Efavirenz and 8-hydroxyefavirenz induce cell death via a JNK- and BimEL-dependent mechanism in primary human hepatocytes

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Abstract

Chronic use of efavirenz (EFV) has been linked to incidences of hepatotoxicity in patients receiving EFV to treat HIV-1. While recent studies have demonstrated that EFV stimulates hepatic cell death a role for the metabolites of efavirenz in this process has yet to be examined. In the present study, incubation of primary human hepatocytes with synthetic 8-hydroxyEFV (8-OHEFV), which is the primary metabolite of EFV, resulted in cell death, caspase-3 activation and reactive oxygen species formation. The metabolite exerted these effects at earlier time points and using lower concentrations than were required for the parent compound. In addition, pharmacological inhibition of cytochrome P450-dependent metabolism of EFV using 1-aminobenzotriazole markedly decreased reactive oxygen species formation and cell death. Treatment of primary human hepatocytes with EFV and 8-OHEFV also stimulated phosphorylation of c-Jun N-terminal kinase (JNK) as well as phosphorylation of the JNK substrate c-Jun. Further, the mRNA and protein expression of an isoform of Bim (Bcl-2 interacting mediator of cell death) denoted as BimEL, which is proapoptotic and has been shown to be modulated by JNK, was increased. Inhibition of JNK using SP600125 prevented the EFV- and 8-OHEFV-mediated cell death. Silencing of Bim using siRNA transfected into hepatocytes also prevented cell death resulting from 8-OHEFV-treatment. These data suggest that the oxidative metabolite 8-OHEFV is a more potent inducer of hepatic cell death than the parent compound EFV. Further, activation of the JNK signaling pathway and BimEL mRNA expression appear to be required for EFV- and 8-OHEFV-mediated hepatocyte death.

Highlights

► 8-Hydroxyefavirenz is a more potent stimulator of cell death than efavirenz. ► Efavirenz and 8-hydroxyefavirenz increase JNK activity and BimEL mRNA expression. ► JNK and Bim are required for efavirenz- and 8-hydroxyefavirenz-mediated cell death. ► Efavirenz and 8-hydroxyefavirenz may be novel modulators of Bim.

Introduction

Efavirenz is the most widely prescribed non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1. EFV is extensively metabolized by the cytochrome P450 (P450) to yield several metabolites including its primary metabolite 8-hydroxyefavirenz (8-OHEFV), which is catalyzed by P450 2B6 (Ward et al., 2003, Bumpus et al., 2006, Ogburn et al., 2010). The EFV to 8-OHEFV ratio decreases over time with chronic use of EFV such that the concentration of 8-OHEFV surpasses that of EFV (Ngaimisi et al., 2010). This is due to the fact that EFV induces the expression of P450 2B6 at the transcriptional level (Mouly et al., 2002, Faucette et al., 2007). Although this drug is therapeutically beneficial it is estimated that up to 10% of patients receiving EFV have to discontinue treatment due to liver toxicity (Kappelhoff et al., 2005). While functional outcomes in patients receiving EFV have been well described, the molecular bases of these toxicities remain largely undefined. Recently, it has been shown in vitro that EFV induces apoptosis in human hepatic cells by compromising mitochondrial function (Apostolova et al., 2010, Blas-Garcia et al., 2010, Apostolova et al., 2011). However, roles for metabolites of EFV in these adverse events have yet to be examined. Since chronic use of EFV results in increased 8-OHEFV formation, the present study tested whether this metabolite also has the ability to stimulate cell death. In addition, experiments were performed to identify the signaling pathways that may play a causal role in EFV-induced hepatic cell death.

c-Jun n-terminal kinases (JNK) are a family of serine/threonine kinases that are important regulators of cellular stress response including modulation of cell death. Sustained activation of JNK has been demonstrated to result in apoptosis in hepatic cells in response to a variety of stimuli including the hepatotoxic drug acetominophen (Liu et al., 2002, Gunawan et al., 2006). Although it is likely that JNK activation induces cell death through a number of mechanisms, there is evidence supporting a role for JNK-mediated increased activity of proapoptotic proteins such as Bcl-2 interacting mediator of cell death (Bim) (Lei and Davis, 2003). Bim functions in stimulating apoptosis by binding to and antagonizing anti-apoptotic Bcl-2 family members including Bcl-xL, Mcl-1 and Bcl-w (Czabotar et al., 2009). Alternative splicing results in the generation of three Bim isoforms, namely BimL (15 kDa), BimS (12 kDa) and BimEL (23 kDa), with BimEL being the most abundant form detected in most cell types including hepatocytes (Ramesh et al., 2009). In addition, Bim isoforms have gained increasing attention as this molecule has been proposed as an attractive target for tumor therapy (Akiyama et al., 2009). In fact, the chemotherapeutic agent paclitaxel has been demonstrated to upregulate BimEL expression which contributes to its mechanism of action (Sunters et al., 2003). The present study tested a role for JNK and BimEL in EFV-mediated cell death. Three novel findings are presented: 1) 8-OHEFV stimulates cell death, caspase-3 activation and increased reactive oxygen species (ROS) production at concentrations that are lower than those of the parent compound that induce similar responses; 2) treatment of primary human hepatocytes with either EFV or 8-OHEFV results in the activation of JNK activity and BimEL mRNA expression; 3) hepatic cell death mediated by both of these compounds is JNK- and Bim-dependent. Taken together these data suggest that 8-OHEFV may play a role in EFV-mediated hepatotoxicity. Further, JNK and BimEL could represent potential therapeutic targets for the prevention of this toxicity. In addition, EFV and 8-OHEFV may be novel pharmacological tools that can be used to modulate the expression and activity of the chemotherapeutic target BimEL.

Section snippets

Materials

6-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (Invitrogen, Carlsbad, CA); 1-aminobenzotriazole (ABT) (Santa Cruz Biotechnology, Santa Cruz, CA); SP600125, an anthrapyrazolone selective inhibitor of JNK (Sigma-Aldrich, St. Louis, MO); N-acetyl cysteine (NAC) and PMSF (phenylmethanesulfonyl fluoride) (Sigma-Aldrich, St. Louis, MO). EFV and 8-OHEFV were purchased from Toronto Research Chemicals (North York, Ontario, Canada).

Primary human hepatocytes

Primary human hepatocytes were obtained

EFV and 8-OHEFV stimulate hepatic cell death

Efavirenz has previously been reported to cause cell death in liver-derived cell lines. With this in mind, experiments were performed to test whether treatment of primary human hepatocytes with the oxidative metabolite of EFV, 8-OHEFV results in apoptotic cell death. Incubation with 10 μM 8-OHEFV increased cell death by 3.4-fold at 6 h following treatment with a maximum effect observed at 24 h (4.1-fold; p < 0.01) (Fig. 1A). In contrast, although EFV increased cell death in a time- and

Discussion

Non-nucleoside reverse transcriptase inhibitors are a structurally diverse class of small (< 600 Da) hydrophobic molecules that specifically inhibit HIV-1 reverse transcriptase (de Clercq et al., 1998) thereby preventing replication of the virus. Efavirenz is the most commonly prescribed non-nucleoside reverse transcriptase inhibitor in part due to the fact that it can be given once daily as a result of its long half-life of 40–55 h after multiple doses (Vrouenraets et al., 2007). Non-nucleoside

Conflict of interest statement

None declared.

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    This work was supported in part by a PhRMA Foundation Research Starter Grant awarded to N.B.

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