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.

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.

Developmental toxicity of TCBPA on the nervous and cardiovascular systems of zebrafish (Danio rerio): A combination of transcriptomic and metabolomics.

Tetrachlorobisphenol A (TCBPA), a widely used halogenated flame retardant, is frequently detected in environmental compartments and human samples. However, unknown developmental toxicity and mechanisms limit the entire understanding of its effects. In this study, zebrafish (Danio rerio) embryos were exposed to various concentrations of TCBPA while a combination of transcriptomics, behavioral and biochemical analyzes as well as metabolomics were applied to decipher its toxic effects and the potential mechanisms. We found that TCBPA could interfere with nervous and cardiovascular development through focal adhesion and extracellular matrix-receptor (ECM-receptor) interaction pathways through transcriptomic analysis. Behavioral and biochemical analysis results indicated abnormal swimming behavior of zebrafish larvae. Morphological observations revealed that TCBPA could cause the loss of head blood vessels. Metabolomic analysis showed that arginine-related metabolic pathways were one of the main pathways leading to TCBPA developmental toxicity. Our study demonstrated that by using omics, TCBPA was shown to have neurological and cardiovascular developmental toxicity and the underlying mechanisms were uncovered and major pathways identified.

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.

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.

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.

The perturbation of parabens on the neuroendocrine system in zebrafish larvae.

Parabens, as the synthetic preservatives, have caused universal environmental contamination and human exposure. Whether parabens could disturb neuroendocrine system was still ambiguous. In this study, the effects of four commonly-used parabens, i.e. methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP), were tested on the neuroendocrine system of zebrafish larvae by investigating the swimming behavior, the related hormones and biomarkers in the hypothalamic-pituitary-interrenal (HPI) axis. The results showed that all test chemicals significantly reduced the swimming distance and mean velocity of zebrafish larvae. The adrenocorticotropic hormone (ACTH) levels in zebrafish larvae were significantly increased, while the cortisol levels were obviously decreased by paraben exposure. The transcriptional analysis showed that the expressions of the target genes including gr, mr and crhr2 in the HPI axis were mostly down-regulated. The exploration of the initial molecular event showed that parabens could bind with the glucocorticoid receptor (GR) and trigger its transactivation, according to MDA-kb2 luciferase assay and molecular docking analysis. The interaction of parabens with the GR included the hydrogen bond and hydrophobic interaction. The findings herein revealed the potential deleterious effects of parabens on the neuroendocrine system of zebrafish larvae, thus accumulating the in vivo toxicological data on this kind of food preservatives.

Zebrafish larvae acute toxicity test: A promising alternative to the fish acute toxicity test.

Aquatic toxicity is a mandatory component in risk assessment of chemicals. The currently recommended used acute fish toxicity (AFT) test requires a large test system, bringing onerous experimental operation and discharge of much experimental wastewater. In this study, we established a more convenient and efficient test defined as the zebrafish larvae acute toxicity (FLT) test, which employed zebrafish larvae at four days post fertilization as the test organisms and implemented a 48-hour exposure in 6-well plates. Based on validated reproducibility, we applied this test to evaluate the acute toxicity of 35 chemicals. By comparing the results with the existing acute toxicity data reported in the literature, we found that most chemicals exhibited highly positive correlated LC50 in the FLT and the AFT test, with the same or similar toxicity grade. The FLT test showed more comparable sensitivity with the current AFT test than the previously recommended fish embryo acute toxicity test (FET). Moreover, the FLT test is easier to implement than the FET test which requires microscopic observation to identify the fertilization and development status of the embryos. Despite a limitation similar to the FET test in terms of detecting neurotoxicants, the FLT test could be a more promising alternative to the AFT test relative to the FET test.

Bisphenol chemicals disturb intestinal homeostasis via Notch/Wnt signaling and induce mucosal barrier dysregulation and inflammation.

Emerging evidence has shown that bisphenol A (BPA) can exert adverse effects on intestinal barrier in rodents, but little is known about its underlying mechanisms. We previously found BPA and its substitute bisphenol F (BPF) disrupted Notch signaling and altered intestinal histological structures in Xenopus laevis tadpoles. The present study aimed to determine whether BPA and BPF could affect intestinal homeostasis via Notch/Wnt signaling and induce intestinal barrier dysregulation in adult mammals, given the fundamental roles of the two conserved signaling pathways in intestinal homeostasis and regulation of intestinal barrier. We found that following 7-day administration with BPA or BPF through drinking water at the reference dose of 50 µg/kg/d and no observed adverse effect level of 5 mg/kg/d (NOAEL) of BPA, adult male mice displayed no alterations at histological and cellular levels in colons, but high dose of both BPA and BPF downregulated the expression of Notch- and Wnt-related genes as well as key genes responsible for intestinal homeostasis. When administration was extended to 14 days, all treatments significantly suppressed the expression of all tested Notch- and Wnt-related genes; correspondingly, administrated colons exhibited downregulated expression of key genes responsible for intestinal homeostasis and reduced cell proliferation in crypts. Importantly, all treatments suppressed secretory cell differentiation, reduced mucin protein levels and downregulated expression of tight junction markers, implicating mucosal barrier dysregulation. Furthermore, inflammatory cell infiltration and upregulated expression of inflammatory cytokine genes in colons, coupled with increased serum inflammatory cytokine levels, were observed in all treatments. All results show that both BPA and BPF at the reference dose disrupted Notch/Wnt signaling and intestinal homeostasis, thereby leading to mucosal barrier dysregulation and intestinal inflammation in mice. This is the first study revealing the adverse influences of BPF on mammal intestines and underlying mechanisms for bisphenol-caused intestinal injury.