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, 287 (52), 43533-42

Alcohol-induced One-Carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain

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Alcohol-induced One-Carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain

Anna-Kate Fowler et al. J Biol Chem.

Abstract

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr(+/-) mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

Figures

FIGURE 1.
FIGURE 1.
3-week ethanol exposure induces neuronal cell death in the mouse cerebral cortex. A, photomicrographs represent cells double-labeled with TUNEL and MAP-2 (arrows) in the cerebral cortex of mice. The brain sections were stained using In Situ Cell Death detection kit for TUNEL labeling and antibody to oxidative neuronal marker MAP-2 and counterstained with Mayer's hematoxylin. Scale bar, 100 μm; for inset, scale bar, 50 μm. B, double-labeled cells were quantified by stereological counting. Values are means ± S.E.; *p < 0.05. Note significantly higher density of TUNEL-labeled neurons in ethanol-exposed brains. Neither the number of MAP-2-positive cells (neurons) nor cerebral cortex volume was affected by chronic ethanol exposure (data not shown). Con, control.
FIGURE 2.
FIGURE 2.
Ethanol induces ROS- and acetaldehyde-induced DNA lesions in the mouse brain. A, photomicrographs represent cells double-labeled with antibody to oxidative DNA modification oxo8dG and neuronal marker MAP-2 (arrows) and counterstained with Methyl Green in the cerebral cortex. Scale bar, 100 μm; for inset, scale bar, 50 μm. B, double-labeled cells were quantified by stereological counting. Values are means ± S.E.; *, p < 0.01. Note significantly higher density of oxo8dG-labeled neurons in ethanol-exposed brains. C, quantitative analysis of acetaldehyde-derived N2-ethyl-dG content in DNA from brain tissue of mice after 3 week-exposure to the Lieber-Decarli liquid diet with or without ethanol (5%) using LC/MS/MS. Values are means ± S.E.; **p < 0.001. Note that N2-ethyl-dG levels are significantly increased in DNA of ethanol-exposed mice. Con, control.
FIGURE 3.
FIGURE 3.
Ethanol exposure induces different modes of cell death and DNA damage response (PARylation). The brain sections (prefrontal cerebral cortex) of mice chronically exposed for 3 weeks (long term) or 4 days (short term) to the Lieber-Decarli liquid diet with or without ethanol (5%) were triple-labeled with neuronal marker NeuN (purple), TUNEL (green), and cleaved caspase-3 (A), AIF (B), or antibody to PAR (C), all red. Hoescht (blue) was used to identify all cell nuclei. Fluorescence was visualized by confocal microscopy. Note increased number of cells in which TUNEL is colocalized with NeuN and active caspase-3, AIF, or PAR (yellow double arrows) in the prefrontal cerebral cortex of mice exposed to long term ethanol compared with control mice and mice exposed to short term ethanol. Of particular interest is the translocation of active caspase-3 into the nucleus (A) and the movement of both AIF (B) and PAR (C) to the periphery of the nucleus after ethanol, especially after long term exposure. Single arrowhead, non-neuronal cell; double arrowhead, neuronal cell (NeuN positive); white, normal; green, TUNEL-positive; red, active caspase-3/AIF/PAR-positive concentrated around nucleus; yellow, TUNEL and active capase-3/AIF/PAR concentrated around nucleus; blue, TUNEL and active caspase-3 nuclear location. Scale bar, 20 μm.
FIGURE 4.
FIGURE 4.
Chronic ethanol exposure compromises DNA base excision repair in the brain. A, qPCR analysis of DNA repair in brain extracts from mice after long (3 weeks) or short term (4 days) exposure to the Lieber-Decarli liquid diet with or without ethanol (5%). The quantity of repaired template was calculated by comparing the ΔCt values of the repaired templates and the control template, and relative levels of repaired template were expressed in %. Note that extracts from mice exposed to control diet had significantly lower DNA repair activity compared with mice exposed to ethanol, both during 4 days (short term exposure; p < 0.002) or 3 weeks (long term exposure; p < 0.01). Values are means ± S.E.; #, p < 0.02. DNA repair activity in brains after long term exposure is lower, compared with those after short term exposure. B, quantitative analysis of acetaldehyde-derived DNA lesions, N2-ethyl-dG in DNA from brain tissue of mice chronically or acutely exposed to the Lieber-Decarli liquid diet with or without ethanol (5%), using LC/MS/MS. Note that N2-ethyl-dG levels are significantly increased in DNA from mice after long term ethanol exposure, compared with mice after short term exposure and control (Con) mice. Values are means ± S.E.; *p < 0.05; **p < 0.001.
FIGURE 5.
FIGURE 5.
Chronic ethanol exposure leads to OCM impairment. A, blood Hcy levels in mice exposed to the Lieber-Decarli liquid diet during 3 weeks or 4 days were determined using LC/MS/MS. Note significant increase in blood Hcy levels in mice exposed to ethanol for 3 weeks but not for 4 days. B, global DNA methylation was determined by methylation quantification assay (Epigenetek kit) in DNA isolated from mouse brains after long term exposure to the Lieber-Decarli liquid diet. Note a significant reduction in DNA methylation in brains of ethanol-exposed mice. Values are means ± S.E. *p < 0.01. Con, control.
FIGURE 6.
FIGURE 6.
OCM impairment mediates alcohol-induced DNA repair dysfunction. A, blood Hcy levels in MTHFR and WT mice chronically exposed to the Lieber-Decarli liquid diet with or without ethanol (5%) were determined using LC/MS/MS. Note the effect of ethanol on Hcy levels in MTHFR, compared with WT mice. B, additional OCM impairment in MTHFR mice exaggerates the effect of ethanol on DNA repair activity. Note a significant decrease in DNA repair activity in MTHFR mice after long term ethanol exposure, compared with WT mice. *p < 0.001. Con, control.

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