Assessing the endocrine activity of PAHs using the DRE-, ERE- and PPARγ CALUX bioassays

Introduction
Mineral oils are a complex mixture of organic compounds, consisting of two main types: the saturated hydrocarbons (MOSH) and the aromatic hydrocarbons (MOAH). The occurrence of mineral oil in food has been reported in the past, with concentrations that often reach 0.1% and can even approach 1% in some exceptional cases. These compounds can enter the food chain either intentionally (as an authorized food contact material or additive), or unintentionally (printing inks, recycling, lubricating oils, …). The biggest concern for mineral oil regarding human health is the MOAH fraction, since these compounds are potentially genotoxic, carcinogenic and may have endocrine disrupting properties. An important group of compounds in the MOAH fraction are the polycyclic aromatic hydrocarbons (PAHs). PAHs consist of fused benzene rings, which are formed during incomplete combustion of organic matter (e.g. in engines, incinerators, forest fires…). An important concern linked to PAHs is their potential endocrine disrupting activity. Endocrine disrupting chemicals (EDCs) are a structurally diverse class of both synthetic and natural compounds which can interfere with our hormonal system by binding to endocrine receptors, resulting in adverse health effects such as reproductive damage, developmental impairment, obesity, diabetes and cancers. The aim of this study was to determine the endocrine activity of 9 PAHs using three different CALUX bioassays: DRE-CALUX, ERE-CALUX and PPARγ CALUX.
Materials and methods
Three CALUX (chemically activated luciferase gene expression) bioassays were used to determine the endocrine activity: 1)The PPARy CALUX® system of BioDetection systems (BDS, Amsterdam) uses U‐2OS cells (human osteoblast) that are stably transfected with human PPARy2. 2)DRE-CALUX (dioxin responsive element) uses a third H1L7.5c1 generation cell line, derived from the mouse hepatoma hepa1c1c7 wild‐type cells, which contain the AhR receptor and 3) The ERE-CALUX (estrogen responsive element) uses a human breast cancer cell line (VM7Luc4E2), which contains the estrogen receptor. All three cell lines are also stably transfected with the firefly luciferase reporter gene. Potential activity is determined by measuring light emission after exposure to the compounds and adding luciferin.
Results and discussion
1)PPARy CALUX®: Anthracene, fluoranthene, pyrene and fluorene showed a weak agonistic activity for the PPARy receptor. Only BaP showed a weak antagonistic activity. In both the agonistic and antagonistic tests, no effects were observed in presence of S9 metabolic fraction. Larger compounds like chrysene and benzo(ghi)perylene did not show any activity on the PPARy receptor which may be the result of sterical hindrance preventing them to interact with this receptor (>4 rings). 2)DRE-CALUX: BaP is known to have an AhR agonistic effect and was used as reference compound. Out of the other eight non‐alkylated PAHs, only chrysene exhibited significant agonistic activity. 3) The ERE-CALUX: So far, only preliminary range finding results were obtained, showing that phenanthrene, naphthalene, chrysene, fluoranthene and pyrene all show agonistic estrogenic activity.