Bioaccumulation and Male Reproductive Toxicity of the New Brominated Flame Retardant Tetrabromobisphenol A-Bis(2,3-dibromo-2-methylpropyl ether) in Comparison with Hexabromocyclododecane.

Tetrabromobisphenol A-bis(2,3-dibromo-2-methylpropyl ether) (TBBPA-DBMPE) has come into use as an alternative to hexabromocyclododecane (HBCD), but it is unclear whether TBBPA-DBMPE has less hazard than HBCD. Here, we compared the bioaccumulation and male reproductive toxicity between TBBPA-DBMPE and HBCD in mice following long-term oral exposure after birth. We found that the concentrations of TBBPA-DBMPE in livers significantly increased with time, exhibiting a bioaccumulation potency not substantially different from HBCD. Lactational exposure to 1000 µg/kg/d TBBPA-DBMPE as well as 50 µg/kg/d HBCD inhibited testis development in suckling pups, and extended exposure up to adulthood resulted in significant molecular and cellular alterations in testes, with slighter effects of 50 µg/kg/d TBBPA-DBMPE. When exposure was extended to 8 month age, severe reproductive impairments including reduced sperm count, increased abnormal sperm, and subfertility occurred in all treated animals, although 50 µg/kg/d TBBPA-DBMPE exerted lower effects than 50 µg/kg/d HBCD. Altogether, all data led us to conclude that TBBPA-DBMPE exerted weaker male reproductive toxicity than HBCD at the same doses but exhibited bioaccumulation potential roughly equivalent to HBCD. Our study fills the data gap regarding the bioaccumulation and toxicity of TBBPA-DBMPE and raises concerns about its use as an alternative to HBCD.

The brominated flame retardant tetrabromobisphenol A-bis(2,3-dibromo-2-methylpropyl ether) as well as hexabromocyclododecane lead to lipid disorders in mice.

The brominated flame retardant tetrabromobisphenol A-bis(2,3-dibromo-2-methylpropyl ether) (TBBPA-DBMPE) is a recommended substitute for hexabromocyclododecane (HBCD), a banned persistent organic pollutant, yet its potential toxicities remains largely unexplored. Here, we investigated the effects of a long-term exposure to TBBPA-DBMPE at nominal doses of 50 and 1000 µg/kg/d on lipid homeostasis in CD-1 mice, in comparison with 50 µg/kg/d HBCD as a positive control. Male pups received chemical treatments through maternal administration via drinking water from postnatal day 0-21, followed by direct administration through drinking water after weaning. On the 23rd week after treatment, the oral lipid tolerance test revealed that low-dose TBBPA-DBMPE as well as HBCD affected lipid tolerance, although the fasting serum triglyceride (TG) levels were not altered. When chemical treatment was extended to the 32nd week, TBBPA-DBMPE-treated animals displayed adipocyte hypertrophy in both white adipose tissue (eWAT) and brown adipose tissue (BAT) and hepatic steatosis, which was largely consistent with the effects of HBCD. These findings indicate that like HBCD, TBBPA-DBMPE led to increased lipid load in mice. Interestingly, we also observed intestinal histological changes, coupled with increased expression of lipid absorption-related genes in both HBCD and TBBPA-DBMPE treatments, suggesting increased lipid absorption. This was supported by in vitro findings that both HBCD and TBBPA-DBMPE promoted lipid accumulation in IEC-6 cells under the stress of oleic acid for 6 h, implying that altered lipid absorption by the intestine may partly contributed to increased lipid load in mice. Overall, the effects of 50 µg/kg/d TBBPA-DBMPE in terms of some parameters were comparable with 50 µg/kg/d HBCD, suggesting that TBBPA-DBMPE may not be an ideal substitute of HBCD.

Extended exposure to tetrabromobisphenol A-bis(2,3-dibromopropyl ether) leads to subfertility in male mice at the late reproductive age.

Tetrabromobisphenol A-bis(2,3-dibromopropyl ether) (TBBPA-BDBPE), a commonly used brominated flame retardant as a decabromodiphenyl ether substitute, has been detected in various environmental compartments, but its health hazards remain largely unknown. Our recent study showed that low-dose exposure of male mice to TBBPA-BDBPE from postnatal day (PND) 0 to 56 caused remarkable damage to the microtubule skeleton in Sertoli cells and the blood-testis barrier (BTB) but exerted little effect on conventional reproductive endpoints in adulthood. To investigate whether TBBPA-BDBPE may cause severe reproductive impairments at late reproductive age, here, we extended exposure of historically administrated male mice to 8-month age and allowed them to mate with non-treated females for the evaluation of fertility, followed by a general examination for the reproductive system. As expected, we found that 8-month exposure to 50 µg/kg/d as well as 1000 µg/kg/d TBBPA-BDBPE caused severe damage to the reproductive system, including reduced sperm counts, increased sperm abnormality, histological alterations of testes. Moreover, microtubule damage and BTB-related impairment were still observed following 8-month exposure. Noticeably, high-dose TBBPA-BDBPE-treated mice had fewer offspring with a female-biased sex ratio. All results show that long-term exposure to TBBPA-BDBPE caused severe reproductive impairment, including poor fertility at late reproductive age. It is therefore concluded that slight testicular injuries in early life can contribute to reproductive impairment at late reproductive age, highlighting that alterations in certain non-conventional endpoints should be noticed as well as conventional endpoints in future reproductive toxicity studies.

Methimazole and sodium perchlorate exert anti-thyroidal effects in the T3-induced Xenopus laevis metamorphosis assay: A rapid assay for screening thyroid disrupting chemicals.

Thyroid disrupting chemicals (TDCs) have received much attention due to their potential adverse effects on animal and human health, which calls for rapid screen assays to identify them. The triiodothyronine (T3)-induced Xenopus metamorphosis assay (TiXMA) we developed previously has been successfully applied to the detection of the TDCs disrupting thyroid hormone (TH) signaling. Here, we attempted to expand the application of the TiXMA to the screening of the TDCs interfering with the hypothalamic-pituitary-thyroid (HPT) axis. Two well-known TH synthesis inhibitors methimazole (MMI) and sodium perchlorate (SP) were employed to test the sensitivity of the TiXMA to the TDCs interfering with the HPT axis. As expected, we observed that the two chemicals concentration-dependently antagonized T3-induced morphological changes and body weight reduction of X. laevis tadpoles following 96 h-exposure, in parallel with blocked thyroid development and down-regulated tshß expression in the brain. All the data show that both MMI and SP exert inhibitory effects on T3-induced metamorphosis, indicating that the TiXMA is capable of screening the TDCs interfering with the HPT axis. In comparison with Amphibian Metamorphosis Assay (AMA), a 21-day assay for screening the TDCs interfering with the HPT axis, the TiXMA has a remarkable advantage of shorter exposure duration (96 h).

Low concentrations of benzophenone-type UV-filters impair testis development in the amphibian Xenopus laevis.

Benzophenone-type UV filters (BPs) are ubiquitous contaminants in aquatic environments, possibly posing ecological risks to aquatic populations. So far, little is known about the potential adverse effects of BPs on amphibians. Given their potential estrogenic property, we investigated the detrimental effects of the commonly used BPs, BP-3, BP-2, and BP-1, on testis development in amphibians using Xenopus laevis as a model species. Following exposure to 10, 100, 1000 nM BP-3, BP-2, or BP-1 from stages 45/46 to 52, tadpoles presented morphological abnormal testes, characterized by reduced gonomere size and testis area, coupled with suppressed cell proliferation. Meanwhile, the downregulation of testis-biased gene expression and the upregulation of ovary-biased gene expression were observed in BPs-treated testes. Moreover, the estrogen receptor (ER) antagonist ICI 182780 significantly antagonized ovary-biased gene upregulation caused by BPs, suggesting that the effects of BPs on testis differentiation could be mediated by ER, at least partially. Of note, the effects of BPs were not concentration-dependent, but the lowest concentration generally exerted significant effects. Altogether, these observations indicate that the three BPs inhibited testis differentiation and exerted feminizing effects. Importantly, when BP-2 exposure was extended to two months post-metamorphosis, testes of froglets were generally less-developed, with relatively fewer spermatocytes, more spermatogonia, and poorly formed seminiferous tubules. Considering the fact that the lowest concentration (10 nM) of BPs in this study are detectable in aquatic environments, we conclude that BP-3, BP-2, and BP-1, even at environmentally relevant concentrations, can retard testis differentiation at pre-metamorphic stages and cause testis dysgenesis after metamorphosis in the amphibian X. laevis. Our findings suggest that ubiquitous BPs in aquatic environments could pose a potential risk to amphibians.

Postnatal exposure to low-dose tetrabromobisphenol A increases the susceptibility of mammal testes to chemical-induced spermatogenic stress in adulthood.

There is increasing data showing that some environmental chemicals can increase susceptibility to follow-up stress or injuries, possibly thereby contributing to certain clinical and subclinical diseases. Previous studies reported that tetrabromobisphenol A (TBBPA), one of the most used brominated flame retardants, exerted little male reproductive toxicity in terms of conventional endpoints but affected testis development and thereby caused testicular alterations at the molecular and cellular levels. Here, we aimed to reveal whether developmental exposure to TBBPA can increase testicular susceptibility to follow-up stress in adulthood. For this purpose, newborn mice were exposed to 50 or 500 µg/kg/d TBBPA for 56 days to confirm adverse effects on testes, followed by a single intraperitoneal injection of 3 mg/kg busulfan (BSF) to induce spermatogenic stress. Four weeks after BSF injection, TBBPA-treated mice exhibited severe pathological alterations, including reduced testis weight, damaged testicular histological structure, declined sperm count, apoptosis of spermatogenic cells, while no remarkable damage was observed in mice without historical exposure to TBBPA. These results demonstrate that historical exposure to TBBPA, either 50 or 500 µg/kg/d, increased the susceptibility of mouse testes to BSF-induced spermatogenic stress, resulting in severe adverse reproductive outcomes. Further analysis indicates that TBBPA-caused microtubule and microfilament damage, along with spermatogonia and spermatocyte reduction, could contributed to the increased susceptibility of testes, suggesting that these non-conventional reproductive lesions caused by chemicals should not be ignored. This is the first study to investigate the reproductive hazard of chemicals from the perspective of testicular susceptibility to stress, thereby opening a new avenue to identify environmental chemicals possibly contributing to male infertility and subfertility.

Comparison of Dechlorane Plus Concentrations in Sequential Blood Samples of Pregnant Women in Taizhou, China.

To develop an appropriate sampling strategy to assess the intrauterine exposure to dechlorane plus (DP), we investigated DP levels in sequential maternal blood samples collected in three trimesters of pregnancy, respectively, from women living in Taizhou. The median concentration of DPs (sum of syn-DP and anti-DP) in all samples was 30.5 pg g−1 wet-weight and 5.01 ng g−1 lipid-adjusted weight, respectively. The trimester-related DP concentrations were consistently strongly correlated (p < 0.01), indicating that a single measurement of DP levels could represent intrauterine exposure without sampling from the same female repeatedly; however, the wet-weight levels significantly increased across trimesters (p < 0.05), while the lipid-adjusted levels did not significantly vary. Notably, whether lipid-adjusted weight or wet-weight levels, the variation extent of DP across trimesters was found to be less than 41%, and those for other persistent organic pollutants (POPs) reported in the literature were also limited to 100%. The limitation in variation extents indicated that, regardless of the time of blood collection during pregnancy and how the levels were expressed, a single measurement could be extended to screen for exposure risk if necessary. Our study provides different strategies for sampling the maternal blood to serve the requirement for assessment of in utero exposure to DP.

Bisphenol B disrupts testis differentiation partly via the estrogen receptor-mediated pathway and subsequently causes testicular dysgenesis in Xenopus laevis.

There is growing concern about adverse effects of bisphenol A alternatives including bisphenol B (BPB) due to their estrogenic activity. However, limited data are available concerning the influences of BPB on male reproductive development in vertebrates, especially in amphibians, which are believed to be susceptible to estrogenic chemicals. The present study investigated the effects of 10, 100 and 1000 nM BPB (2.42, 24.2 and 242 µg/L) on testis development in Xenopus laevis, a model amphibian species for studying gonadal feminization. We found that exposure to BPB from stages 45/46 to 52 resulted in down-regulation of testis-biased gene expression and up-regulation of ovary-biased gene and vitellogenin (vtgb1) expression in gonad-mesonephros complexes (GMCs) of tadpoles at stage 52, coupled with suppressed cell proliferation in testes and reduced gonadal metameres, resembling the effects of 17ß-estradiol. Moreover, an estrogen receptor (ER) antagonist ICI 182780 antagonized BPB-caused up-regulation of ovary-biased gene and vtgb1 expression to some degree, indicating that the effects of BPB on X. laevis testis differentiation could be partly mediated by ER. All observations demonstrate that early exposure to BPB inhibited testis differentiation and exerted certain feminizing effects during gonadal differentiation. When exposure was extended to post-metamorphosis, testes exhibited histological and morphological abnormalities including segmented, discontinuous and fragmented shapes, besides altered sex-dimorphic gene expression. Notably, most of BPB-caused alterations were not concentration-dependent, but the lowest concentration indeed exerted significant effects. Overall, our study for the first time reveals that low concentrations of BPB can disrupt testis differentiation partly due to its estrogenic activity and subsequently cause testicular dysgenesis after metamorphosis, highlighting its reproductive risk to amphibians and other vertebrates including humans. Our finding also implies that estrogenic chemicals-caused testis differentiation inhibition at tadpole stages could predict later testicular dysgenesis after metamorphosis, meaning a possibility of early detection of abnormal testis development caused by estrogenic chemicals.

Measurement of polychlorinated biphenyls with hand wipes and matched serum collected from Chinese E-waste dismantling workers: Exposure estimates and implications.

To date, dermal/hand-to-mouth exposure to chemicals in the e-waste recycling environment has not been sufficiently understood, and the importance of dermal absorption of chemicals in e-waste dismantling workers remains controversial. In this study, we utilized hand wipes and matched sera to characterize dermal/hand-to-mouth exposure to PCBs for e-waste dismantling workers, and potential effects on thyroid hormones were also assessed. PCB loadings in hand wipes varied from 0.829-265 ng wipe-1 (11.3-2850 ng m-2 wipe-1), with 37.2 ng wipe-1 (432 ng m-2 wipe-1) as the median value. Serum concentrations of PCBs ranged from 32.3-3410 ng g-1 lipid weight (lw) with 364 ng g-1 lw as the median value. Between wipes and sera, lower-chlorinated congeners (e.g. CB-28, -66, -74, -99,-105 and -118) showed significant associations (p < 0.01), but higher-chlorinated congeners (e.g. CB-138, -153, -156, -170, and -180) did not. These lower-chlorinated CBs were the major contributors to estimated dermal/hand-to-mouth average daily doses (ADDs) and the hazard index (HI). Correspondingly, their estimated contributions to serum levels by dermal absorption were also significant, with the contribution of CB-28 being as high as 21.4%. As a consequence, dermal absorption of some low-chlorinated congeners was a non-negligible route for e-waste dismantling workers. Although insignificant association was shown between serum PCBs and thyroid hormones, the potential health risk should be of concern due to the high levels of PCBs observed in workers' sera.

Transfer of dechlorane plus between human breast milk and adipose tissue and comparison with legacy lipophilic compounds.

In this study, levels of dechlorane plus (DP) in breast milk and matched adipose tissue samples were measured from 54 women living in Wenling, China. Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) were measured simultaneously for comparison. The levels of ∑DPs/∑PBDEs varied from less than one to several dozens of ng g-1 lipid weight (lw) in matrices and the levels of ∑PCBs varied between several to hundreds of ng g-1 lw. In the same matrix, ∑DPs and ∑PCBs/∑PBDEs showed a significant relationship (p < 0.05), indicating that they shared common sources. Accordingly, there was a strong association of lipid-adjusted concentrations of individual compounds (BDE-209 excluded) between matrices (p < 0.001), suggesting that breast milk could be a proxy for adipose tissue in human bioburden monitoring of these compounds. The predicted lipid-adjusted milk/adipose ratios varied from 0.62 to 1.5 but showed significant differences (p<0.001) between compounds, suggesting a compound-specific transfer between milk lipids and adipose tissue lipids. Specifically, the milk/adipose ratios for syn-DP and anti-DP (-1.40 and 1.3, respectively) were significantly higher than those of CB congeners and hexa/hepta-BDE congeners (p < 0.05). In addition, unlike PCBs/PBDEs (excluding BDE-209), DP's hydrophobicity might not be responsible for its preferable distribution in milk lipids. Instead, the interaction with nonlipid factors played a key role. The fraction of anti-DP between the two kinds of matrices was not significantly different, suggesting that the biochemical transfer processes may not be efficient enough to distinguish DP isomers. Nevertheless, the congener patterns of PCBs/PBDEs gave a clue about the compound-specific transfer between milk and adipose tissue. To our knowledge, this is the first to report the relationships of DP between adipose tissue and breast milk. These results could provide useful and in-depth information on biomonitoring of DP and facilitate the understanding of the accumulation and excretion potentials of DP and its distribution-related mechanism in humans.

2,2',4,4'-tetrabromodipheny ether (BDE-47) disrupts gonadal development of the Africa clawed frog (Xenopus laevis).

Previous studies have shown that BDE-47, one of the most abundant polybrominated diphenyl ethers (PBDEs) congeners, has a weak estrogenic activity, but it has remained unclear whether BDE-47 disrupts gonadal development and causes male-to-female sex reversal in lower vertebrates, with limited and controversial data. The present study aimed to determine the effects of BDE-47 on gonadal development in Xenopus laevis, a model amphibian species for studying adverse effects of estrogenic chemicals on reproductive development. X. laevis at stage 45/46 were exposed to BDE-47 (0.5, 5, 50 nM) in semi-static system, with 1 nM 17ß-estradiol (E2) as the positive control. When reaching stage 53, tadpoles were examined for gonadal morphology, histology and sex-dimorphic gene expression. The phenotypic sex (gonadal morphology and histology) of each BDE-47-treated tadpole matched its genetic sex, showing no sex-reversal, whereas one half of genetic males treated with E2 displayed ovarian-like features. However, some genetic males (26%) in the 50 nM BDE-47 treatment group were found to contain more germ cells clumping together in the medulla, along with an increasing tendency of the gonad length/kidney length ratio in males, resembling feminizing outcomes of E2. These observations seem to suggest that BDE-47 exerted weak feminizing effects. However, BDE-47 induced increases in expression of both female-biased genes and male-biased genes in two sexes, which disagrees with feminizing outcomes, suggesting complicated effects of BDE-47 on gonadal development. Taken together, all results demonstrate that nanomolar BDE-47 disrupted gonadal development and exerted weak feminizing effects, but not resulted in male-to-female sex reversal in X. laevis.

Evaluation of the effects of low concentrations of bisphenol AF on gonadal development using the Xenopus laevis model: A finding of testicular differentiation inhibition coupled with feminization.

Developmental exposures to estrogenic chemicals possibly cause structural and functional abnormalities of reproductive organs in vertebrates. Bisphenol AF (BPAF), a bisphenol A (BPA) analogue, has been shown to have higher estrogenic activity than BPA, but little is known about the effects of BPAF on gonadal development, particularly gonadal differentiation. We aimed to determine whether low concentrations of BPAF could disrupt gonadal differentiation and subsequent development using Xenopus laevis, a model species for studying feminizing effects of estrogenic chemicals. X. laevis tadpoles were exposed to BPAF (1, 10, 100 nM) or 17ß-estradiol (E2, positive control) from stages 45/46 to 53 and 66 in a semi-static exposure system, with a prolonged treatment with the highest concentration to the eighth week post-metamorphosis (WPM8). Gonadal morphology and histology as well as sexually dimorphic gene expression were examined to evaluate the effects of BPAF. All concentrations of BPAF caused changes in testicular morphology at different developmental stages compared with controls. Specifically, at stage 53, BPAF like E2 resulted in decreases in both the size and the number of gonadal metameres (gonomeres) in testes, looking like ovaries. Some of BPAF-treated testes remained segmented and even became discontinuous and fragmented at subsequent stages. Histological abnormalities were also observed in BPAF-treated testes, such as ovarian cavity at stages 53 and 66 and poorly developed seminiferous tubules on WPM8. At the molecular level, BPAF inhibited expression of male highly expressed genes in testes at stage 53. Correspondingly, BPAF, like E2, inhibited cell proliferation in testes at stage 50. All results show that low concentrations of BPAF inhibited testicular differentiation and subsequent development in X. laevis, along with feminizing effects to some degree. Our finding implies a risk of BPAF to the male reproductive system of vertebrates including humans.

Bioaccumulation and transfer characteristics of dechlorane plus in human adipose tissue and blood stream and the underlying mechanisms.

In this study, bioaccumulation and transfer characteristics of dechlorane plus (DP) were examined between human adipose tissue and matched maternal serum, and the possible transfer mechanism between tissues was further discussed. The median level of total DP was 971 pg g-1 wet weight (ww) and 1.22 ng g-1 lipid weight (lw) in adipose tissue, respectively, and was 34.7 pg g-1 ww and 3.98 ng g-1 lw for serum, respectively. DP wet levels' positive association with fat contents of five types of human tissues indicated that DP distribution might be related to lipid-driven mechanism. However, the lipid-adjusted adipose-serum partitioning ratios were estimated to be 0.35 for syn-DP and 0.35 for anti-DP, accordingly, which implied that the DP distribution between serum and adipose tissues, was not only regulated by the tissue lipid contents. Both the internal mono-dechlorination of anti-DP, and stereo-selective behavior of DP isomers were not found in DP transfer from blood to adipose tissue. The marginal positive relationship was observed between serum levels and apolipoprotein A concentrations (p = 0.095 for total DP and 0.045 for syn-DP), and neither association was found between serum levels and thyroid hormone concentrations (THs). To our best knowledge, this is the first report about the accumulation relationship of DP between human adipose tissue and blood stream with the corresponding distribution-related mechanism.

Gonadal differentiation and its sensitivity to androgens during development of Pelophylax nigromaculatus.

Our previous observations proposed Pelophylax nigromaculatus as a model species for studying the masculinizing effects of androgenic EDCs in amphibians. To better develop this model species, we studied the process of the gonadal differentiation/development and the sensitive stage to androgens. We found that the earliest sexual dimorphism in gonads at morphological and histological levels occurred at stages 38-40 and stage 36 respectively. Further examination of molecular markers for testicular and ovarian differentiation during development revealed that the cyp17 and cyp19 expressions were sexually dimorphic from stage 32 and stage 36 respectively. Further, we investigated the sex-reversal induced by 100 ng/L 5α-dihydrotestosterone (DHT) when exposures were initiated at stages 24, 26 and 28. We found that when exposed from stage 24, DHT resulted masculinization of all tadpoles with no typical ovaries, whereas exposures from stage 26 or 28 dramatically reduced the effect of DHT. Our findings show that gonads of P. nigromaculatus are bipotential at stage 24, in the process of differentiation at stage 26 and determined to become either testis or ovary at stage 28. Altogether, exposure of P. nigromaculatus should begin at stage 24 in order to sensitively detect masculinizing effects of EDCs. Present study provides useful information about the gonadal differentiation and development in P. nigromaculatus for effectively evaluating masculinizing effects of EDCs on gonads.

Bisphenol F Disrupts Thyroid Hormone Signaling and Postembryonic Development in Xenopus laevis.

The safety of bisphenol A (BPA) alternatives has attracted much attention due to their wide use. In this study, we investigated the effects of bisphenol F (BPF), an alternative to BPA, on thyroid hormone (TH) signaling and postembryonic development in vertebrates using T3-induced and spontaneous Xenopus metamorphosis as models. We found that in the T3-induced metamorphosis assay, higher concentrations of BPF (100-10000 nM) antagonized T3-induced TH-response gene transcription and morphological changes including intestinal remodeling in a concentration-dependent manner, whereas 10 nM BPF exerted stimulatory effects on T3-induced integral metamorphosis when inhibited T3-induced TH-response gene transcription, demonstrating TH signaling disrupting effects of BPF. In the spontaneous metamorphosis assay, correspondingly, BPF inhibited development at metamorphic climax (with high endogenous TH levels), but promoted pre- and pro-metamorphic development (with low endogenous TH levels), displaying a developmental stage-dependent manner. Importantly, we observed agonistic actions of BPF on Notch signaling in intestines, showing that BPF disrupts vertebrate development possibly via multi pathways besides TH signaling. Thus, we infer the biphasic concentration-response relationship between BPF exposure and T3-induced metamorphosis could result from the interactions of TH signaling with other signaling pathways such as Notch signaling. Our study highlights the adverse influences of BPF on vertebrate development.

Bisphenol A alternatives bisphenol S and bisphenol F interfere with thyroid hormone signaling pathway in vitro and in vivo.

The wide use of the alternatives to bisphenol A (BPA) has raised concerns about their potential toxicities. Considering the disrupting activity of BPA on thyroid hormone (TH) signaling, we investigated whether bisphenol S (BPS) and bisphenol F (BPF), two leading alternatives, could interfere with TH signaling pathway using a series of assays in vitro and in vivo. In the fluorescence competitive binding assay, we found BPS and BPF, like BPA, bound to TH receptors (TRα and TRß), with the binding potencies an order of magnitude lower than BPA (BPA > BPF > BPS). Molecular docking data also show their binding potencies to TRs. In the coactivator recruitment assay, BPS and BPF recruited coactivator to TRß but not TRα, with weaker potencies than BPA. Correspondingly, agonistic actions of the three bisphenols in the absence or presence of T3 were observed in the TR-mediated reporter gene transcription assay. Also, all the three bisphenols induced TH-dependent GH3 cell proliferation, whereas BPA and BPF inhibited T3 induction in the presence of T3. As for in vivo assay, the three bisphenols like T3 induced TH-response gene transcription in Pelophylax nigromaculatus tadpoles, but in the presence of T3 altered T3-induced gene transcription in a biphasic concentration-response manner. These results for the first time demonstrate that BPS and BPF, like BPA, have potential to interfere with TH signaling pathway, i.e., they generally activate TH signaling in the absence of T3, but in the presence of TH, display agonistic or/and antagonistic actions under certain condition. Our study highlights the potential risks of BPS and BPF as BPA alternatives.

Tetrabromoethylcyclohexane affects gonadal differentiation and development in the frog Pelophylax nigromaculatus.

Tetrabromoethylcyclohexane (TBECH), an additive brominated flame retardant, has been shown to have an androgenic activity in vitro. In the present study, we aimed to investigate the effects of TBECH on gonadal differentiation and development in the frog Pelophylax nigromaculatus, an amphibian species sensitive to androgenic chemicals, and to assess the androgenic activity of TBECH in vivo. P. nigromaculatus tadpoles were exposed to TBECH (1, 10, 100nM) from Gosner stage 24 to complete metamorphosis, and to 5α-dihydrotestosterone (DHT) as a positive control. We found that 1nM DHT resulted in 100% males, while the sex ratio in the solvent control group was close to 1:1. In all the TBECH treatment groups, sexually ambiguous gonads based on gross morphology and intersexualities with testicular and ovarian histological structures were found, but no abnormality occurred in the solvent control. In the 1, 10, 100nM TBECH treatment groups, the female percentages were 52%, 31%, 17%, with 36%, 56%, 66% for males and 12%, 13%, 17% for abnormal sexes, respectively. X2-test revealed significant differences in sex ratios between the three TBECH groups and the solvent control group, and the sex ratios in the two higher concentration groups were male-biased. These observations show that TBECH has a masculinizing effect on gonadal differentiation and development in P. nigromaculatus, suggesting an androgenic activity of TBECH in vivo. To our knowledge, this is the first study demonstrating that TBECH could induce gonadal masculinization in an animal, which raises new concerns for reproductive risk of TBECH exposure.

Determining the optimal developmental stages of Xenopus laevis for initiating exposures to chemicals for sensitively detecting their feminizing effects on gonadal differentiation.

Xenopus laevis is an important model for detecting feminizing effects of endocrine disrupting chemicals (EDCs) on amphibians because its genetic males can be induced to phenotypic females by estrogenic chemicals. It is crucial that chemical exposures begin at sensitive developmental stages for gonadal sex-reversal in X. laevis. To determine the optimal stages for initiating exposures, we investigated gonadal sex-reversal induced by low concentrations of 17α-ethinylestradiol (EE2) when exposures were initiated at different stages (3/4, 45/46, 48 and 50) until stage 58. We found that 0.1nM EE2 resulted in 85%, 86%, 43%, and 19% intersex, whereas 1nM EE2 caused 77%, 81%, 17%, and 8% phenotypic females, when genetic male tadpoles were exposed from stages 3/4, 45/46, 48 and 50, respectively. The data show the sensitivity of X. laevis gonads to EE2 at stages 45/46 is similar with that at stages 3/4, but the sensitivity decreases at stage 48 and stage 50, displaying a developmental stage-dependent manner. In another experiment using the offspring of another pair of frogs, we confirmed high sensitivity of X. laevis gonads at stages 45/46 to low concentrations of EE2. Considering that stages 45/46 tadpoles are easier to manipulate and have higher survival rates than earlier embryos, we propose that stages 45/46 are the optimal stages for initiating exposure for detecting feminizing effects of EDCs on gonadal differentiation in X. laevis. The developmental stages for initiating exposures we determined will guarantee the high sensitivity for detecting feminizing effects of EDCs with low estrogenic activities on gonadal differentiation in X. laevis. Also, our study suggests that gonadal differentiation in X. laevis possibly begins at stages 45/46, but not at later stages.

Low concentrations of dihydrotestosterone induce female-to-male sex reversal in the frog Pelophylax nigromaculatus.

Previous studies have demonstrated that some amphibian species can be sex-reversed by high concentrations of androgens. Little attention has focused on the effects of androgenic endocrine-disrupting chemicals (EDCs) on amphibians. The present study aimed to investigate the effects of lower concentrations of the androgenic EDC 5α-dihydrotestosterone (DHT) on gonadal differentiation and development in Pelophylax nigromaculatus, a true frog distributed widely in East Asia. Tadpoles at Gosner stage 24/25 were exposed to nominal concentrations of 40 ng/L, 400 ng/L, and 4000 ng/L DHT to complete metamorphosis. In all DHT treatment groups, males and ambiguous sexes were identified based on gonadal morphology, whereas no females were found; thus, all treatment groups exhibited male-skewed ratios compared with the control group. Gonadal histological examination revealed that ambiguous sexes displayed overall testicular structure with certain ovarian characteristics, demonstrating that DHT-induced sex-ambiguous gonads were incomplete ovary-to-testis reversals (IOTTRs). The expression levels of some ovary-biased genes in the IOTTRs were significantly higher than in the control testes but lower than in the control ovaries. These results show that low concentrations of DHT induced complete or incomplete female-to-male sex reversal in P. nigromaculatus, and incomplete sex reversal retained certain ovarian characteristics not only at gonadal morphological and histological levels but also at the molecular level. They present study highlights potential risks of DHT and other androgenic EDCs for P. nigromaculatus.

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.