Citation:

Serrano, J., R. Kolanczyk, M. Tapper, T. Lahren, B. Sheedy, P. Kosian, AND A. Kubatova. Mass spectrometric identification and estrogenic potential of cyclic phenone metabolites formed in in vitro assays with fish liver slices. American Society for Mass Spectrometry, Atlanta, GA, June 02 - 06, 2019.

Impact/Purpose:

The present research contributed to an overall effort to develop a comprehensive program to evaluate the potential adverse effects of chemicals on vertebrate endocrine systems by advancing metabolic product identification and metabolic pathway elucidation to complement metabolism studies addressing estrogenic activity modulation with the use of in vitro fish assays. Metabolite identification data correlated to chemical exposure and metabolic pathways will facilitate development of additional in vitro and short-term in vivo assays needed to prioritize chemicals for testing and support risk assessment at the USEPA, update metabolism simulators with new mechanistic data, and provide insight into the differential sensitivity of species to chemical exposure.

Description:

Introduction Cyclic phenones are suspected endocrine disruptors used as UV radiation filters, and in the manufacturing of insecticides/pharmaceuticals. Understanding of the estrogenic potential of these chemicals in the context of a defined ER-mediated Adverse Outcome Pathway (AOP) is of interest to the US Environmental Protection Agency (USEPA). The main objectives of this study were to: a. identify the rainbow trout (rt) liver slice-mediated in vitro metabolites of two model parent cyclic phenones (cyclohexylphenylketone and benzophenone), exhibiting disparity between estrogen receptor (rtER) binding and ER-mediated vitellogenin (Vtg) mRNA induction and, b. assess the metabolic competency of fish liver in vitro assays to predict the chemical form (parent and/or metabolite) associated with the biological response, and to help elucidate plausible phenone regulatory pathways. Methods Nine cyclohexylphenylketone (CPK) metabolites (M1-M9), and one benzophenone (DPK) metabolite (benzhydrol) were identified after chemical incubation with rt liver slices by combining GC-MS with and without BSTFA/MSTFA derivatization, LC with tandem MS, and LC with high resolution time of flight (TOF) MS spectra information. Hexane extracts containing test metabolites were either analyzed directly via GC-MS or dried at room temperature under a gentle stream of nitrogen and re-dissolved in appropriate mobile phase for analysis by LC-MS/MS and LC-TOF-MS. Dried CPK metabolites were also analyzed by GC-MS following derivatization. LC fractionation showed relationships between LC- and GC-MS data needed to elucidate CPK metabolite substitution patterns in absence of standards. Metabolite identity was further confirmed with complementary metabolite in vitro conjugation data. Preliminary Data While LC-MS/MS allowed for the investigation of metabolite structural features, LC-TOF-MS provided higher resolution and more accurate molecular mass determination needed to support proposed metabolite structures and identity validation. Results supported that DPK is metabolized by rt slices only to benzhydrol, and that CPK is primarily metabolized through phase I oxidation of the cyclohexyl ring and not the phenyl group as predicted by metabolic simulators. LC-TOF-MS and GC-MS with BSTFA derivatization analyses confirmed the formation of reactive CPK hydroxylated metabolites. Two CPK metabolites (M1 and M2; MW 186) were analyzed by GC-MS and enzyme conjugation studies and proposed to be cyclohexenyl-derivatives. M6-M9 were confirmed as hydroxylated metabolites (MW 204), with the potential for undergoing phase II conjugative metabolism to glucuronides and sulfates. Similarly, M3, M4 and M5 were identified as cyclohexanone-derivatives of CPK (MW 202), resulting from the redox interconversion of the hydroxylated metabolites. Overall, results indicated that the estrogenic effects of cyclic phenones are mediated by the parent chemical structure for DPK, but the metabolic products of CPK. The lack of standards, as well as low metabolite accumulation in media and slices did not allow determination of the specific CPK metabolites or the intra-slice amounts responsible for the observed Vtg gene activation. Nevertheless, the small concentrations of CPK metabolites detected, combined with the Vtg mRNA induction response observed, suggested that CPK metabolites binding to the ER can potentially induce a stronger biological response than the parent chemicals CPK or DPK. Novel aspect Information obtained allowed the proposal of a novel regulatory pathway for a model cyclic phenone in rainbow trout liver slices.

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