Ethanol‐induced oxidative stress precedes mitochondrially mediated apoptotic death of cultured fetal cortical neurons

Abstract

In utero ethanol exposure elicits apoptotic cell death in the fetal brain, and this may be mediated by oxidative stress. Our studies utilize cultured fetal rat cortical neurons and illustrate that ethanol elicits a rapid onset of oxidative stress, which culminates in mitochondrially mediated apoptotic cell death. Cells exposed to ethanol (2.5 mg/ml) remained attached to their polylysine matrix during a 24-hr exposure, but they exhibited distinct signs of oxidative stress, decreased viability, and apoptosis. Confocal microscopy of live cortical neurons pretreated with dichlorodihydrofluorescein diacetate demonstrated an increase in reactive oxygen species (ROS) within 5 min of ethanol exposure. The levels of ROS further increased by 58% within 1 hr (P <.05) and by 82% within 2 hr (P <.05), accompanied by increases of mitochondrial 4-hydroxynonenal (HNE). These early events were followed by decreased trypan blue exclusion of 10% to 32% (P <.05) at the 6- to 24-hr time points, respectively. This culminates in apoptotic death, with increases of Annexin V binding of 43%, 89%, 123%, and 238%, at 2, 6, 12, and 24 hr of ethanol treatment, respectively, as well as DNA fragmentation increases of 50% and 65% by 12 and 24 hr, respectively. Release of cytochrome c by mitochondria increased by 53% at 6 hr of exposure (P <.05), concomitant with activation of caspase 3 (52% at 12 hr, P <.05). Pretreatment with N-acetylcysteine increased cellular glutathione and prevented apoptosis. These studies provide a time line illustrating that oxidative stress and formation of a proapoptotic lipid peroxidation product, HNE, precede a cascade of mitochondrially mediated events in cultured fetal cortical neurons, culminating in apoptotic death. The prevention of apoptosis by augmentation of glutathione stores also strongly supports a role for oxidative stress in ethanol-mediated apoptotic death of fetal cortical neurons.

Ethanol exposure of neonatal rats does not increase biomarkers of oxidative stress in isolated cerebellar granule neurons

“…This was notable based on previous work of other investigators and current hypotheses (Heaton et al, 2000a;Heaton et al, 2003;Heaton et al, 2002;Henderson et al, 1995;Ramachandran et al, 2003;Siler-Marsiglio et al, 2004;Siler-Marsiglio et al, 2005b). However, it is consistent with reports from several investigators suggesting that oxidative stress is not involved in ethanol neurotoxicity (Edwards et al, 2002;Grisel and Chen, 2005;Pierce et al, 2006;Smith et al, 2005;Tran et al, 2005).…”

“…Based on multiple reports, it has been hypothesized that ethanol induces production of ROS and, further, that antioxidants protect neurons from ethanol-induced death by blocking production of ROS (Heaton et al, 2000a;Heaton et al, 2003;Heaton et al, 2002;Henderson et al, 1995;Ramachandran et al, 2003;Siler-Marsiglio et al, 2004;Siler-Marsiglio et al, 2005b). However, other studies have failed to support the hypothesis (Edwards et al, 2002;Grisel and Chen, 2005;Pierce et al, 2006;Smith et al, 2005;Tran et al, 2005).…”

“…These include: altered neuronal metabolism (de la Monte et al, 2005;Li et al, 2002), glutamate excitotoxicity (Ikonomidou et al, 2000), disruption of apoptotic regulators (Heaton et al, 2006;Siler-Marsiglio et al, 2005a), limited neurotrophin support (Heaton et al, 2000b;Light et al, 2002b;Light et al, 2001), restricted synaptic development (Klintsova et al, 2002;West et al, 1994), and oxidative stress (Heaton et al, 2003;Heaton et al, 2002;Henderson et al, 1995;Ramachandran et al, 2003;Reyes et al, 1993;Siler-Marsiglio et al, 2005b;Smith et al, 2005). Oxidative stress can cause mitochondrial membrane depolarization, which is followed by cytochrome c release, caspase activation, and apoptosis (Bernardi et al, 2006;O'Rourke et al, 2005).…”

“…Oxidative stress can cause mitochondrial membrane depolarization, which is followed by cytochrome c release, caspase activation, and apoptosis (Bernardi et al, 2006;O'Rourke et al, 2005). In fact, ethanol treatment in cell culture as well as in the developing brain in vivo increases levels of ROS and free radicals, decreases endogenous antioxidant enzyme and metabolite concentrations, and produces lipid peroxidation (Heaton et al, 2003;Heaton et al, 2002;Henderson et al, 1995;Ramachandran et al, 2003;Siler-Marsiglio et al, 2005a;Siler-Marsiglio et al, 2004;SilerMarsiglio et al, 2005b;Smith et al, 2005).…”

Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the balance

“…HNE can directly induce apoptosis but it can also be a mediator of apoptosis, as it can be generated by ROS (hydrogen peroxide), heat stress, 63 UVA irradiations 64 and pro-oxidant compounds like ethanol. 65 HNE induces the extrinsic apoptotic pathway. The extrinsic apoptotic pathway transmits a death signal from the cell surface to the intracellular compartment and is initiated by the activation of death receptors like receptors of TNF, Fas/CD95 or TRAIL (TNF-related apoptosis-inducing ligand).…”