The role of mitochondrial injury in bromobenzene and furosemide induced hepatotoxicity

Abstract

Bromobenzene (BB) and furosemide (FS) are two hepatotoxicants whose bioactivation to reactive intermediates is crucial to the development of liver injury. However, the events which lead to hepatocellular toxicity following metabolite formation and covalent binding to cellular macromolecules remain unknown. The present study was undertaken to investigate the effect of administered BB and FS on mitochondrial total glutathione (GSH+GSSG, henceforth referred to as glutathione) content and respiratory function as potential initiating mechanisms of the hepatotoxicity of these compounds in the mouse. Bromobenzene (2 g/kg i.p.) significantly decreased mitochondrial glutathione to 48% of control at 3 h post administration, and to 41% at 4 h. This decrease in mitochondrial glutathione was subsequent to a significant decrease in cytosolic glutathione to 64 and 28% of control at 1 and 2 h, respectively. Oxygen consumption supported by complex I (glutamate-supported) of the respiratory chain was not inhibited by BB until 4 h, where state 3 (active) respiration was reduced to 16% of control. This resulted in a decreased respiratory control ratio (RCR) for complex I-supported respiration. Complex II (succinate)-supported state 3 and state 4 respiration were unaffected by BB until 4 h, at which time they were reduced to 57 and 48% of control, respectively. However, the similar reductions in state 3 and state 4 respiratory rates did not alter the corresponding RCR for complex II. Overt hepatic injury was detected at 4 h, with plasma alanine aminotransferase (ALT) activity increasing significantly at this time point. In contrast to the effects of BB, FS administration (400 mg/kg i.p.) did not alter mitochondrial or cytosolic glutathione, and had no effect on respiration supported by complex I or II for up to 5 h following dosing. However, ALT activity was significantly increased 5 h following FS administration. These results suggest that inhibition of mitochondrial respiratory function coinciding with a decrease in mitochondrial glutathione content may be crucial to the initiation of BB-induced hepatotoxicity, while such events are not required for the initiation of FS-induced hepatotoxicity.

Introduction

Bioactivation to reactive intermediates capable of binding to critical cellular macromolecules and disrupting normal cellular function is a common toxicological pathway shared by a wide assortment of xenobiotics (Cohen et al., 1997, Pumford and Halmes, 1997). Bromobenzene (BB) and furosemide (FS) are examples of two such hepatotoxic compounds whose conversion to reactive intermediates is integral to their toxicity (Jollow et al., 1974, Mitchell et al., 1974). However, the subcellular targets associated with the binding of these reactive intermediates, and the subsequent key initiating events for cytotoxicity, have yet to be elucidated.

Bromobenzene and FS both undergo cytochrome P450-mediated phase I metabolism to reactive epoxide intermediates (Jollow et al., 1974, Mitchell et al., 1974), although secondary quinone metabolites have been proposed to play a significant role in the toxicity of BB (Slaughter and Hanzlik, 1991). Postulated mechanisms of the toxicity induced by the reactive metabolites of BB and FS include arylation of critical cellular macromolecules, deregulation of Ca²⁺ homeostasis, and lipid peroxidation (Jollow et al., 1974, Mitchell et al., 1974, Casini et al., 1985, Casini et al., 1987, Massey et al., 1987, Duthie et al., 1994). Despite sharing some common proposed mechanisms, there are notable differences in the events that lead to the hepatotoxicity elicited by these two compounds. Most striking are the effects of these agents on hepatic glutathione content. Depletion of glutathione below a threshold level occurs prior to the onset of BB-induced hepatotoxicity in the mouse (Jollow et al., 1974), whereas hepatic glutathione levels are not altered by FS prior to the hepatotoxic response (Mitchell et al., 1974). Consequently, the hepatotoxicity of BB was shown to be potentiated by pretreatment with the glutathione-depleting agent diethylmaleate (DEM) (Casini et al., 1985), while the hepatotoxicity of FS was unaffected by such treatment (Mitchell et al., 1984). Maintenance of the total cellular glutathione level is critical for overall preservation of cell homeostasis — however intracellular compartmentalization of glutathione has also been shown to be critical in the relationship between glutathione depletion and cellular injury (Smith et al., 1996). The mitochondrial glutathione pool comprises approximately 10% of total hepatic cellular glutathione (Meredith and Reed, 1982), and could conceivably be specifically depleted in response to an hepatotoxic agent, perhaps as a result of local generation of reactive drug metabolites within mitochondria. Depletion of the mitochondrial glutathione pool would render this organelle susceptible to oxidative stress, and consequently, impair cellular respiration and energy production, in turn providing a possible initiating mechanism for cytotoxicity.

Mitochondrial injury has been investigated as a potential initiating factor in various organ toxicities caused by numerous compounds. An example of an early inhibition of mitochondrial respiratory function as a possible initiator of drug-induced hepatotoxicity was reported by Donnelly et al. (1994), who found alterations in mitochondrial oxygen consumption as early as 1 h after administering an hepatotoxic dose of acetaminophen to mice (Donnelly et al., 1994). Several other studies have investigated the effects of BB and FS on functional parameters of mitochondria in various tissues (Manuel and Weiner, 1976, Orita et al., 1983, Maellaro et al., 1990). In particular, membrane potential of mouse liver mitochondria was reported to be decreased as early as 3 h following in vivo BB treatment (Maellaro et al., 1990), with the decrease becoming larger in magnitude at later time points. Additionally, respiration in isolated rat kidney mitochondria was inhibited by FS in vitro, specifically at complex II of the respiratory chain (Manuel and Weiner, 1976). However, whether an inhibition of respiratory function, and an ensuing decrease in cellular energy production, are initiating events in the hepatotoxicities of these compounds has not been addressed.

In order to further elucidate a possible role for mitochondrial injury in the initiation of the hepatotoxicities induced by BB and FS, the objective of the current study was to characterize a time course of the effects of BB and FS administration on mouse liver mitochondrial and cytosolic glutathione levels, mitochondrial respiratory function, and cytotoxicity.