Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite

Abstract

Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

Introduction

Arsenic is associated with multiple liver diseases, including biliary occlusion, fibrosis and cirrhosis, as well as liver cancer (Centeno et al., 2002, Liu et al., 2006). These effects may arise, in part, due to arsenic-mediated decreases in the cytochrome P450s (CYPs), the major drug metabolizing enzymes in the liver (Spink et al., 2002, Jacobs et al., 1999, Noreault et al., 2005a, Noreault et al., 2005b). Such decreases may lead to the accumulation of drugs and toxic chemicals in the liver. In rodents, acute arsenite exposure results in alterations in basal and induced levels of CYPs, depending on the target organ and the chemical inducer that is used (Albores et al., 1992). In studies using cultured rat hepatocytes, arsenite (5 μM) caused major decreases in CYP3A protein induction, with little to no decrease in CYP3A23 mRNA (Jacobs et al., 1999). This decrease in CYP3A protein is partially due to decreased association of CYP3A23 mRNA with polyribosomes, indicating that arsenite can decrease CYP3A translation (Noreault et al., 2005b). This low concentration of arsenite did not decrease the association of total mRNA with polyribosomes. In contrast, high concentrations of arsenite (50 to 100 μM) have been shown to inhibit total protein synthesis in cultured rat hepatocytes, associated with increased phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) on the alpha subunit (Brostrom and Brostrom, 1998).

Arsenite is known to affect many cellular processes (Tseng, 2004 for review). For example, arsenite has been shown to decrease some proteins by activation of the ubiquitin/proteasome pathway of degradation (Chen et al., 2002, Tsou et al., 2005). The proteasome pathway has been suggested to degrade both native and denatured CYP3A (Correia et al., 1992, Roberts, 1997, Wang et al., 1999). Arsenite is known to react with vicinal thiols (Del Razo et al., 2001) and, by this means, may target CYP3A23 protein for degradation. If this were the case, then inhibition of proteasomal degradation would diminish the action of arsenite to decrease induction of CYP3A23. Thus, it was of interest to determine if inhibitors of the proteasome pathway would increase CYP3A induction in the presence of arsenite.

The proteasomal inhibitors, MG132 and lactacystin, are widely used to inhibit proteasome activity in intact cells (Lee and Goldberg, 1998). Neither the reversible inhibitor, MG132, nor the irreversible inhibitor, lactacystin, inhibited MTT reduction, suggesting that these proteasome inhibitors were not toxic. While MG132 effectively inhibited proteasome activity in primary rat hepatocyte cultures, this agent also decreased CYP3A23 protein and mRNA, as well as total protein synthesis. Lactacystin decreased CYP3A23 protein without affecting induction of CYP3A23 mRNA, findings similar to those obtained for arsenite (Noreault et al., 2005b). Therefore, the effect of lactacystin on translational efficiency was investigated by comparison of the amount of DEX-induced CYP3A23 mRNA associated with polyribosomes in the presence and absence of lactacystin.