Influence of Bisphenol Compounds at Nanomolar Concentrations on Chromosome Damage Induced by Metabolically Activated Carcinogens in HepG2 Cells

Abstract

Bisphenol (BP) compounds are endocrine-disrupting organic pollutants. BPs may increase the messenger RNA (mRNA) transcripts of nuclear receptors (NRs) regulating the expression of xenobiotic-metabolizing cytochrome P450 (CYP) enzymes. Their impact on the genotoxicity of metabolically activated carcinogens, however, remains unknown. In this study, effects of the bisphenols A, F, S, and AF on the expression of the aryl hydrocarbon receptor (AhR), the pregnane X receptor (PXR), the constitutive androstane receptor, and individual xenobiotic-metabolizing CYP enzymes in a human hepatoma (HepG2) cell line were investigated, along with in silico binding studies of BPs to each receptor. The results indicated that each BP at 1 to 100 nM concentrations increased the mRNA transcripts and protein levels of AhR, PXR, CYP1A1, 1A2, 1B1, 2E1, and 3A4. The predicted affinities of the BPs for binding AhR were comparable to those of potent agonists. Pretreatment of HepG2 cells with each BP potentiated the induction of micronuclei by benzo­[a]­pyrene, aflatoxin B1, benzene, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; this effect was abolished/reduced by inhibitors of NRs and/or CYPs. Our study suggests that BPs at human exposure levels may aggravate chromosome damage by several impactful carcinogens in human cells by inducing relevant CYP enzymes, mostly via NR modulation.

Impact of Extended and Combined Exposure of Bisphenol Compounds on Their Chromosome-Damaging Effect─Increased Potency and Shifted Mode of Action

“…The results suggest that the activity of AhR, not PXR or CAR, is associated with the potentiating effect of BPA (probably through enhancing CYP1A1 expression) . It seems that through extended/combined exposure the genotoxic effect of BPs may be amplified through AhR stimulation and CYP1A1 induction, as in accordance to our recent report on BP-potentiated effects of several promutagens …”

“…Goat anti-mouse IgG antibodies (DyLight 800, AB2015) and goat anti-rabbit IgG antibodies (DyLight 680, AB2013) were from Amyjet Scientific (Wuhan, China). These antibodies (from the same sources as above) worked well previously in our relevant studies. ,, …”

“…To explore the interactive influence of individual BPs as potentially contributing to the potentiation of genotoxic effects by BP combinations and extended exposure, two segments of cell treatment with chemicals were established. The first segment was pretreatment with each BP at CYP inducing, 23 i.e., nanomolar concentrations; the second segment was subsequent exposure to each BP at micromolar concentrations (for micronucleus formation). Each BP exposing HepG2 cells at 0.6 to 10 μM for 48 h, with or without BP pretreatment, was non-or mildly cytotoxic (indicated by Tables S2−S5).…”

“…17 It seems that through extended/combined exposure the genotoxic effect of BPs may be amplified through AhR stimulation and CYP1A1 induction, as in accordance to our recent report on BPpotentiated effects of several promutagens. 23 Formation of Centromere-Containing Micronuclei by BPs under Extended/Combined Exposure. The micronuclei formed in HepG2 cells by BPs were further analyzed for the presence or absence of centromere(s), by immunofluorescence CENP-B staining, a method valid for discriminating an aneugenic effect from a clastogenic effect.…”

“…However, extended regimes are closer to the real-life BP exposure in humans than a standard regime. We recently observed that BPs at low nanomolar concentrations, which were three orders of magnitude lower than their thresholds for genotoxicity, significantly enhanced the expression of several CYP enzymes (CYP1A1, 1A2, 1B1, 2E1, and 3A4) in a human hepatoma (HepG2) cell line; moreover, this modulation of CYP enzymes led to potentiated genotoxicity of several organic promutagens (such as benzo­(a)­pyrene, aflatoxin B1, and benzene) . Therefore, we were interested in the interaction between/among BPs in regard to their metabolic activation and genotoxicity, the impact of extended (over the standard two-cell cycle covering) and combined BP exposure (possibly favoring metabolic activation) on the potency of BP-induced chromosome damage, the involved mode of action, and their relevance to the activating enzyme (supposedly CYP1A1).…”