Molecular changes associated with alcohol consumption arise from complex interactions between pharmacological effects of alcohol, psychological/placebo context surrounding drinking, and other environmental and biological factors. The goal of this study was to tease apart molecular mechanisms regulated by pharmacological effects of alcohol - particularly at binge-drinking, from underlying placebo effects. Transcriptome-wide RNA-seq analyses were performed on peripheral blood samples collected from healthy heavy social drinkers (N=16) enrolled in a 12-day randomized, double-blind, cross-over human laboratory trial testing three alcohol doses: Placebo, moderate (0.05g/kg (men), 0.04g/kg (women)), and binge (1g/kg (men), 0.9g/kg (women)), administered in three 4-day experiments, separated by minimum of 7-day washout periods. Effects of beverage doses on the normalized gene expression counts were analyzed within each experiment compared to its own baseline using paired-t-tests. Differential expression of genes (DEGs) across experimental sequences in which each beverage dose was administered, as well as responsiveness to regular alcohol compared to placebo (i.e., pharmacological effects), were analyzed using generalized linear mixed-effects models. The 10% False discovery rate-adjusted DEGs varied across experimental sequences in response to all three beverage doses. We identified and validated 22 protein coding DEGs potentially responsive to pharmacological effects of binge and medium doses, of which 11 were selectively responsive to binge dose. Binge-dose significantly impacted the Cytokine-cytokine receptor interaction pathway (KEGG: hsa04060) across all experimental-sequences that it was administered in, and during dose-extending placebo. Medium dose and placebo impacted pathways hsa05322, hsa04613, and hsa05034, in the first two and last experimental sequences, respectively. In summary, our findings add novel, and confirm previously reported data supporting dose-dependent effects of alcohol on molecular mechanisms and suggest that the placebo effects may induce molecular responses within the same pathways regulated by alcohol. Innovative study designs are required to validate molecular correlates of placebo effects underlying drinking.

The authors have declared no competing interest.

NCT02315885

he research was supported by the NIAAA grants K23AA020899 and R01AA026291 (to CS), micro-grants (vouchers) to CS through the UMB Clinical Translational Research Initiative (ICTR program) that partially facilitated human laboratory procedures and statistical analyses through CTSA grant 1UL1TR003098, and in part by the University of Maryland Baltimore, School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014).

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The study was conducted at the University of Virginia CARE center (UVA CARE) from August 2013 to March 2014 and at the General Clinical Research Center (GCRC) of the University of Maryland School of Medicine (UMSOM) from October 2015 to August 2019. The study was approved by the institutional review boards at each institution and monitored by a three-member data and safety monitoring board (DSMB). All participants gave written informed consent prior to starting study procedures.

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All data produced in the present study are available upon reasonable request to the authors following publication of the peer-reviewed article