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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.

Pentachlorophenol (aromatic amine and azo stain metabolic stimulation inhibitor) reduced the hepatocarcinogenic activity of 4-aminoazobenzene and reduced that of ortho-aminoazotoluene in suckling mice. Both 4-aminoazobenzene and ortho-aminoazotoluene exhibited mutagenic activity in Ames' test in vitro on S. typhimurium TA 98 strain with activation with liver enzymes; this mutagenic activity was similarly suppressed by adding pentachlorophenol into activation medium. Induction of xenobiotic metabolism enzymes, stimulating the mutagenic activity of ortho-aminoazotoluene, suppressed its carcinogenic effect on mouse liver. Hence, ortho-aminotoluene (the initial compound), but not its mutagenic metabolites, was the direct active hepatocarcinogen for mice.

It is considered that diethylnitrosamine (DENA) is metabolized mostly in the liver with producing highly reactive compounds alkylating cellular macromolecules and causing toxic and carcinogenic effects. However, competitive inhibition of cytochrome P450 2E1, which metabolizes DENA, by its other substrate, isopropanol, does not weaken, but enhances the toxicity of DENA effect on mice and reduces the number of preneoplastic nodules and liver tumors induced by it. In short-term tests for mutagenicity (in the Ames test and the SOS-hromotest) DENA causes moderate damage of DNA that reduces liver microsomal enzymes, that metabolize nitrosamines. The obtained data indicate the predominant role of the initial compound as compared to its metabolites in toxic, and probably hepatocarcinogenic, effects on DNA in mice.

The effects of five new derivatives of 2,6-dialkyl-4-propylphenole containing in para-radical different ionogenic groups (-SO3Na, -S-SO3Na, -S-(NH2)2Cl) in the presence and in the absence of hydrogen peroxide on the survival of E. coli AB1157 cells and its isogenic strain defective in repair enzyme genes were studied. Cell survival treated with hydrogen peroxide was remarkably increased in the presence of (3-(3,5-dimethyl-4-hydroxyphenyl)propyl)-1-sulphonate of sodium (Cl). Replacement of methyl ortho-radicals in the structure of Cl for tret-buthyl or cyclohexyl groups led to a decrease of the compounds ability to protect the cells from exogenic hydrogen peroxide. Between derivatives of 2,6-di-tret-buthylphenol the compound with thiosulphonate group demonstrated properties comparable with those for its sulphonate analog, then a chloride of isotiurone at concentration 3 mM completely suppressed the growth of cells in presence and in the absence of H2O2. Compound Cl may be considered as most perspective for detail analysis as antioxidant.

Fluorinated derivatives of 1,4-naphthoquinones are highly potent inhibitors of Cdc25A and Cdc25B phosphatases and growth of tumor cells. Eight new derivatives of polyfluoro-1,4-naphthoquinone were synthesized and their cytotoxicity in human myeloma, human mammary adenocarcinoma, mouse fibroblasts and primary mouse fibroblast cells as well as their mutagenic and antioxidant properties in a Salmonella tester strain were studied. The efficiency of suppressing the growth of two lines of tumor cells decreased in the order: 2-(2-hydroxy-ethylamino)-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (1), 2,3-dimethoxy-5,6,7,8-tetrafluoro-1,4-naphthoquinone (2), 2-[2-hydroxyethyl(methyl)amino]-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (3), 2-morpholino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (4), 2-[bis-(2-hydroxyethyl)amino]-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (5), 2-[(2-hydroxy)ethylsulfanyl)]-5,6,7,8-tetrafluoro-1,4-naphthoquinone (6), 2-methoxy-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (7), and 1,4-dioxo-3-(1-pyridinio)-1,4-dihydro-5,6,7,8-tetrafluoronaphthalene-2-olate (8). Taking into account these data together with the better cytotoxic effect against cancer cells as compared with normal mammalian cells, protecting of bacterial cells from spontaneous and H(2)O(2)-dependent mutagenesis, and lower general toxicity of the compounds towards different cells, one can propose that compounds 3-5 may be considered as useful potential inhibitors of growth of tumor cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Effect of seven structurally similar N, N-dimethyl-(4-hydroxyaryl)alkylammonium chlorides in the presence and in the absence of hydrogen peroxide on the survival of E. coli cells AB1157 and its isogenic strain BH910 defective in genes of repair enzymes has been analyzed. Among the studied compounds only chloride of N,N-dimethyl-(3,5-dimethyl-4-hydroxybenzyl)ammonium (C1) has no cytotoxic properties and increases the survive of the cells of both strains in the presence of H2O2 better than trolox (water soluble analog of alpha-tocopherol). C1 analogs: 3-methyl-(5-di(tert-butyl)-4-hydroxybenzyl) and 3-(3,5-di(tert-butyl)-4-hydroxyphenyl)propyl)amines derivatives effectively protected from H2O2 only mutant cells BH910. Among the structural analogs of C1 cytotoxicity increases at substitution of methyl groups in aromatic cycle by tert-butyl and cyclohexyl groups. Only C1 among the seven new compounds is the most promising antioxidant for the subsequent more detailed analysis.

Fluorinated derivatives of 1,4-naphthoquinone are highly potent inhibitors of Cdc25A and Cdc25 phosphatases and growth of tumor cells. Five new N-substituted polyfluorinated derivatives of 2-amino-1,4-naphthoquinone were synthesized and their mutagenic and antioxidant properties in Salmonella cells, as well as cytotoxicity in human myeloma (RPMI 8226), human mammary adenocarcinoma (MCF-7), mouse fibroblasts (LMTK) and primary mouse fibroblast cells (PMF) were studied. 2-tert-Butylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (1) inhibited the growth of normal control and tumor cells at the same concentration. Three compounds: 2-diethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (2), 2-ethylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (3), 2-phenylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (4) exhibited a 50% decrease in the growth of cancer cells at low and comparable concentrations (2.4-8.6 microM) while being remarkably less cytotoxic toward normal LMTK and PMF cells. Quinones (1)-(4), but not 2-phenylamino-3-methyl-5,6,7,8-tetrafluoro-1,4-naphthoquinone (5), efficiently suppressed spontaneous mutagenesis in Salmonella cells, while all compounds 1-5 decreased the mutagenic effect of H2O2 on bacterial cells. Their possible perspectives as anticancer drugs are shortly discussed., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

Four new n-butylamino and two sulfur-containing derivatives of polyfluoro-1,4-naphthoquinone were synthesized and their mutagenic and antioxidant properties in Salmonella cells, as well as the cytotoxicity in human myeloma (RPMI 8226), human mammary adenocarcinoma (MCF-7), mouse fibroblasts (LMTK) and primary mouse fibroblast cells (PMF) were studied. 2-n-Butylamino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (1) showed efficient inhibition of the growth of the tumor cells. 2,8-Di-(n-butyl-amino)-3,5,6,7-tetrafluoro-1,4-naphthoquinone (2) had significantly less growth-inhibiting properties, while 2,6-di-(n-butylamino)-3,5,7,8-tetrafluoro-1,4-naphthoquinone (3) and 2,6,8-tri-(n-butylamino)-3,5,7-tetrafluoro-1,4-naphthoquinone (4), demonstrated very low cytotoxicity. Compounds 1 and 2 were remarkably less cytotoxic while compound 3 and 4 were not cytotoxic toward LMTK and PMF cells as compared with tumor human cell lines. Cytotoxicity of 2-(2'-methylthioethyl)amino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (5) and (2,2'-dithiodi-2)-3,5,6,7,8-pentafluoro-1,4-naphthoquinone-2-ylamino)ethan (6) toward mammalian cells was compared with that for compounds 1 and 2., (Copyright 2009 Elsevier Masson SAS. All rights reserved.)

The antioxidant activity, mutagenicity, and genotoxicity of bis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propyl)sulfide (thiophane) were studied using bacterial tests. The results of both an Ames test and SOS chromotest, as well as those studying the survival of E. coli cells deficient in enzymes responsible for the repair of DNA oxidative damage, testify to the fact that thiophane is not mutagenic and genotoxic, and it protects Salmonella typhimurium cells better than the well-known antioxidant trolox.

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.

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

To study the molecular basis of the origin of complete and mosaic mutants, pBR322 plasmid with one- or two-stranded DNA damage was constructed by limited chemical modification of the plasmid DNA. Damage of one strand of DNA resulted in induction of mosaic mutants. Data were obtained indicating that complete mutations arise as a result of damage of two strands in the region of the mutagenized gene.

The release and extraction of histones from the nuclei of rat liver under the influence of 0.005 M CaCl2 and 0.14 M NaCl was investigated. A considerable loss of nuclear histones, especially lysine-rich ones, was shown biochemically and cytophotometrically. The preliminary incubation of the nuclei in the respective salt solutions resulted in a more complete extraction of histones. This may be explained by a weaker binding capacity of histones to DNA under the latter conditions. Electrophoresis in polyacrylamid gel showed an increased mobility of histone fractions removed from the nuclei by incubation in the salt solutions.

A method of selective modification of certain regions of the genome which may become useful for inactivation of certain genes or for directed mutagenesis is proposed. For this purpose RNA products of certain genes carrying alkylating groupings randomly distributed along the polymer were used. The RNA modified to an extent of 4--5 alkylating residues per 100 nucleotides retains the ability to specific formation of DNA--RNA hybrid complexes. The alkylating molecule is N,N,N'-tri-(beta-chlorethyl), N'-(p-formylphenyl)propylene diamine-1,3. The aliphatic alkylating functions serve for attachment to RNA. The aromatic alkylating function inactivated by the formyl grouping at the para-position of the benzene ring is used for modification of DNA after hybrid formation by reduction of formyl grouping with sodium borohydride. The covalently binding of modified RNA is exhibited to occur in only the case of T7 DNA H-chain, the one complementary to the RNA derivative. L-chain does not hybridize, nor does it undergo alkylation by the RNA product thus indicating high selectivity of alkylation within the hybrid complex.

Corticosterone (8 X 10(-8) g/ml) augmented the amplitude of the rat portal vein phasic contractions within first minutes. The dose 5 X 10(-7) g/ml first augmented and then reduced the amplitude, simultaneously increasing the rate of autorhythmic activity and the vascular tone. Hydrocortisone and dexamethasone exerted no initial effect but reduced the amplitude of phasic contractions within 5-10 min. Glucocorticoids did not affect the rate of contractions, but dexamethasone (5 X 10(-7) g/ml) augmented the vascular tone. The mineral and glucocorticoid action of corticosteroids seems to be actualized through different mechanisms, the initial action of hormones being determined by their membrane effects upon vascular myocytes.

Corticosterone (28 nM/l) sharply increased the phasic tension of the portal vein whereas in dose 140 nM/l it sharply inhibited the phasic activity after its short enhancement, simultaneously, increasing the contraction rate and basal tone of the vessel in the normotensive Wistar-Kyoto rats. The initial response to corticosterone was absent in the spontaneously hypertensive rats, and their system maintaining the tonic component of contraction of the portal vein smooth muscles, was immediately activated. A primary membrane effect of the hormone on the vascular smooth muscle cells seems to be absent in the corticosterone effect on the spontaneous contractile activity of the portal vein in hypertensive animals.

The chromosome number and DNA content in Euglena gracilis cell have been shown by the number of investigators to vary essentially under the influence of some environmental factors (low phosphate and acetate concentrations, density of culture, temperature, light/dark regime, streptomycin treatment). Variability in the nuclear DNA quantity is supposed to be an important peculiarity of the structure and functional activity of the genome of Euglena gracilis.

A new method for obtaining the recombinant DNA based on heteroduplex-initiated site-directed insertion of alien nucleotide sequences is proposed. To generate a single-stranded region, plasmid DNA was nicked with restriction endonuclease in the presence of ethidium bromide with subsequent exonuclease III controlled digestion. The inserted DNA sequences flanked by nucleotide sequences complementary to single-stranded region were annealed with plasmid DNA and E. coli cells were transformed by the resulting heteroduplex molecules. The presented data show the possibility to insert as many as 200 nucleotides. The yield of recombinant DNA varied from 16 to 0.7% as the number of nucleotides inserted correspondingly varied from 15 to 200. The site of insertion does not depend crucially on the localization of the restriction site used.

In the preselected site of pBR322 plasmid DNA related to the Tcr gene mutations were induced by the complementary single-stranded DNA restricts carrying alkylating groups. The alterations of the DNA primary structure in the mutagenized site were studied. It was found and that in the majority of mutants with the impaired Tcr gene function, the tandem direct repeats appeared. The repeats of 7-8 base pairs were localized in a fixed site of the Tcr gene, downstream of the palindrome. It is suggested that tandem repeats appear as a result of D-loops formation when single-stranded DNA forms a hairpin structure, due to the presence of palindromes. In the light of this notion, the tentative schemes of gene conversion and genome evolution are discussed.

The release and extraction of histones from the nuclei of guinea pig liver under the influence of diphteria toxin was investigated in vivo and in vitro. A considerable loss of nuclear histones, increase of easily extracted arginine-rich histones and a more complete extraction of lysine-rich ones have been shown. A possible role of histones in the development of diphteria intoxication is discussed.