Therapeutic effect of S-allylmercaptocysteine on acetaminophen-induced liver injury in mice

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Abstract

S-allylmercaptocysteine is one of the water-soluble organosulfur compounds in ethanol extracts of garlic (Allium sativum L.). We had demonstrated earlier that treatment with S-allylmercaptocysteine before acetaminophen administration protects mice against acetaminophen-induced hepatotoxicity. In this study, we examined the therapeutic effect of S-allylmercaptocysteine treatment after acetaminophen administration. A single dose of S-allylmercaptocysteine (200 mg/kg, p.o.) to mice 0.5 h after acetaminophen administration (500 mg/kg, p.o.) significantly suppressed both the increase in plasma alanine aminotransferase activity and the hepatic necrosis, and also reduced acetaminophen-induced mortality from 43% to 0%. These data indicate that S-allylmercaptocysteine is useful as an antidote for acetaminophen overdose. S-allylmercaptocysteine significantly suppressed hepatic cytochrome P450 2E1 (CYP2E1) activity and induction of inducible 70-kDa heat shock protein, a marker of acetaminophen arylation of protein. These results suggest that S-allylmercaptocysteine exerts its protective effect by inhibition of CYP2E1 activity, which leads to the suppression of acetaminophen arylation of hepatic protein.

Introduction

Since ancient times, garlic (Allium sativum L.) has been used world-wide as a food and a folk medicine. In recent years, many studies have shown that garlic possesses antimicrobial (Cavallito and Bailey, 1944), antithrombotic (Ariga et al., 1981), antitumor (Sumiyoshi and Wargovich, 1989), antihyperlipidemic Kamanna and Chandrasekhara, 1982, Lau et al., 1987, Steiner et al., 1996, antioxidant Horie et al., 1989, Ide et al., 1996, Imai et al., 1994 and hepatoprotective Hikino et al., 1986, Wang et al., 1996 properties.

S-allylmercaptocysteine is one of the water-soluble organosulfur compounds in the aged garlic extract obtained by ethanol extraction of sliced garlic bulbs. S-allylmercaptocysteine has been shown to inhibit cell proliferation (Sigounas et al., 1997) and to have radical scavenging (Imai et al., 1994) and intraocular pressure-lowering effects (Chu et al., 1999). Moreover, several investigators, including our group, have demonstrated that S-allylmercaptocysteine pretreatment protects the liver against a number of hepatotoxicants, such as acetaminophen, carbon tetrachloride and d-galactosamine Hikino et al., 1986, Nakagawa et al., 1989, Sumioka et al., 1998.

Acetaminophen is widely used as an analgesic and antipyretic drug. Although acetaminophen is harmless at therapeutic doses, it can produce hepatic injury in both human and experimental animals when given in high doses. After therapeutic doses, it is biotransformed and eliminated as non-toxic glucuronic acid and sulfate conjugates Black, 1980, Pacifici et al., 1988. Only a small proportion of acetaminophen is converted to N-acetyl-p-benzoquinoneimine (NAPQI) (Dahlin et al., 1984), a cytochrome P450 (CYP)-mediated intermediate metabolite, which is normally detoxified by conjugation with GSH. However, after high doses of acetaminophen, the capacity for its removal by hepatic conjugation with glucuronide and sulfate is exceeded, and more of the reactive metabolite NAPQI is formed. Consequently, more NAPQI is conjugated with GSH, and when the hepatic GSH is depleted, more NAPQI will bind covalently to cellular macromolecules Jollow et al., 1973, Potter and Hinson, 1986. This adduction of proteins by NAPQI is thought to lead to liver injury, and to play a key role in triggering heat shock protein (HSP) induction (Salminen et al., 1998). Mice and hamsters have been shown to be very sensitive to the hepatotoxic effects of acetaminophen, developing fulminant centrilobular necrosis similar to that observed in the human (Hinson, 1980).

Protection against acetaminophen-induced liver injury can be achieved by various mechanisms. Cysteine prodrugs, including N-acetylcysteine, the most widely used antidote for acetaminophen overdose, have been reported to protect the liver against acetaminophen-induced injury. The mechanism responsible for this protection may be metabolism of these prodrugs to l-cysteine, which is incorporated into hepatic GSH Corcoran and Wong, 1986, Hazelton et al., 1986a, Lauterburg et al., 1983, Miners et al., 1984, Roberts et al., 1987. Moreover, several CYP enzymes have been reported to play important roles in the bioactivation of acetaminophen to NAPQI Harvison et al., 1988, Morgan et al., 1983, Patten et al., 1993, Raucy et al., 1989. Studies on CYP2E1 knockout mice have made it clear that CYP2E1 is the most important factor in acetaminophen bioactivation Lee et al., 1996, Zaher et al., 1998. In addition, CYP2E1 inhibitors such as diallyl sulfide, phenethyl isothiocyanate and 2-(allylthio)pyrazine protect the liver against acetaminophen-induced injury Kim et al., 1997, Li et al., 1997, Wang et al., 1996. This evidence supports the crucial role of the CYP2E1 enzyme in acetaminophen-induced liver damage. Furthermore, results of recent studies suggest that acetaminophen-induced liver injury is also caused by cellular oxidative stress, resulting in hepatic lipid peroxidation Albano et al., 1983, Amimoto et al., 1995, Wendel et al., 1979. Several antioxidants and antioxidative enzymes, such as coenzyme Q10, α-tocopherol, ascorbic acid and superoxide dismutase, have been shown to prevent acetaminophen-induced liver injury in vivo Amimoto et al., 1995, Mitra et al., 1991, Nakae et al., 1990. Finally, enhancing the capacity for conjugation with glucuronic acid may protect against acetaminophen-induced liver injury, as shown in an experiment that used butylated hydroxyanisole (Hazelton et al., 1986b).

In a previous study, we demonstrated that S-allylmercaptocysteine pretreatment before acetaminophen administration protected mice against acetaminophen-induced liver injury Nakagawa et al., 1989, Sumioka et al., 1998. Considering the clinical possibility for the use of S-allylmercaptocysteine for acetaminophen overdose, it is very important to examine whether S-allylmercaptocysteine treatment after acetaminophen overdose can relieve hepatotoxicity. In this study, we, therefore, examined the therapeutic effect of S-allylmercaptocysteine against acetaminophen-induced hepatotoxicity. Since the mechanisms responsible for this hepatoprotection may be inhibition of CYP2E1 activity (Sumioka et al., 1998), we assumed that formation of the CYP-mediated reactive metabolite, NAPQI and subsequent acetaminophen arylation of protein would be decreased by S-allylmercaptocysteine treatment. However, we had no data to support our assumption. To clarify this, we examined whether S-allylmercaptocysteine treatment affected acetaminophen-induced hepatic-inducible 70-kDa heat shock protein (HSP70i) induction. This induction is strongly associated with the hepatic intralobular sites of acetaminophen arylation of protein (Salminen et al., 1997a).

Section snippets

Chemicals

S-allylmercaptocysteine was synthesized as described previously (Hikino et al., 1986). Acetaminophen, 2-vinylpyridine and phenylmethylsulfonyl fluoride were purchased from Wako (Osaka, Japan). Glutathione reductase, isocitric dehydrogenase and N-acetylcysteine were purchased from Sigma (St. Louis, MO, USA). NADPH and NADP were purchased from Boehringer Mannheim Biochemicals (Mannheim, Germany). N-nitrosodimethylamine was purchased from Tokyo Kasei Industry (Tokyo, Japan).

Effect of S-allylmercaptocysteine on acetaminophen-induced hepatotoxicity

The effect of S-allylmercaptocysteine on acetaminophen-induced mortality is shown in Table 1. Treatment with only the vehicle 0.5 h after acetaminophen administration gave a mortality of 43% by 48 h after acetaminophen administration. S-allylmercaptocysteine treatment 0.5 h after acetaminophen administration completely blocked acetaminophen-induced mortality. However, S-allylmercaptocysteine treatment 1 h after acetaminophen administration did not improve acetaminophen-induced mortality.

Discussion

In a previous study, we demonstrated that S-allylmercaptocysteine treatment (once a day for 2 days) before acetaminophen administration dramatically protects mice against acetaminophen-induced liver injury Nakagawa et al., 1989, Sumioka et al., 1998. In the current study, we examined the therapeutic effect of S-allylmercaptocysteine given after acetaminophen and its potential application to clinical therapy.

A single dose of S-allylmercaptocysteine 0.5 h after acetaminophen administration

Acknowledgements

We are grateful to Professor K. Sato, Department of Molecular Biology, School of Life Science, Tottori University, for his helpful discussion.

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