Epigenetic Effects of Ethanol

Alcohol use disorder (AUD) is prevalent and associated with significant mortality and socioeconomic costs globally. Despite the tremendous burden of AUD, mechanisms of alcohol (ethanol) action that underlie the development of AUD are still not known. This dissertation addresses the role of epigenetic modifications in heritability of AUD and mechanisms of ethanol by testing three distinct hypotheses: 1) paternal ethanol exposure regulates ethanol drinking and ethanol-related behavior in offspring; 2) acute ethanol induces conserved changes to histone modifications at model gene promoters in the cerebral cortex; 3) chronic intermittent vapor ethanol and withdrawal induce dynamic changes in gene regulation via discrete histone modifications in accumbal dopamine 1 receptor positive medium spiny neurons. To test these hypotheses, this dissertation developed new models for studying heritability of ethanol drinking using paternal vapor ethanol exposure and epigenetic modifications in a neuronal subtype using fluorescence activated cell sorting. The results presented in this thesis provide evidence for epigenetic effects of ethanol in mediating heritability of ethanol drinking and sensitivity to ethanol selectively in male offspring as well as ethanol-induced gene regulation in the cerebral cortex. Additionally, they set up future studies of ethanol’s epigenetic mechanisms in neuronal subtypes, which will increase sensitivity of current assays to detect cell-specific changes in gene regulation. These results are expected to have important implications for the development of drugs that target epigenetic modifying enzymes for treatment of AUD

Offspring were tested for performance on an accelerating rotarod 35 minutes after i.p. injection of 1/kg EtOH or saline.

<p>(A) E-sired male offspring treated with EtOH performed significantly better on the 5^th trial compared to those treated with saline. There are no significant differences associated with EtOH treatment in (B) C-sired male offspring, (C) E-sired female offspring, or (D) C-sired female offspring. n = 7–8/group, data presented as mean ± SEM, *p<0.05.</p

Offspring were tested for EtOH-induced anxiolysis by comparing performance on an elevated plus maze 10 minutes after i.p. injection of 1 g/kg EtOH or saline.

<p>(A) E-sired male offspring spend greater time in open arms after treatment with EtOH compared to C-sired male offspring and E-sired male offspring treated with saline; E-sired male offspring treated with EtOH also have (B) increased percent of open arm entries and (C) total arm entries relative to C-sired male offspring and E-sired male offspring treated with saline. There were no significant differences between E- and C-sired females treated with EtOH or saline on (D) time spent in open arms, (E) percent open arm entries, or (F) total arm entries. n = 6–7/group, data presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001.</p

Offspring were tested for EtOH consumption vs. water on a 2 bottle, free choice drinking assay.

<p>(A) E-sired male offspring (n = 17) had significantly decreased preference for EtOH compared to C-sired male offspring (n = 17) as well as (B) decreased EtOH consumption and (C) no change in total volume consumption per body weight. There were no significant differences between E-sired female offspring (n = 12) and C-sired female offspring (n = 12) on (D) EtOH preference, (E) EtOH consumption, or (F) total volume consumed. Data presented as mean ± SEM, *p<0.05, **p<0.01.</p

Comparison of E-sired and C-sired litters.

<p>(A) There were no differences in number of litters, number of male and female offspring, or (B) number of offspring per litter between E- and C-sires. (C) E-sired male offspring (n = 40) gained significantly more weight after weaning at 3 weeks and maintained increased weight through week 6 compared to C-sired male offspring (n = 61) (p<0.001). (D) There was no significant difference in weight between E- (n = 43) and C-sired (n = 45) female offspring. Data presented as mean ± SEM (Note: Error bars in C and D are obscured by the data points), **p<0.01, ***p<0.001.</p

Expression of <i>Bdnf</i> and <i>Dlk1</i> were measured in the VTA of offspring and DNA methylation was measured at the <i>Bdnf</i> exon IXa promoter in motile sperm and offspring VTA.

<p>(A) E-sired male offspring had significantly increased expression of <i>Bdnf</i> exon IXa but not <i>Dlk1</i> or <i>Bdnf</i> exon IV in the VTA relative to C-sired males. (B) There were no significant differences in <i>Bdnf</i> or <i>Dlk1</i> expression between E- and C-sired females in the VTA. (C) EtOH-exposed sires had decreased DNA methylation at the <i>Bdnf</i> exon IXa promoter relative to Room air exposed sires. (D) Male and (E) female E-sired offspring had decreased DNA methylation at the <i>Bdnf</i> exon IXa promoter relative to C-sired offspring. N.D., none detected, n = 4–7/group, data presented as mean ± SEM, *p<0.05, ***p<0.001.</p

Chronic vapor ethanol exposure in sires.

<p>(A) 8-week-old C57BL/6J mice were exposed to EtOH vapor or room air for 8 hours/day, 5 days/week, for 5 weeks and immediately housed with 2 EtOH-naïve Strain 129Sv/ImJ females for 48 hours; after mating, they were re-exposed for 3 days and motile sperm was collected. (B) Blood EtOH concentrations showed limited variability across 5 weeks and averaged 147.1+/−7.52 mg/dl (mean ± SEM) (n = 25). (C) There was no difference in weight gain between EtOH (n = 25) and Room air (n = 27) exposed sires during the 5 weeks of exposure. Data presented as mean ± SEM bars.</p

Scirrhous carcinoma: A previously undescribed tumor of the oral cavity.

This patient was found to have a scirrhous carcinoma with extensive perineural invasion and without any evidence of minor salivary gland carcinoma. To our knowledge, this is the first report of isolated scirrhous carcinoma of the oral cavity. Treatment was surgery and adjuvant chemoradiation, and there was complete disease response

Scirrhous carcinoma: A previously undescribed tumor of the oral cavity.

This patient was found to have a scirrhous carcinoma with extensive perineural invasion and without any evidence of minor salivary gland carcinoma. To our knowledge, this is the first report of isolated scirrhous carcinoma of the oral cavity. Treatment was surgery and adjuvant chemoradiation, and there was complete disease response

Cerebellar atrophy in human and murine succinic semialdehyde dehydrogenase deficiency

Human succinic semialdehyde dehydrogenase deficiency, an autosomal recessive disorder of γ-aminobutyric acid (GABA) catabolism, was modeled by a murine model sharing the phenotype of ataxia and seizures. Magnetic resonance imaging (MRI) with volumetry was obtained on 7 patients versus controls, and MRI with stereology was derived in 3 murine genotypes: null, wild-type, and heterozygous mutants. All patients had T1 hypointensity and T2 hyperintensity in globus pallidus, and 5 also had similar changes in subthalamic and cerebellar dentate nuclei. There was a trend for patients to have a smaller cerebellar vermis. Homozygous null mice had significantly lower total brain and cerebellar volumes than wild-types and heterozygotes. Stereology confirmed cerebellar atrophy and was otherwise normal in multiple regions. Cerebellar volume loss is present in the murine disorder with a trend for cerebellar atrophy in patients. Reduced cerebellar volume can reflect neurodegeneration and may be related to the clinical manifestations. © 2010 The Author(s)