Protection of HepG2 cells from acrolein toxicity by CDDO-Im via glutathione-mediated mechanism

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. The compound is ubiquitously found in endogenous as well as exogenous environment. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examines the toxic effects and cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause a concentration-dependent decrease in cell viability as measured by MTT and LDH assays. Acrolein exposure was also found to cause apoptotic cell death and an increase in levels of protein carbonyl and TBARS, markers of protein damage and lipid peroxidation, respectively, in HepG2 cells. Acrolein also rapidly depleted intracellular glutathione (GSH), phase II enzyme GSH-linked glutathione-S-transferases (GST) and aldose reductase (AR) -- three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability, which suggests that cellular GSH depletion may be an important event in acrolein-induced cytotoxicity. To further determine the role of cellular GSH in protecting against acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2cells. Furthermore, induction of GSH levels by CDDO-Im, a triterpenoid compound, afforded protection against acrolein toxicity in a concentration-dependent manner. Notably, incubation of HepG2 cells with CDDO-Im at a concentration as low as 10 nM leads to a significant increase in GSH content. To further determine the role of GSH in CDDO-Im-mediated cytoprotection against acrolein-mediated cytotoxicity, BSO was used to inhibit cellular GSH induction by CDDO-Im. Pretreatment of HepG2 cells with BSO and CDDO-Im significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im on acrolein-mediated toxicity. In summary, this study demonstrates that exposure to acrolein results in a faster depletion of GSH, an important phase II defense, and causes an increase in apoptosis, lipid peroxidation and protein carbonylation. Furthermore, the endogenous antioxidant GSH can be induced by CDDO-Im. The CDDO-Im-mediated elevated GSH appears to afford a marked protection against acrolein toxicity suggesting that GSH plays a predominant role in CDDO-Im-mediated protection against acrolein-induced toxicity in HepG2 cells. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity