Sex difference in susceptibility to acetaminophen hepatotoxicity is reversed by buthionine sulfoximine

Abstract

Gender is a factor that influences susceptibility of individuals to drug-induced liver injury in experimental animals and humans. In this study, we investigated the mechanisms underlying resistance of female mice to acetaminophen (APAP)-induced hepatotoxicity. Overnight-fasted male and female CD-1 mice were administered APAP intraperitoneally. A minor increase in serum alanine aminotransferase levels was observed in female mice after APAP administration at a dose that causes severe hepatotoxicity in males. Hepatic glutathione (GSH) depleted rapidly in the both genders prior to development of hepatotoxicity, whereas its recovery was more rapid in female than in male mice. This was consistent with higher induction of hepatic glutamate-cysteine ligase (GCL) in females. Pretreatment of mice with L-buthionine sulfoximine (BSO), an inhibitor of GCL, exaggerated APAP hepatotoxicity only in female mice, resulting in much higher hepatotoxicity in female than in male mice. In addition, hepatic GSH was markedly depleted in BSO-pretreated female mice compared with male mice, which supports severe hepatotoxicity in BSO-pretreated females. APAP treatment highly induced multidrug resistance-associated protein 4 (Mrp4) only in female mice. The resulting high Mrp4 expression could thus contribute to decreased hepatic GSH levels via sinusoidal efflux when GCL is inhibited. In conclusion, resistance to APAP hepatotoxicity in female mice and its reversal by pretreatment with BSO could be attributed to sex differences in disposition of hepatic GSH, which may generally determine susceptibility to drug-induced liver injury.

Introduction

Drug-induced liver injury is often life-threatening. It is a major reason for withdrawal of drugs from the market and cessation of new drug development. In many cases, drug-induced liver injury is classified as an idiosyncratic reaction because a definitive underlying cause of the disease remains to be identified. The relatively low incidence and idiosyncratic nature of most cases of drug-induced liver injury suggest that the susceptibility of impacted individuals could be determined by multiple genetic and/or acquired risk factors, such as cytochrome P450 polymorphisms, age, gender, hepatic and extrahepatic diseases, and drug interactions (Larrey, 2002). Gender is a factor that influences hepatic drug metabolism in experimental animals and in humans (Anderson, 2005, Fujita et al., 1989, Kato, 1974, Waxman and Holloway, 2009), which could thus influence susceptibility of individuals to drug-induced hepatotoxicity. In general, females are more susceptible to drug-induced (Boelsterli and Lim, 2007, Miller, 2001, Zimmerman, 2000) as well as alcoholic liver injuries in humans (Mandayam et al., 2004, Thurman, 1998). However, the mechanism for susceptibility of females remains largely unknown because of the lack of suitable animal models to assess female-dominant, drug-induced liver injury.

Acetaminophen (APAP), a commonly used analgesic, is usually safe when administered in therapeutic doses. However, its overdose causes liver injury in experimental animals and humans. APAP has been used extensively to develop an animal model of drug-induced liver injury. The toxicity is initiated by cytochrome P450 (CYP) metabolism into N-acetyl-p-benzoquinone imine (NAPQI), and the high reactivity of NAPQI with sulfhydryl groups results in depletion of reduced glutathione (GSH) in hepatocytes, followed by covalent binding to intracellular proteins (Dahlin et al., 1984, Jollow et al., 1973, Mitchell et al., 1973). Subsequent studies provided important mechanistic insights into APAP hepatotoxicity, such as targets of NAPQI and multiple pathologic factors both related and unrelated to drug metabolism, which have contributed significantly to understanding the pathogenesis of drug-induced liver injury (Bessems and Vermeulen, 2001, Cohen et al., 1997, Jaeschke et al., 2003, James et al., 2003, Kaplowitz et al., 2008).

Male mice are widely used for developing murine models of APAP-induced liver injury, no sex differences were reported with respect to APAP hepatotoxicity in CD-1 mice (Hoivik et al., 1995). However, recent studies reported that male C57BL/6 mice were more susceptible to APAP hepatotoxicity than females (Botta et al., 2006, Dai et al., 2006, McConnachie et al., 2007). Mechanisms underlying the postulated sex differences remain to be elucidated. However, studies on transgenic and knockout mice of glutamate-cysteine ligase (GCL), a rate-limiting enzyme in glutathione synthesis, suggest that this enzyme is associated with sex differences in APAP hepatotoxicity (Botta et al., 2006, McConnachie et al., 2007). These findings prompted us to identify gender and related key factors influencing host susceptibility to APAP in mice. In this study, we examined APAP-induced liver injury in male and female CD-1 mice and also observed male-dominant hepatotoxicity in this strain. We also examined sex differences of APAP hepatotoxicity in the mice treated with L-buthionine sulfoximine (BSO), an inhibitor of GCL. BSO treatment reversed the sex differences in APAP hepatotoxicity; female mice administered BSO exhibited much higher susceptibility to APAP hepatotoxicity than corresponding male mice.