Abstract
CYP2E1 metabolizes ethanol leading to production of reactive oxygen species (ROS) and acetaldehyde, which are known to cause not only liver damage but also toxicity to other organs. However, the signaling pathways involved in CYP2E1 regulation by ethanol are not clear, especially in extra-hepatic cells. This study was designed to examine the role of CYP2E1 in ethanol-mediated oxidative stress and cytotoxicity, as well as signaling pathways by which ethanol regulates CYP2E1 in extra-hepatic cells. In this study, we used astrocytic and monocytic cell lines, because they are important cells in central nervous system . Our results showed that 100 mM ethanol significantly induced oxidative stress, apoptosis, and cell death at 24 h in the SVGA astrocytic cell line, which was rescued by a CYP2E1 selective inhibitor, diallyl sulfide (DAS), CYP2E1 siRNA, and antioxidants (vitamins C and E). Further, we showed that DAS and vitamin C abrogated ethanol-mediated (50 mℳ) induction of CYP2E1 at 6 h, as well as production of ROS at 2 h, suggesting the role of oxidative stress in ethanol-mediated induction of CYP2E1. We then investigated the role of the protein kinase C/c-Jun N-terminal kinase/specificity protein1 (PKC/JNK/SP1) pathway in oxidative stress-mediated CYP2E1 induction. Our results showed that staurosporine, a non-specific inhibitor of PKC, as well as specific PKCζ inhibitor and PKCζ siRNA, abolished ethanol-induced CYP2E1 expression. In addition, inhibitors of JNK (SP600125) and SP1 (mithramycin A) completely abrogated induction of CYP2E1 by ethanol in SVGA astrocytes. Subsequently, we showed that CYP2E1 is also responsible for ethanol-mediated oxidative stress and apoptotic cell death in U937 monocytic cell lines. Finally, our results showed that PKC/JNK/SP1 pathway is also involved in regulation of CYP2E1 in U937 cells. This study has clinical implications with respect to alcohol-associated neuroinflammatory toxicity among alcohol users.
Highlights
The alcohol metabolism-mediated liver toxicity occurs through the formation of reactive oxygen species (ROS) and the reactive metabolite, acetaldehyde, which cause DNA damage and lipid and protein oxidations.[4]
As previously shown in U937 monocytic cells,[15] we examined whether ethanol induces ROS in SVGA astrocytes at 100 mM ethanol at 12–36 h
To examine whether CYP2E1 is responsible for the generation of ROS, we knocked down CYP2E1 expression through transfection using 10 nM predesigned CYP2E1 siRNA and 10 nM scrambled siRNA as control
Summary
The alcohol metabolism-mediated liver toxicity occurs through the formation of reactive oxygen species (ROS) and the reactive metabolite, acetaldehyde, which cause DNA damage and lipid and protein oxidations.[4]. CYP2E1 is the only enzyme involved in the non-catalase oxidation of ethanol and ROS production.[10] Its induction leads to increased lipid peroxidation and apoptosis, Regulation of CYP2E1 by ethanol through PKC/JNK/SP1 M Jin et al resulting in increased permeability of the blood–brain barrier and neurodegeneration.[11] limited information is available on the role of CYP2E1 in ethanol-mediated effects on human astrocytes, which is the predominant cell type in the brain and its major role is to protect neuronal integrity.[12,13] Activated astrocytes, especially through increased oxidative stress by alcohol, may cause neuronal damage. In this study, we used human SVGA astrocytic and U937 monocytic cell lines to investigate the role of CYP2E1 in ethanol-mediated oxidative stress, apoptosis, cell death, and the mechanism by which ethanol regulates CYP2E1 expression
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