THE EFFECT OF NALOXONE ON THE HEPATOCELLULAR REDOX STATE AND SERUM ETHANOL CONCENTRATIONS FOLLOWING ACUTE ETHANOL ADMINISTRATION

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1 Alcohol & Alcohoiism, Vol. 2, No. 3, pp Printed in Great Britain UHI + () (X) Pcrgamon Prcbs Ltd ( ) 1985 Medical Council on Alcoholism THE EFFECT OF NALOXONE ON THE HEPATOCELLULAR REDOX STATE AND SERUM ETHANOL CONCENTRATIONS FOLLOWING ACUTE ETHANOL ADMINISTRATION PETER R. RYLE*, J. CHAKRABORTYt and ALLAN D. THOMSON* 'Departments of Chemical Pathology and Gastroenterology, Greenwich District Hospital, London SEK) 9HE, U.K. and tclinical Biochemistry Division, Department of Biochemistry, University of Surrey, Guildford. Surrey, U.K. (Received 21 November 1984) Abstract Naloxone hydrochloride (2. mg/kg) has been found to reverse the significant decreases in the hepatic cytosolic and mitochondrial [NAD + ]/[NADH] ratios observed after acute ethanol administration in rats. This correction of the ethanol-induced changes in the hepatocellular redox state by naloxone was, however, not associated with any lowering of serum ethanol concentrations or an observable reduction in the extent of intoxication. This lack of antagonism of alcohol intoxication by naloxone was not affected by the feeding status of the animals, the time point after naloxone administration at which serum ethanol concentration was determined or the method used for ethanol analysis. Thus this study has failed to confirm that naloxone antagonises acute alcohol intoxication, in spite of its potent ability to reverse the ethanol-induced changes in the hepatic redox state. INTRODUCTION A number of clinical studies have indicated that the opiate antagonist, naloxone (17-allyl- 4, 5a-epoxy-3, 14-dihydroxy-normorphinan-6- one), may antagonise the effects of acute alcohol intoxication. Jeffcoate et al. (1979) reported improvement in psychomotor performance in volunteers after giving.4 mg of proprietary naloxone (i.e. less than 6 u.g/kg) intravenously following a low oral ethanol dose (.32 g/kg), without the drug affecting serum ethanol concentrations. Jeffreys et al. (198) reported that proprietary naloxone at doses of up to 15 (j-g/kg was able to reverse the ethanolinduced coma in severely-intoxicated patients on admission to hospital, but only a proportion of subjects responded, degree of intoxication was variable and other drugs were frequently involved. The animal study of Badawy and Evans (1981) showed that proprietary naloxone (1. mg/kg, i.p.) was able to reverse alcohol-induced narcosis in rats following a moderate ethanol dose (2 g/kg, i.p.). This antagonism was associated with reversal of the ethanol-induced changes in the hepatocellular [NADH]/[NAD + ] ratio that results from ethanol oxidation, and a 31% lowering of the blood ethanol concentration measured 3 min after naloxone administration was reported. These authors proposed that naloxone antagonises alcohol intoxication in part by stimulating NADH reoxidation in the liver, thereby generating more NAD + for ethanol metabolism and lowering blood ethanol concentration, since the rate of NADH reoxidation has been considered as a rate-limiting step in ethanol metabolism (Badawy, 1978). However, more recent clinical and experimental studies have failed to demonstrate any influence of proprietary naloxone on acute alcohol intoxication (Nuotto et al., 1984), except at very high doses of the opiate antagonist of 5 mg/kg i.p. in rats (Khanna et al., 1982). Any possible interaction of naloxone with ethanol or its metabolism has been further confused by the finding that methyl-4- hydroxybenzoate, the major preservative present in vials of proprietary naloxone (Narcan) was able to lower the [3-hydroxybutyrate]/ [acetoacetate] ratio and to reverse the inhibition of fatty acid oxidation by ethanol in isolated hepatocyte preparations (Caldecourt et al., 1983). This raised the question as to 287

2 288 P. R. RYLE et al. whether the mechanism of any antagonism of alcohol intoxication by naloxone involved the drug itself or preservatives present in proprietary preparations thereof. The aim of the present study was to assess whether naloxone hydrochloride itself, in the absence of any preservative substances, has any influence on the observable degree of intoxication or possibly lowers blood ethanol levels following acute ethanol administration to rats, and whether it exerts the metabolic effects reported by Badawy and Evans (1981) (i.e. correction of the hepatocellular redox changes) that might account for any antagonistic action of the drug. METHODS Male Wistar albino rats (University of Surrey strain, 18-2 g) were used. They were housed in wire-bottomed cages and maintained on cube diet 41B and water. Animals were starved overnight before intraperitoneal injection of ethanol [2 g/kg, as a 25% (v/v) solution in saline], or an equivalent volume of saline. Pure naloxone hydrochloride powder was dissolved fresh in sterile saline immediately before intraperitoneal injection (2. mg/kg); control animals receiving a similar volume of saline. Naloxone was given 1. hr after ethanol administration, and the animals were killed 3 min after the naloxone injection, usually between 12: and 13: hr. Livers were rapidly isolated by freeze-clamping, and blood was collected from the necks of the animals, allowed to clot and serum prepared. Frozen liver samples were extracted with perchloric acid and the [lactate]/[pyruvate] ratio was determined as described previously (Ryle et al., 1983). 3-Hydroxybutyrate and acetoacetate concentrations were determined by the methods of Williamson and Mellanby (1974) and Mellanby and Williamson (1974) respectively. ATP was assayed using kits supplied by BCL Ltd, Lewes, Sussex, U.K. Cytosolic and mitochondrial free [NAD + ]/[NADH] ratios were calculated from the [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] ratios as described by Williamson et al. (1967). In one series of experiments designed to assess the effect of feeding status on any antagonism of alcohol intoxication by naloxone, this opiate antagonist was given (2. mg/ kg i.p.) 1.5 hr after ethanol (2 g/kg i.p.) to both fed and starved rats, and blood samples collected either.5 or 1.5 hr later (i.e. 2 and 3 hr after ethanol dosing) for ethanol determination. Thus, the effect of naloxone on the serum ethanol concentrations determined at.5 hr after ethanol in fed rats in this experiment can be directly compared with that observed by Badawy and Evans (1981), who used identical experimental conditions, with the exception of the naloxone dose. Serum ethanol concentration was determined both by an enzymatic method (kit supplied by BCL Ltd) and head-space gas chromatography using n-proponal as the internal standard (Von Wartburg and Ris, 1979). Statistical analysis of results was performed using Student's /-test. RESULTS As shown in Table 1, ethanol caused, at 1.5 hr, a 59% decrease in the free cytosolic [NAD + ]/[NADH] ratio, with a simultaneous 41% decrease in the free mitochondrial [NAD + ]/[NADH] ratio. Administration of naloxone.5 hr before testing almost completely abolished these hepatocellular redox state changes in ethanol-treated animals. Naloxone significantly lowered the hepatic 3-hydroxybutyrate and acetoacetate concentrations in both control and ethanol-treated animals. Neither naloxone nor ethanol, alone or in combination, had any effect on hepatic ATP concentrations. Naloxone administration to ethanol-treated rats did not result in any observable reduction in the extent of intoxication (assessed by simple inspection of the animals) in any of the experiments, and the results in Table 1 show that the drug did not affect serum ethanol concentration in the experiment in which the hepatic redox state was assessed, irrespective of the method used to carry out the ethanol analysis. The results in Table 2 show that the feeding status of the animals did not affect the lack of antagonism of alcohol intoxication by naloxone. Thus when fed rats were used as in the previous study of Badawy and Evans (1981), or

3 EFFECT OF NALOXONE 289 Table 1. Effect of naloxone hydrochloride on the hepatic redox state and serum ethanol concentration in acutely-ethanol-intoxicated rats Treatment Pre-treatment A. Control Saline B. Ethanol Naloxone C. Control D. Ethanol Determination Liver ATP Lactate Pyruvate 3-Hydroxybutyrate Acetoacetate [Lactate]/f pyruvate] [3-Hydroxybutyrate]/[acetoacetate] Cytosolic [NAD+MNADH] Mitochondria! [NAD + ]/[NADH] Serum ethanol (Gas chromatography) Serum ethanol (Enzymatic method) 3726 ± 653 ± 42.4 ± 1173 ± 458 ± 16.5 ± 2.58 ± 761 ± 7 95 ± ± 758 ± 219" ± 2.4" ± 458" ± 119 ± 8.*" ±.1*" ± 79*" ±.19*" ± 5. ± ± 669 ± 45 1 ± 95 ± 332 ± 15 7 ± 2.75 ± 86 ± 7.63 ± * 4* ± 644 ± 35.6 ± 76 ± 3()6 ± 18.2 ± 254 ± 625 ± 8 57 ± 22.5 ± 219. ± Experimental details are given in the text. Serum ethanol concentration is expressed as mg/dl, whereas all other determinations (except ratios) are in nmol/g wet wt of liver. Values are expressed as means ± SD (N = 6 rats per group). = P<.25 vs A. = P<.1 vs A. = P<.1 vs A. Table 2 The effect of naloxone hydrochloride on blood ethanol concentrations following acute ethanol administration to fed and starved rats " 59* Fed rats Starved rats Time after ethanol (hr) Serum ethanol (mg/dl) Ethanol only 158. ± ± ± ±11.3 Ethanol + naloxone 161. ± ± ± ± 11.1 Naloxone hydrochloride was given (2. mg/kg i.p.) 1.5 hr after ethanol (2 g/kg i.p.) and the animals were killed either 2 or 3 hr after ethanol administration. Results are means ± SD for groups of four rats each the more recent study of Badawy and Aliyu (1984), no effect of naloxone on serum ethanol concentration was found, even when the time interval after administration of the drug was extended to 1.5 hr (i.e. 3 hr after ethanol). DISCUSSION This study has confirmed the findings of Badawy and Evans (1981) and Badawy and Aliyu (1984) that naloxone itself is able to correct the hepatocellular redox state following acute ethanol administration. However, in contrast to these authors' findings, this correction of the redox state does not appear to influence ethanol metabolism, so that serum ethanol concentrations were unaffected by the drug. This is in spite of an identical ethanol dose being used, similar time intervals between

4 29 P. R. RYLE et al. naloxone administration and blood sampling being employed, and, in addition, double the dose of naloxone was used in the present study as was administered in the aforementioned studies (Badawy and Evans, 1981; Badawy and Aliyu, 1984). Preliminary experiments in which naloxone was given at a dose of 1 mg/kg also failed to show any antagonism of acute alcohol intoxication, although partial correction of the hepatic redox state after ethanol was observed at this dose. Exact duplication of the experimental conditions of Badawy and Evans (1981) and Badawy and Aliyu (1984) (i.e. administering naloxone 1.5 hr after ethanol to fed rats) failed to show an influence of the drug on serum ethanol concentrations, even when the time interval between naloxone administration and blood sampling was extended to 1.5 hr. It might be expected that if naloxone was to influence serum ethanol concentrations in rats, then such an effect would be observed soon after its administration, as it has a half life in the rat of only 16 min (Weinstein et al., 1974). It is difficult to account for this apparent discrepancy between our findings and those of these previous workers. In the present study, serum ethanol was measured by the widely accepted head-space gas chromatography method and, in addition, an enzymatic method was used in the first series of experiments, whereas Badawy and Evans (1981) used the Lion 'Alcolometer' method to determine blood ethanol concentrations, the accuracy of which has recently been called into question (Marks, 1984). However, even using an enzymatic method, Badawy and Aliyu (1984) reported lowering of blood ethanol concentration by naloxone. A number of factors may regulate the rate of ethanol metabolism, including the total liver alcohol dehydrogenase and aldehyde dehydrogenase activities, the rate of transfer of reducing equivalents into the mitochondria, or the rate of oxidative phosphorylation for mitochondrial reoxidation of NADH. It is possible that specific differences in one or more of these factors between the animals used in the present study and those used by the group of Badawy (Badawy and Evans, 1981; Badawy and Aliyu, 1984) could determine whether naloxone has the ability to lower blood ethanol levels in ethanol-treated rats. With respect to the lack of effect of naloxone on serum ethanol concentrations, our findings agree with those of Khanna et al. (1982), who had to give the drug at a dose of 5 mg/kg (s.c.) before any effect on blood ethanol levels was observed. Obviously, such high doses are irrelevant in terms of any potential clinical use of the compound in alcohol intoxication. The apparent lack of effect of naloxone on ethanol metabolism in our study is surprising in view of the potent ability of the drug to correct the ethanol-induced hepatic redox state changes. This suggests that the hepatic [NAD + ]/ [NADH] ratio, or more precisely the rate of NADH reoxidation, is not the critical factor in regulating ethanol metabolism by alcohol dehydrogenase as previously suggested (see, e.g. Badawy, 1978). It is possible that hepatic acetaldehyde concentration during ethanol metabolism may be important in this respect as the equilibrium constant for alcohol dehydrogenase is in favour of the reverse reaction, so that acetaldehyde may act as an inhibitor of ethanol oxidation by this enzyme. This possibility is supported by recent studies in vitro indicating a regulatory role for acetaldehyde in ethanol metabolism (Dawson, 1983) and the finding that thiol compounds such as cysteine, which effectively combine with and lower hepatic acetaldehyde levels during ethanol metabolism, actually accelerate the ethanol disappearance rate in ethanol-intoxicated rats (Hirayama, 1983). The mechanism by which naloxone corrects the hepatic redox state changes after ethanol administration is not clear from this study, but in view of the lack of effect of the drug on ATP concentrations, non-enzymatic oxidation of NADH or simple uncoupling of oxidative phosphorylation do not seem to be involved. Thus, in conclusion, pure naloxone has been shown to correct the hepatocellular redox state after acute alcohol administration to rats. This effect of the drug has not, however, been found to influence the degree of intoxication or serum ethanol concentrations thus suggesting that the hepatic [NAD + ]/[NADH] ratio is not a critical factor in determining the ethanol elimination rate.

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