Impact of co-exposure with lead and decabromodiphenyl ether (BDE-209) on thyroid function in zebrafish larvae

Highlights

• PBDEs and metals are the main contaminants at e-waste recycling sites.

• Combined effects of Pb/BDE-209 on thyroid function in zebrafish larvae were studied.

• Co-exposure of Pb and BDE-209 elicited synergistic effects on T₃ and T₄ levels.

• BDE-209 enhanced Pb uptake, and Pb decreased bioconcentration/metabolism of BDE-209.

• Mixtures of Pb and BDE-209 probably result in an increase in toxicological effects.

Abstract

Polybrominated diphenyl ethers (PBDEs) and metals are the main contaminants at waste electrical and electronic equipment (“e-waste”) recycling sites. However, the potential environmental health effects of mixtures of PBDEs and metals are not known. We investigated co-exposure of lead (Pb) with decabromodiphenyl ether (BDE-209) on thyroid function in zebrafish larvae. Seven groups of embryos/larvae of zebrafish were treated with Pb (0, 2, 5, 10, 15, 20, and 30 μg/L), six groups were exposed to BDE-209 (0, 50, 100, 200, 400, and 800 μg/L), and nine groups of zebrafish larvae were treated with Pb and BDE-209 (5, 10, and 20 μg/L Pb; 50, 100, and 200 μg/L BDE-209). Embryos/larvae were exposed from 2 h post-fertilization (hpf) until 144 hpf, and thyroid hormone (TH) content measured. Pb exposure significantly decreased whole-body TH contents (triiodothyroxine (T₃) and thyroxine (T₄)) but BDE-209 exposure significantly increased T₃ and T₄ levels. Pb or BDE-209 treatment alone caused a predicted downregulation of TH transport (i.e., expression of the mRNA or proteins of transthyretin). Chemical analyses showed Pb uptake to be increased by BDE-209, but BDE-209 bioconcentration was decreased and the ability to metabolize BDE-209 was reduced in the presence of Pb. We also found that a mixture of the two chemicals had a synergistic effect on TH levels in zebrafish.

Introduction

Waste electrical and electronic equipment (“e-waste”) has emerged as a critical global environmental problem, particularly in “developing” countries (Wong et al., 2007, Robinson, 2009). Along with domestic generation of huge amounts of e-waste (Liu et al., 2006), considerable amounts of e-waste are imported from “developed” countries into China for recycling and for use as secondary resources. China now appears to be a “dumping site” for e-waste, receiving 50–80% of the e-waste generated in the rest of the world (Guan et al., 2007, Wei and Liu, 2012). This e-waste has been recycled in towns and villages using inappropriate, primitive, and often hazardous recycling processes for more than a decade. The emission of a wide range of toxic chemicals containing metals and persistent organic pollutants has caused serious contamination of local environments (Huo et al., 2007, Wong et al., 2007, Tang et al., 2010).

Lead (Pb) has been used in televisions and computer monitors containing cathode-ray tubes and has also been used in printed circuit boards and other components (Ramesh et al., 2007). Pb is one of the major metals in e-waste recycling sites. High levels of Pb have been detected in workshop dust, environmental water samples, sediments, and soil samples in e-waste recycling areas in South China (Wong et al., 2007, Leung et al., 2008). For example, ≤400 μg/L of Pb was detected in river water downstream of a recycling plant in Guiyu, South China (Wang and Guo, 2006). Also, a dry weight of Pb of ≤2700 ng/g in fish has been reported (Qiu et al., 2011).

Polybrominated diphenyl ethers (PBDEs) are additive flame-retardants that have been used extensively in furniture, plastics, textiles, television sets, electronic devices, and computers (Alaee et al., 2003). The deca-BDE mixture, which is composed of mainly deca-BDE (BDE-209), has been the most widely used PBDEs (Alaee et al., 2003). It has been used in high-impact polystyrene in televisions, cabinets for computer monitors, and electronic circuit boards (Costa and Giordano, 2011, Noyes et al., 2011). Also, e-waste recycling processes release significant amounts of PBDEs (Luo et al., 2007a, Luo et al., 2007b), of which BDE-209 is, in general, the predominant compound (Bi et al., 2007, Yuan et al., 2008, Chen et al., 2009, Zhang et al., 2011, Wang et al., 2011). For instance, BDE-209 has been detected in mud carp (Cirrhinus molitorella) in rivers around e-waste areas at ≤28,000 ng/g ww (Zhang et al., 2009). Very high concentrations of BDE-209 have been detected among workers employed to dismantle electronic waste (≤3436 ng/g lipid weight) (Qu et al., 2007) as well as in the serum (≤3100 ng/g lipid weight) of residents of the same e-waste dismantling area in Guiyu, South China (Bi et al., 2007). Several reports have focused on the characteristics of toxicants released into the environment from e-waste recycling areas in China. However, the important issue is whether humans or wildlife have been exposed to mixtures of many toxicants, which may have additive or even synergistic effects on them. Until now, quantification of the health effects of exposure to these mixtures (and their potential toxicity) has been lacking.

We evaluated the potential environmental health effects of mixtures of Pb and BDE-209. Thyroid hormones (THs) were selected for study because they have been reported that BDE-209 can disrupt TH function in rodents (Rice et al., 2007, Tseng et al., 2008, Van der Ven et al., 2008, Lee et al., 2010) and fish (Chen et al., 2012, Noyes et al., 2011, Noyes et al., 2013) exposed to BDE-209. It has also been reported that the levels of Pb in blood are negatively associated with the circulating level of THs in a human population occupationally exposed to Pb (López et al., 2000; Dundar et al., 2006). In fish, several studies have reported that Pb exposure can reduce serum levels of THs (Spieler and Weber, 1991; Chaurasia et al., 1996; Chaurasia and Kar, 1999). We hypothesized that BDE-209 and Pb may have additive or synergistic effects on THs.

Zebrafish embryos/larvae have been proposed as powerful alternative models for toxicological research (e.g., for screening chemicals that affect the morphogenesis and/or function of the thyroid gland) (Segner, 2009, Raldúa et al., 2012). The thyroid gland of the zebrafish is derived from endoderm precursor cells. The thyroid primordium develops at ≈24–28 hpf (Rohr and Concha, 2000, Wendl et al., 2002, Elsalini et al., 2003). The thyroid gland is formed from thyroid follicle cells, and the first single follicle differentiates at ≈55–60 hpf (Alt et al., 2006). The first thyroid follicle that produces T₄ is detected at ≈72 hpf, and the number of T₄-producing follicles are increased thereafter and throughout larval life (Alt et al., 2006, Porazzi et al., 2009). Several potential endpoints can be used to assess the function of the thyroid gland in developing embryos/larvae: specific expression in differentiated thyroid follicular cells (e.g., thyroglobulin, slc5a5), markers of activation of the hypothalamus–pituitary–thyroid (HPT) axis (e.g., thyroid-stimulating hormone) and TH transport proteins (e.g., transthyretin, TTR). Levels of THs can also be potential endpoints. Moreover, in the zebrafish, the mechanisms of development of the thyroid gland as well as the molecular functions of thyroid follicles have demonstrated a high degree of conservation and are, in general, comparable with those in humans (Raldúa et al., 2012). Hence, zebrafish embryos/larvae could be alternative models for screening xenobiotics and xenobiotic mixtures that impair thyroid function. THs have fundamental roles in regulation of the development, differentiation and metabolism of vertebrates (Power et al., 2001), and are particularly important for development of the brain. It has been demonstrated that deficiencies in neonatal TH production can affect brain development with irreversible neurologic effects (Boas et al., 2012).

In the present study, embryos and larvae of zebrafish (Danio rerio) were exposed to BDE-209 and Pb alone or in combination. We measured the TH content, bioconcentration of Pb and BDE-209, as well as the metabolism of BDE-209 in zebrafish larvae. Our results suggest that the presence of BDE-209 enhances Pb uptake, but that Pb can also affect the bioconcentration and metabolism of BDE-209. Furthermore, co-exposure of Pb and BDE-209 could have synergistic effects on levels of triiodothyroxine (T₃) and thyroxine (T₄).