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Antimutagenic dietary phenolics as antigenotoxic substances in urothelium of smokers
Mutation Research 402 Ž1998. 219–224
Antimutagenic dietary phenolics as antigenotoxic substances in urothelium of smokers 1 Christian Malaveille
a,)
, Agnes ` Hautefeuille a, Brigitte Pignatelli a, Glenn Talaska b, Paolo Vineis c , Helmut Bartsch d
a
International Agency for Research on Cancer, 150 Cours Albert-Thomas, 69372 Lyon Cedex 08, France Institute for EnÕironmental Health (ML 056), the UniÕersity of Cincinnati, Cincinnati, OH 45267, USA Unit of Cancer Epidemiology, Department of Biomedical Sciences and Human Oncology, UniÕersity and Main Hospital, 10126 Turin, Italy d German Cancer Research Center, D 69120 Heidelberg, Germany b
c
Received 20 May 1997; revised 22 July 1997; accepted 30 July 1997
Abstract Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of PhIP-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified ABP-dG adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. These substances appear to be dietary phenolics andror their metabolites because Ži. the anti-mutagenic activity of urine extracts Ž n s 18. was linearly related to their content in phenolics; Žii. the concentration ranges of these substances in urine extracts were similar to those of various plant phenols Že.g., quercetin, isorhamnetin. for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; Žiii. treatment of urines with b-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; Živ. urinary extracts inhibited non-competitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Overall, our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Dietary phenolic; Antigenotoxicity; Smoker; Urothelial cell; DNA-adduct
Abbreviations: 4-ABP, 4-Aminobiphenyl; ABP-dG, N-Ždeoxyguanosine-8-yl.-4-aminobiphenyl; DMSO, Dimethylsulfoxide; HPLC, High-performance liquid chromatography; MeIQx, 2-Amino-3,8-dimethylimidazow4,5-f xquinoxaline; PhIP, 2-Amino-1-methyl-6-phenylimidazow4,5-b xpyridine ) Corresponding author. Tel.: q33-4-72-73-84-85; fax: q33-4-72-73-85-75; e-mail: [email protected]. 1 Full account of this study has been published. 0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 3 0 0 - X
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C. MalaÕeille et al.r Mutation Research 402 (1998) 219–224
1. Introduction Tobacco smoking causes a major fraction of urinary bladder cancer in humans w1x. Previous studies have implicated primary aromatic and possibly heterocyclic amines as bladder carcinogens which are present in tobacco smoke w2x. We have shown human urine from both smokers and non-smokers to contain substances that strongly inhibit the bacterial mutagenicity of aromatic and heterocyclic amines in vitro w3x. The current report presents data on the characterization of the chemical nature and mode of action of these urinary anti-mutagens. The biological relevance was assessed by examining the relationship between the levels of tobacco-related DNA adducts in exfoliated urothelial cells and the anti-mutagenic activity in the 24-h urine of smoking volunteers. Based on the results, the role of various naturally anti-mutagens occurring in human diet is discussed.
2. Materials and methods 2.1. Preparation of urine extracts Twenty-four hour urine samples from healthy male blood donors were collected in polyethylene bottles which were stored at 48C during the collection period. Part of each sample was used to prepare exfoliated cells as previously described w4x. The remaining urine samples were stored for up to 4 yr at y308C. Nicotine, cotinine and creatinine in urine samples were determined as described w5x. The urine extracts were prepared using Bond Elut cartridges containing 500 mg C-18 sorbent ŽVarian, Harbor City, CA, USA. according to a procedure established in our laboratory w6x. They were dissolved in 500 m l of DMSO and stored at y708C for less than 1 month. 2.1.1. DNA isolation and measurement of tobacco-related carcinogen-DNA adducts DNA was extracted from exfoliated urothelial cells, hydrolysed, 32 P-postlabelled and carcinogenDNA adduct levels measured as described w4x. 2.2. Salmonella mutagenicity assay Salmonella typhimurium TA98 strain was supplied by B.N. Ames ŽBerkeley, CA.. Using a modi-
fied liquid incubation assay as described by Malaveille et al. w5x. The inhibitory effect of each urine extractrphenolicsrHPLC fractionrBond Elut C-18 extract of wine and aqueous food extracts on PhIP mutagenicity was measured in assays containing 20 ng of PhIP Žadded as 10 m l 25% DMSO aqueous solution. and up to 10 m l DMSO solution of anti-mutagens Ž5.7% final DMSO concentration in each assay.. The inhibitory effects were expressed as either percent decrease in revertants per plate per concentrationrfraction assayed or as decrease in revertants per milliliter urine equivalent calculated from the linear part of inhibitory curves. 2.3. Enzym atic deconjugation glucuronides and sulfates
of
urinary
An aliquot of 24-h urine containing 3 mg creatinine Ž1.9–3.3 ml depending on the sample. was mixed with 1 ml 0.5 M Sorensen citrate buffer, pH 6, with or without up to 40 000 Roy units of arylsulfatase and 5000 Fishman units of b-glucuronidase. The final volume was adjusted to 6 ml with deionized water. The mixture was flushed for 30 s with nitrogen, incubated for 1 h at 378C and extracted using a Bond Elut C-18 cartridge as described above for urine; the residue was taken up in 50 m l DMSO and stored at y708C for less than 1 week. 2.3.1. Spectrophotometric analysis of phenolics in urinary and food r beÕerage extracts The amount of phenolics in DMSO solutions of extract or concentrate of urine, wine, HPLC fractions and aqueous food extracts, was measured by analyzing the total phenol concentration by the Folin– Ciocalteu procedure of Singleton and Rossi w7x using gallic acid for the calibration curve. 2.4. Preparation of extracts of aqueous food extracts or wine A total of 50 g of apple were homogenized at room temperature with 20 ml, 50 g of lettuce with 40 ml and 50 g of onion with 50 ml deionized water using a Waring blender. The homogenates were centrifuged for 10 min at 2000 = g; 5 ml of each supernatant were applied on a Bond Elut C-18 cartridge as described for the preparation of urine ex-
C. MalaÕeille et al.r Mutation Research 402 (1998) 219–224
tract. In the case of wine, 5 ml of 10- or 50-fold diluted aqueous solution were applied. The dry extracts of diluted wine, aqueous extract of apple and lettuce were dissolved in up to 100 m l DMSO for the analysis of phenolics and anti-mutagenicity or in acetonitrilerH 2 O Ž50r50. for fractionation by HPLC. 2.5. HPLC separation of urine and food extracts The residues from Bond-Elut C-18 cartridge extractions Žsee above. were dissolved in 100 m l acetonitrilerH 2 O Ž50r50. and separated by HPLC on a C-18 LC column ŽSupercosil, 3 m m; L: 15 cm; ID: 4.6 mm.. Solvent A was 10% acetic acid in H 2 O; solvent B was acetonitrile. The solvents were programmed from an initial concentration of 0% B to 100% B at 60 min and held isocratically until 72 min. The following gradient was applied: 0–12 min 7% B; 12–50 min from 7 to 30% B; 50–60 min from 30 to 100% B. Flow rate was 0.5 mlrmin. The separation was monitored by UV absorption at 254 mm. After injection, six fractions Ž6 ml each. were collected. They were reduced to dryness by vacuum evaporation at room temperature and redissolved in 50 m l DMSO for analysis of anti-mutagenicity and phenolics.
3. Results and discussion To investigate whether urinary anti-mutagens can reduce DNA adduction in the urinary bladder of smokers, arising from exposure to aromatic Žand possibly heterocyclic. amines, we compared urinary anti-mutagenicity measured in 24-h urine samples of 10 smoking volunteers with the level of DNA adducts in exfoliated cells isolated from the same urine. The mean relative adduct labelling ŽRAL. of DNA from these urothelial cell preparations and the association with tobacco smoking have been reported previously w4,8x. Three adducts were related to cigarette smoking, one being the putative N-Ždeoxyguanosine-8yl.-4-aminobiphenyl ŽABP-dG. adduct, and thus were considered for our investigations. Their levels were expressed per the sum of nicotine plus cotinine contents in 24-h urine to adjust for tobacco smoking. Both ABP-dG and total DNA adducts were found to
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be inversely related to bacterial anti-mutagenicity, expressed as the decrease in the number of revertants of S. typhimurium TA98 per ml urine equivalent. The latter was measured in liquid incubation assays using PhIP as a representative mutagen for tobacco smoke-related heterocyclic amines. The logarithms of DNA adduct level vs. anti-mutagenicity were linearly related showing statistical significance Ž r s y0.81, p - 0.01 for both adduct measurements. ŽFig. 1.. Since extracts were prepared from urines of subjects who were also coffee drinkers, we first suspected caffeine and paraxanthine Ža demethylated caffeine metabolite. as inhibitors. Both are known to be substrates for cytochrome P450 IA2-mediated reactions, as are aromatic and heterocyclic amines w9,10x. Caffeine has been reported to inhibit the mutagenicity of various heterocyclic amines in Salmonella w11–13x. However, analysis of urine extracts by HPLC revealed that the concentrations of these methylxanthines in urinary extracts were far too low to account for their inhibitory effect on PhIP mutagenicity, as reported previously w3x. An inhibitory effect of unsaturated fatty acids on the mutagenicity of heterocyclic amines and other carcinogens has been reported w14x and their presence in human urine is well documented w15x. We therefore measured the concentration of the most abundant, linoleic and oleic acid, in urine extracts by gas chromatography wafter methylation, as described by Hayatsu et al. w16xx. The results indicated again that the concentrations of these fatty acids were too low to account for the observed urinary anti-mutagenicity. We then focused on phenolics because Ži. human dietary intake of flavonoids is up to 1 grday w17,18x, Žii. they are partly excreted in urine w19,20x and Žiii. some had been shown to inhibit the mutagenicity of aromatic and heterocyclic amines w11,21–23x. The amount of phenolics was measured in 19 human urine extracts using a spectrophotometric assay w7x. The concentrations were then compared to the urinary anti-mutagenicity, using PhIP as mutagen in S. typhimurium TA98. All the urine extracts contained phenolics, at concentrations of 3.7–12.5 m g per 10 m l DMSO solution of extracts and had anti-mutagenic activities varying by a factor of about 10. A statistically significant linear relationship was found
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C. MalaÕeille et al.r Mutation Research 402 (1998) 219–224
Fig. 1. A, B: Levels of 4-ABP adducts and of tobacco-related adducts of DNA from exfoliated urothelial cells of smokers Žadjusted for tobacco exposure. vs. the anti-mutagenic activity of the corresponding urine extract, using PhIP as representative heterocyclic amine. Relative adduct labellings of DNA were taken from Talaska et al. w8x. The anti-mutagenic activities were calculated from the linear part Žcomprising 2–4 concentrations. of the curve of the inhibition of the liver S9-mediated mutagenicity in S. typhimurium TA98 of PhIP Ž20 ng per assay. as a function of increasing concentration of the urinary extract ŽDMSO solution.. Inhibitory curves were plotted using results from one or two series of duplicate experiments each involving four concentrations. The mutagenicity of 20 ng PhIP after 45 min liquid incubation in the absence of inhibitor ranged from 665 to 880 revertants per plate for nine series of experiments, each involving 2–3-duplicate determinations, the mean value " S.E. was 770 " 30. ŽReproduced with the permission of Oxford University Press..
between the anti-mutagenicity of urine extracts and their content in phenolic substances Ž r s 0.58, p 0.02, n s 18.. In order to examine further the role of plant phenolics as urinary anti-mutagens, we assayed a series of representative flavonoids and other plant phenols to determine whether their inhibitory concentrations match those of the phenolics in urinary extracts. At non-cytotoxic concentrations ranging from 0.2 to 6 m g per assay, which are closed to those of phenolics in assays with urinary extracts, the highest inhibitory effects Žup to 100% inhibition of the liver S9 mediated mutagenicity in S. typhimurium TA98 of PhIP and MeIQx. were obtained with flavonols Žquercetin, isorhamnetin, kaempherol., flavones Ž luteolin, diosm etin . , flavanones Žnaringenin, hesperitin. and one anthocyanidin Žcyanidin-chloride.. All the other substances tested wtwo isoflavones Žgenistein, daidzein., two phenolic acids Žgallic acid, caffeic acid. and one flavanol Ž d-catechin.x had no inhibitory activity or very little. Within a particular class of inhibitory substances, the glycosides Ži.e., O-rhamnoglucoside. were relatively
inactive compared with their aglycone moiety, as shown for quercetin vs. quercitrin and naringenin vs. naringin. Dietary phenolics are partly excreted in urine as glucuronides and sulfates w19,24x. Two urine samples were therefore treated with b-glucuronidase and arylsulfatase before the preparation of extracts. After enzymatic treatment, anti-mutagenicity increased maximally 11-fold and the level of phenolics 2.5-fold. Based on combined results from two extracts, the increase in anti-mutagenicity was linearly related to the concentration of unconjugated phenolics. These data strongly suggest that these free phenolic substances act as inhibitors of liver S9-mediated mutagenicity of PhIP. To gain further evidence for the role of phenolics as urinary anti-mutagens, the mode of inhibition of liver S9-mediated mutagenicity of PhIP was determined for three urinary extracts and compared with that of quercetin. As inferred from double reciprocal plots, both the extracts and the pure flavonoid, quercetin, inhibited the mutagenicity of PhIP in a non-competitive fashion.
C. MalaÕeille et al.r Mutation Research 402 (1998) 219–224
Altogether, the numerous pieces of experimental evidence which were presented above, suggest that much of the urinary anti-mutagenicity is attributable to dietary phenolics and their metabolites. In order to identify the origin of anti-mutagenic substances in urinary extracts, chromatographic separations were performed using conditions reported to be efficient for separation of flavonoids w25,26x. When two urinary extracts were subjected to reverse-phase HPLC, the UV absorption profiles revealed a complex elution pattern. Subsequently, six fractions, covering the entire chromatographic profile, were analysed. All fractions contained substances, in 10 m l DMSO solution ŽSection 2.5., that inhibited liver S9 mutagenicity of PhIP and phenolics at concentrations ranging from 0.3–46 m gr10 m l DMSO solution. The most lipophilic substances displayed the highest inhibitory activity when expressed per microgram of phenolics, i.e., 50% inhibition per 0.2 m g phenolics, which corresponds to the highest antimutagenic effect obtained with the flavonoids assayed w6x. Onion, lettuce, apple and red wine are important dietary sources of flavonoids w17x and these substances are probably responsible for the anti-mutagenicity observed in their water extracts w6x. These extracts were also subjected to reverse phase HPLC as carried out for urinary extracts. The distribution of anti-mutagenic activity in the HPLC fractions of urinary extracts corresponds roughly to a combination of inhibitors present in onion and wine extracts, suggesting that these phenolics and their metabolites comprise a major part of the complex mixture of urinary anti-mutagens. Human diet contains a wide variety of non-nutrient agents, mostly phenolics of plant origin, which inhibit carcinogenesis in animals w27–30x. Epidemiological observations have confirmed a protective effect of vegetable and fruits against the occurrence of many epithelial cancers w31–33x. Phenolics in the human diet could affect the pharmacokinetics of carcinogens by Ži. altering the rates of their absorption and uptake, Žii. reacting with or tightly binding to toxic substances and metabolites and Žiii. modifying cytochrome P450-dependent metabolism and phase II enzymes w14,28,29,34x. The inverse relationship between urinary anti-mutagenicity and level of tobacco-related DNA adducts in the urothelium, reported here for the first time, strongly suggests that
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bladder mucosal cells of smokers are partially protected by dietary phenolics, most probably flavonoids, against the harmful effects of tobacco-derived bladder carcinogens. Studies are warranted to explore further these protective effects of phenolics from vegetables and fruits against cancers in humans as a part of a cancer chemoprevention strategy.
Acknowledgements The authors would like to thank Mrs. P. Collard and Mrs. M. Lezere ´ ` for secretarial help, Dr. J. Cheney for editorial assistance and Dr. J. Esteve ` for helpful discussions. Part of the work was supported by NIEHS 5-P30-ES06096.
References w1x IARC, Tobacco smoking, Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 38, IARC, Lyon, 1986. w2x H. Bartsch, C. Malaveille, M. Friesen, F.F. Kadlubar, P. Vineis, Black Žair-cured. and blond Žflue-cured. tobacco cancer risk: IV. Molecular dosimetry studies implicate aromatic amines as bladder carcinogens, Eur. J. Cancer 29A Ž1993. 1199–1207. w3x C. Malaveille, A. Hautefeuille, G. Brun, P. Vineis, H. Bartsch, Substances in human urine that strongly inhibit bacterial mutagenicity of 2-amino-1-methyl-6-phenylimidazow4,5bxpyridine ŽPhIP. and related heterocyclic amines, Carcinogenesis 13 Ž1992. 2317–2320. w4x G. Talaska, M. Schamer, P. Skipper, S. Tannenbaum, N. Caporaso, L. Unruh, F.F. Kadlubar, H. Bartsch, C. Malaveille, P. Vineis, Detection of carcinogen-DNA adducts in exfoliated urothelial cells of cigarette smokers: association with smoking, hemoglobin adducts, and urinary mutagenicity, Cancer Epidemiol. Biomarkers Prev. 1 Ž1991. 61–66. w5x C. Malaveille, P. Vineis, J. Esteve, H. Ohshima, G. Brun, A. Hautefeuille, P. Gallet, G. Ronco, B. Terracini, H. Bartsch, Levels of mutagens in the urine of smokers of black and blond tobacco correlate with their risk of bladder cancer, Carcinogenesis 10 Ž1989. 577–586. w6x C. Malaveille, A. Hautefeuille, B. Pignatelli, G. Talaska, P. Vineis, H. Bartsch, Dietary phenolics as anti-mutagens and inhibitors of tobacco-related DNA adduction in the urothelium of smokers, Carcinogenesis 17 Ž1996. 2193–2200. w7x S.L. Singleton, J.A. Rossi, Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents, Am. J. Enol. Vitic. 16 Ž1965. 144–188. w8x G. Talaska, M. Schamer, P. Skipper, S. Tannenbaum, N. Caporaso, F. Kadlubar, H. Bartsch, P. Vineis, Carcinogen-
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w20x J.B. Harborne, The Flavonoids: Advance in Research Since 1981, Chapman & Hall, London, 1988. w21x A.J. Alldrick, J. Flynn, I.R. Rowland, Effects of plant-derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food, Mutat. Res. 163 Ž1986. 225– 232. w22x J.H. Weisburger, Practical approaches to chemoprevention of cancer, Drug Metab. Rev. 26 Ž1994. 253–260. w23x R. Edenharder, I. von Petersdorff, R. Rauscher, Antimutagenic effects of flavonoids, chalcones and structurally related compounds on the activity of 2-amino-3-methylimidazow4,5fxquinoline ŽIQ. and other heterocyclic amine mutagens from cooked food, Mutat. Res. 287 Ž1993. 261–274. w24x J.W. Lampe, M.C. Martini, M.S. Kurzer, H. Adlercreutz, J.L. Slavin, Urinary lignan and isoflavonoid excretion in premenopausal women consuming flaxseed powder, Am. J. Clin. Nutr. 60 Ž1994. 122–128. w25x K. Van de Casteele, H. Geiger, C.F. Van Summere, Separation of flavonoids by reverse-phase high-performance liquid chromatography, J. Chromatogr. 240 Ž1982. 81–94. w26x J. Gaspar, A. Laires, M. Monteiro, O. Laureano, E. Ramos, J. Rueff, Quercetin and the mutagenicity of wines, Mutagenesis 8 Ž1993. 51–55. w27x L. Wattenberg, Inhibition of carcinogenesis by minor dietary constituents, Cancer Res. 52 Ž1992. 2085s–2091s. w28x A.E. Rogers, S.H. Zeisel, J. Groopman, Diet and carcinogenesis, Carcinogenesis 14 Ž1993. 2205–2217. w29x C.S. Yang, T.J. Smith, J.Y. Hong, Cytochrome P-450 enzymes as targets for chemoprevention against chemical carcinogenesis and toxicity: opportunities and limitations, Cancer Res. 54 Ž1994. 1982s–1986s. w30x F.P. Guengerich, Influence of nutrients and other dietary materials on cytochrome P-450 enzymes, Am. J. Clin. Nutr. 61 Ž1995. 651S–658S. w31x K.A. Steinmetz, J.D. Potter, Vegetables, fruit, and cancer: I. Epidemiology, Cancer Causes Control 2 Ž1991. 325–357. w32x G. Block, B. Patterson, A. Subar, Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence, Nutr. Cancer 18 Ž1992. 1–29. w33x A. Tavani, C. La Vecchia, Fruit and vegetable consumption and cancer risk in a Mediterranean population, Am. J. Clin. Nutr. 61 Ž1995. 1374S–1377S, Suppl. w34x M.H. Siess, J. Leclerc, M.C. Canivenc Lavier, P. Rat, M. Suschetet, Heterogenous effects of natural flavonoids on monooxygenase activities in human and rat liver microsomes, Toxicol. Appl. Pharmacol. 130 Ž1995. 73–78.
Antimutagenic dietary phenolics as antigenotoxic substances in urothelium of smokers 1 Christian Malaveille
a,)
, Agnes ` Hautefeuille a, Brigitte Pignatelli a, Glenn Talaska b, Paolo Vineis c , Helmut Bartsch d
a
International Agency for Research on Cancer, 150 Cours Albert-Thomas, 69372 Lyon Cedex 08, France Institute for EnÕironmental Health (ML 056), the UniÕersity of Cincinnati, Cincinnati, OH 45267, USA Unit of Cancer Epidemiology, Department of Biomedical Sciences and Human Oncology, UniÕersity and Main Hospital, 10126 Turin, Italy d German Cancer Research Center, D 69120 Heidelberg, Germany b
c
Received 20 May 1997; revised 22 July 1997; accepted 30 July 1997
Abstract Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of PhIP-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified ABP-dG adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. These substances appear to be dietary phenolics andror their metabolites because Ži. the anti-mutagenic activity of urine extracts Ž n s 18. was linearly related to their content in phenolics; Žii. the concentration ranges of these substances in urine extracts were similar to those of various plant phenols Že.g., quercetin, isorhamnetin. for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; Žiii. treatment of urines with b-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; Živ. urinary extracts inhibited non-competitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Overall, our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Dietary phenolic; Antigenotoxicity; Smoker; Urothelial cell; DNA-adduct
Abbreviations: 4-ABP, 4-Aminobiphenyl; ABP-dG, N-Ždeoxyguanosine-8-yl.-4-aminobiphenyl; DMSO, Dimethylsulfoxide; HPLC, High-performance liquid chromatography; MeIQx, 2-Amino-3,8-dimethylimidazow4,5-f xquinoxaline; PhIP, 2-Amino-1-methyl-6-phenylimidazow4,5-b xpyridine ) Corresponding author. Tel.: q33-4-72-73-84-85; fax: q33-4-72-73-85-75; e-mail: [email protected]. 1 Full account of this study has been published. 0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 3 0 0 - X
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C. MalaÕeille et al.r Mutation Research 402 (1998) 219–224
1. Introduction Tobacco smoking causes a major fraction of urinary bladder cancer in humans w1x. Previous studies have implicated primary aromatic and possibly heterocyclic amines as bladder carcinogens which are present in tobacco smoke w2x. We have shown human urine from both smokers and non-smokers to contain substances that strongly inhibit the bacterial mutagenicity of aromatic and heterocyclic amines in vitro w3x. The current report presents data on the characterization of the chemical nature and mode of action of these urinary anti-mutagens. The biological relevance was assessed by examining the relationship between the levels of tobacco-related DNA adducts in exfoliated urothelial cells and the anti-mutagenic activity in the 24-h urine of smoking volunteers. Based on the results, the role of various naturally anti-mutagens occurring in human diet is discussed.
2. Materials and methods 2.1. Preparation of urine extracts Twenty-four hour urine samples from healthy male blood donors were collected in polyethylene bottles which were stored at 48C during the collection period. Part of each sample was used to prepare exfoliated cells as previously described w4x. The remaining urine samples were stored for up to 4 yr at y308C. Nicotine, cotinine and creatinine in urine samples were determined as described w5x. The urine extracts were prepared using Bond Elut cartridges containing 500 mg C-18 sorbent ŽVarian, Harbor City, CA, USA. according to a procedure established in our laboratory w6x. They were dissolved in 500 m l of DMSO and stored at y708C for less than 1 month. 2.1.1. DNA isolation and measurement of tobacco-related carcinogen-DNA adducts DNA was extracted from exfoliated urothelial cells, hydrolysed, 32 P-postlabelled and carcinogenDNA adduct levels measured as described w4x. 2.2. Salmonella mutagenicity assay Salmonella typhimurium TA98 strain was supplied by B.N. Ames ŽBerkeley, CA.. Using a modi-
fied liquid incubation assay as described by Malaveille et al. w5x. The inhibitory effect of each urine extractrphenolicsrHPLC fractionrBond Elut C-18 extract of wine and aqueous food extracts on PhIP mutagenicity was measured in assays containing 20 ng of PhIP Žadded as 10 m l 25% DMSO aqueous solution. and up to 10 m l DMSO solution of anti-mutagens Ž5.7% final DMSO concentration in each assay.. The inhibitory effects were expressed as either percent decrease in revertants per plate per concentrationrfraction assayed or as decrease in revertants per milliliter urine equivalent calculated from the linear part of inhibitory curves. 2.3. Enzym atic deconjugation glucuronides and sulfates
of
urinary
An aliquot of 24-h urine containing 3 mg creatinine Ž1.9–3.3 ml depending on the sample. was mixed with 1 ml 0.5 M Sorensen citrate buffer, pH 6, with or without up to 40 000 Roy units of arylsulfatase and 5000 Fishman units of b-glucuronidase. The final volume was adjusted to 6 ml with deionized water. The mixture was flushed for 30 s with nitrogen, incubated for 1 h at 378C and extracted using a Bond Elut C-18 cartridge as described above for urine; the residue was taken up in 50 m l DMSO and stored at y708C for less than 1 week. 2.3.1. Spectrophotometric analysis of phenolics in urinary and food r beÕerage extracts The amount of phenolics in DMSO solutions of extract or concentrate of urine, wine, HPLC fractions and aqueous food extracts, was measured by analyzing the total phenol concentration by the Folin– Ciocalteu procedure of Singleton and Rossi w7x using gallic acid for the calibration curve. 2.4. Preparation of extracts of aqueous food extracts or wine A total of 50 g of apple were homogenized at room temperature with 20 ml, 50 g of lettuce with 40 ml and 50 g of onion with 50 ml deionized water using a Waring blender. The homogenates were centrifuged for 10 min at 2000 = g; 5 ml of each supernatant were applied on a Bond Elut C-18 cartridge as described for the preparation of urine ex-
C. MalaÕeille et al.r Mutation Research 402 (1998) 219–224
tract. In the case of wine, 5 ml of 10- or 50-fold diluted aqueous solution were applied. The dry extracts of diluted wine, aqueous extract of apple and lettuce were dissolved in up to 100 m l DMSO for the analysis of phenolics and anti-mutagenicity or in acetonitrilerH 2 O Ž50r50. for fractionation by HPLC. 2.5. HPLC separation of urine and food extracts The residues from Bond-Elut C-18 cartridge extractions Žsee above. were dissolved in 100 m l acetonitrilerH 2 O Ž50r50. and separated by HPLC on a C-18 LC column ŽSupercosil, 3 m m; L: 15 cm; ID: 4.6 mm.. Solvent A was 10% acetic acid in H 2 O; solvent B was acetonitrile. The solvents were programmed from an initial concentration of 0% B to 100% B at 60 min and held isocratically until 72 min. The following gradient was applied: 0–12 min 7% B; 12–50 min from 7 to 30% B; 50–60 min from 30 to 100% B. Flow rate was 0.5 mlrmin. The separation was monitored by UV absorption at 254 mm. After injection, six fractions Ž6 ml each. were collected. They were reduced to dryness by vacuum evaporation at room temperature and redissolved in 50 m l DMSO for analysis of anti-mutagenicity and phenolics.
3. Results and discussion To investigate whether urinary anti-mutagens can reduce DNA adduction in the urinary bladder of smokers, arising from exposure to aromatic Žand possibly heterocyclic. amines, we compared urinary anti-mutagenicity measured in 24-h urine samples of 10 smoking volunteers with the level of DNA adducts in exfoliated cells isolated from the same urine. The mean relative adduct labelling ŽRAL. of DNA from these urothelial cell preparations and the association with tobacco smoking have been reported previously w4,8x. Three adducts were related to cigarette smoking, one being the putative N-Ždeoxyguanosine-8yl.-4-aminobiphenyl ŽABP-dG. adduct, and thus were considered for our investigations. Their levels were expressed per the sum of nicotine plus cotinine contents in 24-h urine to adjust for tobacco smoking. Both ABP-dG and total DNA adducts were found to
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be inversely related to bacterial anti-mutagenicity, expressed as the decrease in the number of revertants of S. typhimurium TA98 per ml urine equivalent. The latter was measured in liquid incubation assays using PhIP as a representative mutagen for tobacco smoke-related heterocyclic amines. The logarithms of DNA adduct level vs. anti-mutagenicity were linearly related showing statistical significance Ž r s y0.81, p - 0.01 for both adduct measurements. ŽFig. 1.. Since extracts were prepared from urines of subjects who were also coffee drinkers, we first suspected caffeine and paraxanthine Ža demethylated caffeine metabolite. as inhibitors. Both are known to be substrates for cytochrome P450 IA2-mediated reactions, as are aromatic and heterocyclic amines w9,10x. Caffeine has been reported to inhibit the mutagenicity of various heterocyclic amines in Salmonella w11–13x. However, analysis of urine extracts by HPLC revealed that the concentrations of these methylxanthines in urinary extracts were far too low to account for their inhibitory effect on PhIP mutagenicity, as reported previously w3x. An inhibitory effect of unsaturated fatty acids on the mutagenicity of heterocyclic amines and other carcinogens has been reported w14x and their presence in human urine is well documented w15x. We therefore measured the concentration of the most abundant, linoleic and oleic acid, in urine extracts by gas chromatography wafter methylation, as described by Hayatsu et al. w16xx. The results indicated again that the concentrations of these fatty acids were too low to account for the observed urinary anti-mutagenicity. We then focused on phenolics because Ži. human dietary intake of flavonoids is up to 1 grday w17,18x, Žii. they are partly excreted in urine w19,20x and Žiii. some had been shown to inhibit the mutagenicity of aromatic and heterocyclic amines w11,21–23x. The amount of phenolics was measured in 19 human urine extracts using a spectrophotometric assay w7x. The concentrations were then compared to the urinary anti-mutagenicity, using PhIP as mutagen in S. typhimurium TA98. All the urine extracts contained phenolics, at concentrations of 3.7–12.5 m g per 10 m l DMSO solution of extracts and had anti-mutagenic activities varying by a factor of about 10. A statistically significant linear relationship was found
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Fig. 1. A, B: Levels of 4-ABP adducts and of tobacco-related adducts of DNA from exfoliated urothelial cells of smokers Žadjusted for tobacco exposure. vs. the anti-mutagenic activity of the corresponding urine extract, using PhIP as representative heterocyclic amine. Relative adduct labellings of DNA were taken from Talaska et al. w8x. The anti-mutagenic activities were calculated from the linear part Žcomprising 2–4 concentrations. of the curve of the inhibition of the liver S9-mediated mutagenicity in S. typhimurium TA98 of PhIP Ž20 ng per assay. as a function of increasing concentration of the urinary extract ŽDMSO solution.. Inhibitory curves were plotted using results from one or two series of duplicate experiments each involving four concentrations. The mutagenicity of 20 ng PhIP after 45 min liquid incubation in the absence of inhibitor ranged from 665 to 880 revertants per plate for nine series of experiments, each involving 2–3-duplicate determinations, the mean value " S.E. was 770 " 30. ŽReproduced with the permission of Oxford University Press..
between the anti-mutagenicity of urine extracts and their content in phenolic substances Ž r s 0.58, p 0.02, n s 18.. In order to examine further the role of plant phenolics as urinary anti-mutagens, we assayed a series of representative flavonoids and other plant phenols to determine whether their inhibitory concentrations match those of the phenolics in urinary extracts. At non-cytotoxic concentrations ranging from 0.2 to 6 m g per assay, which are closed to those of phenolics in assays with urinary extracts, the highest inhibitory effects Žup to 100% inhibition of the liver S9 mediated mutagenicity in S. typhimurium TA98 of PhIP and MeIQx. were obtained with flavonols Žquercetin, isorhamnetin, kaempherol., flavones Ž luteolin, diosm etin . , flavanones Žnaringenin, hesperitin. and one anthocyanidin Žcyanidin-chloride.. All the other substances tested wtwo isoflavones Žgenistein, daidzein., two phenolic acids Žgallic acid, caffeic acid. and one flavanol Ž d-catechin.x had no inhibitory activity or very little. Within a particular class of inhibitory substances, the glycosides Ži.e., O-rhamnoglucoside. were relatively
inactive compared with their aglycone moiety, as shown for quercetin vs. quercitrin and naringenin vs. naringin. Dietary phenolics are partly excreted in urine as glucuronides and sulfates w19,24x. Two urine samples were therefore treated with b-glucuronidase and arylsulfatase before the preparation of extracts. After enzymatic treatment, anti-mutagenicity increased maximally 11-fold and the level of phenolics 2.5-fold. Based on combined results from two extracts, the increase in anti-mutagenicity was linearly related to the concentration of unconjugated phenolics. These data strongly suggest that these free phenolic substances act as inhibitors of liver S9-mediated mutagenicity of PhIP. To gain further evidence for the role of phenolics as urinary anti-mutagens, the mode of inhibition of liver S9-mediated mutagenicity of PhIP was determined for three urinary extracts and compared with that of quercetin. As inferred from double reciprocal plots, both the extracts and the pure flavonoid, quercetin, inhibited the mutagenicity of PhIP in a non-competitive fashion.
C. MalaÕeille et al.r Mutation Research 402 (1998) 219–224
Altogether, the numerous pieces of experimental evidence which were presented above, suggest that much of the urinary anti-mutagenicity is attributable to dietary phenolics and their metabolites. In order to identify the origin of anti-mutagenic substances in urinary extracts, chromatographic separations were performed using conditions reported to be efficient for separation of flavonoids w25,26x. When two urinary extracts were subjected to reverse-phase HPLC, the UV absorption profiles revealed a complex elution pattern. Subsequently, six fractions, covering the entire chromatographic profile, were analysed. All fractions contained substances, in 10 m l DMSO solution ŽSection 2.5., that inhibited liver S9 mutagenicity of PhIP and phenolics at concentrations ranging from 0.3–46 m gr10 m l DMSO solution. The most lipophilic substances displayed the highest inhibitory activity when expressed per microgram of phenolics, i.e., 50% inhibition per 0.2 m g phenolics, which corresponds to the highest antimutagenic effect obtained with the flavonoids assayed w6x. Onion, lettuce, apple and red wine are important dietary sources of flavonoids w17x and these substances are probably responsible for the anti-mutagenicity observed in their water extracts w6x. These extracts were also subjected to reverse phase HPLC as carried out for urinary extracts. The distribution of anti-mutagenic activity in the HPLC fractions of urinary extracts corresponds roughly to a combination of inhibitors present in onion and wine extracts, suggesting that these phenolics and their metabolites comprise a major part of the complex mixture of urinary anti-mutagens. Human diet contains a wide variety of non-nutrient agents, mostly phenolics of plant origin, which inhibit carcinogenesis in animals w27–30x. Epidemiological observations have confirmed a protective effect of vegetable and fruits against the occurrence of many epithelial cancers w31–33x. Phenolics in the human diet could affect the pharmacokinetics of carcinogens by Ži. altering the rates of their absorption and uptake, Žii. reacting with or tightly binding to toxic substances and metabolites and Žiii. modifying cytochrome P450-dependent metabolism and phase II enzymes w14,28,29,34x. The inverse relationship between urinary anti-mutagenicity and level of tobacco-related DNA adducts in the urothelium, reported here for the first time, strongly suggests that
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bladder mucosal cells of smokers are partially protected by dietary phenolics, most probably flavonoids, against the harmful effects of tobacco-derived bladder carcinogens. Studies are warranted to explore further these protective effects of phenolics from vegetables and fruits against cancers in humans as a part of a cancer chemoprevention strategy.
Acknowledgements The authors would like to thank Mrs. P. Collard and Mrs. M. Lezere ´ ` for secretarial help, Dr. J. Cheney for editorial assistance and Dr. J. Esteve ` for helpful discussions. Part of the work was supported by NIEHS 5-P30-ES06096.
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