Intracellular inhibition of UDP-glucose dehydrogenase during ethanol oxidation

Chem.-Biol. Interactions, 43 (1943) 283--288

283

Elsevier Scientific Publishers Ireland Ltd.

I N T R A C E L L U L A R INHIBITION OF UDP-GLUCOSE D E H Y D R O G E N A S E D U R I N G E T H A N O L OXIDATION*

T.Y. A W and D.P. J O N E S

Department of Biochemistry, Emory UniversitySchool of Medicine, Atlanta, G A 30322

(U.S.A.) (Received August 1st, 1982) (Accepted September 2nd, 1982)

SUMMARY

The enzymatic basis for inhibition of drug glucuronidation during ethanol oxidation was investigated in isolated rat hepatocytes. The intmcetlular rate of glucuronidation was varied independently b y controlling the steady-state 02 concentration and the concentrations o f UDP-glucose and UDP-glucuronic acid were measured in the absence and presence o f 20 mM ethanol. Ethanol caused substantial inhibition o f the glucuronidation rate which corresponded to a significant decrease in UDP~lucuronic acid concentration b u t not in UDP-glucose concentration. A plot o f glucuronidation rate as a function o f cellular UDP-glucuronic acid concentration yielded a single curve for incubations with or w i t h o u t ethanol; a similar plot of glucuronidation against UDP-glucose concentration gave separate curves for t h e t w o incubation conditions. These results clearly define the UDP-glucose dehydrogenase reaction as the site of inhibition during ethanol oxidation.

Key words: Ethanol -- Acetaminophen -- Glucuronide -- UDP-glucuronic acid -- Oxygen

INTRODUCTION

Substantial inhibition of drug glucuronidation by ethanol has been demonstrated in vivo [1] as well as in studies with isolated hepatocytes [2--4]. The intracellular rate of glucuronidation has been shown to be dependent on UDP~lucuronic acid concentration [5] and the concentration of UDP*This research was supported by N.I.H. grant GM-28176. Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC, high pressure liquid chromatography. 0009-2797/83/0000--0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

284 glucuronic acid is decreased during ethanol metabolism [4]. Molddus et al. [4] proposed that the inhibitory effect of ethanol is due to a decreased UDP-glucuronic acid synthesis caused by increased NADH/NAD ÷ during ethanol oxidation. To test this, we have examined the effect of ethanol on both UDP-glucose and UDP-glucuronic acid concentrations in isolated rat hepatocytes incubated under conditions which provide a wide range of glucuronidation rates. The results clearly define the UDP-glucose dehydrogenase reaction as the site of inhibition of glucuronidation during ethanol metabolism. MATERIALS

AND METHODS

Materials CoUagenase (Type IV) and acetaminophen were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other chemicals used were of reagent grade and purchased locally. Cell preparation and incubation Hepatocytes were prepared from adult male white rats (Charles River, 180--250 g) by the method described by Molddus et al. [6]. Cell viability was 97--99% as judged by exclusion of 0.2% trypan blue. The cells were viable for at least 5 h when maintained at 20°C in a gyrating water bath under air. Incubations (106 cells/ml) were carried out at 37°C in round-bottom flasks in Krebs-Henseleit buffer, containing 12.5 mM N-2-hydroxyethylpipemzine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4) as previously described [6]. Ethanol (20 mM) was added either 1 rain or 5 min prior to initiation of the reaction. Acetaminophen concentration was 5 mM. Oxygen dependences of glucuronidation, UDP-glucose and UDP-glucuronic acid concentrations in the presence of ethanol were studied under different O2 concentrations as previously described [7,8]. Regulation of the partial pressure of O2 in the gaseous phase was achieved with a combination of flow meter and gas mixer. A detailed description of the method has been reported [9]. Analytical methods Acetaminophen glucuronide was measured in the total assay mixtures after removal of proteins by acidification with perchloric acid (3 M, 0.1 ml/0.2 ml assay volume) and centrifugation. The glucuronide was quantitated by high-performance liquid chromatography (HPLC) [7]. Measurements of UDP-glucose and UDP-glucuronic acid concentrations were carried out in heat
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dependence of glucuronidation rate, UDP-glucuronic acid and UDP-glucose concentrations. Hepatocytes (10' cells/ml) were incubated in Krebs-Henseleit buffer containing 12.5 mM HEPES (pH 7.4) under different steady-state 02 concentrations in the presence ( , ) or absence (o) of 20 mM ethanol. Ethanol was added 5 rain before initiation of the reaction. Acetaminophen concentration used was 5 mM and incubations were run for 20 rain. Acetaminophen glucuronides were analyzed in the perchloric acid extracts o f the cell mixtures and concentrations o f UDP-glucose and UDP-glucuronic acid were measured in heat-denatured extracts. Data given were from one cell preparation which is representative of 3 preparations. A: glucuronide formation as a function o f O~. B: UDP-glucuronic acid concentration as a function of 05 . C : UDP-glucose concentration as a function of 02 .

286 RESULTS A N D D I S C U S S I O N

Earlier studies on the interaction of ethanol with drug glucuronidation in isolated hepatocytes [2--4] have shown that (a) ethanol causes significant inhibition of glucuronidation of drugs, (b) ethanol itself does not interact with UDP glucuronyltransferase, (c) inhibition is not due to acetaldehyde, the oxidation product, (d) the inhibitory effect is eliminated by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase and (e) UDP.glucuronic acid concentration is significantly decreased in the presence of ethanol. In this study, we have investigated the UDP-glucose dehydrogenase reaction as a possible crossover point in the ethanol inhibition of drug ~lucuronidation. In the present approach, manipulation of 02 concentrations allowed direct comparisons of the different intracellular rates of acetaminophen glucuroniA

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Fig. 2. Dependence o f rates o f glucuronide formation on UDP-glucose and UDPoglucuronic acid concentrations. Data from Fig. 1 were replotted to express the rates of formation of glucuronide as functions of cellular UDP-glucuronic acid and UDP-glucose concentrations in the presence o f (o) or absence of (o) ethanol. A: formation of glucuronide as a function o f UDP-glucuronic acid concentration. B: formation of glucuronide as a function o f UDP-glucose concentration.

287 dation with changes in the steady-state concentrations of UDP~lucose and UDP-glucuronic acid. Incubation of hepatocytes with ethanol (20 mM) resulted in a 40--60% inhibition in glucuronide formation (Fig. 1A) over the entire range of O~ concentrations. Th~s corresponded to a marked decrease in cellular UDPglucuronic acid concentration over the same range (Fig. 1B). A direct correlation of glucuronidation rate with concentration of UDP-glucuronic acid (Fig. 2A) demonstrates the dependence of the rate on substrate concentration. A single correlation further suggests that inhibition of glucuronidation resulted from a decrease in substrate concentration due to ethanol rather than from an interference with UDP-glucuronyltransferase activity. This establishes that ethanol inhibits formation of drug glucuronide by decreasing the cellular concentrations of UDP-glucuronic acid. To examine whether UDP-glucose dehydrogenase is a crossover point during ethanol inhibition of glucuronidation, the steady-state UDP-glucose concentrations were measured under the conditions as above. Intracellular UDP~glucese concentrations were similar in control and ethanol-treated cells at the higher 02 concentrations (14 and 150 /~M 02, Fig. 1C). The lower UDP-glucose concentrations observed after 5 min preincubation with ethanol at the lower 02 concentrations (~6/~M 02) (Fig. 1C) was presumably due to the effect of ethanol on some other factor which is limiting for the synthesis of UDP-glucose, viz. UTP. The rate of glucuronide formation in control cells correlated directly with UDP~lucose concentrations (Fig. 2B). In a recent study (in preparation), we found that during hypoxia, glucose enhanced the glucuronidation rate with a concomitant increase in UDPglucose concentration. A single correlation of rate with UDP-glucose concentration was obtained for both control and glucose-supplemented cells which indicated that the increased rate resulted from an increase in cellular UDP~glucose. However, in ethanol-treated cells the rate of glucuronidation is much lower than that expected from the UDP~lucose concentration .% (Fig. 2B), clearly showing that flux is inhibited at the UDP-glucose dehydrogenase reaction. The results further show that the point of inhibition by ethanol differs from that which occurs during hypoxia. A reversal of inhibition of drug glucuronidation by 4-methylpyrazole [2--4] indicates that ethanol does not interact directly with UDP-glucose dehydrogenase but rather that inhibition is due to the metabolism of ethanol. Studies with sorbitol, which gave results similar to those for ethanol (data not shown, Ref. 4), supported the suggestion that decrease in UDP-glucose oxidation is due to an increase in NADH/NAD + ratio. At present, it is not resolved whether it is the NADH/NAD + ratio or the absolute concentrations of NAD + and/or NADH that controls UDP~lucose dehydrogenase activity. NADH has been shown to inhibit UDP~glucose dehydrogenase activity [12] but increasing the NAD + supply in the perfused liver by pyruvate infusion did not stimulate glucuronidation [ 13]. In conclusion, the intracellular rate of acetaminophen glucuronidation is dependent on cellular concentrations of both UDP-glucuronic acid and UDP-

288

glucose under control conditions. The significant inhibition of the rate in the presence of ethanol corresponded to a substantial decrease in the cellular UDP-glucuronic acid concentration but not UDP-glucose, thereby defining the UDP-glucose dehydrogenase reaction as the crossover point. These results establish that inhibition by ethanol occurs at the UDP-glucose dehydrogenase-catalyzed reaction. REFERENCES 1 J.H. Str6mme, Metabolism of disulfiram and diethyldithiocarbamate in rats with demonstration of an in vivo ethanol-induced inhibition of the glucuronic acid conjugation of the thiol, Biochem. Pharmacol., 14 (1965) 393. 2 P. Mold6us, H. Vadi and M. Berggren, Oxidative and conjugative metabolism of p-nitroanisole and p-nitrophenol in isolated rat liver cells, Acta Pharmacol. Toxicol., 39 (1976) 17. 3 H. Vadi, Oxidation and conjugation of drugs in hepatocytes with emphasis on benzo[a] pyrene activation, Ph.D. Thesis, University of Stockholm and Karolinska Institute, Stockholm, Sweden, 1977. 4 P. Mold~us, B Andersson and A. Norling, Interaction of ethanol oxidation with glucuronidation in isolated hepatocytes, Biochem. Pharmacol., 27 (1978) 2583. 5 J. Singh and L.R. Schwarz, Dependence of glucuronidation rate on UDP-glucuronic acid levels in isolated hepatocytes, Biochem. Pharmacol., 30 (1981 ) 3252. 6 P. Mold~us, J. HSgberg and S. Orrenius, Isolation and use of liver cells, Methods Enzymol., 52 (1978) 60. 7 T.Y. Aw and D.P. Jones, Secondary Bioenergetic Hypoxia: inhibition of sulfation and glucuronidation reactions in isolated hepatocytes at low O 2 concentration, J. Biol. Chem., 257 (1982) 8997. 8 K. Ormstad, S. Orrenius and D.P. Jones, Preparation and characteristics of isolated kidney cells, Methods Enzymol., 77 (1981) 137. 9 D.P. Jones and H.S. Mason, Apparatus for automatically maintaining cell suspension at low, constant oxygen concentrations: the oxystat, Anal. Bioehem., 90 (1978) 155. 10 J. Zalitis, M. Uram, A.M. Bowser and D.S. Feingold, UDP-glucose dehydrogenase from beef liver, Methods Enzymol., 28 (1972) 430. 11 T.Y. Aw and D_P. Jones, Direct determination of UDP-glucuronic acid in cell extracts by high-performance liquid chromatography, Anal. Biochem. (1982) in press. 12 N.D. Goldberg, Ph.D. Thesis, cited in Ref. 4. 13 R.G. Thurman and F.C. Kauffman, Factors regulating drug metabolism in intact hepatocytes, Pharmacol. Rev., 31 (1980) 229.