Incorporation of a metabolizing system in biodetection assays for endocrine active substances

Abstract

Among the in vitro assays for the detection of endocrine active substances (EAS), the CALUX assay (Chemically Activated LUciferase gene eXpression) allows the detection of substances (i.e., ligands) with the potential to interact with various nuclear receptors and affect the subsequent transcriptional response (Sonneveld et al., 2005). Such assessments generally fail to take into account the role of metabolism. Indeed, the majority of cell-based assays use cells that are not metabolically competent and do not include a metabolizing step. Some attempts to assess the role of metabolism in endocrine bioassays have been made previously. Most of these included 1 Introduction Biodetection is gaining importance, especially for samples for which little toxicological data is available (Krewski et al., 2010). In this context, cell culture-based assays are recognized to play an increasingly important role in the detection of endocrine active substances (EAS). These test systems provide insight into the intrinsic biological properties of both pure chemicals and previously uncharacterized mixtures. These test systems may reduce or even eliminate in vivo testing required for safety assessments.

Combining Different In Vitro Bioassays to Evaluate Genotoxicity of Water-Accommodated Fractions from Petroleum Products

“…All referred in vitro-based assays detecting strong oxidative stress response used (hepatic) cell lines that are capable of active metabolization. Hence, future experiments focusing on petroleum-induced toxicity in the U2-OS cell line should consider the simulation of a vertebrate xenobiotic biotransformation system (e.g., S9 fraction obtained from rat livers) [41,59].…”

The Role of Metabolism in the Estrogenic Activity of Endocrine-Disrupting Chemicals

“…This estrogenic activity was significantly increased when the activated S9 fraction was added to the assay (EC 50 0.15 μM), which suggested the formation of active metabolites of methoxychlor. The pure methoxychlor metabolite 4,4 0 -(2,2,2trichloroethane-1,1-diyl) diphenol was also tested, and its estrogenic activity was comparable with that of methoxychlor treated with the S9 fraction (EC 50 0.05 μM), which suggested that 4,4 0 -(2,2,2-trichloroethane-1,1-diyl) diphenol is the main metabolite formed with the S9 fraction [32]. As the methoxychlor metabolites mono-OH methoxychlor and bis-OH-methoxychlor are formed both in vitro and in vivo, addition of the metabolic enzymes here (i.e., the S9 fraction) improved the in vivo prediction [33].…”

“…While methoxychlor has no estrogenic activity, its mono-demethylated and bis-demethylated metabolites have significant estrogenic activities [31]. Mollergues et al determined the estrogenic activity of methoxychlor using the ER-CALUX assay with and without the β-naphthoflavone-/phenobarbital-induced S9 fraction [32]. In this assay, methoxychlor showed weak estrogenic activity (EC 50 4.6 μM).…”

Incorporation of metabolic enzymes to improve predictivity of reporter gene assay results for estrogenic and anti-androgenic activity

“…In the current study we show that the CALUX system has very low basal metabolic competence. Therefore we have developed a metabolic module using rat liver S9 as an add-on to the CALUX assays; a proof-of-principle of this method was published recently [19]. In genotoxicity testing, mimicking in vivo hepatic metabolism using S9 fractions is common practice [20][21][22].…”