Effects of repeated alcohol abstinence on within-subject prefrontal cortical gene expression in rhesus macaques.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

Profiling of extracellular vesicle-bound miRNA to identify candidate biomarkers of chronic alcohol drinking in nonhuman primates.

BACKGROUND: Long-term alcohol drinking is associated with numerous health complications including susceptibility to infection, cancer, and organ damage. However, due to the complex nature of human drinking behavior, it has been challenging to identify reliable biomarkers of alcohol drinking behavior prior to signs of overt organ damage. Recently, extracellular vesicle-bound microRNAs (EV-miRNAs) have been found to be consistent biomarkers of conditions that include cancer and liver disease. METHODS: In this study, we profiled the plasma EV-miRNA content by miRNA-Seq from 80 nonhuman primates after 12 months of voluntary alcohol drinking. RESULTS: We identified a list of up- and downregulated EV-miRNA candidate biomarkers of heavy drinking and those positively correlated with ethanol dose. We overexpressed these candidate miRNAs in control primary peripheral immune cells to assess their potential functional mechanisms. We found that overexpression of miR-155, miR-154, miR-34c, miR-450a, and miR-204 led to increased production of the inflammatory cytokines TNFα or IL-6 in peripheral blood mononuclear cells after stimulation. CONCLUSION: This exploratory study identified several EV-miRNAs that could serve as biomarkers of long-term alcohol drinking and provide a mechanism to explain alcohol-induced peripheral inflammation.

Effects of graded increases in ethanol consumption on biochemical markers of bone turnover in young adult male cynomolgus macaques.

Chronic heavy alcohol use is often associated with reduced bone mineral density and altered bone turnover. However, the dose response effects of ethanol on bone turnover have not been established. This study examined the effects of graded increases of ethanol consumption on biochemical markers of bone turnover in young adult male cynomolgus macaques (Macaca fascicularis). For this study, 6.6-year-old (95% CI: 6.5, 6.7) male macaques were subjected to three 30-day sessions of increased ethanol intake over a 90-day interval. During the first 30 days, the monkeys drank a predetermined volume of ethanol corresponding to 0.5 g/kg/day, followed by 1.0 g/kg/day and 1.5 g/kg/day. Osteocalcin, a marker of bone formation, and carboxyterminal cross-linking telopeptide of type 1 collagen (CTX), a marker of resorption, were measured during each 30-day session. In addition, the ratio of osteocalcin to CTX was determined as a surrogate measure of global turnover balance. Mean osteocalcin decreased by 2.6 ng/mL (1.8, 3.5) for each one-half unit (0.5 g/kg/day) increase in dose (p < 0.001). Mean CTX decreased by 0.13 ng/mL (0.06, 0.20) for each one-half unit increase in dose (p < 0.001). Furthermore, there was an inverse relationship between dose and the ratio of osteocalcin to CTX, such that the mean ratio decreased by 0.9 (0.3, 1.5) for each one-half unit increase in dose (p = 0.01). In summary, male cynomolgus macaques had decreased blood osteocalcin and CTX, and osteocalcin to CTX ratio during the 90-day interval of graded increases in ethanol consumption, indicative of reduced bone turnover and negative turnover balance, respectively. These findings suggest that over the range ingested, ethanol resulted in a linear decrease in bone turnover. Furthermore, the negative bone turnover balance observed is consistent with reported effects of chronic alcohol intake on the skeleton.