Integrated Studies on Male Reproductive Toxicity of Bis(2-ethylhexyl)-tetrabromophthalate: in Silico, in Vitro, ex Vivo, and in Vivo.

Bis(2-ethylhexyl)tetrabromophthalate (TBPH) has been widely detected in the environment and organisms; thus, its toxic effects on male reproduction were systematically studied. First, we found that TBPH can stably bind to the androgen receptor (AR) based on in silico molecular docking results and observed an antagonistic activity, but not agonistic activity, on the AR signaling pathway using a constructed AR-GRIP1 yeast assay. Subsequently, we validated the adverse effects on male germ cells by observing inhibited androgen production and proliferation in Leydig cells upon in vitro exposure and affected general motility and motive tracks of zebrafish sperm upon ex vivo exposure. Finally, the in vivo reproductive toxicity was demonstrated in male zebrafish by reduced mating behavior in F0 generation when paired with unexposed females and abnormal development of their offspring. In addition, reduced sperm motility and impaired germ cells in male zebrafish were also observed, which may be related to the disturbed homeostasis of sex hormones. Notably, the specifically suppressed AR in the brain provides further evidence for the antagonistic effects as above-mentioned. These results confirmed that TBPH affected male reproduction through a classical nuclear receptor-mediated pathway, which would be helpful for assessing the ecological and health risks of TBPH.

Mechanistic Insights into 1,2-bis(2,4,6-tribromophenoxy)ethane-Induced Male Reproductive Toxicity in Zebrafish.

The novel brominated flame retardant, 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), has increasingly been detected in environmental and biota samples. However, limited information is available regarding its toxicity, especially at environmentally relevant concentrations. In the present study, adult male zebrafish were exposed to varying concentrations of BTBPE (0, 0.01, 0.1, 1, and 10 µg/L) for 28 days. The results demonstrated underperformance in mating behavior and reproductive success of male zebrafish when paired with unexposed females. Additionally, a decline in sperm quality was confirmed in BTBPE-exposed male zebrafish, characterized by decreased total motility, decreased progressive motility, and increased morphological malformations. To elucidate the underlying mechanism, an integrated proteomic and phosphoproteomic analysis was performed, revealing a predominant impact on mitochondrial functions at the protein level and a universal response across different cellular compartments at the phosphorylation level. Ultrastructural damage, increased expression of apoptosis-inducing factor, and disordered respiratory chain confirmed the involvement of mitochondrial impairment in zebrafish testes. These findings not only provide valuable insights for future evaluations of the potential risks posed by BTBPE and similar chemicals but also underscore the need for further research into the impact of mitochondrial dysfunction on reproductive health.

Evaluation and Mechanistic Study of Transgenerational Neurotoxicity in Zebrafish upon Life Cycle Exposure to Decabromodiphenyl Ethane (DBDPE).

The novel brominated flame retardant decabromodiphenyl ethane (DBDPE) has become a ubiquitous emerging pollutant in the environment, which may evoke imperceptible effects in humans or wild animals. Hence in this study, zebrafish embryos were exposed to DBDPE (0, 0.1, 1, and 10 nM) until sexual maturity (F0), and F1 and F2 generations were cultured without further exposure to study the multi- and transgenerational toxicity and underlying mechanism. The growth showed sex-different changing profiles across three generations, and the social behavior confirmed transgenerational neurotoxicity in adult zebrafish upon life cycle exposure to DBDPE. Furthermore, maternal transfer of DBDPE was not detected, whereas parental transfer of neurotransmitters to zygotes was specifically disturbed in F1 and F2 offspring. A lack of changes in the F1 generation and opposite changing trends in the F0 and F2 generations were observed in a series of indicators for DNA damage, DNA methylation, and gene transcription. Taken together, life cycle exposure to DBDPE at environmentally relevant concentrations could induce transgenerational neurotoxicity in zebrafish. Our findings also highlighted potential impacts on wild gregarious fish, which would face higher risks from predators.

Evidence for Adverse Effects on Liver Development and Regeneration in Zebrafish by Decabromodiphenyl Ethane.

Decabromodiphenyl ethane (DBDPE), a ubiquitous emerging pollutant, could be enriched in the liver of organisms, but its effects and mechanisms on liver development and regeneration remain largely unknown. In the present study, we first investigated the adverse effects on liver development and found decreased area and intensity of fluorescence in transgenic zebrafish larvae exposed to DBDPE; further results in wild-type zebrafish larvae revealed a possible mechanism involving disturbed MAPK/Fox O signaling pathways and cell cycle arrest as indicated by decreased transcription of growth arrest and DNA-damage-inducible beta a (gadd45ba). Subsequently, an obstructed recovery process of liver tissue after partial hepatectomy was characterized by the changing profiles of ventral lobe-to-intestine ratio in transgenic female adults upon DBDPE exposure; further results confirmed the adverse effects on liver regeneration by the alterations of the hepatic somatic index and proliferating cell nuclear antigen expression in wild-type female adults and also pointed out a potential role of a disturbed signaling pathway involving cell cycles and glycerolipid metabolism. Our results not only provided novel evidence for the hepatotoxicity and underlying mechanism of DBDPE but also were indicative of subsequent ecological and health risk assessment.

Niclosamide Exposure at Environmentally Relevant Concentrations Efficaciously Inhibited the Growth and Disturbed the Liver-Gut Axis of Adult Male Zebrafish.

In the current study, adult male zebrafish fed a normal diet (ND) or high-fat diet (HFD) were exposed to niclosamide (NIC) at environmentally relevant concentrations to reveal the accumulation and distribution in different tissues and evaluate the effects on liver-gut axis. Chemical analysis indicated that the liver bore a greater burden of NIC compared with the brain and gonads in adult zebrafish, and the HFD-fed fish bore greater burden in their liver and brain than those ND-fed fish. The indications from body weight, growth rate, body mass index, micro-CT images, biochemical and pathological changes confirmed that NIC can efficaciously curb weight gain and improve overloads of in plasma insulin and glucose in HFD-fed zebrafish. However, the potential effects on liver-gut axis in ND-fed zebrafish were also elucidated: NIC disturbed mitochondrial energy production, inhibited the glycemic and triacylglycerol biosynthesis but promoted triacylglycerol and free fatty acid catabolism, therefore reduced lipid accumulation in hepatocytes; NIC also impaired the physical barrier, evoked inflammatory and oxidative stress and led to microbiota dysbiosis in the intestine. There findings highlighted the necessity for evaluating its potential impacts on the health of wild animals as well as human beings upon long-term exposure.

Decabromodiphenyl ethane induced hyperactivity in developing zebrafish at environmentally relevant concentrations.

Decabromodiphenyl ethane (DBDPE), a widely used novel brominated flame retardant, is gaining concerns due to rapidly increased contents in various environmental and biota samples. In the present study, zebrafish (Danio rerio) embryos were exposed to 2.91, 9.71, 29.14 and 97.12 µg/L of DBDPE until 120 h post-fertilization (hpf) to investigate the potential developmental neurotoxicity and underlying mechanisms. Chemical analysis revealed concentration-dependently increased body burdens of DBDPE in zebrafish larvae, with bioaccumulation factors (BCFs) ranging from 414 to 726. Embryonic exposure to DBDPE caused hyperactivity without affecting the development of secondary motoneuron axons and muscle fibers. However, further results implicated that DBDPE may affect the locomotor regulatory network via different mechanisms at lower and higher concentrations. On the one hand, embryonic exposure to 2.91 µg/L DBDPE transiently promoted spontaneous coiling contractions, but showed no effects on touch-response and swimming activity in zebrafish larvae. The whole-body contents of neurotransmitters were significantly decreased. Significant decreased protein abundances of α1-TUBULIN and SYN2a and molecular docking results pointed out possible interactions of DBDPE with these two proteins. However, these changes may be unconcerned with the transient hyperactivity, and the exact molecular mechanisms need further investigation. On the other hand, 29.14 and 97.12 µg/L DBDPE exposure caused longer-lasting effects in promoting spontaneous coiling contractions, and also touch-response and swimming activity. At the same time, increased ACh contents (without changes of other neurotransmitters) and ChAT activity and inhibited transcription of nAChRs were observed at higher concentrations. Molecular docking indicated direct interaction of DBDPE with ChAT. The results suggested that DBDPE induced hyperactivity at higher concentrations was probably involved with disrupted cholinergic system, with ChAT as a potential target. Given that the body burden of DBDPE in lower concentration group was comparable with those detected in wild fish, the current results may provide useful information for ecological risk assessment.

Effects of SiO2 nanoparticles on the uptake of tetrabromobisphenol A and its impact on the thyroid endocrine system in zebrafish larvae.

The coexistence of nanoparticles and organic toxicants in the environment modifies pollutant bioavailability and toxicity. This study investigated the influence of silicon dioxide nanoparticles (n-SiO2) on the uptake of tetrabromobisphenol A (TBBPA) and its impact on the thyroid endocrine system in zebrafish larvae. Zebrafish (Danio rerio) embryos were exposed to TBBPA at different concentrations (50, 100, and 200 µg/L) alone or in combination with n-SiO2 (25 mg/L) until 120 h post-fertilization (hpf). Chemical measurements showed that both TBBPA and n-SiO2 were bioconcentrated in zebrafish larvae, and the uptake of TBBPA was enhanced by n-SiO2. Furthermore, zebrafish larvae exposed to 200 µg/L TBBPA alone exhibited significantly increased T4 contents and decreased T3 contents, whereas n-SiO2 treatment alone did not have a detectable effect. Furthermore, the thyroid hormone levels changed more upon treatment with 200 µg/L TBBPA combined with 25 mg/L n-SiO2 than upon TBBPA treatment alone. Alterations in gene transcription along the related hypothalamic-pituitary-thyroid (HPT) axis were observed, and expression of the binding and transport protein transthyretin (TTR) was significantly decreased for both TBBPA alone and co-exposure with n-SiO2. Thus, the current study demonstrates that n-SiO2, even at the nontoxic concentrations, increases thyroid hormone disruption in zebrafish larvae co-exposed to TBBPA by promoting its bioaccumulation and bioavailability.

Dysregulation of Intestinal Health by Environmental Pollutants: Involvement of the Estrogen Receptor and Aryl Hydrocarbon Receptor.

To determine how environmental pollutants induce dysbiosis of the gut microbiota, we exposed adult zebrafish to model pollutants with varied modes of action (atrazine, estradiol, polychlorinated biphenyl [PCB]126, and PCB153) for 7 days. Subsequently, metagenomic sequencing of the intestines was performed to compare the gut microbiomes among the groups. We observed clear compound- and sex-specific responses to xenobiotic stress. Principal component analysis revealed involvement of the aryl hydrocarbon receptor (AhR) and, to a lesser extent, the estrogen receptor (ER) in the dysregulation of the intestinal microbiota. The model pollutants differentially impaired intestinal and hepatic physiological activities, as indicated by assessments of gut motility, epithelial permeability, inflammation, and oxidative stress. Correlation analysis showed that abnormal Aeromonas reproduction, especially in the PCB126 groups, was significantly positively associated with oxidative damage. Aeromonas closely interacted with Mannheimia and Blastococcus to regulate intestinal permeability. In summary, we demonstrated that ER and AhR signaling regulated the dynamics of the gut microbiota. Our findings provide new mechanistic insight into the complex interactions between the host metabolism and gut microbiota, which may contribute to the grouped assessment of environmental pollutants in future.

Endocrine disruption in Chinese rare minnow (Gobiocypris rarus) after long-term exposure to low environmental concentrations of progestin megestrol acetate.

Synthetic progestins are widely used pharmaceutical agents that have become common contaminants in the aquatic environment. The potential adverse effects of long-term exposure on aquatic wildlife, however, are not fully understood. The aim of this study was to investigate the endocrine disruption in Chinese rare minnow (Gobiocypris rarus) in response to megestrol acetate (MTA) exposure. Newly-hatched Chinese rare minnow larvae were exposed to MTA at a nominal concentration of either 1 ng/L (detected concentrations ranged from 0.18 to 0.93 ng/L) or 10 ng/L (detected concentrations ranged from 4.27 to 9.64 ng/L) for 6 months and the effects on growth, sex steroid hormones, gonadal histology, and steroidogenic genes expression were determined. After 6 months of exposure to a nominal concentration of 10 ng/L MTA, the body weight and condition factors were significantly increased in fish of both sexes. Exposure to a nominal concentration of 10 ng/L MTA significantly reduced plasma concentrations of estradiol and 11-ketotestosterone in female fish while also reducing testosterone and 11-ketotestosterone in male fish. Gonad histology revealed significantly reduced proportions of vitellogenic oocytes in female fish exposed to a nominal concentration of 10 ng/L MTA and induction of atretic follicles in female fish exposed to both nominal concentrations of MTA. The expression of cyp19a1a and cyp17a1 in the gonads was up-regulated in the ovaries while down-regulated in the testes. Our results indicate that MTA can induce endocrine disruption in Chinese rare minnow at the low concentrations found in contaminated environments. This indicates a potentially high ecological risk from MTA to fish populations in MTA-contaminated aquatic environments in China and may also in other regions.

Enhanced Bioconcentration of Bisphenol A in the Presence of Nano-TiO2 Can Lead to Adverse Reproductive Outcomes in Zebrafish.

Titanium dioxide nanoparticles (n-TiO2) and bisphenol A (BPA) are widespread environmental contaminants in the aquatic environment. We hypothesized that n-TiO2 may adsorb BPA, and thus modify its bioavailability and toxicity to aquatic organisms. In this study, the bioavailability and toxicity of BPA (0, 2, 20, 200 µg/L) was investigated in the presence of n-TiO2 (100 µg/L). The n-TiO2 sorbed BPA and the resulting nanoparticles were taken up by zebrafish, where they translocated to the liver, brain, and gonad tissues. Increased tissue burdens of both BPA and n-TiO2 were observed following coexposure, and they also caused a reduction in plasma concentrations of estradiol (E2), testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Plasma vitellogenin (VTG) concentrations were significantly increased in males and females upon exposure to BPA. Histological examination of the ovary and testes did not show obvious morphological alterations; however, inhibition of egg production was noted in the presence of n-TiO2. The results indicated that n-TiO2 acts as a carrier of BPA and enhances its bioconcentration in zebrafish, leading to endocrine disruption and impairment of reproduction.

Interactions between intestinal microbiota and metabolites in zebrafish larvae exposed to polystyrene nanoplastics: Implications for intestinal health and glycolipid metabolism.

Previous studies have shown the harmful effects of nanoscale particles on the intestinal tracts of organisms. However, the specific mechanisms remain unclear. Our present study focused on examining the uptake and distribution of polystyrene nanoplastics (PS-NPs) in zebrafish larvae, as well as its toxic effects on the intestine. It was found that PS-NPs, marked with red fluorescence, primarily accumulated in the intestine section. Subsequently, zebrafish larvae were exposed to normal PS-NPs (0.2-25 mg/L) over a critical 10-day period for intestinal development. Histopathological analysis demonstrated that PS-NPs caused structural changes in the intestine, resulting in inflammation and oxidative stress. Additionally, PS-NPs disrupted the composition of the intestinal microbiota, leading to alterations in the abundance of bacterial genera such as Pseudomonas and Aeromonas, which are associated with intestinal inflammation. Metabolomics analysis showed alterations in metabolites that are primarily involved in glycolipid metabolism. Furthermore, MetOrigin analysis showed a significant correlation between bacterial flora (Pedobacter and Bacillus) and metabolites (D-Glycerate 2-phosphate and D-Glyceraldehyde 3-phosphate), which are related to the glycolysis/gluconeogenesis pathways. These findings were further validated through alterations in multiple biomarkers at various levels. Collectively, our data suggest that PS-NPs may impair the intestinal health, disrupt the intestinal microbiota, and subsequently cause metabolic disorders.

Behavioral impairments and disrupted mitochondrial energy metabolism induced by polypropylene microplastics in zebrafish larvae.

Polypropylene microplastics (PP-MPs) are emerging pollutant commonly detected in various environmental matrices and organisms, while their adverse effects and mechanisms are not well known. Here, zebrafish embryos were exposed to environmentally relevant concentrations of PP-MPs (0.08-50 mg/L) from 2 h post-fertilization (hpf) until 120 hpf. The results showed that the body weight was increased at 2 mg/L, heart rate was reduced at 0.08 and 10 mg/L, and behaviors were impaired at 0.4, 10 or 50 mg/L. Subsequently, transcriptomic analysis in the 0.4 and 50 mg/L PP-MPs treatment groups indicated potential inhibition on the glycolysis/gluconeogenesis and oxidative phosphorylation pathways. These findings were validated through alterations in multiple biomarkers related to glucose metabolism. Moreover, abnormal mitochondrial ultrastructures were observed in the intestine and liver in 0.4 and 50 mg/L PP-MPs treatment groups, accompanied by significant decreases in the activities of four mitochondrial electron transport chain complexes and ATP contents. Oxidative stress was also induced, as indicated by significantly increased ROS levels and significant reduced activities of CAT and SOD and GSH contents. All the results suggested that environmentally relevant concentrations of PP-MPs could induce disrupted mitochondrial energy metabolism in zebrafish, which may be associated with the observed behavioral impairments. This study will provide novel insights into PP-MPs-induced adverse effects and highlight need for further research.

DE-71 affected the cholinergic system and locomotor activity via disrupting calcium homeostasis in zebrafish larvae.

Polybrominated diphenyl ethers (PBDEs) can induce neurotoxicity, but the mechanism of their toxicity on the cholinergic system and locomotion behavior remains unclear. In this paper, zebrafish embryos were exposed to DE-71 (0, 1, 3, 10, 30, and 100 µg/L) until 120 h post fertilization, and its effects on the behavior and cholinergic system of zebrafish larvae and its possible mechanism were investigated. Results indicated a general locomotor activity impairment in the light-dark transition stimulation without affecting the secondary motoneurons. However, with the extension of test time in the dark or light, the decreased locomotor activity was diminished, a significant decrease only observed in the 100 µg/L DE-71 exposure groups in the last 10 min. Furthermore, whole-body acetylcholine (ACh) contents decreased after DE-71 exposure, whereas no changes in NO contents and inducible nitric oxide synthase activity were found. The expression of certain genes encoding calcium homeostasis proteins (e.g., grin1a, camk2a, and crebbpb) and the concentrations of calcium in zebrafish larvae were significantly decreased after DE-71 exposure. After co-exposure with calcium channel agonist (±)-BAY K8644, calcium concentrations, ACh contents, and locomotor activity in the light-dark transition stimulation was significantly increased compared with the same concentrations of DE-71 exposure alone, whereas no significant difference was observed compared with the control, indicating that calcium homeostasis is involved in the impairment of cholinergic neurotransmission and locomotor activity. Overall, our results suggested that DE-71 can impair the cholinergic system and locomotor activity by impairing calcium homeostasis. Our paper provides a better understanding of the neurotoxicity of PBDEs.

Endocrine disrupting effects induced by levonorgestrel linked to altered DNA methylation in rare minnow (Gobiocypris rarus).

Progestins are worldwide environmental contaminants, however, their ecotoxicological risks and underlying molecular mechanisms of effects are not fully understood. In this study, newly hatched rare minnow (Gobiocypris rarus) larvae were exposed to environmentally realistic concentrations (1 and 10 ng/L) of levonorgestrel (LNG) for 6 months. The sex ratios were not affected by LNG at both concentrations, but the growth was significantly inhibited at 10 ng/L while promoted at 1 ng/L. Histological analysis revealed impaired gonadal development. Plasma concentrations of estradiol in females and testosterone in both sexes were significantly induced after exposure to 1 ng/L LNG; plasma concentrations of 11-ketotestosterone were markedly increased in females exposed to 10 ng/L LNG and in males exposed to both concentrations of LNG. The transcription of cyp19a1a was significantly up-regulated in ovaries exposed to LNG at both concentrations, while cyp17a1 was down-regulated in testes exposed to 10 ng/L LNG. The global DNA methylation level was significantly decreased in testes exposed to 10 ng/L LNG, which might be associated with inhibited spermatogenesis. Gender-specific changes in CpG methylation patterns were induced by LNG in the 5' flanking region of cyp19a1a, with hypomethylation in ovaries but hypermethylation in testes, which was linked to the regulation of cyp19a1a transcription. The results suggest that LNG could induce endocrine disrupting effects in fish at environmentally realistic concentrations, which may be linked to altered DNA methylation. This study indicates potentially high ecological risk of LNG to fish populations, and warrants researches on regulatory mechanisms of epigenetic modifications in progestin-induced effects.

Neurotoxicity of tetrabromobisphenol A and SiO2 nanoparticle co-exposure in zebrafish and barrier function of the embryonic chorion.

Silicon dioxide nanoparticles (n-SiO2) absorb tetrabromobisphenol A (TBBPA) and modify its bioavailability and toxicity in the aquatic phase; embryonic chorion is an efficient barrier against nanoparticles (e.g., SiO2) and influences their toxicity. However, few studies have investigated developmental neurotoxicity in fish after co-exposure to TBBPA and n-SiO2, especially considering the barrier function of the chorion. In the present study, zebrafish embryos were exposed to TBBPA (50, 100, and 200 µg/L) alone or in combination with n-SiO2 (25 mg/L) until 24 or 120 h post fertilization (hpf), in the presence and absence of the chorion. The results confirmed that TBBPA exposure alone significantly downregulated the expression of neurodevelopment marker genes (mbp, alpha-tubulin, shha, and gfap), altered acetylcholinesterase activity and acetylcholine content, and affected locomotor behavior at different developmental stages. Moreover, the results indicated that n-SiO2 promoted TBBPA-induced neurotoxic effects in zebrafish larvae at 120 hpf, including further repression of the transcription of CNS-related genes, disruption of the cholinergic system, and decrease in the average swimming speed under dark/light stimulation. However, scanning electron microscopy/energy dispersive spectroscopy analysis revealed that at 24 hpf, the embryonic chorion efficiently blocked n-SiO2 and consequently decreased the bioaccumulation of TBBPA and TBBPA-induced neurotoxicity in dechorionated zebrafish embryos. Taken together, the results demonstrate that n-SiO2 affected the bioavailability and neurodevelopmental toxicity of TBBPA, and their combined toxicity to zebrafish embryos was mitigated by embryonic chorion, which will facilitate risk assessment on n-SiO2 and TBBPA and improve understanding the function of the fish embryonic chorion.

Parental co-exposure to bisphenol A and nano-TiO2 causes thyroid endocrine disruption and developmental neurotoxicity in zebrafish offspring.

The coexistence of organic toxicants and nanoparticles in the environment influences pollutant bioavailability and toxicity. Using chronic co-exposure to an adult zebrafish model, this study investigated the transfer kinetics and transgenerational effects of bisphenol A (BPA) and titanium dioxide nanoparticles (n-TiO2) exposure in F1 offspring. When single and combined exposure to BPA (0, 2, and 20 µg/L) and n-TiO2 (100 µg/L) were compared, combined exposure was found to reciprocally facilitate bioaccumulation in adult fish while enhancing maternal transfer to offspring. Thyroid endocrine disruption and developmental neurotoxicity were observed in larval offspring by parental exposure to BPA alone or in combination with n-TiO2. Exposure to 20 µg/L BPA significantly decreased the thyroxine (T4) concentration in adult plasma, leading to less transfer into the eggs. The presence of 20 µg/L BPA with n-TiO2 further decreased the level of T4 compared to BPA exposure alone. Additionally, offspring larvae derived from exposed parents exhibited lethargic swimming behavior. Overall, this study examined the interactions of BPA and n-TiO2 with regard to their bioaccumulation, maternal transfer, and developmental effects, which highlighted that co-exposure dynamics are important and need to be considered for accurate environmental risk assessment.

Acute exposure to PBDEs at an environmentally realistic concentration causes abrupt changes in the gut microbiota and host health of zebrafish.

Contamination from lower brominated PBDEs is ubiquitous in the environments. However, their effects on gut microbiota and intestinal health have not yet been investigated. This study exposed adult zebrafish to an environmentally realistic concentration of pentaBDE mixture (DE-71) at 5.0 ng/L for 7 days, after which metagenomic sequencing of the intestinal microbiome was conducted and host physiological activities in the intestine and liver were also examined. The results showed that acute exposure to DE-71 significantly shifted the gut microbial community in a sex-specific manner. Certain genera (e.g., Mycoplasma, Ruminiclostridium, unclassified Firmicutes sensu stricto, and Fusobacterium) disappeared from the DE-71-exposed intestines, resulting in decreased bacterial diversity. Bacterial metabolic functions in guts were also affected by DE-71, namely those covering energy metabolism, virulence, respiration, cell division, cell signaling, and stress response. In addition, measurement of diverse sensitive biomarkers showed that the health of male intestines was remarkably compromised by the DE-71 exposure, as indicated by the disruption to its neural signaling (serotonin), epithelial barrier integrity (tight junction protein 2), inflammatory response (interleukin 1ß), oxidative stress and antioxidant capacity, as well as detoxifying potential (ethoxyresorufin-O-deethylase activity). However, female intestines maintained intact physiological activities. Compared to the direct impact on intestines, a latent effect of DE-71 was observed in livers. Co-occurrence network analysis demonstrated that the gut bacteria vigorously interacted to establish the fittest community under DE-71 stress by promoting the reproduction of favorable genera, while diminishing the survival of unfavorable ones. Significant correlations between the zebrafish gut microbiota and physiological activities (e.g., oxidative stress, detoxification, neurotransmission, and epithelial integrity) were also observed. Overall, this study has demonstrated, for the first time, the high susceptibility of gut microbiota and intestinal health of zebrafish to DE-71, thus warranting more work to reveal its mode of toxicity.

The binary mixtures of megestrol acetate and 17α-ethynylestradiol adversely affect zebrafish reproduction.

Synthetic progesterones and estrogens are broadly used bioactive pharmaceutical agents and have been detected in aquatic environments. In the present study, we investigated the combined reproductive effects of megestrol acetate (MTA) and 17α-ethinylestradiol (EE2) on zebrafish. Adult zebrafish were exposed to MTA (33, 100 or 333 ng/L), EE2 (10 ng/L) or a mixture of both (MTA + EE2: 33 + 10, 100 + 10 or 333 + 10 ng/L) for 21 days. Results demonstrated that egg production was significantly reduced by exposure to 10 ng/L EE2, but not MTA. However, a combined exposure to MTA and EE2 caused further reduction of fish fecundity compared to EE2 exposure alone, suggesting an additive effect on egg production when EE2 is supplemented with MTA. Plasma concentrations of 17ß-estradiol and testosterone in the females and 11-ketotestosterone in the males were significantly decreased in the groups exposed to EE2 or MTA alone compared with the solvent control, and the plasma concentrations of the three hormones were further reduced in the co-exposure groups relative to the MTA exposure group, but not the EE2 exposure group. These data indicate that the inhibitory effects on plasma concentrations in the co-exposures were predominantly caused by EE2. Furthermore, exposure to MTA and EE2 (alone or in combination) led to histological alterations in the ovaries (decreased vitellogenic/mature oocytes), but not in the testes. This study has important implications for environmental risk assessment of synthetic hormones that are concurrently present in aquatic systems.

The progestin levonorgestrel affects sex differentiation in zebrafish at environmentally relevant concentrations.

Synthetic progestins have become widespread environmental contaminants and may cause adverse effects on fish. In the present study, we investigated the effects of levonorgestrel (LNG) on sex differentiation in zebrafish (Danio rerio). Embryos were exposed to LNG at environmentally relevant concentrations (0, 1, 10, 33, and 100ng/L) and allowed to develop until sexual maturity. Histological examination at 63 days post fertilization (dpf) caused complete sex reversal and 100% males were observed in the 10, 33 and 100ng/L treatments; gross morphological and histological examination of gonads at 142dpf further confirmed 100% males at these exposure concentrations. The results indicate androgenic activity of LNG, and masculinization during zebrafish gonadal differentiation. The mRNA expression levels of genes involved in fish sex differentiation and gonadal development were examined at 28 and 42dpf. Down-regulation of the mRNA expression of aromatase (e.g., cyp19a1a, cyp19a1b), the forkhead transcription factor gene L2 (foxl2) and the Fushi tarazu factor-1d (nr5a1b) were observed. In contrast, transcription of the doublesex and mab-3-related transcription factor 1 (dmrt1) gene was up-regulated. Androgen receptor (ar) mRNA expression was significantly down-regulated at 28 and 42dpf. Co-exposure to flutamide (an androgen antagonist) and LNG, led to a decrease in the sex inversion potency of LNG. Our study has demonstrated that environmentally relevant concentrations of LNG could alter sex differentiation and gonadal development in zebrafish. Our results also suggest a potentially high ecological risk of LNG to fish populations in LNG-contaminated aquatic environments.