Mechanism of Action of N-Acetylcysteine in the Protection Against the Hepatotoxicity of Acetaminophen in Rats In Vivo BERNHARD H. LAUTERBURG, GEORGE B. CORCORAN, and JERRY R. MITCHELL, Baylor College of Medicine, Department of Internal Medicine, Sections on Gastroenterology and Clinical Pharmacology, Houston, Texas 77030 A B S T R A C T N-Acetylcysteine is the drug of choice exogenous N-acetylcysteine significantly increased the for the treatment of an acetaminophen overdose. It is formation of the acetaminophen-glutathione adduct thought to provide cysteine for glutathione synthesis from 57 to 105 nmol/min per 100 g. Although the ex- and possibly to form an adduct directly with the toxic cretion of acetaminophen sulfate increased from 85±15 metabolite of acetaminophen, N-acetyl-p-benzoqui- to 211±17 iimol/100 g per 24 h after N-acetylcysteine, noneimine. However, these hypotheses have not been kinetic simulations showed that increased sulfation tested in vivo, and other mechanisms of action such does not significantly decrease formation of the toxic as reduction of the quinoneimine might be responsible metabolite. Reduction of the benzoquinoneimine by for the clinical efficacy of N-acetylcysteine. After the N-acetylcysteine should result in the formation of N- administration to rats of acetaminophen (1 g/kg) in- acetylcysteine disulfides and glutathione disulfide via traduodenally (i.d.) and of [35S]-N-acetylcysteine (1.2 thiol-disulfide exchange. Acetaminophen alone de- g/kg i.d.), the specific activity of the N-acetylcysteine pleted intracellular glutathione, and led to a progres- adduct of acetaminophen (mercapturic acid) isolated sive decrease in the biliary excretion of glutathione from urine and assayed by high pressure liquid chro- and glutathione disulfide. N-Acetylcysteine alone did matography averaged 76±6% of the specific activity not affect the biliary excretion of glutathione disulfide. of the glutathione-acetaminophen adduct excreted in However, when administered after acetaminophen, N- bile, indicating that virtually all N-acetylcysteine- acetylcysteine produced a marked increase in the bil- acetaminophen originated from the metabolism of the iary excretion of glutathione disulfide from 1.2±0.3 glutathione-acetaminophen adduct rather than from nmol/min per 100 g in control animals to 5.7±0.8 a direct reaction with the toxic metabolite. N-Acetyl- nmol/min per 100 g. Animals treated with acetamin- cysteine promptly reversed the acetaminophen-in- ophen and N-acetylcysteine excreted 2.7±0.8 nmol/ duced depletion of glutathione by increasing gluta- min per 100 g of N-acetylcysteine disulfides (measured thione synthesis from 0.54 to 2.69 gmol/g per h. Ex- by high performance liquid chromatography) com- ogenous N-acetylcysteine did not increase the formation pared to 0.4±0.1 nmol/min per 100 g in rats treated of the N-acetylcysteine and glutathione adducts of with N-acetylcysteine alone. In conclusion, exogenous acetaminophen in fed rats. However, when rats were N-acetylcysteine does not form significant amounts of fasted before the administration of acetaminophen, conjugate with the reactive metabolite of acetamino- thereby increasing the stress on the glutathione pool, phen in the rat in vivo but increases glutathione syn- thesis, thus providing more substrate for the detoxi- Dr. Lauterburg is a recipient of a Pharmaceutical Man- fication of the reactive metabolite in the early phase ufacturers Association Foundation Faculty Development of an acetaminophen intoxication when the critical Award in Clinical Pharmacology. reaction with vital macromolecules occurs. Address reprint requests to Dr. Lauterburg. Dr. Mitchell is a Burroughs Wellcome Scholar in Clinical Pharmacology. Dr. Corcoran is now located at the Department of Phar- INTRODUCTION maceutics, The State University of New York at Buffalo, Amherst, NY 14260. N-Acetylcysteine and other sulfhydryl donors such as Received for publication 24 February 1982 and in revised cysteine, methionine, and cysteamine have been shown form 29 November 1982. to be effective antidotes protecting against the hepa- 980 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/83/04/0980/12 $1.00 Volume 71 April 1983 980-991 totoxicitv of acetaminophen both in animal models As a third possibility, N-acetylcysteine might protect (1-4) and clinical practice (a, 6). Because of its ease by increasing the availability of inorganic sulfate, thus of administration and low toxicity, N-acetylcvsteine increasing the formation of acetaminophen-sulfate is currently the favorite compound for the treatment and simultaneously decreasing the fraction of acet- of acetaminophen overdose in man (6, 7). Despite its amninophen metabolized to the toxic intermiedi- widespread clinical use, however, the mechanism(s) ate (16). of action of N-acetvlcvsteine in vivo remain to be (1em- Fourthlv, N-acetylcysteine could reduce the reac- onstrated. tive intermediate of acetaminophen, N-acetyl-p-ben- N-Acetvlcvsteine could conceivably protect against zoquinoneimine, back to acetaminophen, thereby pre- the hepatotoxicity of acetaminophen in several ways. venting its reaction with glutathione and vital mac- First, N-acetvlcvsteine ma) serve as a precursor for romolecules. We have recently demonstrated that the glutathione synthesis. Glutathione plays a critical role reduction of the reactive metabolite by cysteine or in the protection against hepatic necrosis produced by ascorbic acid is a quantitatively important reaction in acetaminophen (1). Hepatocellular necrosis occurs vitro and prevents covalent binding of the toxic in- only when the hepatic content of glutathione falls be- termediate to microsomal proteins (17). low a critical threshold concentration (1). A stimula- From data obtained mostly in vitro, therefore, a tion of glutathione synthesis following the administra- number of potential mechanisms of action of N-ace- tion of N-acetylcysteine and thus a greater availability tvleysteine have been demonstrated. However, these of glutathione for the detoxification of the toxic acet- mechanistic studies have not evaluated the importance aminophen intermediate should protect against liver of the potential interactions of N-acetylcvsteine with injury. In isolated hepatocytes, N-acetylcysteine is in- the toxic process in vivo. To elucidate the mechanism deed capable of supporting glutathione synthesis when of action in vivo, we have administered radioactively it is the only source of cysteine (8). However, in iso- labeled N-acetvlcysteine together with acetaminophen lated cells incubated with acetaminophen, very high to rats to determine the contribution of exogenous N- concentrations of N-acetylcysteine or preincubation acetyrlcsteine to the formation of the N-acetylcvsteine with the sulfhvdrvl are required to increase the for- alduct of acetaminophen (mercapturic acid) following mation of acetaminophen-glutathione (9). Our recent a toxic dose of acetaminophen. In addition, we have studies of glutathione kinetics following a toxic dose tested the hypothesis that N-acetylcysteine may pro- of acetaminophen further questioned the importance tect by reducing the toxic metabolite of acetamino- of this mechanism of action (10). The synthesis of glu- phen. tathione following a large dose of acetaminophen was found to be suppressed, rather than stimulated, as one METHODS would expect from a depletion of hepatic glutathione (11). Furthermore, some investigators have reported [35S]-N-Acetyl-L-cvsteine was prepared by treating [3S]iJ cys tine (11.4 mCi/mmol, Amersham Corp., Arlington Heights, that N-acetylcysteine does not prevent the acetamin- IL) with two equivalents of acetic anhydride and reducing ophen-induced depletion of glutathione suggesting the peracetylated disulfide intermediate with zinc and acetic that the antidote does not support glutathione synthesis acid according to the method of Sheffner et al. (18). Follow- in vivo (12). ing the addition of unlabeled compound (Sigma Chemical A Co., St. Louis, MO), [355]-N-acetylcysteine was recrystallized second potential mechanism of action is direct from water to a constant specific activity (58.1 dpm/nmol). adduct formation between N-acetylcysteine and the Radiochemical purity exceeded 99% judging from thin-layer reactive intermediate of acetaminophen, thereby pre- chromatography(of the material on Avicel (Analtech Inc., venting covalent binding and cell injurv. In micro- Newark, DE) developed in n-butanol/acetic acid,/w ater, somal incubations, the reactive metabolite of acet- 4:1:1 (retardation factor, Rf, 0.61). The aqueous portion of the mobile phase also contained 1 g/liter Na2EDTA and 0.1 aminophen indeed readily forms adducts with a galiter KCN to inhibit metal-catalyzed oxidation of thi- variety of sulfhvdrvl anions including the N- ols (18). acetylcysteine anion (13). However, the incubation of In order to test the hypothesis that N-acetvlcysteine pro- isolated mouse hiepatocytes with acetaminophen and tects in vivo by formation of an adduct with the reactive N-acetyslcsteine does not result in the formation of intermediate of acetaininophen we compared the specific activities of the glutathione adduct excreted in bile and the the N-acetvlcvsteine adduct of acetaminiophen (8, 9). N-acetylcvsteine adduct (mercapturic acid) excreted in In vivo, the administration of N-acetylcysteine only urine with the specific activity of the administered N-ace- moderately increases the formation of the N-acetvl- tvlcvsteine. Male Sprague