Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum

Abstract

The purpose of the present work was to study the mechanisms involved in apoptosis induced by oxidative stress in rat hepatocytes. We focused on the apoptotic signaling molecules cytochrome c, Bcl-2 and Bax. Rat hepatocytes were exposed for 1 h to increasing concentrations of tert-butylhydroperoxide (t-BHP). Using lactate dehydrogenase (LDH) leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, we observed that a concentration of t-BHP of 0.4-0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored. Malondialdehyde and 8-oxo-guanine formation indicates that t-BHP induces oxidative stress and damage. However, at 0.4 mM t-BHP, these oxidative molecular changes as well as LDH leakage no longer progress after the first hour of t-BHP exposure, suggesting the activation of some defense mechanisms. Western blot analysis of cytochrome c shows that its level increases in the cytosol while that of Bax decreases in this fraction as a result of t-BHP treatment. Moreover, there is a loss of Bcl-2 from mitochondria while, in contrast, Bax accumulates in this organelle following t-BHP treatment. However, cytochrome c appears to be relocalized to the endoplasmic reticulum as its presence in microsomes is greatly enhanced. We suggest that t-BHP triggers apoptosis through a step that involves cytochrome c release from mitochondria. This event is stimulated by Bcl-2 disappearance from mitochondria and Bax recruitment. Neutralization of excess cytosolic cytochrome c is achieved by its relocalization to the endoplasmic reticulum, hence triggering the down-regulation of apoptotic signals.

Essential Roles of Receptor-Interacting Protein and TRAF2 in Oxidative Stress-Induced Cell Death

“…In order to strengthen our findings, we also tested the effects of other oxidants, such as tert-butyl hydroperoxide, an organic analog of H 2 O 2 (21), and menadione, which is an intracellular superoxide generator (47). In the cell death induced by these two agents, the different gene disruptions had the same effect as in H 2 O 2 -induced cell death ( Fig.…”

Modulation of the age‐related nuclear factor‐κB (NF‐κB) pathway by hesperetin

“…The inactivation of t-BHP is predominantly accomplished by glutathione peroxidase (Aruoma et al, 1991;Termini, 2000). Both t-BHP and H 2 O 2 generate ROS that are responsible for lipid peroxidation, DNA adduct formation and the induction of apoptosis (Haidara et al, 2002). Therefore, in our study, cells were treated with t-BHP to induce oxidative stress.…”

Preventive effects of quercetin on liver damages in high-fat diet-induced obesity

“…These adaptive responses include the stimulation of Bcl-2 gene family proteins, which contain both proapoptotic (such as Bax) and antiapoptotic members (such as Bcl-2) [48]. These proteins play an important role in deciding whether a cell will live or die [49]. An important ratio of Bax/Bcl-2 proteins plays a crucial role in mitochondrial outer-membrane permeabilization, release of cytochrome C, and, initiation of apoptosis [50].…”