Bioconcentration and endocrine disruption effects of diazepam in channel catfish, Ictalurus punctatus C.L. Overturf a,b, ⁎, M.D. Overturf a , D.B. Huggett a a Department of Biological Sciences & Institute of Applied Science, University of North Texas, 1155 Union Circle, Denton, TX 76203, USA b GHD Services Inc., 5551 Corporate Blvd. Suite 200, Baton Rouge, LA 70808, USA abstract article info Article history: Received 20 October 2015 Received in revised form 23 January 2016 Accepted 9 February 2016 Available online 11 February 2016 Recently, the detection of pharmaceuticals in surface waters has increased worldwide. Pharmaceuticals are typically found in the environment at concentrations well below therapeutic levels in humans; however, their mechanisms of action may be largely unknown in non-target organisms, such as teleost species. Thus, chronic ex- posure to these types of compounds warrants further investigation. The goal of this study was to examine the po- tential for diazepam, a model benzodiazepine drug, to bioconcentrate in tissues of channel catfish and to examine its ability to interact with the endocrine system through modulation of steroid hormones and/or steroidogenic genes. To investigate the bioconcentration potential of diazepam, channel catfish (Ictalurus punctatus) were ex- posed to 1 ng/mL diazepam for seven days, followed by clean water for another seven days, using an abbreviated OECD 305 Fish Bioconcentration Test study design. This concentration of diazepam is well below environmental- ly relevant concentrations of diazepam (ng/L). To evaluate steroidogenic effects, fish were exposed to 1 ng/mL diazepam for seven days only. Steroid hormone concentrations were analyzed for various tissues, as well as expression of selected steroidogenic genes. Calculated bioconcentration factors for diazepam were well below regulatory threshold values in all tissues analyzed. No changes in steroid hormone concentration were detected in any tissue analyzed; however, the steroidogenic gene cytochrome P450 side chain cleavage (P450scc) was significantly down-regulated at day 5 and 3β-hydroxy steroid dehydrogenase (3β-HSD) was significantly down-regulated at day 7 in the gonad. These results indicate that although diazepam does not significantly bioconcentrate, low-level chronic exposure to diazepam may have the potential to interact with endocrine function by altering gene expression. © 2016 Elsevier Inc. All rights reserved. Keywords: BCF Benzodiazepine Bioconcentration Channel catfish Diazepam Endocrine disruption Polymerase chain reaction Steroidogenesis 1. Introduction Uptake of pharmaceuticals by fish can occur through various routes, resulting in the accumulation of compounds over time (Corcoran et al., 2010). Accumulation of compounds through gill uptake only (bioconcentration) can be evaluated using the Organization for Economic Cooperation and Development (OECD) 305 Fish Bioconcentration Test (Woodburn et al., 2008). The gill uptake model tends to accurately predict the bioconcentration of moderately non- polar compounds (log K OW b 5), such as pharmaceuticals, and works under the assumption that one route of absorption (gill uptake) deter- mines the total accumulation of the compound in fish species using a flow-through test design (Barber, 2008, Erickson et al., 2006, Nichols et al., 2015). The model results in the calculation of the bioconcentration factor (BCF), which is a unit less ratio of the concentration of the contaminant in fish to that in the water (Barber, 2008). However, the significance of the bioconcentration value depends on the country that the test was conducted and the associated regulatory agency, and can range anywhere from 500 to 5000 (Cowan-Ellsberry et al., 2008). Due to the high cost and lengthy time associated with conducting a full OECD 305 study, bioconcentration data is available for only a limited number of pharmaceuticals. Thus, there is a need to acquire BCF data for a large number of chemicals. A model benzodiazepine pharmaceutical, diazepam, is one of the top 200 drugs dispensed in the U.S. and is also frequently abused; thus, wastewater treatment plants across the United States continually receive diazepam (Lamb, 2009, SAMSA, 2008). Diazepam, as well as its metabolites, has been detected in the environment at concentrations ranging from b 1 ng/L in surface waters to 200 ng/L in wastewater (Phillips et al., 2007; Hummel et al., 2006; Baker and Kasprzyk-Hordern, 2011). However, one metabolite of diazepam, oxaz- epam, has been detected in wastewater at concentrations of up to 482 ng/L (Hummel et al., 2006). Benzodiazepine pharmaceuticals, such as diazepam, act on the central nervous system (CNS) via interac- tion with the ionotropic GABA receptor, specifically GABA A , which re- sults in modulation of the endogenous inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Activation of the GABA receptor Comparative Biochemistry and Physiology, Part C 183–184 (2016) 46–52 ⁎ Corresponding author at: Department of Biological Sciences & Institute of Applied Science, University of North Texas, 1155 Union Circle, Denton, TX 76203, USA. Tel.: +1 225 296 6601. E-mail address: carmen.overturf@ghd.com (C.L. Overturf). http://dx.doi.org/10.1016/j.cbpc.2016.02.001 1532-0456/© 2016 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc