Original Contribution
Mechanism of alcohol-induced oxidative stress and neuronal injury

https://doi.org/10.1016/j.freeradbiomed.2008.08.030Get rights and content

Abstract

Neuro-cognitive deficits, neuronal injury, and neurodegeneration are well documented in alcoholics, yet the underlying mechanisms remain elusive. Oxidative damage of mitochondria and cellular proteins intertwines with the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. Here, we present the evidence that metabolism of ethanol in primary human neurons by alcohol dehydrogenase (ADH) or cytochrome P450-2E1 (CYP2E1) generates reactive oxygen species (ROS) and nitric oxide (NO) via induction of NADPH/xanthine oxidase (NOX/XOX) and nitric oxide synthase (NOS) in human neurons. The acetaldehyde-mediated increase in NOX, XOX, or NOS activity is regulated as a transcriptional rather than a translational process. Marked increase in the lipid peroxidation product (4-hydroxynonenal) and enhanced ROS generation coincides with decreased neuronal viability and diminished expression of neuronal marker (neurofilaments). Novel quantitative methods of ROS and NO detection help dissect the mechanisms of alcohol-induced neurodegeneration. Uncovering the basic mechanisms of oxidative neuronal injury will serve as the basis for development of new therapies.

Introduction

Millions of alcoholics exhibit neuro-cognitive deficits and neuronal injury associated with neuronal degeneration [1], [2], [3]. Although oxidative stress and mitochondrial damage are implicated in tissue injury, the underlying mechanisms of alcohol-induced neurological disorders remain elusive. Oxidative stress-induced mitochondrial damage is involved in Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [4], [5]. Increased microglia prior to development of brain atrophy [6], enhanced neuroinflammation, and oxidative damage [7] after chronic ethanol (EtOH) administration in animal models support the putative mechanism for neurodegeneration in alcoholics. Postmortem examination of brain tissues from alcoholics shows cerebral edema, neuronal loss, and blood–brain barrier (BBB) dysfunction [8], associating with the increased risk of hemorrhagic and ischemic stroke among alcoholics [9], [10], [11]. We observed that alcohol-induced oxidative stress causes BBB dysfunction [12], [13] via an activation of myosin light chain kinase (MLCK) with subsequent phosphorylation of MLC and tight junction proteins [14], activation of inositol 1,4,5-triphosphate receptor-gated intracellular Ca2+ release [15], and activation of matrix metalloproteinases by protein tyrosine kinases [16]. Thus, oxidative stress emerges as the underlying cause of BBB damage, neuroinflammation, and neurological diseases.

It is known that during oxidative stress conditions the levels of oxidants are higher than the levels of antioxidants [17], [18]. Besides mitochondrial oxidative phosphorylation, the cellular activation of NADPH/xanthine oxidase (NOX/XOX) or nitric oxide synthase and copper/iron-catalyzed Fenton-Weiss-Haber (FWH) reaction of H2O2 [19] also produce oxidants. Alcohol-induced ROS production is believed to be specific to EtOH metabolism by cytochrome P450-2E1 (CYP2E1), which produces H2O2 in addition to acetaldehyde (Ach), while alcohol dehydrogenase (ADH) mediated produces only Ach. Interaction of H2O2 with copper/iron produces ROS during EtOH oxidation by alcohol-inducible liver microsomal cytochromes P-450 enzymes [19], [20]. Cederbaum and colleagues showed that metabolism of EtOH by CYP2E1 causes oxidative liver damage by increased ROS levels [21], [22] and by reduction of glutathione and superoxide dismutase activity [23], [24].

While oxidative stress as an underlying cause of many neurological diseases is well documented [25], [26], the role of alcohol-induced oxidative stress in the CNS is largely unknown. Recent high-throughput neuroproteomic studies of protein expression profiles indicated increased levels of ammonia and ROS in the CNS of alcoholics [27]. Chronic alcohol administration resulted in microencephaly and neuronal loss in juvenile mice [28] and impacted behavior [29] through modulation of neuronal nitric oxide synthase, suggesting the involvement of oxidative stress. Protective effects of vitamin E on neuronal loss against alcohol-induced oxidative product in neonatal rat hippocampus [30] and decrease in glutathione level in fetal cortical neurons suggest the role of alcohol-mediated oxidative stress in the CNS [31].

Chronic administration of an alcohol diet enhanced CYP2E1 protein expression in rat brain tissues [32], [33]. Upadhya and colleagues demonstrated the constitutive expression of CYP2E1 protein in neurons within cerebral cortex, Purkinje, or granule cell layers of cerebellum, and induction of CYP2E1 protein level in the cortex of rat or human brain after chronic ethanol consumption [34]. We demonstrated the induction of CYP2E1 activity after EtOH exposure in primary human brain endothelial cells paralleling elevated ROS production and BBB dysfunction [12], [13], [15]. BBB dysfunction could be important to neurological disorders in the context of FWH reaction within the endothelium and copper deficiency in the brain. Copper accumulation in brain endothelial cells increases the rate of FWH reaction and prevents the transport of essential metal ions into the brain cells (astrocytes and neurons) as observed in Menkes' disease [35]. Although accumulation of iron in livers [36] and copper in kidneys/heart [37] has been reported in rats fed chronic alcohol diets, changes in metal ion concentrations in alcoholic brains are currently unknown. Here, we hypothesize that metabolism of EtOH by ADH and CYP2E1 generates ROS and NO in human neurons due to activation of NOX/XOX and inducible nitric oxide synthase (iNOS) by Ach. We observed that an induction of CYP2E1 activity parallels increased ROS and NO production in neurons after EtOH treatment. High levels of lipid peroxidation product, 4-hydroxynonenal, indicating cellular oxidative damage are accompanied by diminished expression of neuronal markers (neurofilaments) and enhanced neuronal death.

Section snippets

Human neuronal isolation

Primary cortical neurons are isolated from human fetal brain tissue (elective abortus specimens) routinely obtained from our neural tissue core facility and are cultured as described [38]. The tissues are obtained in full compliance with the ethical guidelines of both the National Institutes of Health and the University of Nebraska. Briefly, we incubated dissociated tissue with 0.25% trypsin for 30 min, neutralized with 10% fetal bovine serum, and further dissociated by triturating. Resulting

Results

The idea that alcohol abuse causes neuronal injury is well accepted; however, its underlying mechanisms are still poorly understood. Previously, we demonstrated that EtOH metabolism in primary human brain endothelial cells resulted in ROS generation and intracellular calcium release leading to BBB dysfunction and enhanced leukocyte migration across the BBB. We hypothesized that EtOH metabolism in primary human neurons causes ROS/NO production leading to neurodegeneration. To verify this idea,

Discussion

Our findings point to a novel pathway of neurodegeneration associated with alcohol abuse stemming from alcohol-induced oxidative stress. We found that both alcohol-metabolizing enzymes (ADH and CYP2E1) are active in human neurons. While ADH is modestly expressed, EtOH exposure significantly up-regulates the CYP2E1 activity and protein content in primary human neuronal cultures. The present study demonstrates that EtOH metabolism by these enzymes up-regulates the production of ROS and NO via the

Acknowledgments

The authors appreciate the excellent administrative support from Ms. Robin Taylor. This work was supported in part by NIH grants (AA016403, AA015913, and AA017398).

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