Lipoperoxidation products and thiol antioxidants in chromium exposed workers.

Hexavalent chromium is an established carcinogenic agent, which is not directly reactive with DNA. Its genotoxicity involves a reduction step, producing reactive oxygen species and radicals, and also lower valence forms which form stable complexes with intracellular macromolecules. The trivalent form of chromium may directly react with the genetic material and has also been shown to generate oxidative damage in vitro. To further evaluate the importance of in vivo oxidative DNA damage in the toxicity of each valence form, we conducted a comparative study on hexavalent and trivalent chromium-exposed workers (manual metal arc stainless steel welders and leather tanning workers), focusing on the total oxidative status by quantifying the level of lipoperoxidation products in urine. Thiol antioxidants are important in response to oxidative stress, and therefore, the concentration of glutathione and cysteine in peripheral blood lymphocytes was also determined. Chromium exposure was evaluated by quantifying total chromium in plasma and urine. Both groups had a significant increase in lipid peroxidation products expressed as malondialdehyde (MDA) in urine (tanners 1.42 +/- 0.61 micromol/g creatinine, welders 1.67 +/- 1.13 micromol/g creatinine versus controls 0.81 +/- 0.26 micromol/g creatinine, P < 0.005 in both cases) but only welders had a significant decrease in glutathione concentration in lymphocytes. There was a positive correlation between chromium in plasma and urinary MDA in welders, but not in tanners. This work is part of a larger study of which major results have been published previously including cytogenetics and DNA-protein cross-links in workers exposed to the two different forms of chromium. These results are compared with the results of oxidative damage from this study.

Catechols from abietic acid synthesis and evaluation as bioactive compounds.

Catechols from abietic acid were prepared by a short and good yielding chemical process and further evaluated for several biological activities namely, antifungal, antitumoral, antimutagenic, antiviral, antiproliferative and inhibition of nitric oxide. Their properties were compared with those of carnosic acid (6), a naturally occurring catechol with an abietane skeleton and known to possess potent antioxidant activity, as well as anticancer and antiviral properties. From all the synthetic catechols tested compound 2 showed the best activities, stronger than carnosic acid.

Elevated levels of DNA-protein crosslinks and micronuclei in peripheral lymphocytes of tannery workers exposed to trivalent chromium.

DNA-protein crosslinks (DPC) are a promising biomarker of exposure to hexavalent chromium, a known human carcinogen. Although trivalent chromium is considered to have much lower toxicity, the risk involved in chronic exposure is uncertain. DPC may be a useful tool in clarifying this risk, by signaling an exposure of body tissues to biologically active forms of chromium. DPC quantification was carried out in lymphocytes of a group of tannery workers exposed to trivalent chromium, a small group of manual metal arc stainless steel welders exposed to hexavalent chromium and a control group. This biomarker was compared with the frequency of micronuclei in cytokinesis blocked peripheral lymphocytes as a biomarker of cytogenetic lesions and total plasma and urine chromium levels as an index of exposure. The results indicate a significant increase in the formation of DPC in tannery workers compared with controls (0.88 +/- 0.19 versus 0.57 +/- 0.21%, P < 0.001, Mann-Whitney test) and an even higher level of DPC in welders (2.22 +/- 1.12%, P = 0.03). Tanners showed a significant increase in micronucleated cells compared with controls (6.35 +/- 2.94 versus 3.58 +/- 1.69 per thousand, P < 0.01), whereas in welders this increase was not significant (5.40 +/- 1.67 per thousand ). Urinary chromium was increased in both groups, with a greater increase observed in tanners compared with controls (2.63 +/- 1.62 versus 0.70 +/- 0.38 microg/g creatinine, P < 0.001) than in welders (1.90 +/- 0.37 microg/g creatinine, P < 0.005). Plasma chromium was also increased in both groups (tanners 2.43 +/- 2.11 microg/l, P < 0.001, welders 1.55 +/- 0.67 microg/l, P < 0.005 versus controls 0.41 +/- 0.11 microg/l). In summary, chronic occupational exposure to trivalent chromium can lead to a detectable increase in lymphocyte DNA damage which correlates with a significant exposure of the cells to the metal.

No evidence of increased chromosomal aberrations and micronuclei in lymphocytes from nonfamilial thyroid cancer patients prior to radiotherapy.

The relationship between the presence of high frequencies of chromosomal aberrations in peripheral lymphocytes and predisposition to cancer has been suggested for some cancer diseases. In nonfamilial thyroid cancer, the few reports available are equivocal. The aim of this study was to assess the possible chromosomal instability in peripheral blood lymphocytes from 22 patients suffering from nonfamilial thyroid cancer. For this purpose, 2 classic cytogenetic assays, the chromosomal aberrations assay and cytokinesis-blocked micronucleus assay, were chosen. The frequency of chromosomal aberrations excluding gaps (%) was 1.68 +/- 1.39 (mean value +/- SD) for the patients group versus 2.20 +/- 1.87 for the control group. The frequency of binucleated lymphocytes with micronuclei ( per thousand) was 5.41 +/- 3.51 (mean value +/- SD) for the patients group versus 5.37 +/- 3.21 for the control group. The results obtained revealed no significant differences between both groups. The present study reinforces the idea that constitutional chromosomal instability in peripheral blood lymphocytes is not visible in nonfamilial thyroid carcinomas.

Genotoxicity of instant coffee and of some phenolic compounds present in coffee upon nitrosation.

Instant coffee exhibits genotoxic activity upon nitrosation at acidic pH values in the Ames tester strain TA100. Using adsorption chromatography (Amberlit XAD-2) it was observed that the major fraction of molecules responsible for the genotoxic activity upon nitrosation was not retained on this resin, suggesting that the polar molecules present in instant coffee could be responsible for the genotoxicity observed upon nitrosation. Some phenolic molecules present in instant coffee (catechol, caffeic acid, and chlorogenic acid) were also genotoxic upon nitrosation under the same experimental conditions. The concentrations of nitrosatable phenolic compounds in the studied coffee were determined by HPLC and their contributions to the total genotoxicity observed were studied. The results obtained suggest that besides phenolic compounds other molecules were also involved in the genotoxicity of this beverage upon nitrosation. Teratogenesis Carcinog. Mutagen. 20:241-249, 2000.

Heterologous expression of xenobiotic mammalian-metabolizing enzymes in mutagenicity tester bacteria: an update and practical considerations.

There is an increasing need for metabolic competent cell systems for the mechanistic studies of biotransformation of xenobiotics in toxicology in general and in genotoxicology in particular. These cell systems combine the heterologous expression of a particular mammalian biotransformation enzyme with a specific target/ end point by which a functional analysis of the expressed gene product in the (geno)toxicity of chemicals can be performed. cDNAs of an increasing number of mammalian biotransformation enzymes is being cloned. The construction of specific expression vectors permits their heterologous expression in laboratory bacteria, such as Escherichia coli strains. This development does not only allow biochemical and enzymatic studies of (pure) enzyme preparations but also facilitates the engineering of metabolically competent mutagenicity tester bacteria, thereby providing new tools for genotoxicity testing and for studying of the roles of biotransformation in chemical carcinogenesis. In this review, we describe an update as well as an evaluation of enzymes expressed in mutagenicity tester bacteria. Four types of biotransformation enzymes are now expressed in these bacteria, namely, GSTs, CYPs, NATs, and STs. The expression of these enzymes in the tester bacteria and their subsequent application in mutagenicity assays demonstrates that heterologous expression in this type of bacteria has a number implications for the functionality of the biotransformation enzymes as well as for the functioning of the tester bacteria in mutagenicity detection. We also describe here a number of practical considerations in this regard.

Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity.

Patulin is a mycotoxin produced by several species of Penicillium, Aspergillus and BYSSOCHLAMYS: Patulin is a common contaminant of ripe apples used for the production of apple juice concentrates and is also present in other fruits, vegetables and food products. Patulin has been reported to have mutagenic, carcinogenic and teratogenic properties. Nevertheless, these properties are still a matter of debate. In this report, we further investigated the genotoxicity of patulin in mammalian cells by two different approaches. Firstly, we evaluated the induction of micronuclei in cytokinesis-blocked human lymphocytes. This approach is important because available data concerning the genetic toxicity of patulin in human cells is sparse. Secondly, we chose an established model for patulin genotoxicity, i.e. the chromosomal aberration assay in V79 Chinese hamster cells, to clarify whether concomitant exposure to ascorbic acid with the mycotoxin modulates or not the clastogenicity of patulin. The results unequivocally show induction of DNA-damaged cells by patulin as assessed by both cytogenetic assays. In addition, an almost complete abolition of patulin (0.8 microM) clastogenicity was observed in the presence of 80 microM ascorbic acid (P < 0.05), showing that although a genetic risk is present, ascorbic acid could somehow partially modulate this problem.

Cytogenetic alterations and oxidative stress in thyroid cancer patients after iodine-131 therapy.

This study aimed to assess two end-points of DNA damage, namely chromosomal aberrations and micronuclei in peripheral lymphocytes, and their possible relationship with oxidative stress (which may be related to DNA damage and repair) in thyroid cancer patients receiving therapeutic doses of (131)I. Nineteen patients receiving 2590 MBq (70 mCi) were studied. Chromosomal aberrations were scored using standard cytogenetic methods and micronuclei scored in cytokinesis-blocked lymphocytes. Oxidative stress was assessed by determining thiobarbituric acid-reactive substances in blood, total plasma antioxidant status and serum uric acid levels. All parameters were assessed before treatment and 1 and 6 months after (131)I administration. The frequency of micronucleated cells per 1000 binucleated cells scored (mean +/- SEM) increased significantly from 5.21 +/- 0.80 to 9.68 +/- 1.22 1 month after treatment (P < 0.01) and to 8.42 +/- 1.28 6 months after treatment (P < 0.05). The frequency of cells with chromosomal aberrations, excluding gaps, per 100 cells, increased significantly from 1.68 +/- 0.41 to 3.47 +/- 0. 55 1 month after treatment (P < 0.01) and to 4.05 +/- 0.46 6 months after treatment (P < 0.01). Oxidative stress parameters showed slight modifications over the time period studied, but the differences were not significant except for a decrease in thiobarbituric acid-reactive products 6 months after therapy (P < 0. 05) and in serum uric acid concentration 1 and 6 months after therapy (P < 0.01). This report demonstrates slight but significant and persistent DNA damage in (131)I-treated patients as assessed by cytogenetic assays. There was no clear correlation between the cytogenetic findings and oxidative stress parameters studied.

Escherichia coli MTC, a human NADPH P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 isoforms 1A1, 1A2, 2A6, 3A4, or 3A5: catalytic activities and mutagenicity studies.

We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The four new strains were compared for CYP- and RED-expression levels and CYP activities with the formerly developed CYP1A2 expressing strain. CYP1A2 and CYP2A6 were expressed at the highest, CYP1A1 at the lowest and CYP3A4 and CYP3A5 at intermediate expression levels. Membranes of all five tester bacteria demonstrated similar RED-expression levels, except for the two CYP3A-containing bacteria which demonstrated slightly increased RED-levels. CYP-activities were determined as ethoxyresorufin deethylase (CYP1A1 and CYP1A2), coumarin 7-hydroxylase (CYP2A6) and erythromycin N-demethylase (CYP3A4 and CYP3A5) activities. Reaction rates were comparable with those obtained previously for these CYP-enzymes, except for CYP3A5 which demonstrated a lower activity. Benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene demonstrated mutagenicity in the CYP1A1 expressing strain with mutagenic activities, respectively, approximately 10-fold and 100-fold higher as compared with those obtained with the use of rat liver S9 fraction. Aflatoxin B1 demonstrated a significant mutagenicity with all CYP expressing strains, albeit lower as compared to those obtained with the use of rat liver S9. CYP1A2 was approximately 3-fold more effective in generating a mutagenic response of AFB1 as compared to CYP3A4. CYP3A5 and CYP3A4 demonstrated comparable capacities in AFB1 bioactivation which was equal as found for CYP1A1. It is concluded that these four new strains contain stable CYP- and RED-expression, significant CYP-activities and demonstrated significant bioactivation activities with several diagnostic carcinogens.

Expression of human cytochrome P450 1A2 in Escherichia coli: a system for biotransformation and genotoxicity studies of chemical carcinogens.

In this study we describe the development of strain BMX100, a new Escherichia coli K12 tester strain, derived from MX100, a strain which was constructed for detection of mutagens and for mechanistic studies of chemical carcinogens. We demonstrate here that strain BMX100 can be used for stable expression of human CYP1A2 or human CYP1A2 fused to rat liver NADPH cytochrome P450 reductase. Mutagenicity of precarcinogens known to be bioactivated by CYP1A2, namely 2-aminoanthracene (2-AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), could be detected. The mutagenic activity of 2-AA using BMX100 expressing CYP1A2 alone and in combination with rat CYP reductase was respectively 10 and 20 times higher than in BMX100 with the standard metabolic activation system, rat liver S9 fraction. Furthermore, the mutagenicity of 2-AA could be nullified by alpha-naphthoflavone, a known inhibitor of CYP1A2. IQ responded equally in BMX100 expressing the CYP1A2-reductase fusion protein as compared with usage of rat liver S9 fraction. Rat liver S9 fraction was much more potent in generating a mutagenic response to AFB1 in BMX100 than in the strain expressing human CYP1A2 alone or CYP1A2 fused to rat reductase. The results described in this study demonstrate that this new E.coli strain can function as a human CYP1A2-competent prokaryotic mutagenicity test system and they seem to characterize BMX100 as a strain of interest for studies to identify individual human CYPs involved in bioactivation and bioinactivation reactions of putative genotoxins.

Characterization of enzyme activities and cofactors involved in bioactivation and bioinactivation of chemical carcinogens in the tester strains Escherichia coli K12 MX100 and Salmonella typhimurium LT2 TA100.

MX100 is an Escherichia coli K12 genotoxicity tester strain, especially developed for mechanistic studies of chemical mutagens and carcinogens. For the study of the role of specific enzymes in the bioactivation and bioinactivation of carcinogens, it is necessary to characterize MX100 as far as its metabolic bio(in)activation capacities are concerned. In this study such a characterization is performed in two types of cell-free lysates, one derived from stationary phase cells, grown in rich medium (SR-lysates) and one from exponentially growing cells (log phase), cultured in minimal medium (LM-lysates). Six Phase I enzyme activities of aromatic NADPH hydroxylase, NADH hydroxylase, flavin-containing monooxygenase (FMO), nitroreductase, DT-diaphorase and NADPH ferredoxin:oxidoreductase were determined. Activities of six Phase II enzymes glutathione S-transferases (GSTs), N-aryl acetyltransferase (NAT), arylamine sulphotransferase, UDP-glucuronyltransferase and epoxide hydratase and of the Phase III enzyme cysteine conjugate beta-lyase were subsequently assessed. In addition, five antioxidant enzymes: superoxide dismutase (SOD), catalase, glutathione (GSH)-reductase, GSH-peroxidase and alkyl hydroperoxide reductase; as well as concentrations of glutathione (GSH) and its disulphide (GSSG), were measured. The activity parameters of all enzymes were compared with those obtained in similar lysates of the Salmonella strain TA100 and in rat liver preparations. The results indicate that MX100 as well as TA100 contain relatively low oxidative but high reductase Phase I activities. Both strains demonstrated low activities for the Phase II conjugation enzymes except for GSTs. In MX100, relatively high activities were detected for all antioxidative enzymes, activities which were lower in TA100. Significant differences in activities were observed between the SR-lysates derived from stationary phase/rich medium and LM-lysates from log phase/minimal medium cells for nitroreductase, GST, SOD, catalase, NADPH ferredoxin:oxidoreductase as well as in GSH content. In general, we described for the first time a metabolic characterization of the E.coli tester strain MX100 and the Salmonella typhimurium strain TA100 and discussed the results in terms of its significance for carcinogen bioactivation and bioinactivation capacities.

MX100, a new Escherichia coli tester strain for use in genotoxicity studies.

The development of a new Escherichia coli tester strain for use in metabolic and mechanistic studies of genotoxins, strain MR2101/pKR11, has recently been reported. This strain, a derivative of the E. coli K12 laboratory strain AB1157, has sensitivity towards the detection of base-substitution mutagenesis, monitored by the reversion of arginine auxotrophy [argE3, (ochre)]. Besides arginine, MK2101/pKR11 is auxotrophic for histidine (hisG4), leucine (leuB6), proline (DeltaproA) and threonine (thr-1). MX100 was developed to overcome the auxotrophy for four amino acids of MR2101/pKR11 which are non-essential for the mutagenic responsiveness of the strain. We restored the biosynthesis for these four amino acids in MR2101/pKR11, resulting in strain MX100. This strain showed an almost 2-fold increase in mutagenic activity relative to MR2101/pKR11 with a set of diagnostic mutagens (aflatoxin B1, benzo[a]pyrene, 4-nitroquinoline-1-oxide, 2,7-dimethyl-benz[a]anthracene and others) and was further characterized with other types of mutagens in which it showed sensitivity towards the detection of oxidative (H2O2t-butyl-hydroperoxide, cumene-hydroperoxide, KO2) and carbonyl mutagens (methylglyoxal, malondialdehyde). As MX100 seems to have the right characteristics of a versatile genotoxicity tester strain and due to the extensive genetic and physiological knowledge of E. coli K12 in general and AB1157 in particular, we propose that MX100 could serve as mother strain for the development of specialized tester strains, of interest in studies of metabolism and/or mechanism of action of genotoxic carcinogens.

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Mechanisms of myricetin mutagenicity in V79 cells: involvement of radicalar species.

Myricetin is a flavonol that is widely distributed in edible plants and although it has been proved to be genotoxic in bacteria and to induce significant concentration-dependent nuclear DNA degradation concurrent with lipid peroxidation, very little is known about its mechanisms of genotoxicity. In this work we tried to evaluate the role of rat cytochromes P450 in the genotoxicity of myricetin and to study the role that radicalar species may have in its mutagenicity. The results obtained show that the genotoxicity of myricetin as assessed by the induction of chromosomal aberrations is not different in V79 cells lines genetically engineered for the expression of rat cytochromes P450 1A1, 1A2, and 2B1, compared to parental cell lines. We have also been able to show that reactive oxygen species resulting from the autooxidation of myricetin at pH values above neutrality have an important role in its mutagenicity. Therefore, under some conditions, myricetin can act as a prooxidant.

Mutagenic activity of glycine upon nitrosation in the presence of chloride and human gastric juice: a possible role in gastric carcinogenesis.

The mutagenic activity of glycine upon nitrosation was studied in the Ames tester strains TA98, TA100, TA102, and TA104. The results obtained show that glycine at acidic pH values and in the presence of Cl- can react with nitrite giving rise to genotoxic compounds to the tester strains used. When these experiments were carried out in the presence of gastric juice the genotoxicity observed was associated with the Cl- concentration in the different gastric juice samples. The nature and the mechanism of genetic lesion induced by the ultimate genotoxicant arising from the nitrosation of glycine are not fully understood. Primary amines (e.g., amino acids) have been described as potential alkylating agents after nitrosation. However, in our experimental conditions these alkylating activities were not detected, suggesting that other mechanisms could be involved in the genetic lesion induced by nitrosated glycine. The influence of Cl- in the genotoxic activity of glycine and other primary amines upon nitrosation and its possible involvement in the etiology of gastric cancer are discussed.

Mutagenicity of kaempferol in V79 cells: the role of cytochromes P450.

The flavonol kaempferol is widely found in the diet and is directly mutagenic in some short-term tests, such as the induction of chromosomal aberrations in eukaryotic cells. The presence of exogenous metabolizing systems enhances its mutagenicity. We have evaluated the role of cytochromes P450 in the induction of chromosomal aberrations by kaempferol in V79 cells. The results obtained suggest that there is a time-dependent biotransformation of kaempferol to quercetin, by cytochromes P450, as assessed by high pressure liquid chromatography. Quercetin seems to contribute to the mutagenicity of kaempferol in the presence of microsomal metabolizing systems. On the other hand, the direct induction of chromosomal aberrations by kaempferol does not seem to depend on the production of reactive oxygen species.

Structural requirements for mutagenicity of flavonoids upon nitrosation. A structure-activity study.

A wealth of promutagens can damage DNA provided metabolic/chemical reactions take place before an ultimate mutagen is formed. Nitrosation reactions are amongst those chemical reactions which may take place to render some chemical classes of promutagens as ultimate mutagens. Flavonoids are amongst chemicals which can be rendered mutagenic upon nitrosation. In this study, 22 flavonoids were tested in the Ames assay for their mutagenicity upon nitrosation and the respective structural requirements for nitrosation-dependent mutagenicity were established. Nitrosatable chemicals present in the diet may play a role in the aetiology of gastric cancer and flavonoids are amongst the common molecules present in a variety of food items. Flavonoids such as quercetin and catechin were predicted to be non-mutagenic upon nitrosation by the CASE methodology and were shown in this study to be strong nitrosatable mutagens.

Genotoxicity of quercetin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes.

Quercetin, a mutagenic flavonoid widely distributed in edible plants, was studied for the induction of micronuclei (MN). We have carried out the MN assay in bone marrow polychromatic erythrocytes in mice, in cytokinesis-blocked human lymphocytes and in cytokinesis-blocked V79 cells. MN assay in vitro was performed in the presence and in the absence of S9. To further extend the study, an antikinetochore antibody (CREST staining) was used to distinguish MN containing whole chromosomes (kinetochore positive) from those containing acentric fragments (kinetochore negative). When tested in vivo quercetin failed to induce micronuclei, a result which is in agreement with other published reports. When tested in vitro in V79 cells quercetin clearly induces micronuclei in the absence of S9 and also in the presence of S9 for the highest dose used. When tested in vitro in human lymphocytes quercetin shows a significant induction of micronuclei in the absence and in the presence of S9. The presence of S9 compared to its absence is not significant for any of the systems used. Both in the presence and absence of S9, quercetin appears to behave as a clastogenic agent in human lymphocytes inducing a significant majority of kinetochore-negative MN.

On the mechanisms of genotoxicity and metabolism of quercetin.

Quercetin has been the subject of numerous studies on its genetic toxicity and carcinogenicity. Despite its well-proven genetic damaging activity for various genetic end-points (reverse mutations, induction of SOS functions, induction of sister chromatid exchanges, chromosomal aberrations and micronuclei), the mechanisms of genetic damage by quercetin remain, by and large, unknown. The present study aims to further extend the observations on the possible active oxygen species mediated DNA-damaging activity of quercetin and the role of cytochrome P450-dependent metabolism on the genotoxicity of quercetin. The results reported in this work show that quercetin can produce the OH. radical, as assessed by deoxyribose degradation in the presence of Fe3+/EDTA (ethylenediaminetetraacetic acid), and that it induces strand breakage in isolated plasmidic DNA (pUC18). The data support the hypothesis that the production of OH. is mediated by H2O2. The results with genetically engineered V79 cells expressing rat cytochromes 1A1, 1A2 and 2B1 failed to demonstrate metabolism of quercetin, as indicated by the fact that neither an enhancement nor a decrease in the genotoxicity of quercetin was observed. Results obtained on the pH dependence of the induction of chromosomal aberrations by quercetin in V79 cells show that, as the pH value of the medium is increased to 8.0, there is a significant increase in the number of aberrant cells, as expected if oxygen radicals are responsible for the formation of chromosomal aberrations.