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Effects of sulphur-containing compounds on paracetamol activation and covalent binding in a mouse hepatic microsomal system
Toxicology Letters, 5 (1980) 339-344 o Elsevier/North-Holland Biomedical Press
339
EFFECTS OF SULPHUR-CONTAINING COMPOUNDS ON PARACETAMOL ACTIVATION AND COVALENT BINDING IN A MOUSE HEPATIC MICROSOMAL SYSTEM
J. MICHAEL TREDGER*, WILLIAMS
HEATHER M. SMITH, MICHAEL DAVIS AND ROGER
Liver Unit, King’s College Hospital and Medical School, Denmark Hill, London SE5 9RS, (U.K.) (Received August 23rd, 1979) (Accepted December 12th, 1979)
SUMMARY
The interactions of cysteamine, N-acetylcysteine, 2-mercaptopropionylglycine and methionine with N-acetyl4-aminophenol (paracetamol) have been examined during its metabolism to a covalently bound product in an in vitro mouse hepatic microsomal system. Of the compounds used only methionine failed to reduce the amount of covalently bound [ 3H]paracetamol-derived radioactivity. These results indicate that the effectiveness of methionine in reducing paracetamol hepatotoxicity in vivo is achieved by mechanisms other than those involving direct interactions with the hepatic mixed-function oxidase system or its oxidation products. In contrast, cysteamine, N-acetylcysteine and 2-mercaptopropionylglycine can directly inhibit the binding of [3H]paracetamol-derived radioactivity in vitro, and may do so by processes which affect both the formation and the subsequent binding of a reactive paracetamol metabolite.
INTRODUCTION
The hepatic metabolism of therapeutic quantities of paracetamol occurs principally by conjugative pathways, although some of the drug is transformed by the cytochrome P-450-dependent mixed-function oxidase system to an unstable derivative which is normally detoxified by conjugation with glutathione [ 1, 21. After ingestion of large amounts of paracetamol the capacity for its removal by hepatic conjugation with sulphate or glucuronide is exceeded, the rate of production of its oxygenated metabolite is dis*To whom correspondence should be addressed. Abbreviation:
Paracetamol,
N-acetyl 4-aminophenol
(acetaminophen).
340
proportionately increased, cellular glutathione is depleted and liver damage develops [l-4] in association with the covalent binding of a paracetamol metabolite to cellular macromolecules [ 5, 61. Evidence from animal studies using mixed-function oxidase inhibitors and inducing agents suggested that this covalent binding may be quantitatively related to paracetamol toxicity [ 5, 61. Consistent with this observation is the demonstration that cysteamine, cysteine and 2-mercaptopropionylglycine, representative of a number of thiols and sulphur-containing amino acids which are effective in experimentally induced or clinical overdose situations [6--111, can reduce covalent binding in vivo [6, 7, 111. Nonetheless, it is unclear as to what mechanisms are involved in this inhibition of covalent binding and whether all those compounds prevent toxicity in a similar manner. The present communication reports on the effects of four protective agents - cysteamine, N-acetylcysteine, methionine, and 2-mercaptopropionylglycine - on the activation and subsequent covalent binding of (3H) paracetamol in an in vitro hepatic microsomal system. Materials and methods Materials. Glucose 6-phosphate,
N-acetylcysteine, cysteamine, methionine and glucose 6-phosphate dehydrogenase were purchased from Sigma London. NADP’ was bought from International Enzymes Ltd.; 2-Mercaptopropionylglycine was obtained from Santen Pharmaceuticals Co. Ltd., Milan, Italy. [ 3H]Paracetamol (spec. act. 5.74 pCi/pmol) was provided by Sterling Winthrop Ltd., and was synthesized via reaction of [3H](ring) 4-aminophenol with acetic anhydride. Using mass spectrometry, nuclear magnetic resonance spectrometry, ultraviolet spectrophotometry and thin-layer chromatography, it was shown to be identical with authentic paracetamol. Preparation of microsomes. Adult male albino mice (13 weeks old, 30-38 g body weight) from a colony bred at King’s College Hospital Medical School were used. Mice were killed by cervical dislocation, the livers from at least four animals were pooled per sample, and a 25% liver homogenate was prepared in ice-cold 1.15% KC1 containing 10 mM Hepes, pH 7.6. The homogenate was centrifuged at 10 000 X g for 20 min to obtain a post-mitochondrial supernatant from which a washed microsomal pellet was obtained by centrifugation at 132 000 X g for 48 min, resuspension of the pellet in KClHepes and recentrifugation at 160 000 X g for 32 min. The resulting pellet was resuspended in KCl-Hepes and the protein content of this washed microsomal suspension was determined by the method of Lowry et al. [ 121 using a standard of bovine serum albumin. Binding assay. The covalent binding of activated paracetamol to perchloric acid-precipitable microsomal macromolecules was determined under optimal conditions using a modified version of the assay described by Potter et al. [13]. Each incubation contained 60 pmol Hepes pH 7.7, 5 * 105dpm, [3H] paracetamol, the sulphur-containing compound under test, and unlabelled paracetamol to the concentrations described elsewhere, 3 mg microsomal
341
protein and water to 1.5 ml. After a 5-min preincubation, the reaction was initiated by the addition of 0.5 ml of NADPH generating system containing 5 pmol glucose 6-phosphate, 10 E.tmolMgC12,0.5 pmol NADP’, 1 unit of glucose 6-phosphate dehydrogenase and 40 pmol Hepes pH 7.7. The glucose 6-phosphate dehydrogenase was omitted from those incubations in which the amount of mixed-function oxidase-independent binding was being measured. After a 20-min incubation at 37”C, the reaction was terminated by the addition of 2 ml of 1 M perchloric acid. After centrifugation, the resulting supernatant was removed by aspiration and the residual precipitate was left overnight in 0.5 M perchloric acid. Sequential washes were then made with 0.5 M perchloric acid (once at room temperature, twice at 50” C), chloroform: diethyl ether: ethanol (1:2:2) (twice) and diethyl ether. The resulting pellet was dried, re-dissolved in 2 M NaOH and aliquots were removed for determination of protein content [ 121 and radioactivity (using a triton-toluene based scintillation fluid). Counting efficiencies were determined by the internal standardization method using [ 3H]toluene and results are presented as radioactivity bound (nmol paracetamol equivalents) mg microsomal protein-’ min-’ . Calculation of ISo inhibitory constants was made by linear regression analysis of the semi-logarithmic plot of nmol binding mg microsomal protein-’ min-’ vs. concentration of the protective agent. Values are presented f one standard error of estimate [14]. RESULTS
AND DISCUSSION
At the paracetamol concentrations used, methionine was the only compound of the four tested which was ineffective in reducing the amount of mixed-function oxidase-dependent covalent binding. Thus, at methionine concentrations of 0.1 mM or 1.0 mM, covalent binding was not significantly different from that found in control incubations (Fig. 1). These findings indicate that methionine did not inhibit the mixed-function oxidation of paracetamol, nor did it prevent the subsequent binding of a reactive product of this oxygenation with cellular macromolecules. Consequently, the protection afforded by methionine against paracetamol toxicity in vivo [ 91 must arise independently of direct interactions between the two compounds, and may instead be mediated by metabolites of the amino acid not formed in an in vitro microsomal system which may have lacked the appropriate constituents. Methionine metabolites which may be effective in vivo include sulphate anions, homocysteine, cysteine and glutathione [ 151 and these compounds could act by increasing the capacity of detoxication pathways of paracetamol metabolism, such as sulphate conjugation, or by inhibiting the formation or reactivity of the electrophilic paracetamol metabolites. Cysteamine, N-acetylcysteine and 2-mercaptopropionylglycine substantially reduced the in vitro microsomal binding of [ 3H]paracetamol-derived radioactivity, in contrast to methionine (Fig. 1). At an assay concentration
342
l
As
50
-%
l
1
CONTROL
CYSTEAh
N-ACETYLCYSTEINE
PROTECTIVE
AGENT
AND
Z-MERCAPTOPROPIONYLGLYCINE
CONCENTRATION
METHIONINE
ImM)
Fig. 1. Effects of protective agents on paracetamol covalent binding in vitro. Covalent binding (nmol paracetamol equivalents bound - mg microsomal protein-’ - min-‘) in the presence of protective agents (mean * s.e.m; n = 3) is presented as a percentage of that found in control incubations containing 1 mM paracetamol(l.38 ?r 0.10 nmol * mg” * min-r ; mean + s.e.m., n = 45). Asterisks denote significantly different results (paired t test;P < 0.05).
of paracetamol of 1.0 mM the reduction in binding varied between 20% and 100% depending on the concentrations of the thiols used. Cysteamine was the most effective agent and reduced binding by 88% when present at a concentration of 0.1 mM, while the same concentrations of N-acetylcysteine and 2-mercaptopropionylglycine were less potent inhibitors of binding and produced a 60% and 48% inhibition respectively. Quantitative estimates of the overall efficacy of the thiols were derived from calculations of the amount of sulphydryl compound required to reduce paracetamol-derived covalent binding by 50% (ISo concentrations) and are shown in Table I for assay concentrations of paracetamol substrate of 0.1 mM and 1.0 mM. At both substrate concentrations cysteamine was the most potent inhibitor of covalent binding, 2-mercaptopropionylglycine was the least effective and N-acetylcysteine was of intermediate efficacy. As well as demonstrating the different efficacies of the protective agents in vitro, Table I shows that the ISo concentrations of the 3 thiols did not change proportionately with changes in paracetamol concentration. Thus, despite 4-g-fold increases in total covalent binding, the ISo of cysteamine was similar at both 0.1 mM and 1.0 mM concentrations of paracetamol while those of N-acetylcysteine and 2-mercaptopropionylglycine were increased at the higher concentration by around 50% and 100% respectively. One possible explanation for these findings is that the 3 thiols do not act exclusively at a single stage during the formation and binding of the reactive paracetamol
343 TABLE I I,, CONCENTRATIONS (PM) OF PROTECTIVE AND 1.0 mM PARACETAMOL I,, concentrations
AGENTS IN VITRO USING 0.1 mM
were determined as described under MATERIALS
Protective agent Methionine 2-Mercaptopropionylglycine N-Acetylcysteine Cysteamine
I,, NW
At paracetamol concentrations 0.1 mM 1.0 mM >> 1000 61.9 f 4.1 37.7 f 1.6 8.9 f 0.2
AND METHODS
of
>> 1000 121 *3 57.1 + 2.3 9.5 f 0.35
metabolite. Instead, interactions may occur during the attachment of paracetamol to cytochrome P-450, during the reduction of this cytochrome P450-paracetamol (substrate) complex, during the oxygenation of this reduced P-450-substrate complex and by detoxication of the reactive metabolite generated by this oxidation. Inhibition at the first three of these stages has been demonstrated using sulphydryl compounds and other P-450 substrates [16,17] but only at concentrations 10-100 times those used in the present study. Consequently, the efficacy of cysteamine, iV-acetylcysteine and 2mercaptopropionylglycine is probably achieved primarily from direct interactions between the thiols and the preformed reactive paracetamol metabolite. The observation that paracetamol can form adducts with nucleophilic sulphydryl compounds such as glutathione, cysteine or N-acetylcysteine in vitro apparently endorses this hypothesis [ 18, 191. The results of the present study suggest that differences may exist between the mechanism of action of thiols and protective agents such as methionine which contain no free sulphydryl group. Thus, it is possible that the co-administration of methionine and thiol may be a more effective protective combination against paracetamol toxicity than either compound used separately. Moreover, this regimen may avoid the potential toxicity of large doses of a single antidote. ACKNOWLEDGEMENTS
We are grateful to Sterling-Winthrop Ltd. for generous support including provision of [ ‘Hlparacetamol, to Santen Pharmaceutical Co., Ltd. for 2-mercaptopropionylglycine and financial help, and to the King’s College Hospital Joint Research Committee for financial assistance.
344 REFERENCES 1 R.S. Andrews, C.C. Bond, J. Burnett, A. Saunders and K. Watson, Isolation and identification of paracetamol metabolites, J. Int. Med. Res., 4 Suppl. (4) (1976) 34-39. 2 M. Davis, C.J. Simmons, N.G. Harrison and R. Williams, Paracetamol overdose in man: relationship between pattern of urinary metabolites and severity of liver damage, Quart. J. Med., 45 (1976) 181-191. 3 E.M. Boyd and G.M. Bereczky, Liver necrosis from paracetamol, Br. J. Pharmacol., 26 (1966) 606-614. 4 D.G.S. Davidson and W.N. Eastham, Acute liver necrosis following overdose of paracetamol, Br. Med. J., 2 (1966) 497-499. 5 D.J. Jollow, J.R. Mitchell, W.Z. Potter, D.C. Davis, J.R. Gillette and B.B. Brodie, Acetaminophen-induced hepatic necrosis, II. Role of covalent binding in vivo, J. Pharmacol. Exp. Ther., 187 (1973) 195-202. 6 J.R. Mitchell, D.J. Jollow, W.Z. Potter, J.R. Gillette and B.B. Brodie, Acetaminopheninduced hepatic necrosis, IV. Protective role of glutathione, J. Pharmacol. Exp. Ther., 187 (1973) 211-217. 7 J.R. Mitchell, S.S. Thorgeirsson, W.Z. Potter, D.J. Jollow and H. Keiser, Acetaminophen-induced hepatic injury: protective role of glutathione in man and rationale for therapy, Clin. Pharmacol. Ther., 16 (1974) 676-684. 8 0. Strubelt, C.P. Siegers and A. Schutt, The curative effects of cysteamine, cysteine and dithiocarb in experimental paracetamol poisoning, Arch. Toxicol., 33 (1974) 55-64. 9 A.E.M. McLean and P. Day, The effect of diet on the toxicity of paracetamol and the safety of paracetamol-methionine mixtures, Biochem. Pharmacol., 24 (1975) 37-42. 10 E. Piperno and D.A. Berssenbruegge, Reversal of experimental paracetamol toxicosis with N-acetylcysteine, Lancet, i (1976) 739. 11 D. Labadarios, M. Davis, B. Portmann and R. Williams, Paracetamol-induced hepatic necrosis in the mouse - relationship between covalent binding, hepatic glutathione depletion and the protective effect of ol-mercaptopropionylglycine, Biochem. Pharmacol., 26 (1977) 31-35. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 13 W.Z. Potter, D.C. Davis, J.R. Mitchell, D.J. Jollow, J.R. Gillette and B.B. Brodie, Acetaminophen-induced hepatic necrosis, III. Cytochrome P-450-mediated covalent binding in vitro. J. Pharmacol. Exp. Ther., 187 (1973) 203-210. 14 M.J. Moroney, Facts from Figures, Penguin Books, London, 1974, p. 295. 15 A. White, P. Handler and E.L. Smith, Principles of Biochemistry, McGraw, London, 1968, pp. 531-618. 16 R. Mull, W. Winkelbein, J. Gertz and K. Flemming, A comparative study on the influence of cysteamine and metyrapone on mixed-function oxygenase activities in variously pretreated liver microsomes from rats and mice, Biochim. Biophys. Acta, 481(1977) 407-419. 17 J.A. Castro, E.C. de Ferreyra, C.R. de Castro, M.I. Diaz-Gomez, N. D’Acosta and O.M. de Fenos, Studies on the mechanism of cystamine prevention of several liver structural and biochemical alterations caused by carbon tetrachloride, Toxicol. Appl. Pharmacol., 24 (1973) l-19. 18 A.R. Buckpitt, D.E. Rollins, S.D. Nelson, R.B. Franklin and J.R. Mitchell, Quantitative determination of the glutathione cysteine and N-acetylcysteine conjugates of acetaminophen by high-pressure liquid chromatography, Anal. Biochem., 83 (1977) 168-177. 19 P. Moldeus, Paracetamol metabolism and toxicity in isolated hepatocytes from rat and mouse, Biochem. Pharmacol., 27 (1978) 2859-2863.
339
EFFECTS OF SULPHUR-CONTAINING COMPOUNDS ON PARACETAMOL ACTIVATION AND COVALENT BINDING IN A MOUSE HEPATIC MICROSOMAL SYSTEM
J. MICHAEL TREDGER*, WILLIAMS
HEATHER M. SMITH, MICHAEL DAVIS AND ROGER
Liver Unit, King’s College Hospital and Medical School, Denmark Hill, London SE5 9RS, (U.K.) (Received August 23rd, 1979) (Accepted December 12th, 1979)
SUMMARY
The interactions of cysteamine, N-acetylcysteine, 2-mercaptopropionylglycine and methionine with N-acetyl4-aminophenol (paracetamol) have been examined during its metabolism to a covalently bound product in an in vitro mouse hepatic microsomal system. Of the compounds used only methionine failed to reduce the amount of covalently bound [ 3H]paracetamol-derived radioactivity. These results indicate that the effectiveness of methionine in reducing paracetamol hepatotoxicity in vivo is achieved by mechanisms other than those involving direct interactions with the hepatic mixed-function oxidase system or its oxidation products. In contrast, cysteamine, N-acetylcysteine and 2-mercaptopropionylglycine can directly inhibit the binding of [3H]paracetamol-derived radioactivity in vitro, and may do so by processes which affect both the formation and the subsequent binding of a reactive paracetamol metabolite.
INTRODUCTION
The hepatic metabolism of therapeutic quantities of paracetamol occurs principally by conjugative pathways, although some of the drug is transformed by the cytochrome P-450-dependent mixed-function oxidase system to an unstable derivative which is normally detoxified by conjugation with glutathione [ 1, 21. After ingestion of large amounts of paracetamol the capacity for its removal by hepatic conjugation with sulphate or glucuronide is exceeded, the rate of production of its oxygenated metabolite is dis*To whom correspondence should be addressed. Abbreviation:
Paracetamol,
N-acetyl 4-aminophenol
(acetaminophen).
340
proportionately increased, cellular glutathione is depleted and liver damage develops [l-4] in association with the covalent binding of a paracetamol metabolite to cellular macromolecules [ 5, 61. Evidence from animal studies using mixed-function oxidase inhibitors and inducing agents suggested that this covalent binding may be quantitatively related to paracetamol toxicity [ 5, 61. Consistent with this observation is the demonstration that cysteamine, cysteine and 2-mercaptopropionylglycine, representative of a number of thiols and sulphur-containing amino acids which are effective in experimentally induced or clinical overdose situations [6--111, can reduce covalent binding in vivo [6, 7, 111. Nonetheless, it is unclear as to what mechanisms are involved in this inhibition of covalent binding and whether all those compounds prevent toxicity in a similar manner. The present communication reports on the effects of four protective agents - cysteamine, N-acetylcysteine, methionine, and 2-mercaptopropionylglycine - on the activation and subsequent covalent binding of (3H) paracetamol in an in vitro hepatic microsomal system. Materials and methods Materials. Glucose 6-phosphate,
N-acetylcysteine, cysteamine, methionine and glucose 6-phosphate dehydrogenase were purchased from Sigma London. NADP’ was bought from International Enzymes Ltd.; 2-Mercaptopropionylglycine was obtained from Santen Pharmaceuticals Co. Ltd., Milan, Italy. [ 3H]Paracetamol (spec. act. 5.74 pCi/pmol) was provided by Sterling Winthrop Ltd., and was synthesized via reaction of [3H](ring) 4-aminophenol with acetic anhydride. Using mass spectrometry, nuclear magnetic resonance spectrometry, ultraviolet spectrophotometry and thin-layer chromatography, it was shown to be identical with authentic paracetamol. Preparation of microsomes. Adult male albino mice (13 weeks old, 30-38 g body weight) from a colony bred at King’s College Hospital Medical School were used. Mice were killed by cervical dislocation, the livers from at least four animals were pooled per sample, and a 25% liver homogenate was prepared in ice-cold 1.15% KC1 containing 10 mM Hepes, pH 7.6. The homogenate was centrifuged at 10 000 X g for 20 min to obtain a post-mitochondrial supernatant from which a washed microsomal pellet was obtained by centrifugation at 132 000 X g for 48 min, resuspension of the pellet in KClHepes and recentrifugation at 160 000 X g for 32 min. The resulting pellet was resuspended in KCl-Hepes and the protein content of this washed microsomal suspension was determined by the method of Lowry et al. [ 121 using a standard of bovine serum albumin. Binding assay. The covalent binding of activated paracetamol to perchloric acid-precipitable microsomal macromolecules was determined under optimal conditions using a modified version of the assay described by Potter et al. [13]. Each incubation contained 60 pmol Hepes pH 7.7, 5 * 105dpm, [3H] paracetamol, the sulphur-containing compound under test, and unlabelled paracetamol to the concentrations described elsewhere, 3 mg microsomal
341
protein and water to 1.5 ml. After a 5-min preincubation, the reaction was initiated by the addition of 0.5 ml of NADPH generating system containing 5 pmol glucose 6-phosphate, 10 E.tmolMgC12,0.5 pmol NADP’, 1 unit of glucose 6-phosphate dehydrogenase and 40 pmol Hepes pH 7.7. The glucose 6-phosphate dehydrogenase was omitted from those incubations in which the amount of mixed-function oxidase-independent binding was being measured. After a 20-min incubation at 37”C, the reaction was terminated by the addition of 2 ml of 1 M perchloric acid. After centrifugation, the resulting supernatant was removed by aspiration and the residual precipitate was left overnight in 0.5 M perchloric acid. Sequential washes were then made with 0.5 M perchloric acid (once at room temperature, twice at 50” C), chloroform: diethyl ether: ethanol (1:2:2) (twice) and diethyl ether. The resulting pellet was dried, re-dissolved in 2 M NaOH and aliquots were removed for determination of protein content [ 121 and radioactivity (using a triton-toluene based scintillation fluid). Counting efficiencies were determined by the internal standardization method using [ 3H]toluene and results are presented as radioactivity bound (nmol paracetamol equivalents) mg microsomal protein-’ min-’ . Calculation of ISo inhibitory constants was made by linear regression analysis of the semi-logarithmic plot of nmol binding mg microsomal protein-’ min-’ vs. concentration of the protective agent. Values are presented f one standard error of estimate [14]. RESULTS
AND DISCUSSION
At the paracetamol concentrations used, methionine was the only compound of the four tested which was ineffective in reducing the amount of mixed-function oxidase-dependent covalent binding. Thus, at methionine concentrations of 0.1 mM or 1.0 mM, covalent binding was not significantly different from that found in control incubations (Fig. 1). These findings indicate that methionine did not inhibit the mixed-function oxidation of paracetamol, nor did it prevent the subsequent binding of a reactive product of this oxygenation with cellular macromolecules. Consequently, the protection afforded by methionine against paracetamol toxicity in vivo [ 91 must arise independently of direct interactions between the two compounds, and may instead be mediated by metabolites of the amino acid not formed in an in vitro microsomal system which may have lacked the appropriate constituents. Methionine metabolites which may be effective in vivo include sulphate anions, homocysteine, cysteine and glutathione [ 151 and these compounds could act by increasing the capacity of detoxication pathways of paracetamol metabolism, such as sulphate conjugation, or by inhibiting the formation or reactivity of the electrophilic paracetamol metabolites. Cysteamine, N-acetylcysteine and 2-mercaptopropionylglycine substantially reduced the in vitro microsomal binding of [ 3H]paracetamol-derived radioactivity, in contrast to methionine (Fig. 1). At an assay concentration
342
l
As
50
-%
l
1
CONTROL
CYSTEAh
N-ACETYLCYSTEINE
PROTECTIVE
AGENT
AND
Z-MERCAPTOPROPIONYLGLYCINE
CONCENTRATION
METHIONINE
ImM)
Fig. 1. Effects of protective agents on paracetamol covalent binding in vitro. Covalent binding (nmol paracetamol equivalents bound - mg microsomal protein-’ - min-‘) in the presence of protective agents (mean * s.e.m; n = 3) is presented as a percentage of that found in control incubations containing 1 mM paracetamol(l.38 ?r 0.10 nmol * mg” * min-r ; mean + s.e.m., n = 45). Asterisks denote significantly different results (paired t test;P < 0.05).
of paracetamol of 1.0 mM the reduction in binding varied between 20% and 100% depending on the concentrations of the thiols used. Cysteamine was the most effective agent and reduced binding by 88% when present at a concentration of 0.1 mM, while the same concentrations of N-acetylcysteine and 2-mercaptopropionylglycine were less potent inhibitors of binding and produced a 60% and 48% inhibition respectively. Quantitative estimates of the overall efficacy of the thiols were derived from calculations of the amount of sulphydryl compound required to reduce paracetamol-derived covalent binding by 50% (ISo concentrations) and are shown in Table I for assay concentrations of paracetamol substrate of 0.1 mM and 1.0 mM. At both substrate concentrations cysteamine was the most potent inhibitor of covalent binding, 2-mercaptopropionylglycine was the least effective and N-acetylcysteine was of intermediate efficacy. As well as demonstrating the different efficacies of the protective agents in vitro, Table I shows that the ISo concentrations of the 3 thiols did not change proportionately with changes in paracetamol concentration. Thus, despite 4-g-fold increases in total covalent binding, the ISo of cysteamine was similar at both 0.1 mM and 1.0 mM concentrations of paracetamol while those of N-acetylcysteine and 2-mercaptopropionylglycine were increased at the higher concentration by around 50% and 100% respectively. One possible explanation for these findings is that the 3 thiols do not act exclusively at a single stage during the formation and binding of the reactive paracetamol
343 TABLE I I,, CONCENTRATIONS (PM) OF PROTECTIVE AND 1.0 mM PARACETAMOL I,, concentrations
AGENTS IN VITRO USING 0.1 mM
were determined as described under MATERIALS
Protective agent Methionine 2-Mercaptopropionylglycine N-Acetylcysteine Cysteamine
I,, NW
At paracetamol concentrations 0.1 mM 1.0 mM >> 1000 61.9 f 4.1 37.7 f 1.6 8.9 f 0.2
AND METHODS
of
>> 1000 121 *3 57.1 + 2.3 9.5 f 0.35
metabolite. Instead, interactions may occur during the attachment of paracetamol to cytochrome P-450, during the reduction of this cytochrome P450-paracetamol (substrate) complex, during the oxygenation of this reduced P-450-substrate complex and by detoxication of the reactive metabolite generated by this oxidation. Inhibition at the first three of these stages has been demonstrated using sulphydryl compounds and other P-450 substrates [16,17] but only at concentrations 10-100 times those used in the present study. Consequently, the efficacy of cysteamine, iV-acetylcysteine and 2mercaptopropionylglycine is probably achieved primarily from direct interactions between the thiols and the preformed reactive paracetamol metabolite. The observation that paracetamol can form adducts with nucleophilic sulphydryl compounds such as glutathione, cysteine or N-acetylcysteine in vitro apparently endorses this hypothesis [ 18, 191. The results of the present study suggest that differences may exist between the mechanism of action of thiols and protective agents such as methionine which contain no free sulphydryl group. Thus, it is possible that the co-administration of methionine and thiol may be a more effective protective combination against paracetamol toxicity than either compound used separately. Moreover, this regimen may avoid the potential toxicity of large doses of a single antidote. ACKNOWLEDGEMENTS
We are grateful to Sterling-Winthrop Ltd. for generous support including provision of [ ‘Hlparacetamol, to Santen Pharmaceutical Co., Ltd. for 2-mercaptopropionylglycine and financial help, and to the King’s College Hospital Joint Research Committee for financial assistance.
344 REFERENCES 1 R.S. Andrews, C.C. Bond, J. Burnett, A. Saunders and K. Watson, Isolation and identification of paracetamol metabolites, J. Int. Med. Res., 4 Suppl. (4) (1976) 34-39. 2 M. Davis, C.J. Simmons, N.G. Harrison and R. Williams, Paracetamol overdose in man: relationship between pattern of urinary metabolites and severity of liver damage, Quart. J. Med., 45 (1976) 181-191. 3 E.M. Boyd and G.M. Bereczky, Liver necrosis from paracetamol, Br. J. Pharmacol., 26 (1966) 606-614. 4 D.G.S. Davidson and W.N. Eastham, Acute liver necrosis following overdose of paracetamol, Br. Med. J., 2 (1966) 497-499. 5 D.J. Jollow, J.R. Mitchell, W.Z. Potter, D.C. Davis, J.R. Gillette and B.B. Brodie, Acetaminophen-induced hepatic necrosis, II. Role of covalent binding in vivo, J. Pharmacol. Exp. Ther., 187 (1973) 195-202. 6 J.R. Mitchell, D.J. Jollow, W.Z. Potter, J.R. Gillette and B.B. Brodie, Acetaminopheninduced hepatic necrosis, IV. Protective role of glutathione, J. Pharmacol. Exp. Ther., 187 (1973) 211-217. 7 J.R. Mitchell, S.S. Thorgeirsson, W.Z. Potter, D.J. Jollow and H. Keiser, Acetaminophen-induced hepatic injury: protective role of glutathione in man and rationale for therapy, Clin. Pharmacol. Ther., 16 (1974) 676-684. 8 0. Strubelt, C.P. Siegers and A. Schutt, The curative effects of cysteamine, cysteine and dithiocarb in experimental paracetamol poisoning, Arch. Toxicol., 33 (1974) 55-64. 9 A.E.M. McLean and P. Day, The effect of diet on the toxicity of paracetamol and the safety of paracetamol-methionine mixtures, Biochem. Pharmacol., 24 (1975) 37-42. 10 E. Piperno and D.A. Berssenbruegge, Reversal of experimental paracetamol toxicosis with N-acetylcysteine, Lancet, i (1976) 739. 11 D. Labadarios, M. Davis, B. Portmann and R. Williams, Paracetamol-induced hepatic necrosis in the mouse - relationship between covalent binding, hepatic glutathione depletion and the protective effect of ol-mercaptopropionylglycine, Biochem. Pharmacol., 26 (1977) 31-35. 12 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 13 W.Z. Potter, D.C. Davis, J.R. Mitchell, D.J. Jollow, J.R. Gillette and B.B. Brodie, Acetaminophen-induced hepatic necrosis, III. Cytochrome P-450-mediated covalent binding in vitro. J. Pharmacol. Exp. Ther., 187 (1973) 203-210. 14 M.J. Moroney, Facts from Figures, Penguin Books, London, 1974, p. 295. 15 A. White, P. Handler and E.L. Smith, Principles of Biochemistry, McGraw, London, 1968, pp. 531-618. 16 R. Mull, W. Winkelbein, J. Gertz and K. Flemming, A comparative study on the influence of cysteamine and metyrapone on mixed-function oxygenase activities in variously pretreated liver microsomes from rats and mice, Biochim. Biophys. Acta, 481(1977) 407-419. 17 J.A. Castro, E.C. de Ferreyra, C.R. de Castro, M.I. Diaz-Gomez, N. D’Acosta and O.M. de Fenos, Studies on the mechanism of cystamine prevention of several liver structural and biochemical alterations caused by carbon tetrachloride, Toxicol. Appl. Pharmacol., 24 (1973) l-19. 18 A.R. Buckpitt, D.E. Rollins, S.D. Nelson, R.B. Franklin and J.R. Mitchell, Quantitative determination of the glutathione cysteine and N-acetylcysteine conjugates of acetaminophen by high-pressure liquid chromatography, Anal. Biochem., 83 (1977) 168-177. 19 P. Moldeus, Paracetamol metabolism and toxicity in isolated hepatocytes from rat and mouse, Biochem. Pharmacol., 27 (1978) 2859-2863.