In vitro-in vivo biotransformation and phase I metabolite profiling of benzo[a]pyrene in Gulf killifish (Fundulus grandis) populations with different exposure histories.

Chronic exposure to pollution may lead populations to display evolutionary adaptations associated with cellular and physiological mechanisms of defense against xenobiotics. This could result in differences in the way individuals of the same species, but inhabiting different areas, cope with chemical exposure. In the present study, we explore two Gulf killifish (Fundulus grandis) populations with different exposure histories for potential differences in the biotransformation of benzo[a]pyrene (BaP), and conduct a comparative evaluation of in vitro and in vivo approaches to describe the applicability of new approach methodologies (NAMs) for biotransformation assessments. Pollution-adapted and non-adapted F. grandis were subjected to intraperitoneal (IP) injections of BaP in time-course exposures, prior to measurements of CYP biotransformation activity, BaP liver concentrations, and the identification and quantification of phase I metabolites. Additionally, substrate depletion bioassays using liver S9 fractions were employed for measurements of intrinsic hepatic clearance and to evaluate the production of metabolites in vitro. Pollution-adapted F. grandis presented significantly lower CYP1A activity and intrinsic clearance rates that were 3 to 4 times lower than non-adapted fish. The metabolite profiling of BaP showed the presence of 1­hydroxy-benzo[a]pyrene in both the in vitro and in vivo approaches but with no significant population differences. Contrarily, 9­hydroxy-benzo[a]pyrene and benzo[a]pyrene-4,5-dihydrodiol, only identified through the in vivo approach, presented higher concentrations in the bile of pollution-adapted fish relative to non-adapted individuals. These observations further the understanding of the evolutionary adaptation of F. grandis inhabiting heavily polluted environments in the Houston Ship Channel, TX, USA, and highlight the need to consider the evolutionary history of populations of interest during the implementation of NAMs.

Drug interaction potential of toremifene and N-desmethyltoremifene with multiple cytochrome P450 isoforms.

Toremifene is an effective agent for the treatment of breast cancer in postmenopausal women and is being evaluated for its ability to prevent bone fractures in men with prostate cancer taking androgen deprivation therapy. Due to the potential for drug-drug interactions, the ability of toremifene and its primary circulating metabolite N-desmethyltoremifene (NDMT) to inhibit nine human cytochrome P450 (CYP) enzymes was determined using human liver microsomes. Induction of CYP1A2 and 3A4 by toremifene was also investigated in human hepatocytes. Toremifene did not significantly inhibit CYP1A2 or 2D6. However, toremifene is a competitive inhibitor of CYP3A4, non-competitive inhibitor of CYP2A6, 2C8, 2C9, 2C19 and 2E1 and mixed-type inhibitor of CYP2B6. CYP inhibition by NDMT was similar in magnitude to toremifene. Toremifene did not induce CYP1A2 but increased CYP3A4 monooxygenase activity and gene expression in drug-exposed human primary hepatocytes. Although clinical doses of toremifene produce steady state exposures to toremifene and NDMT that may be sufficient to cause pharmacokinetic drug-drug interactions with other drugs metabolised by CYP2B6, CYP2C8, CYP3A4, CYP2C9 and CYP2C19, these data indicate that toremifene is unlikely to play a role in clinical drug-drug interactions with substrate drugs of CYP1A2 and CYP2D6.