Templates for qPCR-based BER activity assay.

<p>Whole cell extracts were isolated from brain tissue and exposed to a template DNA containing a single nucleotide lesion (A) and control template (B). BER activity was calculated by comparing the ΔΔCt values (Ct is the number of cycles required for the fluorescent signal to cross the threshold) of the repaired and control templates.</p

OCM impairment is involved in ethanol-induced oxidative DNA damage and neuronal apoptosis effects in the PFC.

<p>The brain sections (PFC) of WT and <i>Mthfr+/−</i> mice exposed for 3 weeks or 4 days (acute) to the Lieber-DeCarli liquid diet with- or without ethanol (5%) were triple-labeled with NeuN (purple), TUNEL (green) and cleaved caspase-3 (red). Hoechst 33342 (blue) was used to identify all cell nuclei. Fluorescence was visualized by confocal microscopy. Scale bar = 20 µm. Note increased number of TUNEL/caspase-3-positive neurons in PFC of <i>Mthfr+/−</i> mice chronically exposed to ethanol, compared with corresponding WT mice (arrows). Also, note a higher density of TUNEL/caspase-3-positive neurons in PFC of chronically, compared with acutely exposed to ethanol WT mice (arrows).</p

OCM impairment is involved in ethanol impact on DNA repair in the PFC.

<p>(A) Blood Hcy levels in WT and <i>Mthfr+/−</i> mice following acute or chronic 3- week (3 w) or 5-week (5 w) ethanol exposure. Values are means ± SEM; *p<0.01; **p<0.001. Note chronic alcohol-induced increase in blood Hcy levels, compared with the acute exposure and the heightening this increase by MTHFR deficiency. (B) DNA repair activity in the PFC of WT and <i>Mthfr+/−</i> mice exposed to acute and chronic ethanol. Values are means ± SEM; *p<0.01; **p<0.005; ***p<0.002. Note a decrease in DNA repair activity in the PFC following 3-week (3 w) and even more so following 5 week (5 w) exposure, compared with acute alcohol exposure and a significant exaggeration of this decrease by MTHFR deficiency.</p

PFC is more vulnerable to ethanol-induced neuronal apoptosis than hippocampus.

<p>Brain sections double-labeled with TUNEL and MAP-2 in the PFC and hippocampus (Hip) and quantified by stereological counting; Values are means ± SEM; *p<0.01. Neither the number of MAP-2-positive cells (neurons) nor volumes of the brain structures were affected by chronic 3-week ethanol exposure. Note significantly higher density of TUNEL- positive neurons in PFC than in hippocampus of ethanol-exposed mice.</p

PFC is more vulnerable to ethanol-induced oxidative stress than hippocampus.

<p>(A) oxo-8dG expression in neurons (neuronal marker MAP-2) was quantified in PFC and hippocampus (Hip) by stereological counting. Neither the number of neurons nor volumes of the brain structures were affected. Values are mean ± SEM; *p<0.01. (B) Oxo-8dG expression in neurons normalized to corresponding controls (Δ). Note significantly higher density of oxo-8dG -labeled neurons and Δ in PFC, compared with hippocampus of ethanol-exposed mice. (C) DNA repair activity in response to oxidative DNA damage (oxo-8dG) assessed by qPCR in whole cell extracts obtained from PFC and hippocampus (Hip) of control mice and mice exposed to acute or chronic ethanol. Values are means ± SEM; *p<0.05, **p<0.01. Note the response to oxidative DNA damage by PFC lysate is significantly stronger than those in the hippocampus.</p