Ethanol (E) Impairs Fetal Brain GSH Homeostasis by Inhibiting Excitatory Amino-Acid Carrier 1 (EAAC1)-Mediated Cysteine Transport

Abstract

Central among the fetotoxic responses to in utero ethanol (E) exposure is redox-shift related glutathione (GSH) loss and apoptosis. Previously, we reported that despite an E-generated Nrf2 upregulation, fetal neurons still succumb. In this study, we investigate if the compromised GSH results from an impaired inward transport of cysteine (Cys), a precursor of GSH in association with dysregulated excitatory amino acid carrier1 (EAAC1), a cysteine transporter. In utero binge model involves administration of isocaloric dextrose or 20% E (3.5 g/kg)/ by gavage at 12 h intervals to pregnant Sprague Dawley (SD) rats, starting gestation day (gd) 17 with a final dose on gd19, 2 h prior to sacrifice. Primary cerebral cortical neurons (PCNs) from embryonic day 16–17 fetal SD rats were the in vitro model. E reduced both PCN and cerebral cortical GSH and Cys up to 50% and the abridged GSH could be blocked by administration of N-acetylcysteine. E reduced EAAC1 protein expression in utero and in PCNs (p < 0.05). This was accompanied by a 60–70% decrease in neuron surface expression of EAAC1 along with significant reductions of EAAC1/Slc1a1 mRNA (p < 0.05). In PCNs, EAAC1 knockdown significantly decreased GSH but not oxidized glutathione (GSSG) illustrating that while not the sole provider of Cys, EAAC1 plays an important role in neuron GSH homeostasis. These studies strongly support the concept that in both E exposed intact fetal brain and cultured PCNs a mechanism underlying E impairment of GSH homeostasis is reduction of import of external Cys which is mediated by perturbations of EAAC1 expression/function.

Role for Cystathionine γ Lyase (CSE) in an Ethanol (E)-Induced Lesion in Fetal Brain GSH Homeostasis

“…In a setting of reduced Cys availability as shown by us earlier [ 26 ] with increased accumulation of cystathionine levels (in the current study), we next determined whether this might be caused by an altered expression of CSE, a rate-limiting enzyme that metabolizes cystathionine to Cys. Immunoblotting experiments for CSE revealed that CSE protein was reduced with the treatment of E in fetal PCNs by 36%, 53% ( p < 0.05), and 64% ( p < 0.05) at 6, 12, and 24 h, respectively.…”

“…Consistent with this concept, we observed that alcohol caused a significant accumulation of cystathionine levels in the PCNs that was also replicated in the 2nd trimester equivalent in utero binge alcohol model and the 3rd trimester equivalent postnatal day (PN7) ethanol model as indicated by HPLC analysis. The resultant downstream impact of cystathionine accumulation on Cys and GSH levels were recently demonstrated by us [ 26 ]. Notably, previous reports in humans have shown that mutations of Cse gene causes an accumulation of cystathionine in liver, kidney, brain, and cerebrospinal fluid, leading to cystathionuria and decreased plasma cysteine concentration.…”

“…Earlier we have shown that E exposure decreases Cys and GSH content in both PCNs and binge alcohol model [ 26 ]. To gain a better understanding of the E-induced GSH loss and the role of transulfuration pathway (TSP), we first assessed the levels of cystathionine, an intermediate of TSP and an immediate precursor of Cys.…”

“…Further, alcohol treatment decreased the expression of CSE protein in fetal brain cortices of binge alcohol exposed prenatal animals and PCNs exposed to alcohol without a change in the CBS expression, which supplies the CSE with cystathionine with serine and homocysteine condensation. A decreased CSE expression in the TSP could explain previously observed reductions of Cys and subsequent GSH synthesis with E exposure [ 26 ]. Consistent with this concept, we observed that alcohol caused a significant accumulation of cystathionine levels in the PCNs that was also replicated in the 2nd trimester equivalent in utero binge alcohol model and the 3rd trimester equivalent postnatal day (PN7) ethanol model as indicated by HPLC analysis.…”

Role of Excitatory Amino Acid Carrier 1 (EAAC1) in Neuronal Death and Neurogenesis After Ischemic Stroke

“…Furthermore, experiments using ethanol in neurons, which impairs the expression of EAAC1 by 60-70%, revealed that the level of GSH and cysteine was reduced by up to 50% in primary cerebral cortical neurons. This effect was reversed by administration of NAC [22]. To increase the synthesis of GSH, EAAC1 is translocated to the plasma membrane and transports cysteine and glutamate into the neuron.…”