Gene alteration in zebrafish exposed to a mixture of substances of abuse

Abstract

A recent surge in the use and abuse of diverse prescribed psychotic and illicit drugs necessitates the surveillance of drug residues in source water and the associated ecological impacts of chronic exposure to the aquatic organism. Thirty-six psychotic and illicit drug residues were determined in discharged wastewater from two centralized municipal wastewater treatment facilities and two wastewater receiving creeks for seven consecutive days in Kentucky. Zebrafish (Danio rerio) larvae were exposed to the environmental relevant mixtures of all drug residues, all illicit drugs, and all prescribed psychotic drugs. The extracted RNA from fish homogenates was sequenced, and differentially expressed sequences were analyzed for known or predicted nervous system expression, and screened annotated protein-coding genes to the true environmental cocktail mixture. Illicit stimulant (cocaine and one metabolite), opioids (methadone, methadone metabolite, and oxycodone), hallucinogen (MDA), benzodiazepine (oxazepam and temazepam), carbamazepine, and all target selective serotonin reuptake inhibitors including sertraline, fluoxetine, venlafaxine, and citalopram were quantified in 100% of collected samples from both creeks. The high dose cocktail mixture exposure group revealed the largest group of differentially expressed genes: 100 upregulated and 77 downregulated (p ≤ 0.05; q ≤ 0.05). The top 20 differentially expressed sequences in each exposure group comprise 82 unique transcripts corresponding to 74% annotated genes, 7% non-coding sequences, and 19% uncharacterized sequences. Among 61 differentially expressed sequences that corresponded to annotated protein-coding genes, 23 (38%) genes or their homologs are known to be expressed in the nervous system of fish or other organisms. Several of the differentially expressed sequences are associated primarily with the immune system, including several major histocompatibility complex class I and interferon-induced proteins. Interleukin-1 beta (downregulated in this study) abnormalities are considered a risk factor for psychosis. This is the first study to assess the contributions of multiple classes of psychotic and illicit drugs in combination with developmental gene expression.

Keywords: Illicit drugs; Nervous system; Next generation gene sequencing; Psychotic drugs; RNA-Seq; Zebrafish.

Figure 1.

Concentration of illicit and prescribed antipsychotic drug residues in two treated wastewater receiving creeks in Eastern Kentucky (this study) and surface water elsewhere in the U.S.

Figure 2.

The number of common and unique differentially expressed genes across comparisons. The differentially expressed genes were mostly unique to the high dose mixture groups, though some genes were also differentially expressed in the other groups with varying degrees of overlap. ADR: mixture of all drug residues quantified in creeks A and B; PPD: mixture of all prescribed psychotic drug residues quantified in creeks A and B; ID: mixture of all illicit drug residues quantified in creeks A and B; HD: high dose mixture of all target drug residues reported in surface water elsewhere at the highest concentrations; C: control.

Figure 3.

Of the top differentially expressed sequences, approximately 25% represent novel or non-protein-coding genes (A). Of the annotated differentially expressed sequences, most have verified or predicted nervous system function (B).