Emerging impacts of ionic liquids on eco-environmental safety and human health.

Owing to their unique physicochemical properties, ionic liquids (ILs) have been rapidly applied in diverse areas, such as organic synthesis, electrochemistry, analytical chemistry, functional materials, pharmaceutics, and biomedicine. The increase in the production and application of ILs has resulted in their release into aquatic and terrestrial environments. Because of their low vapor pressure, ILs cause very little pollution in the atmosphere compared to organic solvents. However, ILs are highly persistent in aquatic and terrestrial environments due to their stability, and therefore, potentially threaten the safety of eco-environments and human health. Specifically, the environmental translocation and retention of ILs, or their accumulation in organisms, are all related to their physiochemical properties, such as hydrophobicity. Based on results of ecotoxicity, cytotoxicity, and toxicity in mammalian models, the mechanisms involved in IL-induced toxicity include damage of cell membranes and induction of oxidative stress. Recently, artificial intelligence and machine learning techniques have been used in mining and modeling toxicity data to make meaningful predictions. Major future challenges are also discussed. This review will accelerate our understanding of the safety issues of ILs and serve as a guideline for the design of the next generation of ILs.

Long-term exposure to bisphenol S damages the visual system and reduces the tracking capability of male zebrafish (Danio rerio).

Bisphenol S (BPS) is widely detected in aquatic environments and in human bodies. BPS has reproductive and thyroid disrupting effects, but its effect on the visual system remains unknown. In the present study, zebrafish embryos were exposed to BPS at concentrations of 1, 10, 100 and 1000 µg l-1 until 120 days post-fertilization in a semistatic system, and the effect of BPS on the visual behavior was examined using the optokinetic response and the optomotor response tests in male zebrafish. The retinal histology, mRNA expression of photoreceptor opsin genes (zfrho, zfblue, zfgr1, zfred and zfuv) and apoptosis-related genes (bax and bcl-2) were also assessed. Long-term BPS exposure decreased the tracking capability of male zebrafish, consistent with structural damage to the retina. BPS induced different amounts of vacuoles in the retinal pigment epithelium, and 1000 µg l-1 BPS exposure decreased the length of the inner plexiform layer, ganglion cell layer and retina, and induced an irregular arrangement of photoreceptor cells. The expression levels of the opsin genes (zfred, zfgr1 and zfrho) were significantly elevated, indicating an enhanced spectral sensitivity to red, green and dim light to compensate for the reduction of the optomotor response. Together, the results showed for the first time that long-term exposure to BPS damaged the structure of male zebrafish retina and reduced their tracking capability.

Bisphenol S exposure impairs glucose homeostasis in male zebrafish (Danio rerio).

Bisphenol S (BPS) is a substitute of the plastic additive bisphenol A (BPA). Its concentrations detected in surface waters and urine samples are on the same order of magnitude as BPA. Human exposure to BPA has been implicated in the development of diabetes mellitus; however, whether BPS can disrupt glucose homeostasis and increase blood glucose concentration remains unclear. We extensively investigated the effects of environmentally relevant concentrations of BPS on glucose metabolism in male zebrafish (Danio rerio) and the underlying mechanisms of these effects. Male zebrafish were exposed to 1, 10, or 100µg/L of BPS for 28 d. Fasting blood glucose (FBG) levels, glycogen levels in the liver and muscle, and mRNA levels of key glucose metabolic enzymes and the activities of the encoded proteins in tissues were evaluated to assess the effect of BPS on glucose metabolism. Plasma insulin levels and expression of preproinsulin and glucagon genes in the visceral tissue were also evaluated. Compared with the control group, exposure to 1 and 10µg/L of BPS significantly increased FBG levels but decreased insulin levels. Gluconeogenesis and glycogenolysis in the liver were promoted, and glycogen synthesis in the liver and muscle and glycolysis in the muscle were inhibited. Exposure to 100µg/L of BPS did not significantly alter plasma insulin and blood glucose levels, but nonetheless pronouncedly interfered with gluconeogenesis, glycogenolysis, glycolysis, and glycogen synthesis. Our data indicates that BPS at environmentally relevant concentrations impairs glucose homeostasis of male zebrafish possibly by hampering the physiological effect of insulin; higher BPS doses also pronouncedly interfered with glucose metabolism.

Impairment of bisphenol F on the glucose metabolism of zebrafish larvae.

Bisphenol F (BPF) is a substitute of bisphenol A in the production of epoxy resin and polycarbonate. Its extensive use in consumer products leads to a wide human exposure at high levels. Although the adverse effects of BPF on animal health are of increasing public concern, its risks on systematic glucose metabolism and blood glucose concentrations still remain largely unknown. Using zebrafish larvae as the model animal, we investigated the disturbance of BPF exposure on glucose metabolism and the underlying mechanisms. Zebrafish larvae at 96 h post fertilization were exposed to 0.1, 1, 10, and 100 µg/L of BPF for 48 h. Compared with the control group, glucose levels of larvae increased significantly in the 10 and 100 µg/L exposure groups, which are associated with enhancement of gluconeogenesis and suppression of glycolysis induced by high doses of BPF. Additionally, both mRNA expressions and protein levels of insulin increased significantly in the 10 and 100 µg/L exposure groups, while transcription levels of genes encoding insulin receptor substrates decreased significantly in these groups, indicating a possibly decreased insulin sensitivity due to impairment of insulin signaling transduction downstream of insulin receptor. Further, compared with BPF alone, co-exposure of larvae to BPF and rosiglitazone, an insulin sensitizer, significantly attenuates increases in both glucose levels and mRNA expressions of a key gluconeogenesis enzyme. Our data therefore indicate impairing insulin signaling transduction may be the main mechanism through which BPF disrupts glucose metabolism and induces hyperglycemia. Results of the present study inform the health risk assessment of BPF and also suggest the use of zebrafish larvae in large-scale screening of chemicals with possible glucose metabolism disturbing effect.

Estrogenic effects associated with bisphenol a exposure in male zebrafish (Danio rerio) is associated with changes of endogenous 17ß-estradiol and gene specific DNA methylation levels.

The binding affinity of bisphenol A (BPA) to estrogen receptors (ERs) is much lower than that of 17ß-estradiol (E2), and whether there are other molecular mechanisms responsible for the estrogenic action of BPA in vivo currently remains unknown. The objective of this study was to explore the potential association between the estrogenic effect induced by bisphenol A in vivo and changes of endogenous E2 and gene specific DNA methylation levels. After a waterborne exposure of male zebrafish to 500, 1000, or 1500µg/L of BPA for 21d, vitellogenin (VTG) concentration in whole body homogenate, plasma E2 and testosterone levels, hepatic ERs mRNA expressions, gonadal cyp19a1a and cyp17a1 mRNA expressions, and methylation levels of hepatic esr1 and gonadal cyp19a1a's promoters were determined. Our results indicated that for the 500 and 1500µg/L treatment groups, VTG might be induced mainly by the elevated E2 levels; increases of E2 levels could be partly explained by the up-regulated expression of gonadal aromatase, mRNA levels of which were found to be negatively related to the methylation levels of both its promoter and one CpG site. In addition, upon BPA exposure, hepatic esr1 mRNA levels were also negatively related to the methylation levels of both its promoter and one CpG site. These observations provide evidence for the non-ERs mediated mechanisms underlying the estrogenic action of BPA on male zebrafish.

Effects of environmentally relevant concentrations of florfenicol on the glucose metabolism system, intestinal microbiome, and liver metabolome of zebrafish.

Florfenicol, a widely used veterinary antibiotic, has now been frequently detected in various water environments and human urines, with high concentrations. Accordingly, the ecological risks and health hazards of florfenicol are attracting increasing attention. In recent years, antibiotic exposure has been implicated in the disruption of animal glucose metabolism. However, the specific effects of florfenicol on the glucose metabolism system and the underlying mechanisms are largely unknown. Herein, zebrafish as an animal model were exposed to environmentally relevant concentrations of florfenicol for 28 days. Using biochemical and molecular analyses, we found that exposure to florfenicol disturbed glucose homeostasis, as evidenced by the abnormal levels of blood glucose and hepatic/muscular glycogen, and the altered expression of genes involved in glycogenolysis, gluconeogenesis, glycogenesis, and glycolysis. Considering the efficient antibacterial activity of florfenicol and the crucial role of intestinal flora in host glucose metabolism, we then analyzed changes in the gut microbiome and its key metabolite short-chain fatty acids (SCFAs). Results indicated that exposure to florfenicol caused gut microbiota dysbiosis, inhibited the production of intestinal SCFAs, and ultimately affected the downstream signaling pathways of SCFA involved in glucose metabolism. Moreover, non-targeted metabolomics revealed that arachidonic acid and linoleic acid metabolic pathways may be associated with insulin sensitivity changes in florfenicol-exposed livers. Overall, this study highlighted a crucial aspect of the environmental risks of florfenicol to both non-target organisms and humans, and presented novel insights into the mechanistic elucidation of metabolic toxicity of antibiotics.

Thyroid endocrine disruption induced by [C8mim]Br: An integrated in vivo, in vitro, and in silico study.

Conventional thyroid-disrupting chemicals (TDCs) such as polybrominated diphenyl ethers, polychlorinated biphenyls, and bisphenols perturb animal's thyroid endocrine system by mimicking the action of endogenous thyroid hormones (THs), since they share a similar backbone structure of coupled benzene rings with THs. 1-methyl-3-octylimidazolium bromide ([C8mim]Br), a commonly used ionic liquid (IL), has no structural similarity to THs. Whether it interferes with thyroid function and how its mode of action differs from conventional TDCs is largely unknown. Herein, zebrafish embryo-larvae experiments (in vivo), GH3 cell line studies (in vitro), and molecular simulation analyses (in silico) were carried out to explore the effect of [C8mim]Br on thyroid homeostasis and its underlying mechanism. Molecular docking results suggested that [C8mim]+ likely bound to retinoid X receptors (RXRs), which may compromise the formation of TH receptor/RXR heterodimers. This then perturbed the negative regulation of thyroid-stimulating hormone ß (tshß) transcription by T3 in GH3 cell line. The resulting enhancement of tshß expression further caused hyperthyroidism and developmental toxicity in larval zebrafish. These findings provided a crucial aspect of the ecological risks of ILs, and presented a new insight into the thyroid-disrupting mechanisms for emerging pollutants that do not have structural similarity to THs.

Safety assessment of organic micropollutants in reclaimed water: Chemical analyses, ecological risk assessments, and in vivo endocrine-disrupting studies.

Reclaimed water from municipal wastewater has great potential in mitigating the water resource crisis, while the inevitable residue of organic micropollutants (OMPs) challenges the safety of reclaimed water reuse. Limited information was available regarding the overall adverse effects of mixed OMPs in reclaimed water, especially the endocrine-disrupting effects on living organisms. Herein, chemical monitoring in two municipal wastewater treatment plants showed that 31 of 32 candidate OMPs including polycyclic aromatic hydrocarbons (PAHs), phenols, pharmaceuticals and personal care products (PPCPs) were detected in reclaimed water, with a concentration ranging from ng/L to µg/L. Then, based on the risk quotient value, phenol, bisphenol A, tetracycline, and carbamazepine were ranked as high ecological risks. Most PAHs and PPCPs were quantified as medium and low risks, respectively. More importantly, using aquatic vertebrate zebrafish as an in vivo model, the endocrine-disrupting potentials of OMP mixtures were comprehensively characterized. We found that a realistic exposure to reclaimed water induced estrogen-like endocrine disruption and hyperthyroidism in zebrafish, abnormal expression of genes along the hypothalamus-pituitary-thyroid (-gonad) axes, reproductive impairment, and transgenerational toxicity. Based on the chemical analyses, risk quotient calculations, and biotoxicity characterization, this study contributed to understanding the ecological risks of reclaimed water and developing the control standards for OMPs. In addition, application of the zebrafish model in this study also highlighted the significance of in vivo biotoxicity test in water quality evaluation.

Bisphenol AF exposure causes fasting hyperglycemia in zebrafish (Danio rerio) by interfering with glycometabolic networks.

Bisphenol AF (BPAF), one of the main alternatives to bisphenol A, has been frequently detected in various environmental media, including the human body, and is an emerging contaminant. Epidemiological investigations have recently shown the implications of exposure to BPAF in the incidence of diabetes mellitus in humans, indicating that BPAF may be a potential diabetogenic endocrine disruptor. However, the effects of BPAF exposure on glucose homeostasis and their underlying mechanisms in animals remain largely unknown, which may limit our understanding of the health risks of BPAF. To this end, zebrafish (Danio rerio), an emerging and valuable model in studying animal glycometabolism and diabetes, were exposed to environmentally relevant concentrations (5 and 50 µg/L) and 500 µg/L BPAF for 28 d. Several key toxicity endpoints of blood glucose metabolism were detected in our study, and the results showed significantly increased fasting blood glucose levels, hepatic glycogen contents and hepatosomatic indexes and decreased muscular glycogen contents in the BPAF-exposed zebrafish. The results of quantitative real-time PCR showed the abnormal expression of genes involved in glycometabolic networks, which might promote hepatic gluconeogenesis and inhibit glycogenesis and glycolysis in the muscle and/or liver. Furthermore, the failure of insulin regulation, including plasma insulin deficiency and impaired insulin signaling pathways in target tissues, may be a potential mechanism underlying BPAF-induced dysfunctional glycometabolism. In summary, our results provide novel in vivo evidence that BPAF can cause fasting hyperglycemia by interfering with glycometabolic networks, which emphasizes the potential health risks of environmental exposure to BPAF in inducing diabetes mellitus.

Long-term exposure of zebrafish to bisphenol S impairs stress function of hypothalamic-pituitary-interrenal axis and causes anxiety-like behavioral responses to novelty.

Bisphenol S (BPS), a main substitute of bisphenol A, has been reported to induce multiple endocrine disrupting effects on animals, however, whether it can interfere with the corticosteroid-endocrine system still remains unknown. Furthermore, previous studies mainly investigated the influences of environmental pollutants on corticosteroid levels and gene expressions of hypothalamic-pituitary-interrenal/adrenal (HPI/A) axis, while the downstream toxic effects caused thereafter have not yet been fully elucidated. Considering the key role of cortisol, a primary corticosteroid hormone in teleost, in mediating stress adaptation and the highly positive correlation between cortisol level and anxious phenotype in the novel environment, we hypothesized that an imbalanced cortisol homeostasis due to environmental pollutant exposure may further affect the behavioral responses to novelty stress. In the present study, zebrafish, a valuable model in studying human stress physiology and anxiety behavior, were exposed to BPS from embryos to adults (120 days) at environmentally relevant concentrations (1 and 10 µg/L) and 100 µg/L. Results found that long-term exposure to BPS increased whole-body cortisol levels and caused abnormal expressions of HPI axis genes. Moreover, the excessive cortisol levels may be due to the inhibition of cortisol catabolism and excretion, as evidenced by the down-regulated expressions of hydroxysteroid 11-beta dehydrogenase 2 and hydroxysteroid 20-beta dehydrogenase 2 genes. More importantly, as we speculated, excessive cortisol levels may be responsible for the occurrence of anxiety-like behavioral responses indicated by longer latency, fewer time spent in the upper half, and more erratic movements in a 6-min novel tank test. Overall, our study provides basic data for the comprehensive understanding of BPS toxicity, and emphasizes environmental health risks of BPS in inducing anxiety syndrome at environmentally realistic concentrations.

Long-term bisphenol S exposure induces fat accumulation in liver of adult male zebrafish (Danio rerio) and slows yolk lipid consumption in F1 offspring.

Bisphenol S (BPS), as a substitute for bisphenol A, was frequently detected in human urine and blood. It has been reported that BPS could disrupt fat metabolism in vivo and vitro although mechanisms remain unclear. Additionally, there is no study that the disruptive effect of BPS on parental fat metabolism indirectly interferes with the lipid metabolism of offspring. Here, after 120-d exposure to 1, 10, 100, and 1000 µg/L BPS, the transcription level of genes involved in lipid metabolism in liver and feeding regulation of brain-gut axis, as well as the hepatic triacylglycerol (TAG) and plasma lipid levels were investigated in both male and female zebrafish. Results showed that in male liver, fatty acid synthesis and degradation were inhibited by reducing transcription levels of srebp1 and pparα, and the synthesis of TAG was significantly increased using fatty acid as a precursor by elevating agpat4 and dgat2 mRNA expression levels. As a consequence, fat accumulation and the increased TAG levels were observed in male liver, and lipid levels were also elevated in male plasma. In female liver, there was no excessive fat accumulation and BPS exposure had a non-monotonic effect on the gene expression of fasn, dagt2, and pparα. Notably, the unexposed offspring showed a large amount of yolk lipid remain at 5 days post fertilization. This study obviously demonstrated that long-term BPS exposure increases the risk of non-alcoholic fatty liver disease in male zebrafish and life-cycle exposure hazard on offspring is noteworthy.

Transgenerational thyroid endocrine disruption induced by bisphenol S affects the early development of zebrafish offspring.

Maternal thyroid hormones (THs) play an essential role in the embryonic and larval development of fish. Previous studies in fish have reported that parental exposure to thyroid disrupting chemicals (TDCs) changed maternal TH levels in the offspring; however, whether this transgenerational thyroid endocrine disruption can further disturb the early development of the offspring still remains largely unknown. Bisphenol S (BPS), a substitute of bisphenol A, has been reported to be a potential TDC. In this study, zebrafish (F0) were exposed to environmentally relevant concentrations (1, 10, and 100 µg/L) of BPS from 2 h post-fertilization to 120 days post-fertilization and then paired to spawn. Plasma levels of thyroxine (T4) were significantly decreased in F0 females while 3,5,3'-triiodothyronine (T3) plasma levels were significantly increased in F0 females and males; moreover, TH content in eggs (F1) spawned by exposed F0 generation exhibited similar changes as the F0 females, with significant decreases in T4 and increases in T3, demonstrating BPS-induced maternal transfer of thyroid endocrine disruption. Further, excessive levels of maternal T3 in the offspring resulted in delayed embryonic development and hatching, swim bladder inflation defect, reduction in motility, developmental neurotoxicity, and lateral stripe hypopigmentation in non-exposed F1 embryos and larvae. These results highlight the adverse effects on the early development of offspring induced by transgenerational thyroid endocrine disruption, which have been ignored by previous studies. Therefore, these results can further improve our understanding of the ecological risks of TDCs.