Glutathione content as a potential mediator of the vulnerability of cultured fetal cortical neurons to ethanol‐induced apoptosis

Abstract

Ethanol ingestion during pregnancy elicits damage to the developing brain, some of which appears to result from enhanced apoptotic death of neurons. A consistent characteristic of this phenomenon is a highly differing sensitivity to ethanol within specific neuron populations. One possible explanation for this "selective vulnerability" could be cellular variations in glutathione (GSH) homeostasis. Prior studies have illustrated that ethanol elicits apoptotic death of neurons in the developing brain, that oxidative stress may be an underlying mechanism, and that GSH can be neuroprotective. In the present study, both multiphoton microscopy and flow cytometry demonstrate a striking heterogeneity in GSH content within cortical neuron populations. Ethanol differentially elicits apoptotic death and oxidative stress in these neurons. When neuron GSH content is reduced by treatment with butathione sulfoxamine, the ethanol-mediated enhancement of reactive oxygen species is exacerbated. Sorting of cells into high- and low-GSH populations further exemplifies ethanol-mediated oxidative stress whereby apoptotic indices are preferentially elevated in the low-GSH population. Western blot analysis of the low-GSH subpopulations shows higher ethanol-mediated expression of active caspase 3 and 24-kDa PARP-1 fragments compared with the high-GSH subpopulation. In addition, neuronal content of 4-hydroxynonenal adducts is higher in low-GSH neurons in response to ethanol. These studies suggest that GSH content is an important predictor of neuronal sensitivity to ethanol-mediated oxidative stress and subsequent cell death. The data support the proposition that the differences in proapoptotic responses to ethanol within specific neuron populations reflect a heterogeneity of neuron GSH content.

Overexpression of Nrf2 Protects Cerebral Cortical Neurons from Ethanol-Induced Apoptotic Death

“…A functional consequence of this is a selective vulnerability of cerebral cortical neurons to ETOH, which is dependent on heterogeneity of GSH homeostasis (Maffi et al, 2008). The present study supports our earlier findings that a 24-h ETOH exposure dramatically reduced the high and medium GSH populations (Fig.…”

“…Previous findings have illustrated that prevention of the ETOH-related reduction of GSH in PCNs mitigates subsequent cell death and that GSH heterogeneity in a single cell type can dictate PCN sensitivity to ETOH (Ramachandran et al, 2003;Watts et al, 2005;Maffi et al, 2008). Cultured PCNs express a striking heterogeneity of GSH content and overexpression of Nrf2 clearly influences ETOH-induced changes in this GSH heterogeneity (Fig.…”

“…A major determinant of the neurotoxicity of ETOH is depletion of neuronal GSH content (Watts et al, 2005;Maffi et al, 2008), and modulation of the Nrf2/ARE/GSH pathways (Yang et al, 2005) has been shown to prevent ethanol-induced liver injury in mice (Gong and Cederbaum, 2006). In our current study, we assessed the involvement of Nrf2 and the effect of heterologous Nrf2 overexpression in regulating GSH-mediated protection against ETOH in fetal neurons.…”

“…Monochlorobimane, a nonfluorescent reagent that reacts with GSH to form a highly fluorescent derivative, was used to detect free GSH in individual viable cells by flow cytometry (Maffi et al, 2008). At the end of treatment, neurons were incubated with 10 M MCB for 30 min in a cell culture incubator, and cells were scraped, washed, and resuspended in icecold PBS.…”

“…Thus, a key issue is that although ETOH exposure increases Nrf2 protein expression and subsequent early DNA/ARE binding (Figs. 1-3), a portion of these neurons will develop oxidative stress that ultimately elicits apoptotic death (Maffi et al, 2008) (Fig. 4).…”