Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique.

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.

Foci of aberrant crypts in the colons of mice and rats exposed to carcinogens associated with foods.

Aberrant crypt foci can be identified in the colons of rodents treated 3 wk earlier with azoxymethane, a known colon carcinogen. These crypts can easily be visualized in the unsectioned methylene blue-stained colons under light microscopy, where they are distinguished by their increased size, more prominent epithelial cells, and pericryptal space. They occur as single aberrant crypts or as two, three, or four aberrant crypts in a cluster. We compared the reported ability of carcinogens associated with the human diet to induce colon cancer with the measured rate of induction of aberrant crypts in female CF1 mice and Sprague-Dawley rats. The carcinogens used were 1,2-dimethylhydrazine, methyl nitrosourea, N-nitrosodimethylamine, benzo(a)pyrene, aflatoxin B1, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 2-amino-3-methylimidazo[4,5-P]quinoline, 2-amino-3,4-dimethylimidazo[4,5-P]quinoline, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Graded doses of these compounds were given to the animals by gavage twice with a 4-day interval, and the animals were terminated 3 wk later. All colon carcinogens induced aberrant crypts in a dose-related fashion. N-Nitrosodimethylamine and 3-amino-1-methyl-5H-pyrido[4,3-b]indole, carcinogenic compounds that do not induce colon cancer, did not induce them. The ability of the studied compounds to induce aberrant crypts was species specific; e.g., aflatoxin B1 and 2-amino-3,4-dimethylimidazo[4,5-P]quinoline induce about 20 times more in rats than mice. This relationship was consistent with their reported ability to induce colon cancer in these species. Results of the present study support the use of the aberrant crypt assays to screen colon-specific carcinogens and to study the process of colon carcinogenesis.

Inflammation increases oxidative DNA damage repair and stimulates preneoplastic changes in colons of newborn rats.

Oxidative DNA damage may be a risk factor for development of various pathologies, including malignancy. We studied inflammation triggered modulation of repair activity in the intestines of three weeks old rats injected i.p. with E.coli or S. typhimurium lipopolysaccharides (LPS) at doses of 1, 5 or 10 mg/kg. Subsequent formation in these animals of colonic preneoplastic lesions, aberrant crypt foci (ACF) was also investigated. Five days after LPS administration no differences were observed in repair rate of 1,N(6)-ethenoadenine (εA), 3,N(4)-ethenocytosine (εC) and 8-oxoguanine (8-oxoG) in intestines of these rats, as measured by the nicking assay. However a significant increase in all three repair activities was found within one and two months after S. typhimurium LPS treatment. E. coli LPS significantly increased only the 8-oxoG repair. S. typhimurium LPS stimulated mRNA transcription of pro-inflammatory proteins, lipooxygenase-12 and cyclooxygenase-2, as well as some DNA repair enzymes like AP-endonuclease (Ape1) and εC-glycosylase (Tdg). mRNA level of DNA glycosylases excising εA (MPG) and 8-oxoG (OGG1) was also increased by LPS treatment, but only at the highest dose. Transcription of all enzymes increased for up to 30 days after LPS, and subsequently decreased to the level observed before treatment, with the exception of APE1, which remained elevated even two months after LPS administration. Thus, the repair efficiency of εA, εC and 8-oxoG depends on the availability of APE1, which increases OGG1 and TDG turnover on damaged DNA, and presumably stimulates MPG. One and two months after administration of E. coli or S. typhimurium LPS, the number of aberrant crypt foci in rat colons increased in a dose and time dependent manner. Thus, inflammation stimulates the repair capacity for εA, εC and 8-oxoG, but simultaneously triggers the appearance of preneoplastic changes in the colons. This may be due to increased oxidative stress and imbalance in DNA repair.

Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases.

Hypoxanthine¿xanthine oxidase¿Fe3+¿ethylenediaminetetraacetate (EDTA) was used to modify ss M13 mp18 phage DNA. The dominant base modifications found by GC/IDMS-SIM were FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol. Analysis of in vitro DNA synthesis on oxidatively modified template by three DNA polymerases revealed that T7 DNA polymerase and Klenow fragment of polymerase I from Escherichia coli were blocked mainly by oxidized pyrimidines in the template whereas some purines that were easily bypassed by the prokaryotic polymerases constituted a block for DNA polymerase beta from calf thymus. DNA synthesis by T7 polymerase on poly(dA) template, where FapyAde content increased 16-fold on oxidation, yielded a final product with a discrete ladder of premature termination bands. When DNA synthesis was performed on template from which FapyAde, FapyGua, and 8OHGua were excised by the Fpg protein new chain terminations at adenine and guanine sites appeared or existing ones were enhanced. This suggests that FapyAde, when present in DNA, is a moderately toxic lesion. Its ability to arrest DNA synthesis depends on the sequence context and DNA polymerase. FapyGua might possess similar properties.

Conformation of plasmid DNA from Escherichia coli deficient in the repair systems protecting DNA from 8-oxyguanine lesions.

8-Oxyguanine (8ohG) is a major oxidation product of guanine and a biomarker of oxidative stress in mammal. We have attempted to estimate the level of 8ohG residues in plasmid DNA (pGW2123 and pBR322) grown in various bacterial strains (fpg, mutY, or mutT, plus mutT fpg and mutT mutY double mutants) differing in the system protecting cells against the mutagenic effects of 8ohG in DNA. The method was based on digestion of plasmid DNA with Fpg, and agarose gel electrophoresis. Fpg converts pDNA from covalently closed circular to the open circular (ccc-->oc) form of pDNA when there is at least one 8ohG, or apurinic site, per ccc pDNA molecule. It was found that: i) the content of 8ohG in pDNA grown in any of the tested bacteria is below one 8ohG per 10(4) base pairs; ii) a substantial part of pGW2123 is isolated from the bacteria in the oc form; iii) the ratio of oc/ccc in pGW2123 depends on the bacterial host and is the lowest when the plasmid was harvested from mutY- deficient cells; iv) pBR322, unlike pGW2123, is isolated predominantly in the ccc form; and v) of the pBR322 grown in the tested bacteria apparently the most resistant to Fpg digestion was pBR322 grown in the mutY strain. It is proposed that this reflects the compact structure of pDNAs when they are grown in bacteria deficient in mutY gene product.

Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA.

The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.

Contribution of E. coli AlkA, TagA glycosylases and UvrABC-excinuclease in MMS mutagenesis.

MMS, an S(N)2 alkylating agent, is a moderate inducer of SOS mutagenesis and adaptive response. Our previous studies have shown that transient starvation of Escherichia coli AB1157argE3 strain causes a decrease of MMS-induced argE3-->Arg(+) reversions and this decrease is accompanied by the disappearance of the Fpg protein sensitive sites on plasmids isolated from MMS-treated and subsequently starved bacteria. This suggests that in such cells the mutation frequency decline (MFD) repair takes place. Here, we study the relation between MMS-induced mutagenesis as well as mutation frequency decline during starvation, and the repair of alkylated bases and AP-sites by base and nucleotide excision repair systems. In the AB1157alkA(-) strain, MMS-induced mutagenesis was over five-fold higher than in the wild type strain and no MFD repair occurred during starvation. Surprisingly, the lack of TagA glycosylase diminished MMS mutagenesis and accelerated the MFD effect. However, in double tagA(-)alkA(-) mutant, the frequency of Arg(+) reversions increased over 10-fold during 60 min of aminoacid starvation after MMS-treatment. Lack of the uvrA gene function did not affect the MMS-induced mutation rate and MFD in AB1157alkA(+)tagA(+). Starvation of MMS treated AB1157tagAalkAuvrA triple mutant caused a decrease of mutation frequency almost to the level of spontaneous mutation rate. Examination of the repair of 3-MeAde, 7-MeGua and AP sites during starvation using repair glycosylases and plasmids isolated from MMS-treated and starved bacteria revealed that in E. coli uvr(+) but tagAalkA strain, neither 3-MeAde nor 7-MeGua were repaired during 60 min starvation and these persistent lesions could be responsible for the induction of the SOS system and an increase in mutation rate during starvation. In the triple tagAalkAuvrA mutant the repair of 3-MeAde, 7-MeGua and AP sites was carried out effectively and this could explain the observed decrease in the mutation rate during starvation. These results suggest that only in the absence of the "first choice" repair enzymes TagA, AlkA glycosylases and UvrABC excinuclease, a third error-free repair system of alkylated bases is activated. In the absence of only TagA and AlkA glycosylases, UvrABC excinuclease mediates activation of the SOS response, and this results in an increase of mutagenesis induced by the presence of alkylated bases in DNA.

Tobacco BY-2 cells excise both 3-methyladenine and 7-methylguanine from methylated DNA.

Two of the major products in DNA resulting from exposure to alkylating agents are 7-methylguanine and 3-methyladenine. N-methylpurine-DNA-glycosylase is required for excision of these lesions. Recently, the 3-methyladenine-DNA-glycosylase gene of Arabidopsis thaliana was cloned and shown to be involved only in repair of 3-methyladenine. In BY-2 tobacco cells, we showed an enzymatic activity which excised both 3-methyladenine and 7-methylguanine from methylated DNA.

Studies on the effect of ascorbic acid and selenium on the genotoxicity of nitrofurans: nitrofurazone and furazolidone.

The genotoxic properties of nitrofurazone and furazolidone were studied using the Ames test and SOS-chromotest. Both compounds were found to act as strong mutagens on the TA97 and TA102 strains of S. typhimurium and to induce the SOS-repair system in the PQ37 strain of E. coli. A good concordance was found between the mutagenic activity and the ability to induce the SOS system. Ascorbic acid and sodium selenite only very slightly lowered the genotoxic effect of the 2 nitrofurans studied both in the Ames test and in the SOS-chromotest.

Mutagenic activity of thiram in Ames tester strains of Salmonella typhimurium.

The mutagenic activity of thiram was investigated in 4 histidine-requiring strains of Salmonella typhimurium (TA1535, TA100, TA1538, TA98) with and without activation by liver microsomes. In strains TA1535 and TA100, thiram induces mutations without metabolic activation. The presence of rat-liver microsome fraction, cysteine or glutathione abolish its mutagenic activity in these strains. In contrast, thiram requires metabolic activation for the expression of its mutagenic activity in TA1538 and TA98 strains. The compounds containing the sulphydryl group abolish mutagenic activity of thiram in these strains, too.

Mutagenic activity of furfural in Salmonella typhimurium TA100.

The mutagenic activity of furfural was tested in Salmonella typhimurium strains TA98 and TA100. Furfural produced mutations in the TA100 strain, but not in the TA98 strain. A rat-liver microsomal fraction did not increase the mutagenic activity of furfural in either strain. Mutagenic activity of furfural in the TA100 strain was not increased by benzo[alpha]pyrene in the presence of metabolic activation.

SOS-dependent A-->G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA.

Gas chromatography/isotope dilution-mass spectrometry with selected ion monitoring (GC/IDMS-SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C-->A (approximately 20-fold increase in SOS-conditions), G-->A (9-fold increase) and G-->C (4.5-fold increase). Very few G-->T transitions were found. A particularly large group of A-->G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A-->G transitions in SOS-induced bacteria.

Screening for genotoxic activity of amitraz with short-term bacterial assays.

Genotoxic effects of both amitraz and its metabolites made by S9 fraction were reevaluated in short-term bacterial assays. Neither amitraz nor its metabolites induced frameshift mutation or caused base-pair substitution as detected by the Ames test. They also did not introduce any damages into DNA recognized by correndonuclease II as shown by the repair test. Metabolites of amitraz (but not amitraz itself) induced the SOS-repair system in E. coli strain PQ 243 tagA, alkA which was deficient in N-glycosylases. It is concluded that neither amitraz nor its metabolites have mutagenic activity. In contrast to amitraz, its metabolites alkylate DNA in the N3-position of adenine.

Antimutagenic effects of several subfractions of extract from wheat sprout toward benzo[a]pyrene-induced mutagenicity in strain TA98 of Salmonella typhimurium.

The aqueous extract from wheat sprouts contains some antimutagenic factor(s). The factor(s) abolish(es) the activity of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) in the S9 fraction from Aroclor-treated rat livers and also inhibit(s) the mutagenic activity of benzo[a]pyrene (B(a)P) in the Ames test. The extract (fraction S30) was subjected to initial fractionation by thermal treatment, 3 24-h cycles of dialysis and ultrafiltration. The antigenotoxic activity of fraction S30 amounted to 98% and was unchanged by thermal treatment (100 degrees C, 10 min). Both the dialysate and the dialysis fluid inhibited the mutagenic effect of B(a)P by 48.4 and 48% respectively. The microsomal subfraction inhibited the mutagenicity only in 10%, and the postmicrosomal subfraction in 68%. It is concluded that the extract from wheat sprouts contains at least 2 heat-resistant compounds (or groups of compounds) located within the cell cytosol and showing antimutagenic activity: one group is of low molecular weight and another of high MW. Alternatively, low-molecular compounds could either be free or bound to high-molecular compound(s).

Activity of Escherichia coli DNA-glycosylases on DNA damaged by methylating and ethylating agents and influence of 3-substituted adenine derivatives.

Methylating and ethylating agents are used in the chemical industry and produced during tobacco smoking. They generate DNA base damage whose role in cancer induction has been documented. Alkylated bases are repaired by the base excision repair pathway. We have established the repair efficiency of methylated and ethylated bases by various Escherichia coli repair proteins, namely 3-methyladenine-DNA-glycosylase I (TagA protein), which excises 3-methyladenine and 3-methylguanine, 3-methyladenine-DNA-glycosylase II (AlkA protein), which has a broad substrate specificity including 3- and 7-alkylated purines and the formamidopyrimidine(Fapy)-DNA-glycosylase (Fpg protein) repairing imidazole ring-opened 7-methylguanine. The comparison of the Km values of these various enzymes showed that methylated bases were excised more efficiently than ethylated bases. Several 3-alkyladenine derivatives have been synthesized and examined for their ability to inhibit the activity of the various repair proteins. We have shown that 3-ethyl-, 3-propyl-, 3-butyl- and 3-benzyladenine were much more efficient inhibitors of TagA protein than 3-methyladenine. The inhibitory effect was increased with the increase of the size of alkyl-group and IC50 for 3-benzyladenine was 0.4 +/- 0.1 microM as compared to 1.5 +/- 0.3 mM for 3-methyladenine. These compounds inhibited neither the AlkA protein nor human 3-methyladenine-DNA-glycosylase (ANPG protein). Moreover, 3-hydroxyethyladenine did not affect the activity of any of these enzymes. Taken together, these results suggest that hydrophobic interactions are involved in the mechanism of inhibition and/or recognition and excision of alkylated purines by TagA protein.

Urine mutagenicity of petroleum plant workers.

The mutagenicity of urinary extracts from workers employed in a petroleum plant was analyzed by means of the plate test using S. typhimurium strains TA98 and TA100. Mean urinary mutagenic activities in TA98 were 2-14 times higher in the petroleum plant workers than in the control group. In TA100 these differences were even bigger, the mutagenicity in petroleum plant workers' urine being 3-42 times higher than in the control group. These results suggest that the environmental exposure of people to mutagenic substances is markedly increased in a petrochemical plant.

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs.

Quercetin, rhamnetin, isohamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation. Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3' and 4' positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.

The effect of wheat sprout extract on benzo(a)pyrene and 7,2-dimethylbenz(a)anthracene activity.

Subcutaneous application of aqueous wheat sprout extract to mice resulted in a slight decrease of the ability of fraction S-9 from their skin to activate DMBA to metabolites mutagenic for S. typhimurium TA 98. Induction by benzo(a)pyrene of sperm abnormalities in mice was diminished after oral administration of the wheat sprout extract; however, even high doses of the extract did not completely abolish the effect of benzo(a)pyrene on spermatozoa. In the carcinogenicity studies, the wheat sprout extract, when applied to mouse skin during the initiation phase, enhanced fourfold the induction of papillomas by DMBA and shortened the period of latency from 9 to 5 weeks.