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Effect of onion consumption by rats on hepatic drug-metabolizing enzymes
Food and Chemical Toxicology 39 (2001) 981–987 www.elsevier.com/locate/foodchemtox
Research section
Effect of onion consumption by rats on hepatic drug-metabolizing enzymes C. Teyssiera, M.-J. Amiotb, N. Mondyc, J. Augerc, R. Kahaned, M.-H. Siessa,* a
UMR de Toxicologie Alimentaire, INRA-Universite´ de Bourgogne, 17 rue Sully, 21065 Dijon Cedex, France b UMR A408, INRA-Universite´ d’Avignon, Site Agroparc, 84914 Avignon Cedex 9, France c I.R.B.I. UMR CNRS, Universite´ F. Rabelais, Faculte´ des Sciences et Techniques, Parc Grandmont, 37200 Tours, France d Coopd’Or R&D, Station de Ge´ne´tique et d’Ame´lioration des Plantes, INRA, 21110 Bretenie`res, France Accepted 15 May 2001
Abstract Fruits and vegetables or their natural constituents which increase detoxication enzymes and/or reduce activating enzymes are considered as good candidates to prevent chemically-induced carcinogenesis. In this study, rats were fed a diet supplemented with 20% onion powder for 9 days. Several cytochrome P450 (CYP)s enzymes (CYP 1A, 2B, 2E1, 3A), which are involved in carcinogen activation, were determined by measuring their enzyme activities using specific substrates. In addition, phase II enzymes activities such as UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST), involved in detoxication of carcinogens, were measured. Protein levels of CYPs and GST A1/A2, A3/A5, Ml, M2 and P1 were measured using antibodies in Western blots. Consumption of onion induced CYP 1A and CYP 2B activities while it decreased CYP 2E1 activity. This later modification was accompanied by a decrease of CYP 2E1 levels. The same dietary treatment caused a slight increase of the total GST activity. The relative proportions of GST subunits were modified. GST Al/A2 subunits were increased while GST A3/A5 and GST M2 subunits were decreased and GST M1 and P1 were not modified. Onion consumption also increased p-nitrophenol UGT activity. Taken together, these results suggest that the decrease of CYP 2E1 and the increase of phase II enzymes by onion can afford protection against some carcinogens, while the decrease of some GST subunits could increase the genotoxic effects of other chemicals. The modulating effect of onion could be ascribed to alk(en)yl polysulphides and/or glycosides of flavonols, which were identified in the onion powder. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Onion; Flavonols; Sulphur compounds; Cytochrome P450; Glutathione S-transferase; UDP-glucuronosyltransferase; Rat; Liver
1. Introduction Onion is grown and consumed worldwide. It has been considered for centuries as beneficial for health, and is recommended for curing or preventing a wide variety of diseases. The beneficial effects of onion have only recently been validated in many experimental studies (Dorsch, 1996). Several effects were described, such as Abbreviations: CYP, cytochrome P450; EROD, ethoxyresorufin O-deethylase; GST, glutathione S-transferase; MROD, methoxyresorufin O-demethylase; NO, nifedipine oxidase; PNPH, p-nitrophenol hydroxylase; PROD, pentoxyresorufin O-dealkylase; SPF, specified pathogen-free; SPME, solid phase microextraction; UGT, UDP-glucuronosyltransferase. * Corresponding author. Tel.: +33-3-8069-3221; fax: +33-3-80693225. E-mail address: [email protected] (M.-H. Siess).
anti-inflammatory, anti-asthmatic, antimicrobial and cardiovascular effects. Moreover, epidemiological studies indicated that consumption of onion and other Allium species can reduce the risk of cancer at specific sites. Several case control studies reported a decreasing risk for stomach cancer (Haenszel et al., 1972; Buiatti et al., 1989; Boeing et al., 1991), colon cancer (Graham et al., 1988; Steinmetz and Potter, 1993) or breast cancer (Levi et al., 1993; Challier et al., 1998) with increasing consumption of onion. A recent cohort study provided evidence for a strong inverse relationship between onion consumption and stomach carcinoma incidence (Dorant et al., 1996). Although the preventive effects of onion have been clearly demonstrated in epidemiological studies, their mechanisms of action have not been yet specified. Most carcinogens are not active in themselves but require bioactivation to reactive metabolites that
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bind covalently to DNA. Drug-metabolizing enzymes play a key role in this transformation. These enzymes have been classified into phase I and phase II enzymes. Phase I enzymes such as cytochromes P450 (CYPs) are responsible for carcinogen activation, while phase II enzymes such as UDP-glucuronosyltransferase (UGT) or glutathione S-transferase (GST) are more responsible for detoxication. Enhancement or induction of phase II enzyme activities and/or inhibition of CYP activities are generally described as efficient mechanisms of cancer prevention (Guengerich, 1992; Wattenberg, 1993). Onions contain a wide variety of microconstituents such as trace elements, vitamins, flavonoids and sulphur compounds (Breu, 1996), which may have protective effects against cancer. Several studies have shown that the administration of pure components such as organosulphur compounds or flavonoids to animals was able to affect the biotransformation of xenobiotics and could influence the toxicity and carcinogenicity of environmental chemicals (Smith and Yang, 1994; Suschetet et al., 1998). Allyl sulphides or alkyl sulphides, derived from Allium species, have been shown to increase phase II enzymes such as GST, UGT (Sparnins et al., 1988; Hu et al., 1996; Kim et al., 1996; Guyonnet et al., 1999) and/or to modulate CYP activities (Brady et al., 1991; Reicks and Crankshaw, 1996; Siess et al., 1997). The effects of the flavonoid quercetin on drug-metabolizing enzymes have been widely demonstrated. Many studies showed an in vitro inhibition of CYP activities by quercetin (Miniscalco et al., 1992; Tsyrlov et al., 1994; Siess et al., 1995) whereas administered in vivo, quercetin caused no effect on CYP enzymes (Siess et al., 1989). However, quercetin incorporated at 1% in a diet produced an increase of GST activity in the intestine of rats (Nijhoff et al., 1993). As isolated components issued from onion have been found to influence drug-metabolizing enzymes, it is predictable that the whole vegetable would influence the same enzymes. This experiment was designed to study the effects of feeding rats with onion on drug-metabolizing enzymes in order to better understand the anticarcinogenic effects of this food plant. In the present study rats were fed a diet containing onion powder for 9 days. A chemical analysis (HPLC/ UV and GC/MS) was performed to identify the flavonoids and sulphur compounds present in the onion powder. Several CYP enzyme activities that are important in carcinogen metabolism were determined: ethoxyresorufin O-deethylase (EROD) as a marker of CYP 1Al which is active towards hydrocarbons, methoxyresorufin O-demethylase (MROD) as a marker of CYP 1A2 active towards heterocyclic amines, pentoxyresorufin O-dealkylase (PROD) as a marker of CYP 2B1/2 active in aflatoxin B1 activation, p-nitrophenol hydroxylase (PNPH) as a marker of CYP 2E1 able to metabolize nitrosamines and numerous low molecular weight chemicals, and nifedipine oxidase (NO) as a marker of
CYP 3A. Phase II enzymes activities such as UGT and GST, involved in detoxication of carcinogens, were also measured. In addition, immunoblot analyses were performed to assess the microsomal levels of CYP isoenzymes (2B1/2, 2E1, 3A) and the cytosolic levels of GST subunits (Al /A2, A3/A5, Ml, M2 and P1).
2. Materials and methods 2.1. Chemicals The onion powder was obtained from Socie´te´ de Transformation Le´gumie`re (Auxonne, France). This powder was prepared from long-day yellow onions (Allium cepa) grown in the plain of Dijon (France). Onions were washed and sliced before dehydrating through a conventional hot-air oven (temperature gradient from 85 to 45 C) until the product retained less than 6% moisture. The polyclonal antibodies against rat CYP 2B were a gift from Professor A.-M. Batt (Centre du Me´dicament, Nancy, France) and the polyclonal antibody against rat CYP 2E1 was a gift from Professor P. Beaune (Universite´ Paris V, Paris, France). The polyclonal antibodies against rat GSTAI/A2, GSTA3/A5, GSTM1, GSTM2 and GSTP1 were obtained from Biotrin International (Dublin, Ireland). Other chemicals were of the highest quality available. 2.2. Characterisation and quantification of flavonoids The onion powder (5 g) was mixed with 30 ml of methanol. The alcoholic fraction was collected and filtrated and made up to 50 ml methanol. Flavonoids were separated and characterised by HPLC diode array detection. Separation of flavonoids was performed using a Alltima C18 (1504.6 mm, 5 m) at 35 C. The mobile phase (flow rate 1 ml/min) consisted in acidified distilled water adjusted to pH: 2.6 with o-phosphoric acid and acetonitrile. The separation was obtained using a linear gradient from 5 to 40% acetonitrile during 24 min. Flavonoids were identified by the comparison of their retention times and their spectra with the structures described by Price and Rhodes (1997) in onions analyzed under similar conditions. The same mass spectral measurements have been obtained using a Micromass detector-Platform LCZ- (data not shown). Flavonoids were quantified using external calibration at 365 nm and were expressed in mg/kg onion powder as equivalent quercetin. 2.3. Separation and characterisation of sulphur compounds The onion powder (2 g) was mixed with 2 ml distilled water. Compounds emitted in a closed 4-ml vial at room
C. Teyssier et al. / Food and Chemical Toxicology 39 (2001) 981–987
temperature were trapped for 60 min and transferred to the GC–MS injector by headspace SPME (solid phase microextraction, polyacrylate fibre 85 m). GC–MS analysis was carried out on a benchtop Perkin-Elmer Turbomass system with a split-splitless injector and a fusedsilica capillary column (10 m0.32 mm) with a 4 m methylsilicone coating. This column was chosen because it can analyse polysulphides, thiophenes and even very labile sulphur compounds such as the lacrymatory factor, without degradation (Arnault et al., 2000). The carrier gas was helium (99.999%) and the column temperature was programmed from 70 to 250 C at a rate of 5 C/min. The injection port temperature was 200 C. Total ion chromatograms and mass spectra were recorded in the electron impact ionisation mode at 70 eV. The transfer line and the source temperature were maintained at 150 C. The identification of sulphur compounds was accomplished by comparing the retention time and the mass spectra data with those of compounds available from NIST library and from previous data (Ferary and Auger, 1996; Ferary et al., 1996). 2.4. Animals and dietary treatment The experiment was performed with male SPF Wistar rats. 5-Week-old rats were purchased from Iffa Credo (L’Arbresle, France) and were housed in individual stainless-wire cages, maintained at 22 C with a 12-h light/dark cycle. They had free access to water. They were fed a purified diet for 2 weeks. The composition is reported in Table 1. Afterwards, the onion powder was incorporated into the diet at the expense of sucrose and cellulose (Table 1). After 9 days of feeding rats, were killed after 16 h of fasting. 2.5. Preparation of microsomal and cytosolic fractions The animals were killed by exsanguination. Livers were immediately removed. Microsomes and cytosols
Table 1 Composition of the experimental diets (g/kg of diet)
were prepared by differential centrifugation and stored in small aliquots at 80 C (Haber et al., 1994). The protein levels of the microsomal and the cytosolic fractions were measured by the method of Bradford (1976) adapted for the use of a Cobas Fara II centrifugal analyser (Roche Instruments) using serum albumin as standard. 2.6. Enzyme assays Total microsomal P450 content was quantified according to Omura and Sato (1964). Determination of phenoxazone O-dealkylase activities (EROD, MROD and PROD) was adapted from the method of Burke et al. (1985). Reactions were carried out in a fluorimeter at 37 C using a Cobas Fara II centrifugal analyser (Roche Instruments). The concentrations of substrates ethoxy-, methoxy- and pentoxyresorufin were respectively 5, 5 and 10 mm. Assay for PNPH activity was determined by HPLC according to Tassaneeyakul et al. (1993). The concentration of the substrate p-nitrophenol was 0.1 mm and the microsomal protein was 0.5 mg/ml. The product of the reaction, p-nitrocatechol was monitored at 340 nm. NO activity was determined by HPLC using a method adapted from Guengerich et al. (1986). The concentration of the substrate nifedipine was 0.2 mm and the microsomal protein was 1 mg/ml. UV detection of the product dehydronifedipine was performed at 254 nm. Total GST activity was measured with 1-chloro-2,4dinitrobenzene (CDNB) as substrate according to Habig et al. (1974). The reaction mixture contained 1 mm GSH and 1 mm CDNB. The formation of the conjugate was continuously monitored at 340 nm. The UGT activity was determined with p-nitrophenol as a substrate by the method of Mulder and van-Doorn (1975) using 3 mm UDP-glucuronic acid and 0.15 mm p-nitrophenol. Microsomes were activated with Triton X- 100 so that the ratio of Triton to protein concentration was 0.2. The measurement of GST and UGT activities were adapted for the use of a Cobas Fara II centrifugal analyser (Roche Instruments). 2.7. Immunoblot analyses
Components
Control
Onion
Casein Sucrose Starch Cellulose Mineral mixture Onion powder d,l-Methionine Corn oil Vitamin mixture+water (ml)a
230 270 360 40 50 – 1 50 500
210 130 360 – 50 200 1 50 500
a
983
The dry matter was mixed with water containing the vitamin mixture.
Immunoblot procedures were performed as previously described by Haber et al. (1994). The quantification of the individual bands was done by comparing blot density between treated and control rats, using an image analyser (Bioscan Optimetric, Edmonds, WA, USA). 2.8. Statistical analysis The Student’s t-test was used for comparing the onion-treated group to the control group. The level of significance was P < 0.05.
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3. Results 3.1. Flavonols and sulphur compounds present in the onion powder The following flavonols were identified in the onion powder: quercetin 3,40 -diglucoside, quercetin 40 -glucoside, isorhamnetin diglucoside, isorhamnetin 40 -glucoside and free quercetin (Table 2). Quercetin 3,40 diglucoside and quercetin 40 -glucoside represented the major components (more than 90%). The ratio of quercetin 3,40 -diglucoside:quercetin 40 -glucoside was 1:1. These results are in good agreement with those reported by Price and Rhodes (1997) and Fossen et al. (1998). The total content of flavonols was 8450 mg equivalent quercetin /kg dry weight. Fig. 1 shows the GC chromatographic separation of sulphur compounds of the onion powder. A total of 15 compounds were identified (Table 3). Most of them Table 2 Flavonol content of the onion powder Flavonols
Amounta
Quercetin 3,40 -diglucoside Quercetin 40 -glucoside Quercetin Isorhamnetin diglucoside Isorhamnetin 40 -glucoside Total
4170 3820 290 70 100 8450
a
mg/kg dry weight of the onion powder.
were alk(en)yl polysulphides: disulphides, trisulphides and tetrasulphides. In addition, two dimethyl thiophenes (2,4- and 3,4-dimethyl) were detected. Polysulphides had all the possible combinations of the 1propenyl, propyl and methyl thiomoieties. Dipropyl disulphide, methyl 1-propenyl trisulphide and propyl 1propenyl trisulphide were predominant. During the process of dehydrating, polysulphides and dimethylthiophenes were formed from lacrymatory factor, thiosulphinates and zwiebelanes, which are volatile compounds present in fresh onion. Therefore the pattern of onion powder is very different from that of fresh onion analysed in the same conditions (Arnault et al., 2000). Table 3 Sulphur compounds identified in the onion powder by GC–MS Peak no.
Compounds
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Lacrymatory factor 2,4-Dimethylthiophene 3,4-Dimethylthiophene Dimethyl trisulphide Dipropyl disulphide Propyl 1-propenyl disulphide Methyl propyl trisulphide Methyl 1-propenyl trisulphide Dimethyl tetrasulphide Dipropyl trisulphide Propyl 1-propenyl trisulphide Di(1-propenyl) trisulphide Methyl 1-propenyl tetrasulphide Dipropyl tetrasulphide Propyl 1-propenyl tetrasulphide
Fig. 1. GC chromatogram of the onion powder.
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C. Teyssier et al. / Food and Chemical Toxicology 39 (2001) 981–987 Table 4 Effect of onion consumption on body weights and liver weights of rats
Body weightsa Liver weightsa Relative liver weightb
Control
Onion
2084 7.60.2 3.670.05
1983* 7.20.2 3.660.04
Rats received the onion powder into the diet (20%) for 9 days. Values are meansS.E.M. of six rats. a g. b g liver per 100 g body weight. * Differs significantly from control mean (P40.05) Student’s t-test
Table 5 Effect of onion consumption on liver drug-metabolizing enzymes Control
Onion
a
0.520.03 0.570.01 Cytochrome P450 Ethoxyresorufin O-deethylase (CYP 1A1)b 361 502* 263 Methoxyresorufin O-demethylase (CYP 1A2)b 212 Pentoxyresorufin O-dealkylase (CYP 2B1/2)b 162 254* p-Nitrophenol hydroxylase (CYP 2E1)c 0.730.03 0.480.07* 0.790.09 0.770.08 Nifedipine oxidase (CYP 3A)c Glutathione S-transferased 88638 106751* UDP-glucuronosyltransferased 11.50.5 21.20.7* Rats received the onion powder into the diet (20%) for 9 days. Values are means S.E.M. of six rats. a nmol/mg microsomal protein. b pmol/min/nmin of CYP. c nmol/min/nmol of CYP. d nmol/min/mg of protein. * Differs significantly from control mean (P40.05) Student’s t-test.
3.2. Effects of consumption of the onion powder on drugmetabolizing enzymes At the end of the experimental period there was a slight decrease of the body weights of rats but no significant difference in the relative liver weights (Table 4). The total CYP content was slightly increased, although not significantly (Table 5). EROD, MROD and PROD activities were increased by 39, 24 and 56%, respectively. The increase was significant with EROD and PROD activities (Table 5). PNPH activity was decreased by 34% in rats consuming onion. Therefore, it was of interest to determine the levels of these CYP isoforms. Table 6 showed that the levels of CYP 2E1 were decreased by 15% while CYP2B1/2 isoforms were not detected in either group. UGT and GST activities were increased in rats fed with the onion powder (Table 5). GST levels of different classes a, m and p were analysed by immunodetection. GST A1/A2 was increased by feeding onion, whereas GST A3/A5 and GST M2 were decreased, and GST M1 was unchanged (Table 6). GST P1 subunit was not detected in the both groups.
Table 6 Effect of onion consumption on the expression of CYP isoenzymes and GST subunits (% of control) Control CYP isoenzymes CYP 2B1/2 CYP 2E1 GST subunits GST A1/A2 GST A3/A5 GST M1 GST M2 GST P1
Onion
nd 100
nd 8515
100 100 100 100 nd
20010* 687* 10418 569* nd
Rats received the onion powder into the diet (20%) for 9 days. Values represent % of control rat for each isoform. Values are means S.E.M. of six rats. For immunoblotting with anti CYP 2B diluted (1:500), 25 mg of microsomal proteins were loaded. For immunoblotting with anti CYP 2E1 diluted (1:1000), 40 mg of microsomal proteins were loaded. For immunoblotting with anti GST A1/A2 and anti A3/ A5 diluted (1:200), 10 mg of cytosolic proteins were loaded and for immunoblotting with anti GST M1, M2, P diluted (1:400), 10 mg of cytosolic proteins were loaded. nd: not detectable. * Differs significantly from control mean (P40.05) Student’s t-test
4. Discussion In this study we have demonstrated that feeding rats with a diet supplemented with onion powder for 9 days modulates drug-metabolizing enzymes. Consumption of onion produced an increase of EROD and PROD activity markers of CYP 1A and CYP 2B. The increase of PROD activity was not accompanied by an increase of CYP 2B1/2 levels. This could be explained by the fact that this activity is supported by other forms of CYPs. In addition, the increase of this activity remains very low when compared to the increase obtained with the classical inducer phenobarbital (Burke et al., 1985). Consumption of onion produced no significant effect on NO activity, while it decreased PNPH activity by 34%. This latter decrease was accompanied by a 15% decrease of CYP 2E1 levels. This result suggests the possible action of onion on reducing the activation of low molecular weight carcinogens or nitrosamines which are metabolized by this CYP isoform (Guengerich et al., 1991). We also examined the effects of onion consumption on phase II enzymes. Total GST activity was slightly but significantly increased. Therefore, it was of interest to check the variations of the relative proportions of different subunits of GST. GST subunits Al/A2 were increased. These results suggest that onion could prevent the genotoxic action of carcinogens detoxified by GST A1/A2, which were reported to be involved in the detoxication of heterocyclic amines and benzo[a]pyrene (Singh et al., 1985; Lin et al., 1994). Conversely, GST A3/A5 and GST M2 were lowered. This would mean
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that onion could increase the genotoxic effect of carcinogens which are detoxified by these subunits (Hayes and Pulford, 1995). UGT activity was measured with p-nitrophenol assumed to represent the UGT 1A6 isoform. This isoform is actively involved in the detoxification of chemical carcinogens such as polycyclic hydrocarbons and heterocyclic aromatic amines (Bock, 1991). p-Nitrophenol-UGT activity was significantly increased. Therefore, the protective effect of onion against the carcinogenic action of these compounds could be expected. However, biotransformation of carcinogens by glucuronidation or conjugation with glutathione can sometimes lead to activation (Monks et al., 1990). Therefore, it would be interesting to know whether onion consumption could modify these enzymes involved in the activation. Previous studies have shown that microconstituents of onion such as flavonols (quercetin) or organosulphur compounds (polysulphides) were able to modify the levels of phase I and phase II enzymes when administered in isolation (Nijhoff et al., 1993; Siess et al., 1997). In a previous study, we showed that quercetin when incorporated in a diet at 3000 ppm produced no effect on liver drug-metabolizing enzymes (Siess et al., 1989). In the present study, we identified in the onion powder quercetin and also glycosides of quercetin. Taking account of the levels of quercetin glycosides in the onion powder (8280 mg/kg dry weight, Table 2), the level of quercetin in the diet is around 1600 ppm. Therefore, the involvement of quercetin in the inducing effect of onion is probably not possible. However, it cannot be excluded that the bioavailability of quercetin would be better when ingested through the onion. It was actually demonstrated by Hollman et al. (1997) that conjugating quercetin with glucose enhanced its absorption from the small gut. Therefore the role of quercetin from onion cannot be excluded. The modulating effect of onion consumption on drug-metabolizing enzymes could be attributed partly to the sulphur compounds. Among the organosulphur compounds identified in the onion powder, dipropyl disulphide, methyl 1-propenyl trisulphide and propyl 1-propenyl trisulphide were predominant. Although it is difficult to attribute the effect to a specific component, the induction pattern observed with onion is similar to that produced by different propyl or methyl sulphides fed alone: decrease of CYP 2E1 and induction of UGT and GST (Siess et al., 1997). This suggests that polysulphides are probably involved in the overall effect of onion. Sulphur compounds and glycosides of quercetin are present simultaneously in onion. It was reported that the combination of a sulphur compound and quercetin was more efficient in reducing the mammary cancer than the single administration of these compounds (Ip and Ganther, 1991). Thus, the different components present in onion could have probably an additive action.
In this experiment, the modulating effect of onion was observed with administered doses that are much higher than the possible intake through a normal human diet. Therefore, it would be of great interest to explore whether smaller doses may have produced the same effects. Further studies are needed to determine the dose of onion in the diet and the duration of animal feeding necessary to obtain such drug-metabolizing enzyme modifications and thereby a possible chemoprotective effect of this vegetable. Moreover, it would be interesting to appreciate the effect of onion consumption in humans. This information could be useful in the development of a strategy for cancer prevention by eating fruits and vegetables.
Acknowledgements The authors thank M-F. Vernevaut for excellent technical assistance and P. Goupy for analyzing flavonoids. This work was supported by a grant of Conseil Re´gional de Bourgogne (France) and a grant of the french Ministe`re de Ia Recherche (no. 98 P0470).
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DNA binding of the N-acetoxy derivatives of heterocyclic amine carcinogens in vitro: a possible mechanism of organ specificity in their carcinogenesis. Cancer Research 54, 4920–4926. Miniscalco, A., Lundahl, J., Regardh, C.G., Edgar, B., Eriksson, U.G., 1992. Inhibition of dihydropyridine metabolism in rat and human liver microsomes by flavonoids found in grapefruit juice. Journal of Pharmacology and Experimental Therapeutics 261, 1195–1199. Monks, T.J., Anders, M.W., Dekant, W., Stevens, J.L., Lau, S.S., Bladeren, P.J., 1990. Glutathione conjugate mediated toxicities. Toxicology and Applied Pharmacology 106, 1–19. Mulder, G.J., van-Doorn, A.B., 1975. A rapid NAD+-linked assay for microsomal uridine diphosphate glucuronyltransferase of rat liver and some observations on substrate specificity of the enzyme. Biochemical Journal 151, 131–140. Nijhoff, W.A., Groen, G.M., Peters, W.H., 1993. Induction of rat hepatic and intestinal glutathione S-transferases and glutathione by dietary naturally occurring anticarcinogens. International Journal of Oncology 3, 1131–1139. Omura, T., Sato, R., 1964. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. Journal of Biological Chemistry 269, 2370–2378. Price, K.R., Rhodes, M.J.C., 1997. Analysis of the major flavonol glycosides present in four varieties of onion (Allium cepa) and changes in composition, resulting from autolysis. Journal of the Science of Food and Agriculture 74, 331–339. Reicks, M.M., Crankshaw, D.L., 1996. Modulation of rat hepatic cytochrome P-450 activity by garlic organosulfur compounds. Nutrition and Cancer 25, 241–248. Siess, M.H., Guillermic, M., Le Bon, A.M., Suschetet, M., 1989. Induction of monooxygenase and transferase activities in rat by dietary administration of flavonoids. Xenobiotica 19, 1379–1386. Siess, M.H., Leclerc, J., Canivenc-Lavier, M.C., Rat, P., Suschetet, M., 1995. Heterogenous effects of natural flavonoids on monooxygenase activities in human and rat liver microsomes. Toxicology and Applied Pharmacology 130, 73–78. Siess, M.H., Le Bon, A.M., Canivenc-Lavier, M., Suschetet, M., 1997. Modification of hepatic drug-metabolizing enzymes in rats treated with alkyl sulfides. Cancer Letters 120, 195–201. Singh, S.V., Srivastava, S.K., Awasthi, Y.C., 1985. Binding of benzo(a)pyrene to rat lung glutathione S-transferases in vivo. FEBS Letters 179, 111–114. Smith, T.J., Yang, C.S., 1994. Effect of food phytochemicals on xenobiotic metabolism and tumorigenesis. In: Huang, M.T., Osawa, T., Ho, C.T., Rosen, R.T. (Eds.), Food Phytochemicals for Cancer Prevention. I: Fruits and Vegetables. ACS Series, Washington DC, pp. 17–48. Sparnins, V.L., Barany, G., Wattenberg, L.W., 1988. Effects of organosulfur compounds from garlic and onions on benzo[a]pyreneinduced neoplasia and glutathione S-transferase activity in the mouse. Carcinogenesis 9, 131–134. Steinmetz, K.A., Potter, J.D., 1993. Food-group consumption and colon cancer in the Adelaide Case-Control Study. I. Vegetables and fruit. International Journal of Cancer 53, 711–719. Suschetet, M., Siess, M.H., Le Bon, A.M., Canivenc-Lavier, M.C., 1998. Anticarcinogenic properties of some flavonoids. In: Vercauteren, J., Che`se, C., Triaud, J. (Eds.), Polyphenols 96. INRA Editions, Paris, pp. 165–204. Tassaneeyakul, W., Veronese, M.E., Birkett, D.J., Miners, J.O., 1993. High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P450 2E1 activity, in human liver microsomes. Journal of Chromatography 616, 73–78. Tsyrlov, I.B., Mikhailenko, V.M., Gelboin, H.V., 1994. Isozyme- and species-specific susceptibility of cDNA-expressed CYP1A P-450s to different flavonoids. Biochimica et Biophysica Acta 1205, 325–335. Wattenberg, L.W., 1993. Chemoprevention of carcinogenesis by minor non-nutrient constituents of the diet. In: Parke, D.V., loannides, C., Walker, R. (Eds.), Food, Nutrition and Chemical Toxicity. SmithGordon, UK, pp. 287–300.
Research section
Effect of onion consumption by rats on hepatic drug-metabolizing enzymes C. Teyssiera, M.-J. Amiotb, N. Mondyc, J. Augerc, R. Kahaned, M.-H. Siessa,* a
UMR de Toxicologie Alimentaire, INRA-Universite´ de Bourgogne, 17 rue Sully, 21065 Dijon Cedex, France b UMR A408, INRA-Universite´ d’Avignon, Site Agroparc, 84914 Avignon Cedex 9, France c I.R.B.I. UMR CNRS, Universite´ F. Rabelais, Faculte´ des Sciences et Techniques, Parc Grandmont, 37200 Tours, France d Coopd’Or R&D, Station de Ge´ne´tique et d’Ame´lioration des Plantes, INRA, 21110 Bretenie`res, France Accepted 15 May 2001
Abstract Fruits and vegetables or their natural constituents which increase detoxication enzymes and/or reduce activating enzymes are considered as good candidates to prevent chemically-induced carcinogenesis. In this study, rats were fed a diet supplemented with 20% onion powder for 9 days. Several cytochrome P450 (CYP)s enzymes (CYP 1A, 2B, 2E1, 3A), which are involved in carcinogen activation, were determined by measuring their enzyme activities using specific substrates. In addition, phase II enzymes activities such as UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST), involved in detoxication of carcinogens, were measured. Protein levels of CYPs and GST A1/A2, A3/A5, Ml, M2 and P1 were measured using antibodies in Western blots. Consumption of onion induced CYP 1A and CYP 2B activities while it decreased CYP 2E1 activity. This later modification was accompanied by a decrease of CYP 2E1 levels. The same dietary treatment caused a slight increase of the total GST activity. The relative proportions of GST subunits were modified. GST Al/A2 subunits were increased while GST A3/A5 and GST M2 subunits were decreased and GST M1 and P1 were not modified. Onion consumption also increased p-nitrophenol UGT activity. Taken together, these results suggest that the decrease of CYP 2E1 and the increase of phase II enzymes by onion can afford protection against some carcinogens, while the decrease of some GST subunits could increase the genotoxic effects of other chemicals. The modulating effect of onion could be ascribed to alk(en)yl polysulphides and/or glycosides of flavonols, which were identified in the onion powder. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Onion; Flavonols; Sulphur compounds; Cytochrome P450; Glutathione S-transferase; UDP-glucuronosyltransferase; Rat; Liver
1. Introduction Onion is grown and consumed worldwide. It has been considered for centuries as beneficial for health, and is recommended for curing or preventing a wide variety of diseases. The beneficial effects of onion have only recently been validated in many experimental studies (Dorsch, 1996). Several effects were described, such as Abbreviations: CYP, cytochrome P450; EROD, ethoxyresorufin O-deethylase; GST, glutathione S-transferase; MROD, methoxyresorufin O-demethylase; NO, nifedipine oxidase; PNPH, p-nitrophenol hydroxylase; PROD, pentoxyresorufin O-dealkylase; SPF, specified pathogen-free; SPME, solid phase microextraction; UGT, UDP-glucuronosyltransferase. * Corresponding author. Tel.: +33-3-8069-3221; fax: +33-3-80693225. E-mail address: [email protected] (M.-H. Siess).
anti-inflammatory, anti-asthmatic, antimicrobial and cardiovascular effects. Moreover, epidemiological studies indicated that consumption of onion and other Allium species can reduce the risk of cancer at specific sites. Several case control studies reported a decreasing risk for stomach cancer (Haenszel et al., 1972; Buiatti et al., 1989; Boeing et al., 1991), colon cancer (Graham et al., 1988; Steinmetz and Potter, 1993) or breast cancer (Levi et al., 1993; Challier et al., 1998) with increasing consumption of onion. A recent cohort study provided evidence for a strong inverse relationship between onion consumption and stomach carcinoma incidence (Dorant et al., 1996). Although the preventive effects of onion have been clearly demonstrated in epidemiological studies, their mechanisms of action have not been yet specified. Most carcinogens are not active in themselves but require bioactivation to reactive metabolites that
0278-6915/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0278-6915(01)00056-4
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bind covalently to DNA. Drug-metabolizing enzymes play a key role in this transformation. These enzymes have been classified into phase I and phase II enzymes. Phase I enzymes such as cytochromes P450 (CYPs) are responsible for carcinogen activation, while phase II enzymes such as UDP-glucuronosyltransferase (UGT) or glutathione S-transferase (GST) are more responsible for detoxication. Enhancement or induction of phase II enzyme activities and/or inhibition of CYP activities are generally described as efficient mechanisms of cancer prevention (Guengerich, 1992; Wattenberg, 1993). Onions contain a wide variety of microconstituents such as trace elements, vitamins, flavonoids and sulphur compounds (Breu, 1996), which may have protective effects against cancer. Several studies have shown that the administration of pure components such as organosulphur compounds or flavonoids to animals was able to affect the biotransformation of xenobiotics and could influence the toxicity and carcinogenicity of environmental chemicals (Smith and Yang, 1994; Suschetet et al., 1998). Allyl sulphides or alkyl sulphides, derived from Allium species, have been shown to increase phase II enzymes such as GST, UGT (Sparnins et al., 1988; Hu et al., 1996; Kim et al., 1996; Guyonnet et al., 1999) and/or to modulate CYP activities (Brady et al., 1991; Reicks and Crankshaw, 1996; Siess et al., 1997). The effects of the flavonoid quercetin on drug-metabolizing enzymes have been widely demonstrated. Many studies showed an in vitro inhibition of CYP activities by quercetin (Miniscalco et al., 1992; Tsyrlov et al., 1994; Siess et al., 1995) whereas administered in vivo, quercetin caused no effect on CYP enzymes (Siess et al., 1989). However, quercetin incorporated at 1% in a diet produced an increase of GST activity in the intestine of rats (Nijhoff et al., 1993). As isolated components issued from onion have been found to influence drug-metabolizing enzymes, it is predictable that the whole vegetable would influence the same enzymes. This experiment was designed to study the effects of feeding rats with onion on drug-metabolizing enzymes in order to better understand the anticarcinogenic effects of this food plant. In the present study rats were fed a diet containing onion powder for 9 days. A chemical analysis (HPLC/ UV and GC/MS) was performed to identify the flavonoids and sulphur compounds present in the onion powder. Several CYP enzyme activities that are important in carcinogen metabolism were determined: ethoxyresorufin O-deethylase (EROD) as a marker of CYP 1Al which is active towards hydrocarbons, methoxyresorufin O-demethylase (MROD) as a marker of CYP 1A2 active towards heterocyclic amines, pentoxyresorufin O-dealkylase (PROD) as a marker of CYP 2B1/2 active in aflatoxin B1 activation, p-nitrophenol hydroxylase (PNPH) as a marker of CYP 2E1 able to metabolize nitrosamines and numerous low molecular weight chemicals, and nifedipine oxidase (NO) as a marker of
CYP 3A. Phase II enzymes activities such as UGT and GST, involved in detoxication of carcinogens, were also measured. In addition, immunoblot analyses were performed to assess the microsomal levels of CYP isoenzymes (2B1/2, 2E1, 3A) and the cytosolic levels of GST subunits (Al /A2, A3/A5, Ml, M2 and P1).
2. Materials and methods 2.1. Chemicals The onion powder was obtained from Socie´te´ de Transformation Le´gumie`re (Auxonne, France). This powder was prepared from long-day yellow onions (Allium cepa) grown in the plain of Dijon (France). Onions were washed and sliced before dehydrating through a conventional hot-air oven (temperature gradient from 85 to 45 C) until the product retained less than 6% moisture. The polyclonal antibodies against rat CYP 2B were a gift from Professor A.-M. Batt (Centre du Me´dicament, Nancy, France) and the polyclonal antibody against rat CYP 2E1 was a gift from Professor P. Beaune (Universite´ Paris V, Paris, France). The polyclonal antibodies against rat GSTAI/A2, GSTA3/A5, GSTM1, GSTM2 and GSTP1 were obtained from Biotrin International (Dublin, Ireland). Other chemicals were of the highest quality available. 2.2. Characterisation and quantification of flavonoids The onion powder (5 g) was mixed with 30 ml of methanol. The alcoholic fraction was collected and filtrated and made up to 50 ml methanol. Flavonoids were separated and characterised by HPLC diode array detection. Separation of flavonoids was performed using a Alltima C18 (1504.6 mm, 5 m) at 35 C. The mobile phase (flow rate 1 ml/min) consisted in acidified distilled water adjusted to pH: 2.6 with o-phosphoric acid and acetonitrile. The separation was obtained using a linear gradient from 5 to 40% acetonitrile during 24 min. Flavonoids were identified by the comparison of their retention times and their spectra with the structures described by Price and Rhodes (1997) in onions analyzed under similar conditions. The same mass spectral measurements have been obtained using a Micromass detector-Platform LCZ- (data not shown). Flavonoids were quantified using external calibration at 365 nm and were expressed in mg/kg onion powder as equivalent quercetin. 2.3. Separation and characterisation of sulphur compounds The onion powder (2 g) was mixed with 2 ml distilled water. Compounds emitted in a closed 4-ml vial at room
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temperature were trapped for 60 min and transferred to the GC–MS injector by headspace SPME (solid phase microextraction, polyacrylate fibre 85 m). GC–MS analysis was carried out on a benchtop Perkin-Elmer Turbomass system with a split-splitless injector and a fusedsilica capillary column (10 m0.32 mm) with a 4 m methylsilicone coating. This column was chosen because it can analyse polysulphides, thiophenes and even very labile sulphur compounds such as the lacrymatory factor, without degradation (Arnault et al., 2000). The carrier gas was helium (99.999%) and the column temperature was programmed from 70 to 250 C at a rate of 5 C/min. The injection port temperature was 200 C. Total ion chromatograms and mass spectra were recorded in the electron impact ionisation mode at 70 eV. The transfer line and the source temperature were maintained at 150 C. The identification of sulphur compounds was accomplished by comparing the retention time and the mass spectra data with those of compounds available from NIST library and from previous data (Ferary and Auger, 1996; Ferary et al., 1996). 2.4. Animals and dietary treatment The experiment was performed with male SPF Wistar rats. 5-Week-old rats were purchased from Iffa Credo (L’Arbresle, France) and were housed in individual stainless-wire cages, maintained at 22 C with a 12-h light/dark cycle. They had free access to water. They were fed a purified diet for 2 weeks. The composition is reported in Table 1. Afterwards, the onion powder was incorporated into the diet at the expense of sucrose and cellulose (Table 1). After 9 days of feeding rats, were killed after 16 h of fasting. 2.5. Preparation of microsomal and cytosolic fractions The animals were killed by exsanguination. Livers were immediately removed. Microsomes and cytosols
Table 1 Composition of the experimental diets (g/kg of diet)
were prepared by differential centrifugation and stored in small aliquots at 80 C (Haber et al., 1994). The protein levels of the microsomal and the cytosolic fractions were measured by the method of Bradford (1976) adapted for the use of a Cobas Fara II centrifugal analyser (Roche Instruments) using serum albumin as standard. 2.6. Enzyme assays Total microsomal P450 content was quantified according to Omura and Sato (1964). Determination of phenoxazone O-dealkylase activities (EROD, MROD and PROD) was adapted from the method of Burke et al. (1985). Reactions were carried out in a fluorimeter at 37 C using a Cobas Fara II centrifugal analyser (Roche Instruments). The concentrations of substrates ethoxy-, methoxy- and pentoxyresorufin were respectively 5, 5 and 10 mm. Assay for PNPH activity was determined by HPLC according to Tassaneeyakul et al. (1993). The concentration of the substrate p-nitrophenol was 0.1 mm and the microsomal protein was 0.5 mg/ml. The product of the reaction, p-nitrocatechol was monitored at 340 nm. NO activity was determined by HPLC using a method adapted from Guengerich et al. (1986). The concentration of the substrate nifedipine was 0.2 mm and the microsomal protein was 1 mg/ml. UV detection of the product dehydronifedipine was performed at 254 nm. Total GST activity was measured with 1-chloro-2,4dinitrobenzene (CDNB) as substrate according to Habig et al. (1974). The reaction mixture contained 1 mm GSH and 1 mm CDNB. The formation of the conjugate was continuously monitored at 340 nm. The UGT activity was determined with p-nitrophenol as a substrate by the method of Mulder and van-Doorn (1975) using 3 mm UDP-glucuronic acid and 0.15 mm p-nitrophenol. Microsomes were activated with Triton X- 100 so that the ratio of Triton to protein concentration was 0.2. The measurement of GST and UGT activities were adapted for the use of a Cobas Fara II centrifugal analyser (Roche Instruments). 2.7. Immunoblot analyses
Components
Control
Onion
Casein Sucrose Starch Cellulose Mineral mixture Onion powder d,l-Methionine Corn oil Vitamin mixture+water (ml)a
230 270 360 40 50 – 1 50 500
210 130 360 – 50 200 1 50 500
a
983
The dry matter was mixed with water containing the vitamin mixture.
Immunoblot procedures were performed as previously described by Haber et al. (1994). The quantification of the individual bands was done by comparing blot density between treated and control rats, using an image analyser (Bioscan Optimetric, Edmonds, WA, USA). 2.8. Statistical analysis The Student’s t-test was used for comparing the onion-treated group to the control group. The level of significance was P < 0.05.
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3. Results 3.1. Flavonols and sulphur compounds present in the onion powder The following flavonols were identified in the onion powder: quercetin 3,40 -diglucoside, quercetin 40 -glucoside, isorhamnetin diglucoside, isorhamnetin 40 -glucoside and free quercetin (Table 2). Quercetin 3,40 diglucoside and quercetin 40 -glucoside represented the major components (more than 90%). The ratio of quercetin 3,40 -diglucoside:quercetin 40 -glucoside was 1:1. These results are in good agreement with those reported by Price and Rhodes (1997) and Fossen et al. (1998). The total content of flavonols was 8450 mg equivalent quercetin /kg dry weight. Fig. 1 shows the GC chromatographic separation of sulphur compounds of the onion powder. A total of 15 compounds were identified (Table 3). Most of them Table 2 Flavonol content of the onion powder Flavonols
Amounta
Quercetin 3,40 -diglucoside Quercetin 40 -glucoside Quercetin Isorhamnetin diglucoside Isorhamnetin 40 -glucoside Total
4170 3820 290 70 100 8450
a
mg/kg dry weight of the onion powder.
were alk(en)yl polysulphides: disulphides, trisulphides and tetrasulphides. In addition, two dimethyl thiophenes (2,4- and 3,4-dimethyl) were detected. Polysulphides had all the possible combinations of the 1propenyl, propyl and methyl thiomoieties. Dipropyl disulphide, methyl 1-propenyl trisulphide and propyl 1propenyl trisulphide were predominant. During the process of dehydrating, polysulphides and dimethylthiophenes were formed from lacrymatory factor, thiosulphinates and zwiebelanes, which are volatile compounds present in fresh onion. Therefore the pattern of onion powder is very different from that of fresh onion analysed in the same conditions (Arnault et al., 2000). Table 3 Sulphur compounds identified in the onion powder by GC–MS Peak no.
Compounds
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Lacrymatory factor 2,4-Dimethylthiophene 3,4-Dimethylthiophene Dimethyl trisulphide Dipropyl disulphide Propyl 1-propenyl disulphide Methyl propyl trisulphide Methyl 1-propenyl trisulphide Dimethyl tetrasulphide Dipropyl trisulphide Propyl 1-propenyl trisulphide Di(1-propenyl) trisulphide Methyl 1-propenyl tetrasulphide Dipropyl tetrasulphide Propyl 1-propenyl tetrasulphide
Fig. 1. GC chromatogram of the onion powder.
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C. Teyssier et al. / Food and Chemical Toxicology 39 (2001) 981–987 Table 4 Effect of onion consumption on body weights and liver weights of rats
Body weightsa Liver weightsa Relative liver weightb
Control
Onion
2084 7.60.2 3.670.05
1983* 7.20.2 3.660.04
Rats received the onion powder into the diet (20%) for 9 days. Values are meansS.E.M. of six rats. a g. b g liver per 100 g body weight. * Differs significantly from control mean (P40.05) Student’s t-test
Table 5 Effect of onion consumption on liver drug-metabolizing enzymes Control
Onion
a
0.520.03 0.570.01 Cytochrome P450 Ethoxyresorufin O-deethylase (CYP 1A1)b 361 502* 263 Methoxyresorufin O-demethylase (CYP 1A2)b 212 Pentoxyresorufin O-dealkylase (CYP 2B1/2)b 162 254* p-Nitrophenol hydroxylase (CYP 2E1)c 0.730.03 0.480.07* 0.790.09 0.770.08 Nifedipine oxidase (CYP 3A)c Glutathione S-transferased 88638 106751* UDP-glucuronosyltransferased 11.50.5 21.20.7* Rats received the onion powder into the diet (20%) for 9 days. Values are means S.E.M. of six rats. a nmol/mg microsomal protein. b pmol/min/nmin of CYP. c nmol/min/nmol of CYP. d nmol/min/mg of protein. * Differs significantly from control mean (P40.05) Student’s t-test.
3.2. Effects of consumption of the onion powder on drugmetabolizing enzymes At the end of the experimental period there was a slight decrease of the body weights of rats but no significant difference in the relative liver weights (Table 4). The total CYP content was slightly increased, although not significantly (Table 5). EROD, MROD and PROD activities were increased by 39, 24 and 56%, respectively. The increase was significant with EROD and PROD activities (Table 5). PNPH activity was decreased by 34% in rats consuming onion. Therefore, it was of interest to determine the levels of these CYP isoforms. Table 6 showed that the levels of CYP 2E1 were decreased by 15% while CYP2B1/2 isoforms were not detected in either group. UGT and GST activities were increased in rats fed with the onion powder (Table 5). GST levels of different classes a, m and p were analysed by immunodetection. GST A1/A2 was increased by feeding onion, whereas GST A3/A5 and GST M2 were decreased, and GST M1 was unchanged (Table 6). GST P1 subunit was not detected in the both groups.
Table 6 Effect of onion consumption on the expression of CYP isoenzymes and GST subunits (% of control) Control CYP isoenzymes CYP 2B1/2 CYP 2E1 GST subunits GST A1/A2 GST A3/A5 GST M1 GST M2 GST P1
Onion
nd 100
nd 8515
100 100 100 100 nd
20010* 687* 10418 569* nd
Rats received the onion powder into the diet (20%) for 9 days. Values represent % of control rat for each isoform. Values are means S.E.M. of six rats. For immunoblotting with anti CYP 2B diluted (1:500), 25 mg of microsomal proteins were loaded. For immunoblotting with anti CYP 2E1 diluted (1:1000), 40 mg of microsomal proteins were loaded. For immunoblotting with anti GST A1/A2 and anti A3/ A5 diluted (1:200), 10 mg of cytosolic proteins were loaded and for immunoblotting with anti GST M1, M2, P diluted (1:400), 10 mg of cytosolic proteins were loaded. nd: not detectable. * Differs significantly from control mean (P40.05) Student’s t-test
4. Discussion In this study we have demonstrated that feeding rats with a diet supplemented with onion powder for 9 days modulates drug-metabolizing enzymes. Consumption of onion produced an increase of EROD and PROD activity markers of CYP 1A and CYP 2B. The increase of PROD activity was not accompanied by an increase of CYP 2B1/2 levels. This could be explained by the fact that this activity is supported by other forms of CYPs. In addition, the increase of this activity remains very low when compared to the increase obtained with the classical inducer phenobarbital (Burke et al., 1985). Consumption of onion produced no significant effect on NO activity, while it decreased PNPH activity by 34%. This latter decrease was accompanied by a 15% decrease of CYP 2E1 levels. This result suggests the possible action of onion on reducing the activation of low molecular weight carcinogens or nitrosamines which are metabolized by this CYP isoform (Guengerich et al., 1991). We also examined the effects of onion consumption on phase II enzymes. Total GST activity was slightly but significantly increased. Therefore, it was of interest to check the variations of the relative proportions of different subunits of GST. GST subunits Al/A2 were increased. These results suggest that onion could prevent the genotoxic action of carcinogens detoxified by GST A1/A2, which were reported to be involved in the detoxication of heterocyclic amines and benzo[a]pyrene (Singh et al., 1985; Lin et al., 1994). Conversely, GST A3/A5 and GST M2 were lowered. This would mean
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that onion could increase the genotoxic effect of carcinogens which are detoxified by these subunits (Hayes and Pulford, 1995). UGT activity was measured with p-nitrophenol assumed to represent the UGT 1A6 isoform. This isoform is actively involved in the detoxification of chemical carcinogens such as polycyclic hydrocarbons and heterocyclic aromatic amines (Bock, 1991). p-Nitrophenol-UGT activity was significantly increased. Therefore, the protective effect of onion against the carcinogenic action of these compounds could be expected. However, biotransformation of carcinogens by glucuronidation or conjugation with glutathione can sometimes lead to activation (Monks et al., 1990). Therefore, it would be interesting to know whether onion consumption could modify these enzymes involved in the activation. Previous studies have shown that microconstituents of onion such as flavonols (quercetin) or organosulphur compounds (polysulphides) were able to modify the levels of phase I and phase II enzymes when administered in isolation (Nijhoff et al., 1993; Siess et al., 1997). In a previous study, we showed that quercetin when incorporated in a diet at 3000 ppm produced no effect on liver drug-metabolizing enzymes (Siess et al., 1989). In the present study, we identified in the onion powder quercetin and also glycosides of quercetin. Taking account of the levels of quercetin glycosides in the onion powder (8280 mg/kg dry weight, Table 2), the level of quercetin in the diet is around 1600 ppm. Therefore, the involvement of quercetin in the inducing effect of onion is probably not possible. However, it cannot be excluded that the bioavailability of quercetin would be better when ingested through the onion. It was actually demonstrated by Hollman et al. (1997) that conjugating quercetin with glucose enhanced its absorption from the small gut. Therefore the role of quercetin from onion cannot be excluded. The modulating effect of onion consumption on drug-metabolizing enzymes could be attributed partly to the sulphur compounds. Among the organosulphur compounds identified in the onion powder, dipropyl disulphide, methyl 1-propenyl trisulphide and propyl 1-propenyl trisulphide were predominant. Although it is difficult to attribute the effect to a specific component, the induction pattern observed with onion is similar to that produced by different propyl or methyl sulphides fed alone: decrease of CYP 2E1 and induction of UGT and GST (Siess et al., 1997). This suggests that polysulphides are probably involved in the overall effect of onion. Sulphur compounds and glycosides of quercetin are present simultaneously in onion. It was reported that the combination of a sulphur compound and quercetin was more efficient in reducing the mammary cancer than the single administration of these compounds (Ip and Ganther, 1991). Thus, the different components present in onion could have probably an additive action.
In this experiment, the modulating effect of onion was observed with administered doses that are much higher than the possible intake through a normal human diet. Therefore, it would be of great interest to explore whether smaller doses may have produced the same effects. Further studies are needed to determine the dose of onion in the diet and the duration of animal feeding necessary to obtain such drug-metabolizing enzyme modifications and thereby a possible chemoprotective effect of this vegetable. Moreover, it would be interesting to appreciate the effect of onion consumption in humans. This information could be useful in the development of a strategy for cancer prevention by eating fruits and vegetables.
Acknowledgements The authors thank M-F. Vernevaut for excellent technical assistance and P. Goupy for analyzing flavonoids. This work was supported by a grant of Conseil Re´gional de Bourgogne (France) and a grant of the french Ministe`re de Ia Recherche (no. 98 P0470).
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