Cell-Specific Biotransformation of Benzophenone-2 and Bisphenol-S in Zebrafish and Human in Vitro Models Used for Toxicity and Estrogenicity Screening

Abstract

Several human and fish bioassays have been designed to characterize the toxicity and the estrogenic activity of chemicals. However, their biotransformation capability (bioactivation/detoxification processes) is rarely reported, although this can influence the estrogenic potency of test compounds. The fate of two estrogenic chemicals, the UV filter benzophenone-2 (BP2) and the bisphenol A substitute bisphenol S (BPS) was deciphered in eight human and zebrafish in vitro cell models, encompassing hepatic and mammary cellular contexts. BP2 and BPS were metabolized into a variety of gluco- and sulfo-conjugated metabolites. Similar patterns of BP2 and BPS biotransformation were observed among zebrafish models (primary hepatocytes, ZFL and ZELH-zfER cell lines). Interestingly, metabolic patterns in zebrafish models and in the human hepatic cell line HepaRG shared many similarities, while biotransformation rates in cell lines widely used for estrogenicity testing (MELN and T47D-KBLuc) were quantitatively low and qualitatively different. This study provides new data on the comparative metabolism of BP2 and BPS in human and fish cellular models that will help characterize their metabolic capabilities, and underlines the relevance of using in vitro zebrafish-based bioassays when screening for endocrine disrupting chemicals.

Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults

“…This allowed the hypothesis of doubly conjugated (sulfate + glucuronide) metabolites to be ruled out. Finally, the structures of BPS mono-glucuronide ( R T 14.6 min; BPS-monoG) and of BPS mono-sulfate ( R T 24.5 min; BPS-monoS) were confirmed by co-elution assays with their respective authentic standards previously isolated from zebrafish hepatocyte incubations, and whose structure had been confirmed by liquid chromatography coupled to mass spectrometry (LC-MS), as detailed in Le Fol et al, (2015) [8]. In the water samples corresponding to adults as well as larvae assays, only the parent compound was detected (Figure 1, lower section).…”

“…Accordingly, for adult extracts, only the aqueous phase was further analyzed. Based on the radio-HPLC system developed for BPS in a previous work [8], the retention time ( R T ) of parent BPS was 27.7 min (Figure 1). …”

“…Ideally, in vitro and in vivo models should mimic as closely as possible the metabolic fate of candidate EDCs in wildlife species and/or humans. We recently characterized the comparative fate of the candidate EDCs bisphenol S (BPS) and benzophenone-2 (BP2) in human and zebrafish cellular models [8,9,10]. Benzophenones are emerging environmental contaminants used as UV filters in sunscreens and in other products such as perfumes or food packaging, to which they are incorporated to prevent UV degradation.…”

“…BPS, a molecule increasingly used as a substitute for bisphenol A (BPA), was also shown to be an estrogenic compound in fish and human models [15,16,17]. The comparative metabolic study of BP2 and BPS in primary zebrafish hepatocyte cultures, and in ZFL and ZELH-zfERs cell lines, demonstrated that these cellular systems are metabolically competent [8]. Fish embryo assays may provide an alternative small scale analysis system with a complexity close to an adult organism [18,19].…”

“…Notwithstanding, recent in vivo studies demonstrated that biotransformation of some compounds can start very early, indicating that some phase I and phase II enzymes are already functional at early life-stages [26,27]. We previously demonstrated that the metabolic capacities expressed in zebrafish hepatic cellular models are close to that of metabolically competent hepatic human cell lines (HepaRG), at least for BPS and BP2 [8]. However, no evidence was provided (nor is currently available in the literature) to assess whether the biotransformation of model EDCs in zebrafish embryos accurately reflects adult fish metabolism, and the fate of these EDCs in other vertebrates, including human.…”