Aquatic Toxicology 81 (2007) 275–285 Inhibition of CYP1A enzymes by -naphthoflavone causes both synergism and antagonism of retene toxicity to rainbow trout (Oncorhynchus mykiss) Peter V. Hodson ∗ , Kashif Qureshi 1 , Cameron A.J. Noble 2 , Parveen Akhtar 3 , R. Stephen Brown School of Environmental Studies, Queen’s University, Kingston, Ont., Canada K7L 3N6 Received 29 September 2006; received in revised form 13 December 2006; accepted 13 December 2006 Abstract Retene (7-isopropyl-1-methyl phenanthrene) is a polycyclic aromatic hydrocarbon (PAH), that causes dioxin-like toxicity to early life stages of fish, including increased rates of mortality, developmental defects characterized as blue sac disease (BSD), and induction of CYP1A enzymes. This study determined whether toxicity is associated with retene, or with its metabolism by CYP1A enzymes to hydroxylated derivatives. Larval rainbow trout (Oncorhynchus mykiss) were co-exposed to four concentrations of waterborne retene and four concentrations of waterborne -naphthoflavone (ANF), a compound that antagonizes CYP1A induction and inhibits oxygenation reactions. The prevalence of mortality and BSD increased in an exposure-dependent manner for larvae exposed to retene alone. Tissue concentrations of CYP1A protein and retene metabolites also increased, but no un-metabolized retene (i.e., the parent compound) was measurable. At low concentrations of ANF, toxicity increased dramatically, while tissue concentrations of polar hydroxylated metabolites of retene decreased, and concentrations of less polar metabolites, and of parent retene, increased. At the highest concentration of ANF, retene toxicity was eliminated, and parent retene was the predominant form in tissue; no concentration of ANF was toxic by itself. The inhibition of retene hydroxylation and toxicity by ANF suggests that toxicity was caused by specific retene metabolites, and not by parent retene. The potentiation of retene toxicity at low concentrations of ANF, and the antagonism at high concentrations is a unique, non-linear interaction based on modulating CYP1A enzyme activity and retene metabolism. It demonstrates that effects on fish of different complex mixtures of hydrocarbons may not be easily predicted. © 2007 Elsevier B.V. All rights reserved. Keywords: Retene; Toxicity; Alpha naphthoflavone; CYP1A; PAH; Fish; Metabolism 1. Introduction Retene (7-isopropyl-1-methyl phenanthrene) is an alkyl- substituted polynuclear aromatic hydrocarbon (PAH) and a product of the anaerobic metabolism of dehydroabietic acid, a resin acid from conifers (Zender et al., 1994). It can be generated during the treatment of pulp mill effluent (Hawkins et al., 2004) and has been found at concentrations up to 3300 g/g dry weight ∗ Corresponding author. Tel.: +1 613 533 6129; fax: +1 613 533 6090. E-mail address: hodsonp@biology.queensu.ca (P.V. Hodson). 1 Current address: 12-301, 17 Van Order Dr., Kingston, Ont., Canada K7L 4C5. 2 Current address: Box 29131, RPO Okanagan Mission, Kelowna, BC, Canada V1W 4A7. 3 Current address: 9 Elm Court, Apt#9C, Rensselaer, NY 12144, United States. in aquatic sediments near a pulp mill in Finland (Leppanen and Oikari, 1999). Chronic exposure to retene causes dioxin-like toxicity to early life stages of fish, characterized by Blue Sac Disease (BSD) and induction of cytochrome P450 (CYP1A) enzymes (Billiard et al., 1999; Brinkworth et al., 2003). BSD includes elevated rates of mortality associated with pericardial and yolk sac edema, hemorrhaging, craniofacial deformities, impaired cardiac development, and circulatory failure. BSD can also be caused by exposing larval fish to other alkylphenan- threne congeners (Kiparissis et al., 2001), to crude oil rich in alkyl PAH (review, Barron et al., 2004; Carls et al., 1999; Clarke et al., 2004), and to pore water of PAH-contaminated sediments (Meyer et al., 2002). Hence, alkyl PAH such as retene may cause recruitment failure of fish spawning near pulp and paper mills (Sandstrom, 1994), or in shoals contaminated by crude oil (Carls et al., 1999). 0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.12.012