Comp. Biochem. Physiol. Vol. 103B,No. 4, pp. 863-867, 1992 Printed in Great Britain

0305-0491192$5.00+ 0.00 © 1992PergamonPress Ltd

EFFECTS OF PHENOL COMPOUNDS, GLUTATHIONE ANALOGUES AND A DIURETIC DRUG ON GLUTATHIONE S-TRANSFERASE, GLUTATHIONE REDUCTASE AND GLUTATHIONE PEROXIDASE FROM CANINE ERYTHROCYTES MASAAIOKURATA,MASATOSHISUZUKI'~and ~SUKE TAKEDA* Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Gifu University, Gifu 501-1I, Japan (Tel. 0582 30-111I); and *Toxicological Research Laboratories, Chugal Pharmaceutical Co., Ltd., Minowa-machi, Kamiina-gun, Nagano 399-46, Japan (Tel. 0265 79-6691) (Received 15 May 1992; accepted 26 June 1992)

Abstract--l. Phenol compounds (eilagic acid, quercetin and purpurogallin), glutathione analo~_es (S-hexylglutathione and S-octylglutathione) and a diuretic drug (ethacrynic acid) were compared for their inhibitory effects on glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in the canine erythrocytes. 2. All these compounds inhibited GST activity; quercetin was found to be the most potent inhibitor. 3. Ellaglc acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity; S-hexylglutathione and S-octylglutathione had no effect on GR and GSH-Px activities. 4. Quercetin and purpurogaUin inhibited GST non-competitively toward glutathione, whereas ellagic acid showed a competitive inhibition. Ellagic acid and purpurogallin inhibited GR non-competitively toward oxidized glutathione.

metabolizing enzymes such as GSH-Px and GR in the mammalian erythrocytes have not been studied. We have, therefore, examined the effects of six of the GST inhibitors on glutathione metabolizing enzymes of canine erythrocytes, namely GST, G R and GSH-Px.

INTRODUCTION

Glutathione S-transferase (GST), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) are widely distributed in animal tissues, where they serve several physiological functions. GST catalyses a variety of reactions in which reactive electrophilic centres on chemicals including xenobiotics are detoxified by conjugation to the thiol group of giutathione (GSH). GSH-Px catalyses oxidation of GSH to oxidized glutathione (GSSG) by hydrogen peroxide or lipid peroxides. G R promotes the reduction of GSSG by nicotinamide--adenine dinucleotide phosphate (reduced form; NADPH) and H + to GSH. It is generally accepted that these glutathione metabolizing enzymes play important roles in the protection of mammalian cells against oxidative and alkylating agents. A number of compounds including giutathione analogues (Mannervik and Danielson, 1988), diuretics (Ahokas et aL, 1985; Ploemen et al., 1990), phenol compounds (Das et al., 1984, 1986) and anti-inflammatory agents (Wu and Mathews, 1983) have been used as GST inhibitors in liver and other tissues in humans and experimental animals. Das et al. (1986) showed the inhibitory effects of phenol compounds on the GST in human erythrocytes. Dirr and Schabort (1988) reported the inhibitory effects of bromosulphophthalein, S-hexylglutathione, oxidized glutathione and cholate on the GST activity in rat erythrocytes. However, such studies in other animals have not been reported. Also, the GST inhibitory affects of these compounds on other giutathione

MATERLALSAND MErlIODS Chemicals. Ellaglc acid, S-octylglutathione, S-hexylglutathione, ethacryulc acid, purpurogallin, quercefin, GSH, GSSG, l-chloro-2,4-dinitrobenzene (CDNB), t-butyl hydroperoxide (tBH), flavin adenine dinucleotide (FAD) and yeast glutathione reductase were purchased from Sigma, St Louis, MO. NADPH was obtained from The Oriental Yeast Co., Tokyo. All other chemicals were of highest purity commercially available. Ellaglc acid, S-octylglutathione, S-bexylglutathione, purpurogallin and quercetin were dissolved in 0.001-0.050N sodium hydroxide solution and then they were neutralized. Ethacrynic acid was dissolved in 1 M Tris-HCl buffer (pH 8.0) and 0.5 M phosphate buffer (pH 6.5). Enzyme preparation and enzyme assay. Blood was collected from the cephalic vein of male beagle dogs (Beagle/ CSK, Canis familiaris) u~ng heparin as an anticoagulant. The separation of erythrocytes, enzyme preparations and enzyme assays were done by the methods of Beutler (1984). Haemoglobin concentration was determined by the standard laboratory method. Inhibition study. The enzymes were exposed to the inhibiton for five minutes before the sub6trates were added to the reaction solution. The concentration of inhibitor re~ting in 50% inhibition, the Is value, was determined from plots of remaining activity versus the inhibitor concentration. K~ and K~values were calculated from double reciprocal plots (Lineweaver-Burk plot) using various concentrations of substrates.

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Inhibitors for canine erythrocyte enzymes

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and noncompetitive inhibition toward CDNB (B) by 10/~M eUagic acid. RESULTS

In this study, the GST activity was measured using GSH and CDNB as the substrates. The Y~ values of GST for GSH and CDNB were 1 . 2 + O . l x 10-3M (mean-t- SE, n - - 6 ) and 5.5+0.8 x 10-4M, respectively. Figure 1 shows the dose dependent inhibitory effects of S-octylglutathione, S-hexylghitathione, ethacrynic acid, ellagic acid, quercetin and purpurogallin on the GST activity in canine erythrocytes. The I~o values of these GST inhibitors were as follows quercetin (1.5 + 0.3 x 10 -6 M, M + SE, n ffi6) < S-octylglutathione (2.1 + 0.2 x 10-eM) < purpurogallin (2.5 + 0.1 x 10-eM) < ethacrynic acid (6.6 + 0.5 x 10 -6 M) < ellagic acid (7.2 + 0.5 × 10-6M) < S-hexylglutathione (9.5 + 0.6 x 10 -6 M). Thus, quercetin was the most potent inhibitor and S-bexylglutathione the weakest. As shown in Figs 2 and 3, quercetin and purpurogallin caused a

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Fig. 5. Effects of S-octylglutathione, S-hexyll0utathione, ethacrynic acid, eUagicacid, qnercefin and purpurogallin on canine erythrocyte GR activity. Data represent the mean + standard error (n = 6).

noncompetitive inhibition of GST against GSH (the /~. values were a 2.4 + 0.5 x 10 -e M and 4.0 + 0.6 x 10 -6 M, respectively, n ffi 5), while a competitive inhibition with respect to CDNB. The /~. values were 1.2 + 0.2 x 10 -6 M and 1.6 _+0.3 x 10 -6 M, respectively. In contrast, ellagic acid inhibited the GST competitively toward GSH with the /~. value of 4.4 + 0.6 x 10 -6 M and noncompetitively toward CDNB with the Ki value of 9.4 + 1.6 x 10-eM (Fig. 4). T h e / ~ values of canine erythrocyte G R for GSSG and N A D P H were 1.4+0.1 x 10-4M (n =6) and 1.1 + 0.2 x 10 -5 M, respectively. The dose-dependent inhibitory effects of GST inhibitors on the G R activity of canine erythrocytes in the presence of F A D are shown in Fig. 5. Ellagic acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity. The Iso values were 18.2 + 3.9, 45.6 + 3.1, 99.8 + 3.0 and 456.6 + 43.2 x 10-6 M, respectively. Thus, these chemicals were less potent for G R than for GST. Two glutathione analogues, S-octylglutathione and Shexylglutathione, did not cause an obvious inhibition of the G R activity even at a concentration of 5 x 1O-4 M. Ellagic acid and purpurogaUin inhibited the GR activity in a non-competitive inhibition for GSSG with Ki values of 10.1 + 1.1 × 10-6M and 19.3 + 2.2 x 10 -6 M, respectively, while they caused an uncompetitive inhibition for NADPH with /~ values of 9.1 + 1.4 x 10-6 M and 16.4 -l- 1.9 x 10 -6 M, respectively (Figs 6 and 7). S-Octylglutathione, S-hexylglutatione and ethacrynic acid at a concentration of 5 x 10 -+ M were ineffective on GSH-Px activity using tBH as a substrate. The effects of quercetin, purpurogallin and ellagic acid on GSH-Px coul'd not be evaluated, since GR (from yeast) is used for GSH-Px and these phenols inhibit the G R activity. DISCUSSION

Many chemicals including glutathione analogues, phenol compounds and diuretic drugs have been known as the inhibitor of GST in human and rat

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thione metabolizing enzymes, its analogues could inhibit only GST activity. As quercetin, purpurogaUin and ellagic acid inhibited GST and GR by both competitive and uncompetitive manners, indicating that these compounds may affect the active site of the enzyme. The nature of GST inhibition by quercetin and purpurogallin was a competitive inhibition with respect to CDNB. It may easily be explained by the fact that GST utilize many chemicals including CDNB as the substrate. On the other hand, ellagic acid inhibited GST competitively toward GSH, as was also shown by Das et al. (1984) in rat liver GST. If ellagic acid is similar to glutathione in its chemical structure, it should also competitively inhibit GR toward oxidized glutathione. Contrary to this expectation, the inhibition of GR by ellagic acid was in a non-competitive manner with respect to oxidized glutathione. Since the mechanism of the inhibition of GST and GR by phenol

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Inhibitors for canine erythrocyte enzymes compounds is still not clear, further kinetic studies should be carried out. Ellagic acid and quercetin are known as naturally occurring plant phenols, and are widely distributed among many kinds of vegetables and fruits that are regularly consumed by animals (Alldrick et al., 1986; Brown, 1980). Interestingly, ellagic acid enhances the GST of liver by oral administration in mice (Das et aL, 1985). In addition, Pardini et al. (1988) found that the insects eating quercetin-containing plants possess relatively high G R activity. These reports clearly contradict the/!1 vitro data of Das et al. (1984, 1986) and our present data on the inhibition of GST and GR. It is conceivable that the animals may have adapted to the daily exposure of naturally occurring phenols by enhancing the activities of both GST and GR. Acknowledgements--We are grateful to Professor Nihal S. Agar, Department of Physiology, University of New England, Armidale, NSW 2531, Australia, for his comments in the preparation of this manuscript. Helpful discussions with Mr Koulchi Haruta, Tadashi Asano and Kazuya Kimura of Toxicological Research Laboratories, Chugal Pharmaceutical Co., Japan, are also gratefully acknowledged. REFERENCES

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activity of mutagens from cooked food. Mutat. Ices. 163, 225-232. Ikutler E. (1984) Red Cell Metabolism: A Manual of Biochemical Methods Ord edn). Grune and Stratton, New York. Brown J. P. (1980) A review of the genetic effects of naturally occurring fiavonoids, anthraquinones and related compounds. Mutat. Ices.75, 243-277. Das M., BickersD. R. and Mukhtar H. (1984)Plant phenols as in vitro inhibitors of giutathione S-transferase(s). BIOchem. Biophys. ICes. Commun. 1~0, 427--433.

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