Yin-Feng Zhang

Dual body burdens of polychlorinated biphenyls and polybrominated diphenyl ethers among local residents in an e-waste recycling region in Southeast China

E-waste recycling resulted in serious pollution of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in Taizhou of Zhejiang Province, China. The aims of this study were to assess dual body burdens of the two pollutants and potential health risk for local residents. Blood samples were collected from two e-waste recycling sites, Luqiao (where PCBs-containing e-wastes were recycled) and Wenling (where PBDEs-containing e-wastes were recycled). The mean summation SigmaPCBs (CB-105, 118, 153, 183, and 180) and summation SigmaPBDEs (BDE-28, 47, 99, 100, 153, 154, 180, and 209) were 204.20 and 117.58 ng g(-1) lipid in the blood from Luqiao, respectively, while they were 83.80 and 357.44 ng g(-1) lipid from Wenling, respectively. The PCBs levels among Luqiao residents were comparable to the values reported for US populations, while the PBDEs levels among two study populations were higher than the values from US populations. This is the first report to present dual body burdens of PCBs and PBDEs at so high levels. Based on previous epidemiologic data, it is suggested that dual burdens of PCBs and PBDEs at so high levels might pose health risk for local residents. In addition, no correlation between PCBs or PBDEs concentrations and the ages of the volunteers was observed in the two populations, which was explained by similar exposure time. No correlation of PBDEs with PCBs concentrations suggested different pathways of human exposures to PCBs and PBDEs. Our findings have raised concern about human health risk of dual exposure to PCBs and PBDEs resulting from e-waste recycling.

Structure–activity relations in binding of perfluoroalkyl compounds to human thyroid hormone T3 receptor

Perfluoroalkyl compounds (PFCs) have been shown to disrupt thyroid functions through thyroid hormone receptor (TR)-mediated pathways, but direct binding of PFCs with TR has not been demonstrated. We investigated the binding interactions of 16 structurally diverse PFCs with human TR, their activities on TR in cells, and the activity of perfluorooctane sulfonate (PFOS) in vivo. In fluorescence competitive binding assays, most of the 16 PFCs were found to bind to TR with relative binding potency in the range of 0.0003–0.05 compared with triiodothyronine (T3). A structure–binding relationship for PFCs was observed, where fluorinated alkyl chain length longer than ten, and an acid end group were optimal for TR binding. In thyroid hormone (TH)-responsive cell proliferation assays, PFOS, perfluorohexadecanoic acid, and perfluorooctadecanoic acid exhibited agonistic activity by promoting cell growth. Furthermore, similar to T3, PFOS exposure promoted expression of three TH upregulated genes and inhibited three TH downregulated genes in amphibians. Molecular docking analysis revealed that most of the tested PFCs efficiently fit into the T3-binding pocket in TR and formed a hydrogen bond with arginine 228 in a manner similar to T3. The combined in vitro, in vivo, and computational data strongly suggest that some PFCs disrupt the normal activity of TR pathways by directly binding to TR.

Tetrabromobisphenol A Disrupts Vertebrate Development via Thyroid Hormone Signaling Pathway in a Developmental Stage-Dependent Manner

Data concerning effects of tetrabromobisphenol A (TBBPA) on thyroid hormone (TH)-dependent vertebrate development have been limited, although TBBPA has been demonstrated in vitro to disrupt the TH signaling pathway at the transcriptional level. In this study, we investigated the effects of TBBPA on T3-induced and spontaneous Xenopus laevis metamorphosis, which share many similarities with TH-dependent development in higher vertebrates. In a 6-day T3-induced metamorphosis assay using premetamorphic tadpoles, 10-1000 nM TBBPA exhibited inhibitory effects on T3-induced expression of TH-response genes and morphological changes in a concentration-dependent manner, with a weak stimulatory action on tadpole development and TH-response gene expression in the absence of T3 induction. In a spontaneous metamorphosis assay, we further found that TBBPA promoted tadpole development from stage 51 to 56 (pre- and prometamorphic stages) but inhibited metamorphic development from stage 57 to 66 (metamorphic climax). These results strongly show that TBBPA, even at low concentrations, disrupts TH-dependent development in a developmental stage-dependent manner, i.e., TBBPA exhibits an antagonistic activity at the developmental stages when animals have high endogenous TH levels, whereas it acts as an agonist at the developmental stages when animals have low endogenous TH levels. Our study highlights the adverse influences of TBBPA on TH-dependent development in vertebrates.

Molecular characterization and mRNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Like Xenopus laevis, some species of the Rana genus are also used to study endocrine disrupting chemicals (EDCs). Although ribosomal protein L8 (rpl8) is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rana, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rpl8 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rpl8 cDNA consisted of 919bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rpl8 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rpl8 gene was found in all tissues examined (brain, liver, intestine, tail, testis and ovary) during R. nigromaculata development. Finally, we investigated rpl8 expression under exposure to hormones. No change in rpl8 mRNA expression was found under exposure to thyroid hormone (T4) and estrogen (estradiol), whereas expression of the corresponding biomarker genes was induced. Our results show that rpl8 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rpl8 as a reference gene in other Rana species due to the high conservation of rpl8 among the Rana genus.

Molecular characterization and developmental expression patterns of thyroid hormone receptors (TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata

Considering some advantages of Rana nigromaculata as an experimental species, we propose that this species, like Xenopus laevis, could be used to assay thyroid hormone (TH) signaling disrupting actions. To validate the utilizability of R. nigromaculata, we investigated the responsiveness of R. nigromaculata to a TH receptor (TR) agonist (T3) and antagonist (amiodarone) by analyzing expression, based on characterizing TR cDNA and developmental expression patterns. With high levels of identity with the corresponding genes in X. laevis, both TRα and TRβ in R. nigromaculata exhibited roughly similar developmental expression patterns to those of X. laevis, in spite of some species-specific differences. Both TRα and TRβ expression had greater changes in the liver and intestine than in the tail and brain during metamorphosis. T3 exposure for 2days induced more dramatic increases of TRβ expression in stage 27 than in stage 34 tadpoles but not in stage 42 tadpoles, showing that the responsiveness of R. nigromaculata to TH decreased with development and disappeared at the onset of metamorphic climax. Corresponding to greater changes of TRβ expression in the liver and intestine than in the tail and brain during metamorphosis, the liver and intestine had higher responsiveness to exogenous T3 than the tail and brain. Amiodarone inhibited T3-induced TRβ expression. Our results show that R. nigromaculata can be used as a model species for assaying TH signaling disrupting actions by analyzing TRβ expression, and intestine tissues at stage 27 are ideal test materials due to high responsiveness and easy accessibility.

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118-1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.

Optimization of the T3-induced Xenopus metamorphosis assay for detecting thyroid hormone signaling disruption of chemicals

T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone (TH) signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we intend to develop quantitatively morphological endpoints and choose appropriate concentrations and exposure durations for T3 induction. Xenopus laevis at stage 52 were exposed to series of concentrations of T3 (0.31-2.5nmol/L) for 6days. By comparing morphological changes induced by T3, we propose head area, mouth width, unilateral brain width/brain length, and hindlimb length/snout-vent length as quantitative parameters for characterizing T3-induced morphological changes, with body weight as a parameter for indicating integrated changes. By analyzing time-response curves, we found that following 4-day exposure, T3-induced grossly morphological changes displayed linear concentration-response curves, with moderate morphological changes resulting from 1.25nmol/L T3 exposure. When using grossly morphological endpoints to detect TH signaling disruption, we propose 4days as exposure duration of T3, with concentrations close to 1.25nmol/L as induction concentrations. However, it is appropriate to examine morphological and molecular changes of the intestine on day 2 due to their early response to T3. The quantitative endpoints and T3 induction concentrations and durations we determined would improve the sensitivity and the reproducibility of the T3-induced Xenopus metamorphosis assay.

Environmental (anti-)androgenic chemicals affect germinal vesicle breakdown (GVBD) of Xenopus laevis oocytes in vitro

Progesterone-induced germinal vesicle breakdown (GVBD) of Xenopus oocytes in vitro was used to study endocrine disrupting activity of chemicals in previous studies. In this study, we investigated for the first time effects of environmental androgens on oocyte maturation and effects of anti-androgens on androgen-induced oocyte maturation, using Xenopus GVBD in vitro. Trenbolone and nandrolone, two environmental androgens, were found to induce Xenopus GVBD at low concentrations. The potential of trenbolone to induce GVBD was approximately 100-fold lower than that of testosterone, while nandrolone had a several-fold lower potential than testosterone. Our findings have aroused new concerns for effects of environmental androgens on amphibian oocyte maturation at environmentally relevant concentrations, and suggested that Xenopus GVBD can be used to test androgenic activity of suspicious environmental androgens. Androgen receptor (AR) antagonist flutamide at 10 μM only exhibited a weakly inhibitory effect on androgen-induced GVBD, while another known AR antagonist vinclozolin had no effect even at high concentrations. The results show that Xenopus GVBD is not sensitive to AR-mediated environmental anti-androgens. In contrast to flutamide and vinclozolin, methoxychlor (a weaker AR antagonist) inhibited dramatically androgen-induced GVBD, suggesting that androgen-induced Xenopus GVBD can be used to study non-AR-mediated effects of chemicals on oocyte maturation.