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.

Chemical features of flavonols affecting their genotoxicity. Potential implications in their use as therapeutical agents.

Flavonls are natural compounds present in edible plants and possess several biological activities that can be useful in drug design. Conversely some of these compounds have been shown to be genotoxic to prokaryotic and eukaryotic cells. In this study we tried to establish the chemical features responsible for the genotoxicity of flavonols and to study the conditions that can modulate their genotoxicity namely pH, the presence of antioxidants and metabolism. We assessed the induction of revertants in Salmonella typhimurium TA98 and the induction of Chromosomal aberrations in V79 cells by eight different flavonols and one catechin in the presence and in the absence of metabolizing systems. We have also studied the generation of hydroxyl radical by these flavonoids using the deoxyribose degradation assay. The results obtained in this study suggest that flavonols having a free hydroxyl group at position 3 of the C ring, a free hydroxyl group at position 7 of the A ring and a B ring with a catechol or pyrogallol structure, or a structure that after metabolic activation is transformed into a catechol or a pyrogallol, are flavonols whose genotoxicity in eukaryotic cells depends on their autooxidation. These flavonols can autooxidize when the pH value is slightly alkaline, such as in the intestine, and therefore can induce genotoxicity in humans. Given the above mentioned considerations it is necessary to clarify the mechanisms and the conditions that mediate the biological effects of flavonols before considering them as therapeutical agents.

Activation of promutagens by porphyrinic biomimetic systems.

Biomimetic oxidative systems using tetraarylporphyrins, which can bind various metals, and exogenous oxygen donors have been extensively studied as models of the natural heme prosthetic group. Those systems were shown to catalyze oxidations in a manner consistent with cytochromes P-450 and usefully contributed to an understanding of the mechanisms of the cytochromes P-450-dependent reactions when using oxygen donors. The usage of those systems in mutagenicity studies showed that some promutagens could be activated to proximate mutagens. In the present work we report on the activation of benzo[a]pyrene, 3-methylcholanthrene, 7,12-dimethylbenz[a]anthracene; 2-aminofluorene, 2-acetylaminofluorene and the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to Ames assay mutagens using tetraphenylporphinatoiron(III) chloride and various oxygen donors, namely iodosylbenzene, cumene hydroperoxide, tert-butylhydroperoxide and H2O2. Our results demonstrate that IQ could be activated using any of the oxygen donors. However, a pattern of specificity for the oxygen donor could be identified. Polycyclic aromatic hydrocarbons displayed higher levels of mutagenicity with iodosylbenzene, whereas aromatic amines were preferentially activated when tert-butylhydroperoxide was used. For the heterocyclic amine IQ the higher responses were obtained using cumene hydroperoxide. The putative non-carcinogen pyrene and the controversial carcinogen quercetin were not activated irrespective of the oxygen donor used.

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.

Isolation and prevalidation of an Escherichia coli tester strain for the use in mechanistic and metabolic studies of genotoxins.

We have isolated an Escherichia coli tester strain for the use in mechanistic and metabolic studies of genotoxins. We started with one of the more used and better characterized E. coli K-12 laboratory strains, AB1157. We isolated a lipopolysaccharide defective mutant of strain AB1886 which is an excision repair deficient derivative of AB1157 and introduced a newly constructed plasmid pKR11, encoding mucAB, resulting in strain MR2101/pKR11. A genotoxicity assay was designed, monitoring the reversion to arginine prototrophy and a preliminary validation was carried out against Ames tester strain TA100 with a set of diagnostic compounds. The results seem to indicate that strain MR2101/pKR11 is an adequate tester strain which can be a useful tool in mechanistic studies. Moreover, this strain can serve as mother strain to isolate improved and more specialized tester strains.

Oxygen species and the genotoxicity of quercetin.

Quercetin has been extensively studied in various short-term assays for genotoxicity. The patterns of genotoxicity of quercetin for different genetic endpoints are subject to a variety of factors (pH, antioxidants, metabolism) whose precise role in each test remains unclear. In the present study we report on the possible effect of oxygen-derived species on the activity of quercetin in the Ames assay and in the SOS chromotest. Our results seem to suggest that superoxide dismutase (SOD) does not account for the levels of mutagenicity detected in the presence of S9 or S100. The latter may, however, contain other factors of antioxidant defense which may prevent the oxidative degradation of quercetin. Since this degradation occurs at pH values above neutrality and the SOS-inducing activity is higher at pH 6.0, it is concluded that the response of quercetin in the SOS chromotest is due to quercetin itself at acidic pH. The SOS-inducing activity at pH 7.4 is enhanced by SOD, but it cannot be unambiguously concluded that this effect in the SOS chromotest might only be due to protection against the oxidative degradation of quercetin.

Metabolism of galangin by rat cytochromes P450: relevance to the genotoxicity of galangin.

The mutagenicity of flavonols seems to depend on the number and position of hydroxyl groups in the B ring. Galangin is a flavonol that does not have any hydroxyl group in the B ring and has been suggested to be a substrate of cytochromes P450 which, through the hydroxylation of the B ring, could metabolise it to more genotoxic products. The present study was undertaken to test this hypothesis. Using high performance liquid chromatography we show that glangin is sequentially transformed to kaempferol and then to quercetin by a mechanism dependent on cytochrome P450 reactions. The metabolites of galangin are responsible for its mutagenicity in Salmonella typhimurium reversion assay and for the induction of chromosomal aberrations in V79 cells.

Genotoxicity of instant coffee: possible involvement of phenolic compounds.

Instant coffee exhibits direct genotoxic activity in the tester strains TA 98, 100, 102, 104 and YG 1024. In the Ames tester strain TA 100, the presence of S9 mix, S100 mix, S9 mix without cofactors led to a significant decrease of the genotoxicity observed. The decrease observed in the presence of S9 mix seems to be highly correlated with the catalase content of S9 mix. The genotoxicity of instant coffee detected in strain TA 100 was dependent on the pH, with higher genotoxic effects at pH values above neutrality. Also, dependent on the pH was the ability of some phenolic molecules present in coffee promoting the degradation of deoxyribose in the presence of Fe3+/EDTA. These results suggest that apart from other molecules present in instant coffee responsible for their genotoxicity in several short term assays, phenolic molecules could also be implicated in the genotoxicity of coffee, via reactive oxygen species arising from its auto-oxidation.

Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: comparison of three types of expression systems.

Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and the highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.

Genotoxicity assessment of aromatic amines and amides in genetically engineered V79 cells.

A genetically engineered V79 cell line expressing rat CYP1A2 and another cell line expressing rat CYP1A2 as well as endogenous acetyltransferase activity, as well as CYP-deficient parental V79 cell lines, were used to assess the genotoxicity of the aromatic amines and amides 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, 4-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline, with chromosomal aberrations and sister chromatid exchanges as the end-points. None of the test compounds showed a clear effect on the frequency of chromosomal aberrations in any cell line used. Sister chromatid exchanges, however, were induced by 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene in the CYP1A2-proficient cells, but not in the CYP1A2-deficient cells. The presence of acetyltransferase activity enhanced the effect of 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene. 4-Acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline did not induce sister chromatid exchanges in the investigated cell lines. The use of cell lines with defined metabolic capabilities seems to be a valuable tool to study specific metabolic pathways important in the activation of procarcinogens.

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.

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.

Mutagenicity of rutin and the glycosidic activity of cultured cell-free microbial preparations of human faeces and saliva.

Genotoxic testing of flavonol glycosides, which account for most of the human intake of flavonoids, is dependent on the use of enzymatic extracts that exhibit beta-glycosidic activity. This study was aimed at characterizing further the beta-glycosidic activity of cultured cell-free microbial extracts from human faeces (faecalase) and saliva (salivase). Using o-nitrophenyl-beta-D-galactoside as substrate, the optimum pH and apparent Km and energy of activation were shown to be 7.6, 3.5 x 10(-4) M and 8.65 kcal/mol, respectively, for faecalase, and 7.4, 8.7 x 10(-5) M and 3.8 kcal/mol, respectively, for salivase. Rutin (quercetin-3-O-rutinoside) was shown to be a competitive inhibitor for faecalase, whereas no inhibitory activity could be found for salivase. Enzymatic hydrolysis of rutin gave the mutagenic product quercetin that was detected in the Ames assay and using high-performance liquid chromatography.

Genotoxicity of nitrosated red wine and of the nitrosatable phenolic compounds present in wine: tyramine, quercetin and malvidine-3-glucoside.

Phenolic compounds and biogenic amines are known to be present in some foodstuffs which become directly genotoxic after nitrosation in vitro. Red wine has previously been shown to be genotoxic and this activity has been attributed mainly to flavonoids. Besides flavonoids, red wine contains a multiplicity of compounds, including biogenic amines. Using the Ames assay and the SOS chromotest, this study has shown that red wine and some of the nitrosatable molecules present in wine become directly genotoxic on nitrosation in vitro: these include the phenolic molecules tyramine, quercetin and malvidine-3-glucoside, whereas phenylethylamine and histamine were negative on nitrosation. Interestingly, quercetin had been predicted to be negative after nitrosation, using the CASE methodology. The concentrations of these three positive nitrosatable compounds in wine were determined by HPLC. Comparison of these concentrations and their respective levels of genotoxicity suggests that the genotoxicity after nitrosation is probably attributable to other molecules. It is also possible that synergistic effects may occur between various nitrosatable compounds in wine.

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.

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.

Mutagenic activity in the wine-making process: correlations with rutin and quercetin levels.

Mutagenic activity was monitored during the time course of the vinification process of a Portuguese red wine using the Ames assay. Dependence upon faecalase treatment for detection of mutagenicity was evaluated concurrently with HPLC analysis of the flavonoids rutin and quercetin. Rutin (quercetin-o-3-rutinoside) is liberated from the grapes during the first 10 days of the process and is hydrolysed as the vinification process proceeds. This explains the levels of the free quercetin in the wine and mutagenicity in the absence of faecalase treatment. Mutagenicity in the presence of faecalase during the first 26 days of the process correlates with the levels of rutin, and in the absence of faecalase the highest mutagenic activity detected (160th day) coincides with the highest level of free quercetin. Our results do not rule out the possibility that other mutagens are present in wines, in particular oxidative-type mutagens.

Involvement of rat cytochrome 1A1 in the biotransformation of kaempferol to quercetin: relevance to the genotoxicity of kaempferol.

Kaempferol is a flavonoid widely distributed in edible plants and has been shown to be genotoxic to V79 cells in the absence of external metabolizing systems. The presence of an external metabolizing system, such as rat liver homogenates (S9 mix), leads to an increase in its genotoxicity, which is attributed to its biotransformation to the more genotoxic flavonoid quercetin, via the cytochrome P450 (CYP) mono-oxygenase system. In the present work we investigated the mechanisms of the genotoxicity of kaempferol further. Special attention has been given to the role of CYP in the genotoxicity of this flavonoid. We studied the induction of mutations in Salmonella typhimurium TA98 in the presence and in the absence of S9 mix and the induction of chromosomal aberrations (CAs) and micronuclei (MN) by kaempferol in V79 cells in the presence and in the absence of S9 mix. To evaluate the role of different CYP in the biotransformation of kaempferol we studied the induction of CAs and MN in V79 cells genetically engineered for the expression of rat CYP 1A1, 1A2 and 2B1. In addition we performed CYP inhibition studies using the above-mentioned indicators and high performance liquid chromatography (HPLC) analysis. The results obtained in this work suggest that rat CYP 1A1 is, among the cytochromes studied, the one that plays the major role in the transformation of kaempferol into quercetin. The relevance of these findings to the human situation is 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.