An in vitro approach to assess the toxicity of certain food contaminants: Methylmercury and polychlorinated biphenyls

Abstract

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants and food contaminants, and known developmental neurotoxicants. Aim of this study was to evaluate the effects of MeHg, PCB 126 and PCB 153 in a battery of in vitro cell systems. A total of 17 cell types were utilized, including nervous system (neuronal and astroglial) and non-nervous system cells. End-points measured included MTT reduction, Trypan blue exclusion and ³H-thymidine incorporation into DNA. Results indicate that this approach would identify these three compounds as neurotoxicants, and would also point out to the thyroid (for PCB 126 and MeHg) and the prostate (for both PCBs) as important additional targets. Tests of binary combinations of MeHg and PCBs indicated no interaction and an additive response, in agreement with other recent reports. Cerebellar granule neurons from mice with genetically determined low glutathione levels were more sensitive than wild-type neurons to the toxicity of all three compounds, supporting a role for oxidative stress in their neurotoxicity. These findings provide initial evidence that a relatively rapid in vitro screening approach can be developed, that would provide initial information useful for assessing neurotoxicity, as well as indication on potential other targets of biological action or toxicity.

Introduction

Among the several thousand commercial chemicals, less than 10% have been evaluated for neurotoxicity (Landrigan, 1994). Yet, environmental neurotoxicants may contribute to the etiology of neurodegenerative diseases, such as Parkinson's disease (Wright and Keller-Byrne, 2005), and to neurodevelopmental disorders, such as learning disabilities, autism or attention deficit disorder (Grandjean and Landrigan, 2006). Guidelines for neurotoxicity and developmental neurotoxicity testing have been developed, that require cumbersome protocols prescribed for standard tests in animals. Economical and time considerations, as well as ethical considerations for reducing the number of animals, have in recent years argued for the need to develop alternative in vitro methodologies to test for potential neurotoxicity (Costa, 1998, Harry et al., 1998, Gartlon et al., 2006). No in vitro neurotoxicity test has been fully validated so far, and regulatory agencies accept only in vivo animal data to predict potential neurotoxic effects in humans. Yet, in addition to their established role to investigate mechanisms of neurotoxicity, in vitro systems may be useful to screen and prioritize compounds, and to provide initial information useful to guide further in vitro and in vivo studies (Costa, 1998, Harry et al., 1998, Gartlon et al., 2006).

The present study explores the possible development of a series of in vitro screening assays by focussing on three known neurotoxicants, methylmercury (MeHg) and two polychlorinated biphenyls (PCBs). MeHg is a ubiquitous environmental pollutant, responsible in the past for large outbreaks of developmental neurotoxicity (Harada, 1995, Pierce et al., 1972). Current exposure occurs primarily through the consumption of contaminated fish, and epidemiological, animal, and in vitro studies have clearly demonstrated that MeHg is a developmental neurotoxicant (Grandjean et al., 1997, Castoldi et al., 2001, Clarkson and Magos, 2006). PCBs are a class of widespread environmental contaminants, also present in food such as meat, dairy products and fish (Carpenter, 2006). Among the 209 PCB congeners, a handful has a coplanar structure that allows them to bind to, and activate the aryl hydrocarbon (Ah) receptor, similarly to dioxins. Their toxicological effects thus include carcinogenicity, immunotoxicity and reproductive and developmental toxicity (Van den Berg et al., 2006, Lundqvist et al., 2006). Non-dioxin-like PCBs do not significantly interact with the Ah receptor, but cause a variety of toxic effects, including carcinogenicity and developmental neurotoxicity (Ulbrich and Stahlmann, 2004, Knerr and Schrenk, 2006).

MeHg, PCB 126 (3,3′,4,4′,5-pentachlorobiphenyl, a dioxin-like PCB), and PCB 153 (2,2′,4,4′,5,5′-hexachlorobiphenyl, a non-dioxin-like PCB) were chosen in the present study to explore simple in vitro approaches to test their known neurotoxicity. The purpose of the study was threefold: (1) To assess the ability of these compounds to cause cytotoxicity and inhibit cell proliferation in a battery of 17 different cell types. Nervous system-derived cell lines (neuronal and astroglial), primary neuronal and astroglial cells from different brain areas, and non-nervous system cell lines, were chosen for this endeavor. Among these, five cell lines were of human origin. (2) To assess possible interactions between MeHg and PCBs. This was prompted by epidemiological, animal, and in vitro evidence which suggested a possible synergistic interaction between these compounds (Grandjean et al., 2001, Bemis and Seegal, 1999, Roegge et al., 2004). (3) To determine whether neurons with genetically modified glutathione (GSH) levels may be exploited as a screening tool for further mechanistic studies. These experiments were prompted by reports indicating that PCBs and MeHg induce oxidative stress (Sarafian and Verity, 1991, Yee and Choi, 1996, Venkataraman et al., 2007, Lin and Lin, 2006). Cerebellar granule neurons prepared from Gclm (−/−) mice [that lack the modifier subunit of glutamate-cysteine ligase (GCLM), the rate limiting step in the synthesis of GSH], and from wild-type mice were used for this purpose (Giordano et al., 2006). Preliminary results of these studies have been presented (Vitalone et al., 2006, Vitalone et al., 2007).