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.

Developing a novel quantitative parameter for characterizing spatial distribution of fish following exposure to chemicals and wastewater: Behavioral Gini coefficient.

While the spatial distribution pattern of fish is increasingly used for toxicological test of chemicals or wastewater, no ideal parameter is available for quantitative assessment of spatial distribution, especially uneven distribution with multiple hotspots. Here, to develop a quantitative assessment parameter for spatial distribution, the zebrafish were exposed to ethanol, pentylenetetrazole (PTZ), paraquat dichloride (paraquat) and wastewater, followed by a behavioral test in a narrow tank. Behavioral data was acquired and analyzed by idTracker and MATLAB. By comparing the effects of all treatments on behavior parameters, we confirmed that the spatial distribution was more easily altered rather than general locomotor parameters, e.g. 0.7-70 mg/L PTZ and 5-20 mg/L paraquat being effective for altering spatial distribution but having little effects on general locomotor parameters. Based on the heatmap, i.e., the cumulative proportion of grids and that of frequency in grids, we calculated the behavioral Gini coefficient (Gb) for quantitative assessment of fish spatial distribution. The Gini coefficient ranged from zero to 1, with larger values meaning poorer evenness of spatial distribution. Of note, Gb showed smaller coefficient of variations (CV) with 3%-19% between replicate tanks in all treatments than the highest frequency (4%-79%), displaying well robustness. Especially, Gb addressed the challenge of the complicated heatmap with multiple hotspots. Overall, the behavioral Gini coefficient we established is an ideal parameter to quantitatively assess spatial distribution of fish shoal, which is expected to be applied in toxicity testing for chemicals and wastewater and automatic quality monitoring for surface water and aquaculture water.

Transcriptomic analysis reveals the effects of maternal exposure to bisphenol AF on hypothalamic development in male neonatal mice.

Fragmented data suggest that bisphenol AF (BPAF), a chemical widely used in a variety of products, might have potential impacts on the hypothalamus. Here, we employed male neonatal mice following maternal exposure to explore the effects of low-dose BPAF on hypothalamic development by RNA-sequencing. We found that maternal exposure to approximately 50 µg/(kg·day) BPAF from postanal day (PND) 0 to PND 15 altered the hypothalamic transcriptome, primarily involving the pathways and genes associated with extracellular matrix (ECM) and intercellular adhesion, neuroendocrine regulation, and neurological processes. Further RNA analysis confirmed the changes in the expression levels of concerned genes. Importantly, we further revealed that low-dose BPAF posed a stimulatory impact on pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus and induced the browning of inguinal white adipose tissue. All findings indicate that developmental exposure to low-dose BPAF could interfere with hypothalamic development and thereby lead to alterations in the metabolism. Interestingly, 5000 µg/(kg·day) BPAF caused slighter, non-significant or even inverse alterations than the low dose of 50 µg/(kg·day), displaying a dose-independent effect. Further observations suggest that the the dose-independent effects of BPAF might be associated with oxidative stress and inflammatory responses caused by the high dose. Overall, our study highlights a risk of low-dose BPAF to human neuroendocrine regulation and metabolism.

Tetrabromobisphenol A exerts thyroid disrupting effects but has little overt impact on postnatal brain development and neurobehaviors in mice.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant. There is evidence showing that TBBPA can exert thyroid disrupting effects in mammals, but different results were also reported, along with inconsistent reports regarding its neurotoxicity. Here, we investigated thyroid disrupting effects and neurotoxicity of TBBPA (5, 50, 500 µg/(kg·day)) to male mice following maternal and direct exposure through drinking water, with the anti-thyroid drug propylthiouracil (PTU) as the positive control. On postnatal day (PND) 15, we expectedly observed severe thyroid compensatory hyperplasia and cerebellar developmental retardation in PTU-treated pups. The highest dose of TBBPA also caused thyroid histological alteration but had no effects on cerebellar development in terms of Purkinje cell morphology and the thickness of the internal granular layer and the molecular layer of the cerebellum. During puberty and adulthood, the thyroid morphological alterations became more pronounced in the TBBPA-treated animals, accompanied by decreased serum thyroid hormone levels. Furthermore, the 50 and 500 µg/(kg·day) TBBPA groups showed a significant decrease in the serum level of serotonin, a neurotransmitter associated with anxiety behaviors. Correspondingly, the highest dose group displayed anxiety-like behaviors in the elevated plus-maze test on PND 35, but this neurobehavioral alteration disappeared on PND 56. Moreover, no changes in neurobehavioral parameters tested were found in TBBPA-treated animals at puberty and adulthood. Altogether, all observations show that TBBPA can exert thyroid disrupting effects but has little overt impact on brain development and neurobehaviors in mice, suggesting that thyroid disruption does not necessarily cause overtly adverse neurodevelopmental outcomes.

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.

Effects of low-dose bisphenol AF on mammal testis development via complex mechanisms: alterations are detectable in both infancy and adulthood.

Despite growing concern about adverse effects of bisphenol AF (BPAF) due to its endocrine disrupting properties, there is a lack of toxicity data from low-dose studies and direct evidence linking its adverse effects to endocrine disrupting properties. Here, we investigated the effects of gestational and postnatal exposure to BPAF through drinking water (0.15-15 µg/mL, equivalent to the daily intake of ~ 50 and 5 mg/kg/day) on testis development in mice. We found that like mestranol, 5 mg/kg/day BPAF resulted in remarkable decreases in multiple male reproductive parameters in adulthood, such as the sperm number and serum testosterone level. Notably, 50 µg/kg/day BPAF also caused significant decreases in anogenital distance (AGD), the luteinizing hormone level and spermatocyte number, along with declining trends in sperm number and the serum levels of testosterone and follicle-stimulating hormone. In line with the adverse outcomes observed in adulthood, on postnatal day (PND) 9, we also observed BPAF-caused dose-dependent alterations, including reduced AGD, seminiferous tubule area and numbers of total germ cells, spermatocytes and Leydig cells, coupled with down-regulated expression of male-biased genes in testes. Even when exposure to 5 mg/kg/day BPAF as well as MES was initiated from PND 0, similar alterations in male reproductive parameters were also found on PND 9, along with a decrease in the GnRH content in the hypothalamus; moreover, testicular alterations and the reduction in AGD were partly antagonized by the estrogen receptor (ER) antagonist ICI 182,780, but the reduction of GnRH production was not done, showing that the effects of BPAF on testis development may be partially mediated by ER signaling. In conclusion, all the findings demonstrate that low-dose BPAF can partly disrupt mammal testis development and cause adverse testicular outcomes in adulthood, indicating a potential reproductive risk to mammals including humans. Importantly, our finding that developmental alterations elicited by BPAF have been detectable on PND 9 provides important motivation for the development of effective methods for early detection of adverse effects of estrogenic chemicals on testis development.

Effects of postnatal exposure to tetrabromobisphenol A on testis development in mice and early key events.

Whether or not tetrabromobisphenol A (TBBPA) has reproductive developmental toxicity remains controversial. Here, we evaluated the effects of postnatal TBBPA exposure of dams (before weaning) and pups through drinking water (15, 150, 1500 ng/mL) on testis development in mice. On postnatal day (PND) 56, we found that TBBPA exerted little effects on testis weight, anogenital distance, sperm parameters, and the serum testosterone level, but resulted in dose-dependent reductions in the seminiferous tubule area coupled with decreased Sertoli cells and spermatogonia and the number of stage VII-VIII seminiferous tubules, and cytoskeleton damage in Sertoli cells, along with down-regulated expression of marker genes for Sertoli cells, spermatogonia and spermatocyte. Further study revealed that the reduced tubule area coupled decreased Sertoli cell and germ cell numbers and marker gene expression also occurred in TBBPA-treated testes on PND 7, along with reduced cell proliferation and disordered arrangement of Sertoli cell nuclei. On PND 15, most of these testicular alterations were still observed in TBBPA-treated males, and cytoskeleton damage in Sertoli cells became observable. All observations convincingly demonstrate that postnatal exposure to TBBPA disturbed testis development in early life and ultimately caused adverse outcomes in adult testes, and that cell proliferation inhibition, the reduction in the seminiferous tubule area coupled decreased Sertoli cell and germ cell numbers and marker gene expression, and cytoskeleton damage in Sertoli cells, are early events contributing to adverse outcomes in adult testes. Our study improves the understanding of reproductive developmental toxicity of TBBPA, highlighting its risk for human health.

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.

A Multiwell-Based Assay for Screening Thyroid Hormone Signaling Disruptors Using thibz Expression as a Sensitive Endpoint in Xenopus laevis.

There is a need for rapidly screening thyroid hormone (TH) signaling disruptors in vivo considering the essential role of TH signaling in vertebrates. We aimed to establish a rapid in vivo screening assay using Xenopus laevis based on the T3-induced Xenopus metamorphosis assay we established previously, as well as the Xenopus Eleutheroembryonic Thyroid Assay (XETA). Stage 48 tadpoles were treated with a series of concentrations of T3 in 6-well plates for 24 h and the expression of six TH-response genes was analyzed for choosing a proper T3 concentration. Next, bisphenol A (BPA) and tetrabromobisphenol A (TBBPA), two known TH signaling disruptors, were tested for determining the most sensitive TH-response gene, followed by the detection of several suspected TH signaling disruptors. We determined 1 nM as the induction concentration of T3 and thibz expression as the sensitive endpoint for detecting TH signaling disruptors given its highest response to T3, BPA, and TBBPA. And we identified betamipron as a TH signaling agonist, and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) as a TH signaling antagonist. Overall, we developed a multiwell-based assay for rapidly screening TH signaling disruptors using thibz expression as a sensitive endpoint in X. laevis.

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.

Tetrabromobisphenol A Disturbs Brain Development in Both Thyroid Hormone-Dependent and -Independent Manners in Xenopus laevis.

Although tetrabromobisphenol A (TBBPA) has been well proven to disturb TH signaling in both in vitro and in vivo assays, it is still unclear whether TBBPA can affect brain development due to TH signaling disruption. Here, we employed the T3-induced Xenopus metamorphosis assay (TIXMA) and the spontaneous metamorphosis assay to address this issue. In the TIXMA, 5-500 nmol/L TBBPA affected T3-induced TH-response gene expression and T3-induced brain development (brain morphological changes, cell proliferation, and neurodifferentiation) at premetamorphic stages in a complicated biphasic concentration-response manner. Notably, 500 nmol/L TBBPA treatment alone exerted a stimulatory effect on tadpole growth and brain development at these stages, in parallel with a lack of TH signaling activation, suggesting the involvement of other signaling pathways. As expected, at the metamorphic climax, we observed inhibitory effects of 50-500 nmol/L TBBPA on metamorphic development and brain development, which was in agreement with the antagonistic effects of higher concentrations on T3-induced brain development at premetamorphic stages. Taken together, all results demonstrate that TBBPA can disturb TH signaling and subsequently interfere with TH-dependent brain development in Xenopus; meanwhile, other signaling pathways besides TH signaling could be involved in this process. Our study improves the understanding of the effects of TBBPA on vertebrate brain development.

Low Concentrations of Tetrabromobisphenol A Disrupt Notch Signaling and Intestinal Development in in Vitro and in Vivo Models.

Tetrabromobisphenol A (TBBPA) was recently reported to upregulate Notch target gene expression in embryonic stem cells differentiating to neurons in vitro, implying activation on Notch signaling, a crucial signaling involved in multiple organ development and homeostasis.The present study aimed to determine whether TBBPA at low concentrations can disrupt Notch signaling in the intestine and subsequently its development using in vitro and in vivo models, given TBBPA uptake mainly via the intestine. In rat intestinal epithelium cells (IEC-6), an in vitro model for intestinal development and homeostasis, we found 5-500 nM TBBPA upregulated Notch-related gene expression and stimulated cell proliferation as well as the growth of microvilli in a linear concentration-dependent manner. When Notch inhibitor DAPT had no obvious effects on all end points, DAPT significantly antagonized all changes caused by TBBPA, indicating that TBBPA activated Notch signaling in IEC-6 cells and subsequently stimulated cell proliferation and differentiation. Then we employed Xenopus laevis, an ideal model species for intestinal development with the strong similarities to mammals, to further confirm the action of TBBPA in vivo. Expectedly, we observed the stimulatory effects of TBBPA on Notch signaling and cell proliferation and differentiation in X. laevis intestines, which agrees with the results in vitro. Antagonistic actions of Notch inhibitor DBZ on TBBPA-caused intestinal changes show that TBBPA affected intestinal development via disrupting Notch signaling. Interestingly, TBBPA stimulated cell differentiation into secretory cells, which is generally believed to be regulated by Wnt signaling, suggesting disruption of Wnt signaling besides Notch signaling. All the results for the first time demonstrate that TBBPA at low concentrations, including environmentally relevant concentrations, disrupt Notch signaling and subsequently affect intestinal development by altering cell proliferation and differentiation in vertebrates. Our study highlights the intestine as a new target of TBBPA and broaden our understanding of developmental toxicity of TBBPA.

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.

TBBPA and Its Alternatives Disturb the Early Stages of Neural Development by Interfering with the NOTCH and WNT Pathways.

Tetrabromobisphenol A (TBBPA), as well as its alternatives Tetrabromobisphenol S (TBBPS) and Tetrachlorobisphenol A (TCBPA), are widely used halogenated flame retardants. Their high detection rates in human breast milk and umbilical cord serum have raised wide concerns about their adverse effects on human fetal development. In this study, we evaluated the cytotoxicity and neural developmental toxicity of TBBPA, TBBPS, and TCBPA with a mouse embryonic stem cell (mESC) system, at human body fluid and environmental relevant doses. All the three compounds showed similar trends in their cytotoxic effects. However, while TBBPA and TBBPS stimulated ESC neural differentiation, TCBPA significantly inhibited neurogenesis. Mechanistically, we demonstrated that, as far as the NOTCH (positive regulator) and WNT (negative regulator) pathways were concerned, TBBPA only partially and slightly disturbed them, whereas TBBPS significantly inhibited the WNT pathway, and TCBPA down-regulated the expression of NOTCH effectors but increased the WNT signaling, actions which both inhibited neural specification. In conclusion, our findings suggest that TBBPS and TCBPA may not be safe alternatives to TBBPA, and their toxicity need to be comprehensively evaluated.

Effects of triclosan on gonadal differentiation and development in the frog Pelophylax nigromaculatus.

Previous studies have reported that triclosan (TCS) could possess an androgenic activity. We aimed to investigate the effects of TCS on gonadal differentiation and development in the frog Pelophylax nigromaculatus, a sensitive amphibian species to androgenic chemicals. P. nigromaculatus tadpoles at stage 24 were exposed to TCS (3, 30, and 300nmol/L) to stage 46 in a semi-static exposure system. At the end of exposure, gonadal morphology and histology, sex ratio and gonadal expression of sex-biased genes were examined in P. nigromaculatus. In each TCS treatment group, we found several individuals whose gonads exhibited morphological and/or histological abnormalities. Gonadal histological abnormalities were characterized by few oocytes and many somatic cells. Although the percentage of the individuals with abnormal gonads was low (7.8%) among all animals treated with TCS, statistical test revealed the sex ratios in the 3 and 300nmol/L TCS treatment groups were significantly different from the solvent control. In the 30nmol/L TCS treatment group, abnormal gonads were also observed, although the sex ratio was not changed compared with the solvent control, which was possibly due to the smaller sample size in this group. In all the TCS treatment groups, the sex ratios were not obviously male-biased, but the expression levels of some sex-biased genes were significantly altered by TCS. Altogether, our results suggest that TCS, even at environmentally relevant concentrations, could disrupt gonadal differentiation and development in P. nigromaculatus, but we are not sure whether the disrupting effects were associated with masculinization or feminization.

An ex vivo assay for screening glucocorticoid signaling disruption based on glucocorticoid-response gene transcription in Xenopus tails.

There is a pressing need for developing in vivo or ex vivo assays to screen the glucocorticoid (GC) signaling disruption of chemicals. Thus, we aimed to establish an ex vivo assay for screening GC signaling disruption based on the GC-response gene transcription in Xenopus laevis tails cultured ex vivo. Firstly, we investigated effects of corticosterone (CORT, a main GC in frogs) on GC-response gene expression, and determined the six genes as molecular endpoints for assaying the GC signaling disruption. CORT in the range of 1.56-400nmol/L was found to up-regulate transcription of the six GC-response genes, exhibiting comparable or higher sensitivity than previously reported assays. To validate this ex vivo assay, then, we examined effects of dexamethasone (a known GC signaling agonist) on GC-response gene expression. Dexamethasone displayed an agonistic action in a concentration-dependent manner, further demonstrating the efficiency of the established assay. Finally, we applied the ex vivo assay to evaluate the GC signaling disruption of bisphenol A (BPA). In accordance with previous reports, we found a concentration-dependent agonistic activity of BPA, showing that the established assay is effective for detecting the GC signaling disrupting activity of environmental chemicals. Correspondingly, the GC signaling agonistic actions of CORT and BPA in ex vivo tails accorded with the observations in vivo, indicating that the ex vivo assay is able to detect the actions of chemicals in vivo. Overall, we established an ex vivo assay that can effectively screen GC signaling disruption of environmental chemicals.

Discovery of a Novel Polyfluoroalkyl Benzenesulfonic Acid around Oilfields in Northern China.

The existence of more than 3000 per- and polyfluoroalkyl substances (PFASs) on the global market has prompted the identification and hazard characterization of hitherto unknown PFASs. In the present study, a novel PFAS, sodium p-perfluorous nonenoxybenzenesulfonate (OBS), was identified using Orbitrap MS/MS in water samples around a suspected application area, Daqing Oilfield, China. The peak OBS concentration was 3.2 × 103 ng/L in a sample taken near the oil well with the longest production history in Daqing. The concentrations of OBS and contribution to the sum of PFASs in surface waters displayed considerable variation among the three sampling areas (mean levels at 6.9, 50, and 5.6 × 102 ng/L with mean percentages at 9.8%, 45%, and 69% in the background, new and old oilfield areas respectively) confirming that the density of oil wells and the oil production history are important factors influencing OBS contamination in the studied areas. A preliminary assessment of acute toxicity and environmental fate indicates that OBS exhibits similar toxicity and environmental persistence to perfluorooctanesulfonic acid (PFOS). The widespread occurrence of OBS, in conjunction with its potential hazard properties, underscores the need to further study on the bioaccumulation and potential for human exposure.

Re-evaluation of thyroid hormone signaling antagonism of tetrabromobisphenol A for validating the T3-induced Xenopus metamorphosis assay.

We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone (TH) signaling disruption of chemicals. The present study aimed to validate the T3-induced Xenopus metamorphosis assay by re-evaluating the TH signaling antagonism of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. According to the assay we developed, Xenopus tadpoles at stage 52 were exposed to 10-500nmol/L TBBPA in the presence of 1nmol/L T3. After 96hr of exposure, TBBPA in the range of 10-500nmol/L was found to significantly inhibit T3-induced morphological changes of Xenopus tadpoles in a concentration-dependent manner in term of body weight and four morphological endpoints including head area (HA), mouth width (MW), unilateral brain width/brain length (ULBW/BL), and hind-limb length/snout-vent length (HLL/SVL). The results show that these endpoints we developed are sensitive for characterizing the antagonistic effects of TBBPA on T3-induced metamorphosis. Following a 24-hr exposure, we found that TBBPA antagonized expression of T3-induced TH-response genes in the tail, which is consistent with previous findings in the intestine. We propose that the tail can be used as an alternative tissue to the intestine for examining molecular endpoints for evaluating TH signaling disruption. In conclusion, our results demonstrate that the T3-induced Xenopus metamorphosis assay we developed is an ideal in vivo assay for detecting TH signaling disruption.

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.

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.