex].

Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.

[Mammalian carcinogen-induced warts tumor in Drosophilidae: a test sensitive to the blastomogenic action of chemical compounds]. / Opukholi warts u drozofily, vyzvannye kantserogenami mlekopitaiushchikh: test, chuvstvitel'nyi k blastomogennomu deistviiu khimicheskikh soedinenii.

A basically new system has been developed to screen carcinogens in Drosophilidae, which is based on somatic mutagenesis and recombination. The system may induce tumors in Drosophilidae, which are recorded in adult insects. The test uses recessive mutation in the suppressor gene of growth of warts (wts) tumors. The new system is sensitive to a wide spectrum of mutagenic carcinogens that are naturally encountered. The sensitivity of the system to polycyclic aromatic hydrocarbons and aromatic amides is higher than that of classical tests. Dominant p53 gene mutation that ceases mutagen-induced apoptosis has been shown to increase the incidence of wts tumors by many times. The magnitude of the effect depends on the rate of mutant p53 expression. The increasing effect of p53 mutation extends to both somatic recombination and point mutations and deletions at the wts locus.

[The role of duplex ends in the spontaneous interaction of homologous linear DNA fragments]. / Rol' kontsevykh uchastkov dupleksov v spontannom vzaimodeistvii gomologichnykh lineinykh fragmentov DNK.

The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA-DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.

[The effect of transposon P(GUS . p53.259H)on the frequency of tumor clones in Drosophila melanogaster wtsp2/+ heterozygotes]. / Vliianie transpozona P(GUS . p53.259H) na chastoty opukholevykh klonov u geterozigot wtsp2/+ Drosophila melanogaster.

We showed that transposon P(GUS.p53.259H), mapped to chromosome 3 and carrying a dominant mutation p53(259)H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts- in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53(259)H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P(GUS.p53.259H) transposon itself. The effect of the P(GUS.p53.259H) appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo(alpha)pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P(GUS.p53.259H) effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts- clones induced by P(GUS.p53.259H) was established.

[Absence of mutagenic and carcinogenic properties in Mildronate]. / Otsutstvie mutagennykh i kantserogennykh svoistv u Mildronata.

Mutagenic and carcinogenic properties of mildronate (3-[2.2.2.-trimethyl hydrazonium]-propionate) have been studied for its marked immuno-stimulating and anti-ischemic action. When dosage up to 1,000 mg/dish was used no reversal of base-pair substitution or frame shift in S.typhimurium was observed. In drosophila females treated with mildronate, mosaic patches were, on the average, as frequent as in control. Although chronic treatment of female mice B6D2F1, C3H and SHR with mildronate was not followed by any change in tumor incidence, mammary gland adenocarcinoma development was slightly inhibited in mice C3H and SHR. The latter effect was in correlation with the antigonadotropic one of the drug and was not determined by its estrogenous properties. This pointed to the absence of mutagenic and carcinogenic properties which was confirmed in two short-term and one chronic experiments on mice of the three lines.

[Genomic sequences in Drosophila melanogaster, homologous to mammalian oncogenes]. / Genomnye posledovatel'nosti Drosophila melanogaster, gomologichnye onkogenam mlekopitaiushchikh.

Drosophila melanogaster genomic sequences homologous to the mammalian oncogenes ras, src, jun, fos, ets, and wnt are considered. Their structure, expression, and functions are compared.

[The specificity of the genotoxic action of carcinogenic aromatic compounds on Drosophila mus mutants]. / Spetsifichnost' genotoksicheskogo deistviia kantserogennykh aromaticheskikh soedinenii na mus-mutanty drozofily.

Hypersensitivity to the toxic effect of benzo(a)pyrene (B(a)P) was determined in homozygous larvae of two D. melanogaster mus strains (mus 208B2 and mus210). The two others (mus205B1 and mus208B1) were found to be less sensitive and the parent strain was resistant. The lack of correlation between the sensitivity in larvae and the activity of aryl hydrocarbon hydroxylase in S15 fractions from adult flies whole body homogenates of the same strains was demonstrated. The hypertoxic effect of B(a)P and 2-acethylaminofluorene in strain mus210 seems to be rather specific because noncarcinogenic pyrene, benzo(e)pyrene and fluorene did not affect the survival of this most sensitive strain. Perspectives of the strain mus210 use for the environmental genotoxic pollutants screening were discussed.

[Benzo(a)pyrene pretreatment of Drosophila simulans mutant strain results in the induction of aberrant isoform of cytochrome P-450 with increased capacity to metabolize benzo(a)pyrene]. / Predobrabotka benz(a)pirenom mutantnoi linii Drosophila simulans privodit k induktsii abberrantnoi izoformy tsitokhroma P-450 s povyshennoi sposobnost'iu metabolizirovat' benz(a)piren.

The basal level of benzo(a)pyrene monooxygenase, epoxide hydrolase and glutathione S-transferase activity as well as the content of cytochrome P-450 were found the same in both compared benzo(a)pyrene (BP) sensitive D. simulans strain 364yv and BP-resistant wild one (Turku). Phenobarbital pretreatment resulted in the same increase level of these enzyme activities in both strains. BP-pretreatment of 364yv flies decreased the amount of the cytochrome P-450 but raised up the turnover of BP per molecule of cytochrome P-450. SDS-polyacrylamide gel electrophoresis of the microsomal proteins from BP-pretreated 364yv flies (but not from Turku) showed an increased hemoprotein content in the 56000 band. The relationship between BP-sensitivity of the strain 364yv and BP-induced aberrant isoform of the cytochrome P-450 has been discussed.

[A mutant strain of Drosophila simulans sensitive to the toxic and mutagenic action of benz(a)pyrene]. / Mutantnaia liniia Drosophila simulans, chuvstvitel'naia k toksicheskomu i mutagennomu deistviiu benz(a)pirena.

A 364 yv strain sensitive to the toxic effect of benzo(a)pyrene (BP) was identified among 49 mutant strains of Drosophila simulans. Heterozygotes female 364 x male Turku obtained by crossing 364 yv species with those of wild BP-resistant Turku strain were more sensitive than female Turku x male 364 heterozygotes to both toxic and mutagenic effects of the carcinogen in the test of induction of somatic mosaicism with the yellow marker. Non-carcinogenic pyrene appeared weekly toxic and non-mutagenic. Possible mechanisms of 364 yv strain sensitivity to BP as well as vistas in application of the strain for monitoring genotoxic environmental pollution are discussed.

[Direct-action mutagens in exhausts of vehicles with diesel engines]. / Mutageny priamogo deistviia v vybrosakh transportnykh dvigatelei, rabotaiushchikh na dizel'nom toplive.

The genotoxic activity of exhausts from one-shaft gas-turbine GTE-5 engine (30 kW) and a standard D-54A diesel (40 kW) have been studied. Thus, the extracts of soot from GTE-5 and D-54A induced reversions in Salmonella typhimurium both with and without metabolic activation: furthermore, extracts of soot from GTE-5 demonstrated a higher mutagenic activity. The direct mutagenic effect of the exhausts depended neither on the presence of BP nor on the other polycyclic aromatic hydrocarbons (PAHs). Most probably, it was connected with the presence of nitro-PAHs. The need for studying the PAH content in vehicle engines' exhausts and for taking into account their effect in the control and standardization is established.

[The mutagenic activity of coal tar]. / Mutagennaia aktivnost' kamennougol'noi smoly.

Genotoxicity of tar samples collected at the Kemerovo and Altai by-product coke plants has been studied, the contribution of benz(a)pyrene (BP) into the total mutagenic activity of extracts being estimated. Both direct and indirect mutagenicity of the samples is determined in the Ames test. A direct mutagenic effect of coal tar may be attributed neither to the BP action nor to other polycyclic aromatic hydrocarbons. The onset of the His revertant induction in Salmonella typhimurium strains Ta 100 and TA 97 treated with the coal tar activated by the S-9 mixture is observed in doses containing BP an order lower than threshold of its activity found in sole experiment. The highest mutagenic effect of the activated coal tar is observed at a dose containing the minimal active sole dose of BP (0.72-1.12 micrograms per plate).

[Microsomal monooxygenases of the liver in mice with transplanted tumors]. / Mikrosomnye monooksigenazy pecheni u myshei s perevivaemymi opukholiami.

Hepatoma 22a and Ehrlich's tumor growth were shown to be accompanied by decrease in cytochrome P-450 level in liver of noninbred and C3HA mice, these changes being more pronounced as compared to solid neoplasms. Benzo(a)pyrene-hydroxylase and amidopyrine-N-demethylase activity varied with tumor pattern. It was not changed in cases of hepatoma 22a but decreased in mice bearing Ehrlich's tumor, particularly, in those with the ascitic form. The inhibition analysis using metyrapone and 7,8-benzoflavone identified phenobarbital and methylcholanthrene forms of benzo(a)pyrene-hydroxylase in murine liver; isoform profile was not significantly affected by tumor. Liver microsomal monooxygenases of tumor-bearing mice retained inducibility by 3-methylcholanthrene.