Inhibitory effect of phenolic compounds on aflatoxin B1 metabolism and induced mutagenesis

Mutation Research, 177 (1987) 229-239

229

Elsevier MTR 08642

Inhibitory effect of phenolic compounds on aflatoxin B 1 metabolism and induced mutagenesis R.H.C. San and R.I.M. Chan Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3 (Canada)

(Received2,1 February 1986) (Revision received6 October 1986) (Accepted 14 October 1986)

Key words: Phenolic compounds; Aflatoxin B1, inhibition (Salmonella typhimurium); Gallic acid; Chlorogenic acid; Caffeic acid;

Dopamine; p-Hydroxybenzoicacid; Salicyclicacid Summary The interaction between phenolic compounds and the food-borne carcinogenic mycotoxin, aflatoxin Bx (AFB1), was examined. 6 phenolic compounds (gallic acid, chlorogenic acid, caffeic acid, dopamine, p-hydroxybenzoic acid and sahcyclic acid) inhibited AFBl-induced mutagenesis in Salmonella typhimurium strain TA98 in a suspension assay in the presence of rat-liver microsomes ($9). The inhibitory effect was observed when the phenolic compound and the mutagen (AFB 1 plus $9) were administered concurrently, but not when exposure to the mutagen was followed by the phenolic compound. The concentrations of the phenolic compounds used were not mutagenic to S. typhimurium strain TA98 and had no effect on the survival of the bacteria. The inhibition of AFB 1 metabolism was studied using high-pressure liquid chromatography. Increasing the concentration of all 6 phenolic compounds resulted in a dose-dependent reduction of both major AFB 1 metabolite peaks. The results are consistent with the hypothesis that (1) the phenolic compounds do not react covalently with AFB 1, and (2) the inhibitory effect of phenolic compounds on AFBl-induced mutagenesis may be due to the inhibition of the activation enzymes.

Epidemiological evidence suggests that a substantial portion of human cancers are attributable to dietary factors. At the same time, the question arises as t o the role of dietary components in reducing the impact of environmental carcinogens on man and in the prevention of human cancers. The increased consumption of green/yellow vegetables has been associated with a lower risk for cancer at various sites (Graham et al., 1978; Correspondence: Dr. R.H.C. San, EnvironmentalCarcinogenesis Unit, British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3 (Canada).

Hirayama, 1979; Mettlin et al., 1981). Phenolic compounds are commonly present in fruits and vegetables and are consumed daily in gram quantities (Brown, 1980). These compounds are effective in suppressing neoplasia in animals (Wattenberg et al., 1980) and carcinogen-induced mutagenesis (Brown, 1980; Huang et al., 1985; Terwel and van der Hoeven, 1985). However, phenolics are also known to exhibit genotoxic effects (Stich and Powrie, 1982) and to enhance mutagenesis (Kaul and Tandon, 1981; Shimoi et al., 1985). The basis for the observed ambivalent activity of phenolic compounds remains to be explained.

0027-5107/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)

230 In this paper the interaction between phenolic compounds and the food-borne carcinogenic mycotoxin, aflatoxin B1 (AFB1), was examined. Specifically, we report on the effect of plant phenolics on (1) AFBl-induced mutagenesis in strain TA98 of Salmonella typhimurium, and (2) AFB 1 metabolism in the presence of rat-liver microsomes. Materials and methods

Salmonella mutagenicity suspension assay The mutagenic action of the procarcinogen AFB~ was assayed in the presence of liver microsomes ($9) prepared from Aroclor 1254-induced rats, as described by Ames et al. (1975). Mutagenicity studies were conducted in S. typhimurium strain TA98 using the suspension procedure (Rosin and Stich, 1978a,b), a modification of the basic method of Ames et al. (1975). This procedure permits an estimation of the survival as well as the incidence of reverse mutations of the bacteria. An inoculum of the frozen bacterial stock was incubated overnight on a rotary wheel in Difco nutrient broth at 37°C. An aliquot (0.1 ml) of this stationary-phase culture was then reinoculated into 5 ml fresh nutrient broth and incubated for 4 h at 37°C to give a logarithmically-growing culture (5 × 108 cells/ml). 1-ml aliquots of this culture were placed in centrifuge tubes and the bacteria pelleted by centrifugation. The pellets were resuspended in 0.5 ml of phosphate-buffered saline (PBS) at pH 7.4 containing phenolic compound (when required), AFB 1 and $9. AFB 1 was dissolved in dimethyl sulfoxide (DMSO), with the final solvent concentration being 5% in the incubation mixture. Following incubation for 20 min at 37°C in a water-bath, the bacteria were pelleted by centrifugation, resuspended in PBS, centrifuged once more, and resuspended in PBS at approximately 10 9 cells/ml. Aliquots (0.1 ml) of each sample were added to 2.0 ml molten top agar (0.455 mM histidine) and overlaid on minimal glucose agar plates in triplicate. Aliquots of the treated bacterial culture were also diluted with 0.9% sodium chloride solution and plated onto nutrient agar plates in triplicate to determine cell survival. The nutrient agar plates were incubated for one day, while the minimal agar plates were

incubated for two days prior to scoring on an Artek 880 automatic colony counter (Artek Systems Corp., Farmingdale, NY). The mutagenic activities were calculated in terms of the number of his + revertannts per 107 surviving bacterial cells (Rosin and Stich, 1978a,b). Some experiments involved two consecutive incubation periods of 20 min each at 37°C. The bacteria were centrifuged and washed once before they were resuspended in the second treatment mixture. These experiments were conducted in order to determine the effect of treating the bacteria with test chemicals before or after exposure to AFB 1 and $9 on the mutagenic activity of the carcinogen.

Analysis of aflatoxin B 1 metabolism using high-pressure liquid chromatography (HPLC) Treatment mixtures with pH adjusted to 6.5 and consisting of 0.5 ml phenolic compound, 0.5 ml AFB 1 and 0.5 ml standard $9 preparation were incubated at 37 ° C for 20 min in a gyratory shaker. After the incubation period, the samples were processed as described by Lin et al. (1978). To stop the reaction, 1 ml of the treatment mixture was added to 2.0 ml ice-cold ethanol and 0.075 ml 2 M NaC1 in a centrifuge tube, vortexed and allowed to stand on ice for 30 min. At the end of this time, the samples were centrifuged at 3000 rpm for 5 min and the supernatants removed for analysis by HPLC. HPLC was carried out using a Vydac 201 TP, 10-/~m particle reverse phase column with 3.2 × 250 mm inside dimensions (Spruce Herperia, CA) and a Spectro-Physics SP 8700 solvent delivery system (Santa Clara, CA). The solvent was initialized at 0% methanol in double-distilled water with a gradient of 12.5% methanol/rain, raising the methanol concentration of 50% at 4 min, and then held at 50% until 6 min before resetting to 0% methanol. The flow rate was 1 m l / m i n . The samples were monitored by a Varian Fluorichrom fluorescence detector (Palo Alto, CA) using an excitation wavelength of 360 nm and a 400 nm emission cut-off filter. The data were analyzed by a Perkin-Elmer Sigma 10 data station (Montreal, P.Q.). The aflatoxin M 1 (AFM1) standard used for the HPLC analysis was kindly provided by Dr.

231 D.P.H. Hsieh of the U n i v e r s i t y of California at Davis.

Results

Inhibition of aflatoxin Bl-induced mutagenesis by phenolic compounds T h e i n h i b i t o r y effect of a series of phenolic c o m p o u n d s of A F B l - i n d u c e d mutagenesis in the presence of $9 was studied using the Salmonella s u s p e n s i o n test. D o p a m i n e , gallic acid, caffeic acid, chlorogenic acid, p - h y d r o x y b e n z o i c acid a n d salicyclic acid all i n h i b i t e d A F B l - i n d u c e d m u t a genesis (Table 1 a n d Fig. 1). Salicyclic acid a n d p - h y d r o x y b e n z o i c acid were less p o t e n t inhibitors t h a n the other phenolic c o m p o u n d s tested. The c o n c e n t r a t i o n s of the phenolic c o m p o u n d s used were n o t m u t a g e n i c to S. typhimurium strain TA98 a n d had n o effect o n the survival of the bacteria. A F B : at 3 X 10 -5 M was toxic, decreasing the survival of the Salmonella to 70% of that i n the u n t r e a t e d control (data n o t shown). However, the a d d i t i o n of phenolic c o m p o u n d s in all cases red u c e d the toxic effect of AFB1 as well as its m u t a g e n i c activity. The effect of p r e t r e a t m e n t a n d of post-treatm e n t with chlorogenic acid o n the suppression of

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Fig. 1. Effect of phenolic compounds on cell survival and reversion frequency of aflatoxin B:-exposed Salmonella typhimuriumTA98 in the suspension test. AFB1 concentration: 3 X l0 -5 M. Phenolic compounds tested were dopamine (A), gallic acid (e), caffeic acid (Ha), chlorogenic acid (zx), p-hydroxybenzoic acid (O) and salicyclic acid (O). Plotted are +-S.D. based on 3 Expts. Values have been corrected for spontaneous reversion ( < 3 revertants/107 survivors).

TABLE 1 EFFECT OF CHLOROGENIC ACID ON CELL SURVIVAL AND REVERSION FREQUENCY OF AFLATOXIN B1-EXPOSED SalmonellatyphimuriumSTRAIN TA98 IN THE SUSPENSION TEST AFB1

Survivorsper plate a Expt. 1 2 3

Mean Revertants per plate " percent Expt.1 2 3 survival b

Mean revertants per 107 survivors 6

Mean percent mutagenic activity b

3×10 -5 M 9(2.5×10-2M) 6(1.7x10-ZM) 3(8.5X10-3M) l(2.8X10-3M) 0

400+47 391+- 4 468+-27 332+-10 249+-26

432+_52 493+-25 462+-60 428+-34 278+-18

364+-18 399+44 381+51 254+-24 231+-24

104+ 6 112+- 7 115±13 88+-18 66+- 4

5+- 1 12+- 4 23+- 6 51+-19 85+-10

6.0+- 0.9 14.6+- 5.5 26.5+- 6.3 58.7+14.9 100

9(2.5x10 2M) 6(1.7×10-2M) 3(8.5X10-3M) 1(2.8×10-3M) 0

419+-13 394+-15 409+-19 387+-31 363+26

387+-35 378+-30 417+13 460+-27 411+-34

397+-32 402+-55 361+-22 355+-22 370+-38

105+-11 103+-10 104+- 8 105+ 8 100

None

Chlorogenic acid (mg/ml)

72___ 5 110_+15 249+-20 501_+.+ 5 687+-37 28+28+ 24+21+24+-

2 3 5 2 7

55+-16 75+- 6 262+-23 164+- 8 363+-29 357+-19 506+-31 608+-35 710+-20 735+-23 17+- 2 18+- 3 17+- 4 25+- 4 17+- 5

31+38+29+33+30+-

5 5 6 5 6

2+2+2± 2+2+-

1 1 1 1 1

2.2+2.4+2.1+2.3+2.2+-

0.4 0.6 0.5 0.5 0.5

a For survivors per plate and revertants per plate, each value represents mean _+S.D. based on triplicate plating in each experiment. b For mean percent survival, mean revertants per 107 survivors, and mean percent mutagenic activity, each value represents mean + S.D. based on 3 Expts. each with triplicate plating.

232 A F B l - i n d u c e d mutagenesis was also examined. However, bacteria pretreated with PBS or chlorogenic acid and then subsequently exposed to AFB~ resulted in very low cell survival. Consequently, comparison of the effects of pretreatment and concurrent treatment of the inhibitor on AFBx-induced mutagenesis could not be made. It was observed, however, that post-treatment of the bacteria with chlorogenic acid after their exposure to AFB1 had no effect on the mutagenic activity of A F B 1 (Table 2).

Study of the inhibitory effect of phenofic compounds on aflatoxin B1 metabolism using high-pressure liquid chromatography The inhibition of A F B 1 metabolism was studied using high-pressure liquid chromatography. A F B 1 and the activation system $9 were incubated with phenolic c o m p o u n d s for 20 min at 37°C. At the end of this period, the activation of A F B 1 was terminated by precipitating out the enzymes with an N a C 1 / e t h a n o l mixture. The processing of the test materials was essentially identical to that of Lin et al. (1978), except that the samples were not filtered before injection into the H P L C column. The eluates were monitored at wavelengths greater than 400 n m by a fluorescence detector. Using this system, fluorescent c o m p o u n d s were eluted from the column in the order of their decreasing polarity. Figs. 2 a - 2 d show the c h r o m a t o g r a m s of mix-

tures of AFB~ and $9 incubated for various time periods. The first peak eluted from the column appears to be related to polar fluorescent substances associated with the $9 preparation. Incubation of $9 alone resulted in the appearance of this same peak (Fig. 2e). The last peak eluted from the column with a retention time of 7.76 min was the A F B 1 parental c o m p o u n d . Injection of AFB~ alone into the column resulted in the elution of a single peak at this same retention time (Figs. 2f and 2g). The other peaks in the chromatograms (Figs. 2 a - 2 d ) increased in size as the incubation time increased, indicating that they were peaks representing fluorescent AFB~ metabolites. The major metabolite peak with a retention time of 6.79 min had an identical retention time as that of the 4 - h y d r o x y - A F B 1 (AFM1) standard (Fig. 2h). The other peaks remained to be identified. The peak with the retention time of 6.28 min is m o s t likely 2 , 3 - d i h y d r o - 2 , 3 - d i h y d r o x y - A F B 1 (AFBl-dihydrodiol), the major A F B 1 metabolite p r o d u c e d b y rat-liver microsomal preparation (Lin et al., 1978). The other possible identity of this major peak is AFQ1, an A F B 1 metabolite possessing a hydroxyl group on the carbon a t o m fl to the carbonyl of the cyclopentenone ring (Neal and Colley, 1978). However, according to Masri et al. (1974), AFQ1 only represents a very small fraction of the A F B 1 metabolites produced by rat-liver preparation. The fluorescence detector used in this present study also failed to detect an AFQ1 stan-

TABLE 2 EFFECT OF CONCURRENT AND POST-TREATMENT WITH CHLOROGENIC ACID ON AFLATOXIN B~-INDUCED MUTAGENESIS IN Salmonella typhymurium STRAIN TA98 First treatment a

Second treatment a

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Mutagenicity Revertants per plate

Revertants/ 107 survivors c

AFB1 + $9

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2574- 36

69 + 12

414 + 4

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Ch.A.

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83+ 9

7+1d

a Samples of Salmonella were exposed to 1 of 3 treatment combinations: aflatoxin Bl (AFBt) concentration, 3×10 5 M; chlorogenic acid (Ch.A.) concentration, 9 mg/ml, i.e., 2.5 × 10-2 M; rat-liver microsomes ($9); phosphate-buffered saline (PBS). b Cell survival in nutrient agar compared to that observed in bacteria samples exposed to PBS only. ~ _+S.D. based on 3 replicate plates from each of 3 Expts. c .~ in bacteria exposed to PBS only: < 3 revertants per 1 0 7 survivors. d Significantly different from AFBt + $9 controls (P < 0.001).

233

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dard. The failure to detect AFQ1 is most likely due to the fact that AFQ 1 has fluorescent properties different from those of AFB 1, AFM1 and AFBl-dihydrodiol (Neal and Colley, 1978). Despite the appearance of metabolite peaks, the incubation of AFB~ with $9 did not result in any substantial decrease in the level of the AFB~ parental peak (Figs. 2a-2d). This observation suggested that only a small fraction of AFB 1 was actually metabolized by the standard $9 preparation. By increasing the concentration of $9 in the incubation mixture, a greater proportion of AFB 1 was metabolized (Fig. 3). Incubation of AFB 1 alone without $9 did not result in the appearance of any metabolic peaks or in any significant changes in the fluorescent level of the AFB 1 parental peak (Figs. 2f, 2g and 3a). When AFB 1 was incubated with $9 in the pres-

ence of chlorogenic acid, the two AFB~ metabolic peaks became reduced in size, but there were no changes in their respective retention times (Fig. 4b). The amount of reduction in metabolic peak size increased as the concentration of chlorogenic acid was increased (Figs. 4b-4d). This indicates a dose-dependent inhibitory effect of chlorogenic acid on the metabolism of AFB~. The chromatograms of incubation mixtures containing chlorogenic acid exhibited a broad fluorescent peak with approximately the same retention time as that of the $9 peak (Fig. 4e). The size or retention time of this peak was not affected by the addition of either $9 or AFB x (Figs. 4f and 4g). Incubating AFB 1 with chlorogenic acid in the absence of $9 did not result in the appearance of new peaks or changes in the fluorescent properties (peak size, peak height, retention time) of the AFB 1 parental

234 peak (Fig. 4g c o m p a r e d to 4h). These results suggest that A F B 1 a n d chlorogenic acid do n o t react covalently to p r o d u c e fluorescent complexes. N o new peaks appeared when chlorogenic acid, A F B 1 a n d $9 were i n c u b a t e d together, suggesting that covalently b o u n d fluorescent complexes between this phenolic c o m p o u n d a n d A F B t were not

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235

yield any fluorescent peaks. Salicylic acid exhibited a fluorescent peak with similar properties to that of chlorogenic acid. As in the case of chlorogenic acid, all 5 phenolic compounds showed a dose-dependent reduction of both major AFB t metabolite peaks (Fig. 5).

min in the absence of phenolic compounds. The reaction mixture was then analyzed by HPLC. Fig. 6a represents the inhibition of AFB t metabolism in the presence of caffeic acid. Fig. 6b shows that the level of AFM 1 already present in the heat-treated mixture was not decreased by the subsequent addition of increasing concentrations of caffeic acid. Furthermore, the level of AFM 1 present in Fig. 6b is comparable to that present at zero caffeic acid concentration in Fig. 6a. This suggests that the AFM z produced during the first 20 min of incubation of AFB~ with $9 was not affected by the heat-treatment and subsequent incubation with caffeic acid. The level of the other major metabolite peak was diminished substantially by the heat-treatment, probably suggestive of the heat-lability of this metabolite. Further incubation of the heat-treated mixture with caffeic

Interaction between phenolic compounds and preformed aflatoxin Bz metabolites In order to gain some insight into the interaction between phenolic compounds and preformed AFB~ metabolites, an experiment was carried out by incubating AFB t with $9 at 37°C for 20 rain to form metabolites. This incubation mixture was then heat-treated in a water-bath at 80°C for 2 rain to inactivate the enzymes present. The heated AFBz/S9 mixture was then cooled to room temperature with ice and incubated for a further 20

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236

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acid did not have any effect on this peak (Fig. 6b). In another experiment, AFB 1 and $9 were first incubated together to form AFB 1 metabolites and then, without heat-treating the mixture, this was followed by the addition of caffeic acid (Fig. 6c). The two AFB 1 metabolites decreased in level as expected when the concentration of caffeic acid was increased, However, the two metabolites were decreased only to levels comparable to those at zero caffeic acid concentration in Fig. 6a. This suggests that these two metabolites formed during the first 20 min of incubation in the absence of caffeic acid were not affected by the subsequent incubation with caffeic acid. The decrease seen in Fig. 6c may thus be attributed solely to the inhibition of AFB~ metabolism during the second 20-rain incubation period.

Discussion

The present study shows that a number of naturally occurring phenolic compounds inhibited AFBl-induced mutagenesis in S. typhimurium strain TA98. Since AFB 1 is a procarcinogen and promutagen, it requires metabolic activation before its mutagenic effect can be demonstrated (Garner et al., 1972; McCann et al., 1975; Wong and Hsieh, 1976; Campbell and Hayes, 1976; Ong, 1975). Consequently, a reduction in its mutagenic activity on bacteria can be due to interaction with the parental AFB 1 compound, or to an inhibition of the activation process or to the scavenging of the activated AFB 1 metabolite. The HPLC data indicate that AFB 1 metabolism was inhibited by phenolic compounds since the addition of the latter reduced the size of all major and minor metabolite peaks of AFB~. When the

237 concentration of the phenolic compounds was increased to a certain level, all metabolite peaks were completely suppressed. At the same time, the AFB 1 parental peak increased in size as the concentration of phenolic compounds was increased. This indicates that less AFB x was metabolized when phenolic compounds were present so that more AFB 1 was retained in the incubation mixture. Incubation of AFB1 with phenolic compounds in the absence of $9 did not alter the fluorescent level or the retention time of the AFB~ peak. If AFB~ and phenolic compounds could interact covalently to form a complex, it was not detectable by the fluorescent method. Garner et al. (1972) reported that AFB 1 does not bind to nucleic acid covalently in the absence of metabolic activation. Only non-covalent interactions between nonactivated AFB 1 and nucleic acid have been described (Clifford and Rees, 1967; Sporn et al., 1966). All these observations point to the fact that AFB 1 by itself is not a reactive electrophile, and consequently it is unlikely that AFB 1 will form covalent adducts with nucleophilic phenolic compounds. In the attempt to investigate whether phenolic compounds inhibit AFBl-induced mutagenesis by suppressing its metabolism, AFB~ was incubated with $9 at 37°C in the absence of S. typhimurium to form metabolites, then heat-treated in a waterbath at 80°C for 2 min to inactivate the enzymes present, and incubated for a further 20 min at 37°C with the bacteria in the absence or presence of a phenolic compound. However, this heattreated reaction mixture did not elicit any mutagenic activity in the Salmonella assay (data not shown), probably as a result of the destruction of the mutagenic AFB1 metabolites by heat. Based on the HPLC analysis, the data suggest that preformed AFB 1 metabolites did not react with the phenolic compound, chlorogenic acid. The lack of an effect of post-treatment with a phenolic on AFBx-induced mutagenesis in the Salmonella experiments (Table 2) is consistent with this hypothesis. The observations in the present study are supportive of the view that the inhibitory effect of phenolic compounds on AFBl-induced mutagenesis may be due to the inhibition of the activation

enzymes rather than to interaction with the metabolites. Some phenolic compounds have been shown to inhibit mitochondrial respiration (Cheng and Pardini, 1978, 1979), rat-liver mevalonate pyrophosphate decarboxylase and mevalonate phosphate kinase activities (Shama Bhat and Ramasarma, 1979). Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), both phenolic antioxidants, alter the pattern of metabolism of the carcinogen benzo[a]pyrene (Wattenberg, 1979). Not all phenolic compounds exhibit antimutagenic activity. Such effects depend on the potential inhibitor and the mutagen examined. For example, the phenolic antioxidants, BHA, BHT and propyl gallate, enhanced rather than inhibited AFBl-induced mutagenesis in S. typhimurium with rat-liver microsomal activation (Shelef and Chin, 1980). Certain phenolic compounds (e.g., BHA) also enhanced the genotoxic activity of the alkylating agent, propane sultone (Kaul and Tandon, 1981). While tannic acid reduced the mutagenicity of ultraviolet light and 4-nitroquinoline-l-oxide (4NQO) in Escherichia coli, it did not affect the mutagenic activity of "/-ray and N-methyl-N'nitro-N-nitrosoguanidine (MNNG) (Shimoi et al., 1985). The relative concentration of the phenolic compound may be a determinant of its effect on mutagens. Propyl gallate at 10 -2 to 10-4M concentration inhibits the mutagenic activity of 5 × 10 -6 M MNNG, 1.5×10 -5 M N-acetoxy-2acetylaminofluorene and 10-5 M AFB 1. However, propyl gallate at equimolar concentrations causes an enhancement of the mutagenic activities of the carcinogens N-hydroxy-2-acetylaminofluoreneand 4NQO (Rosin and Stich, 1980). The animal species studied may also determine whether the activation of carcinogens/mutagens will be increased or decreased. Buening et al. (1978, 1981) showed that certain naturally occurring flavonoids enhanced the mutagenicity of AFB 1 in the presence of human-liver microsomes, but no enhancement was observed with rat-liver microsomes. Various plant phenolics induce an array of genotoxic effects, including DNA breaks, point mutations, mitotic crossing-over, gene conversion, chromosome aberrations, and sister-chromatid ex-

238

change (reviewed by Stich and Powrie, 1982). Under acid or neutral pH conditions, phenolic compounds are relatively stable and induce a significant genotoxic effect only after the addition of transition metals (Stich et al., 1981) which enhance their oxidation (Martell, 1980). At alkaline pH ranges, a number of plant phenolics (including catechin, gallic acid, pyrogallol and resorcinol) induced gene conversion in Saccharomyces cerevisiae, whereas they lacked this capacity at acid pH levels (Rosin, 1984). The data described in this paper point to the possible preventive action of plant phenolics against carcinogens and mutagens. In order to assess the role of these dietary components in human cancer, there is a need to obtain a better understanding of the conditions which may influence the enhancement versus the inhibition of carcinogen metabolism.

Acknowledgements The authors would like to thank Dr. H.F. Stich for helpful criticism and suggestions. This~study was supported by the Natural Sciences and Engineering Research Council of Canada and by the National Cancer Institute of Canada. R.H.C. San is a staff member of the British Columbia Cancer Foundation of Vancouver, B.C.

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