Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcripts by excessive ethanol drinking.

The underlying genetic and epigenetic mechanisms driving functional adaptations in neuronal excitability and excessive alcohol intake are poorly understood. Small-conductance Ca2+-activated K+ (KCa2 or SK) channels encoded by the KCNN family of genes have emerged from preclinical studies as a key contributor to alcohol-induced functional neuroadaptations in alcohol-drinking monkeys and alcohol dependent mice. Here, this cross-species analysis focused on KCNN3 DNA methylation, gene expression, and single nucleotide polymorphisms including alternative promoters in KCNN3 that could influence surface trafficking and function of KCa2 channels. Bisulfite sequencing analysis of the nucleus accumbens tissue from alcohol-drinking monkeys and alcohol dependent mice revealed a differentially methylated region in exon 1A of KCNN3 that overlaps with a predicted promoter sequence. The hypermethylation of KCNN3 in the accumbens paralleled an increase in expression of alternative transcripts that encode apamin-insensitive and dominant-negative KCa2 channel isoforms. A polymorphic repeat in macaque KCNN3 encoded by exon 1 did not correlate with alcohol drinking. At the protein level, KCa2.3 channel expression in the accumbens was significantly reduced in very heavy drinking monkeys. Together, our cross-species findings on epigenetic dysregulation of KCNN3 represent a complex mechanism that utilizes alternative promoters to impact firing of accumbens neurons. Thus, these results provide support for hypermethylation of KCNN3 as a possible key molecular mechanism underlying harmful alcohol intake and alcohol use disorder.

Increased levels of the acetaldehyde-derived DNA adduct N 2-ethyldeoxyguanosine in oral mucosa DNA from Rhesus monkeys exposed to alcohol.

Alcohol is a human carcinogen. A causal link has been established between alcohol drinking and cancers of the upper aerodigestive tract, colon, liver and breast. Despite this established association, the underlying mechanisms of alcohol-induced carcinogenesis remain unclear. Various mechanisms may come into play depending on the type of cancer; however, convincing evidence supports the concept that ethanol's major metabolite acetaldehyde may play a major role. Acetaldehyde can react with DNA forming adducts which can serve as biomarkers of carcinogen exposure and potentially of cancer risk. The major DNA adduct formed from this reaction is N (2)-ethylidenedeoxyguanosine, which can be quantified as its reduced form N (2)-ethyl-dG by LC-ESI-MS/MS. To investigate the potential use of N (2)-ethyl-dG as a biomarker of alcohol-induced DNA damage, we quantified this adduct in DNA from the oral, oesophageal and mammary gland tissues from rhesus monkeys exposed to alcohol drinking over their lifetimes and compared it to controls. N (2)-Ethyl-dG levels were significantly higher in the oral mucosa DNA of the exposed animals. Levels of the DNA adduct measured in the oesophageal mucosa of exposed animals were not significantly different from controls. A correlation between the levels measured in the oral and oesophageal DNA, however, was observed, suggesting a common source of formation of the DNA adducts. N (2) -Ethyl-dG was measured in mammary gland DNA from a small cohort of female animals, but no difference was observed between exposed animals and controls. These results support the hypothesis that acetaldehyde induces DNA damage in the oral mucosa of alcohol-exposed animals and that it may play role in the alcohol-induced carcinogenic process. The decrease of N (2)-ethyl-dG levels in exposed tissues further removed from the mouth also suggests a role of alcohol metabolism in the oral cavity, which may be considered separately from ethanol liver metabolism in the investigation of ethanol-related cancer risk.

Rapid mitochondrial DNA segregation in primate preimplantation embryos precedes somatic and germline bottleneck.

The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.