Use of the γH2AX assay for assessing the genotoxicity of bisphenol A and bisphenol F in human cell lines

Abstract

Bisphenol A (BPA) and bisphenol F (BPF) are widely used to manufacture plastics and epoxy resins. Both compounds have been shown to be present in the environment and are food contaminants, with, as a result, a low but chronic exposure of humans. However, the fate and possible bioactivation of these compounds at the level of human cell lines was not completely elucidated yet. In this study, we investigated the ability of human cells (intestinal cell line: LS174T, hepatoma cell line: HepG2, and renal cell line: ACHN) to biotransform BPA and BPF, and focused on the cytotoxicity and genotoxicity of these two bisphenols, through the use of a novel and efficient genotoxic assay based on the detection of histone H2AX phosphorylation. BPA and BPF were extensively metabolized in HepG2 and LS174T cell lines, with stronger biotransformation capabilities in intestinal cells than observed in liver cells. Both cell lines produced the glucuronide as well as the sulfate conjugates of BPA. Conversely, the ACHN cell line was found to be devoid of any metabolic capabilities for the two examined bisphenols. Cytotoxicity was tested for BPA, BPF, as well as one metabolite of BPF produced in vivo in rat, namely dihydroxybenzophenone (DHB). In the three cell lines used, we observed similar ranges of toxicity, with DHB being weakly cytotoxic, BPF exhibiting an intermediary cytotoxicity, and BPA being the most cytotoxic compound tested. BPA and DHB were not found to be genotoxic, whatever the cell line examined. BPF was clearly genotoxic in HepG2 cells. These results demonstrate that some human cell lines extensively metabolize bisphenols and establish the genotoxic potential of bisphenol F.

Mutagenicity and DNA Damage of Bisphenol a and its Structural Analogues in Hepg2 Cells

“…However, structural differences alone cannot clearly explain the structure-activity relationship in this series of BPA analogues or why BPF, BPAF, BPZ, and DMBPA did not induce DNA damage, and why BPA and BPS did induce signifi cant DNA damage only after 24 h of exposure. It has recently been demonstrated that BPA and BPF are metabolised in HepG2 cells predominantly to conjugated sulphate metabolites (31,60). However, we still do not know to what extent BPA and its analogues are biotransformed into reactive quinone intermediates in HepG2 cells, and how stable these intermediates are.…”

“…In their recent study, Audebert et al (31) reported that 24-hour exposure to BPA (50 μmol L -1 and 100 μmol L -1 ) and BPF (100 μmol L -1 ) reduced the viability of several cell lines, including HepG2 cells. However, they determined cell viability by measuring DNA content with in-cell western (ICW) assays, while we used an MTT assay that is based on the measurement of the metabolic activity of living cells.…”

“…In turn, BPF has been reported to induce DNA strand breaks, but not micronuclei, in human hepatoma HepG2 cells (30). Audebert et al (31) have recently found that BPF genotoxicity depended on the metabolic capabilities of cells. In human HepG2 cells it induced histone H2AX phosphorylation, an indicator of DNA doublestrand breaks.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…Paradoxically, DSB can also help to kill cancer cells 153. Thus, this method is used in different fields ranging from cancer chemo‐ and radiotherapy154 or drug discovery155 to in vitro toxicology testing of environmental pollutants 150…”

Nanotoxicology and oxidative stress control: cutting-edge topics in toxicology

“…In rat brain tissue, PFOS activates calcium signalling and c-Fos, as well as c-Jun (32) Liu et al (2010a, b) This study applied a genotoxicity assay based on the detection of histone H2AX phosphorylation to compare bisphenol A and bisphenol F. Bisphenol A was not found to be genotoxic, whereas bisphenol F showed positive effects (33) Audebert et al (2011) Sodium fluoride suppresses proliferation and induces apoptosis in cultivated osteoblasts. This effect was caused by decreased insulin-like growth factor-1 expression…”