Hepatotoxicity caused by methylparaben in adult zebrafish.

Parabens are a class of aquatic pollutants of emerging concern, among which methylparaben (MeP) causes severe pollution worldwide. However, aquatic toxicology of MeP remains largely unknown, which hinders ecological risk evaluation. In the present study, adult zebrafish were exposed to environmentally realistic concentrations (0, 1, 3, and 10 µg/L) of MeP for 28 days, with objectives to reveal the hepatotoxicity based on transcriptional, biochemical, metabolomics, and histopathological evidences. The results showed that MeP subchronic exposure induced the occurrence of hepatocellular vacuolization in zebrafish. The most severe symptom was noted in 10 µg/L MeP-exposed female liver, which was characterized by rupture of cell membrane and small nuclei. In addition, MeP exposure disturbed the balance between oxidative stress and antioxidant capacity. Lipid metabolism dynamics across gut, blood, and liver system were significantly dysregulated after MeP exposure by altering the transcriptions of lipid nuclear receptors and concentrations of key metabolites. Metabolomic profiling of MeP-exposed liver identified differential metabolites mainly belonging to fatty acyls, steroids, and retinoids. In particular, hepatic concentration of cortisol was increased in male liver by MeP pollutant, implying the activation of stress response. Exposure to MeP also inhibited the synthesis and conjugation of primary bile acid (e.g., 7-ketolithocholic acid and taurochenodeoxycholic acid) in female liver. Furthermore, degradation of biologically active molecules, including retinoic acid and estradiol, was enhanced in the liver by MeP. Overall, the present study highlights the hepatotoxicity caused by MeP pollutant even at environmentally realistic concentrations, which necessitates an urgent and accurate risk assessment.

Metabolomic profiles in zebrafish larvae following probiotic and perfluorobutanesulfonate coexposure.

Probiotic supplements are able to attenuate the developmental toxicity of perfluorobutanesulfonate (PFBS) pollutant. However, the underlying mechanisms remain unexplored. To this end, the present study acutely exposed zebrafish larvae for 4 days to 0 and 10 mg/L of PFBS, with or without the addition of probiotic Lactobacillus rhamnosus in the rearing water. The metabolomics approach was used to reveal the combined effects of PFBS and probiotics on metabolic dynamics, based on which gene transcriptions, enzymatic activities, and behavioral endpoints were further examined. The results showed that probiotic supplements were the major driver of the metabolomic fingerprints in coexposed zebrafish larvae. The addition of probiotic bacteria significantly decreased the methylation potential whilst up-regulating the demethylation process of genomic DNA, which may globally stimulate the gene expression to improve somatic growth. Acute exposure to PFBS significantly increased the cortisol concentration in zebrafish larvae, subsequently inducing stress response and hyperactive behavior. In contrast, probiotic supplementation promoted the degradation of cortisol, thus alleviating the stressful state. Antagonistic action of probiotics against PFBS developmental toxicity was also noted regarding the locomotor behavior. In addition, gut microbiota-mediated production of secondary bile acids was remarkably enhanced by probiotic supplements regardless of PFBS exposure. Overall, the present study underlines the efficacy of probiotic bacteria to protect zebrafish larvae from the metabolic disturbances of PFBS, thereby providing more evidence to support the application of probiotics in aquaculture and fishery as an environmentally-friendly choice.

Probiotics inhibit the stunted growth defect of perfluorobutanesulfonate via stress and thyroid axes in zebrafish larvae.

Perfluorobutanesulfonate (PFBS) is an emerging pollutant in aquatic environments and potently disrupts the early developmental trajectory of teleosts. Considering the persistent and toxic nature of PFBS, it is necessary to develop in situ protective measures to ameliorate the toxic damage of PFBS. Probiotic supplements are able to mitigate the growth retardation defects of PFBS. However, the interactive mechanisms remain elusive. To this end, this study acutely exposed zebrafish larvae to a concentration gradient of PFBS (0, 1, 3.3 and 10 mg/L) for 4 days, during which probiotic bacteria Lactobacillus rhamnosus were added in the rearing water. After exposure, alterations in gene transcriptions and key hormones along the hypothalamus-pituitary-interrenal (HPI), growth hormone/insulin-like growth factor (GH/IGF) and hypothalamus-pituitary-thyroid (HPT) axes were examined. The results showed that PFBS single exposure significantly increased the cortisol concentrations, suggesting the induction of stress response, while probiotic supplementation effectively decreased the cortisol levels in coexposed larvae in an attempt to relieve the stress of PFBS toxicant. It was unexpected that probiotic additive significantly decreased the larval GH concentrations independent of PFBS, thereby eliminating the contribution of GH/IGF axis to the growth improvement of probiotics. In contrast, probiotic bacteria remarkably increased the concentration of thyroid hormones, particularly the thyroxine (T4), in zebrafish larvae. The pronounced down-regulation of uridinediphosphate glucoronosyltransferases (UDPGT) gene pointed to the blocked elimination process of T4 by probiotics. Furthermore, proteomic fingerprinting found that probiotics were potent to shape the protein expression pattern in PFBS-exposed zebrafish larvae and modulated multiple biological processes that are essential for the growth. In summary, the present findings suggest that HPI and HPT axes may cooperate to enhance the growth of fish larvae under PFBS and probiotic coexposures.

Probiotic supplementation mitigates the developmental toxicity of perfluorobutanesulfonate in zebrafish larvae.

Perfluorobutanesulfonate (PFBS) is an emerging pollutant of international concern, which is found to impair the early embryonic development of fishes. In the context of ubiquitous and persistent pollution, it is necessary to explore mitigatory strategies against the developmental toxicity of PFBS. In this study, zebrafish larvae were acutely exposed to 0, 1, 3.3 and 10 mg/L of PFBS till 168 h post-fertilization (hpf), during which probiotic Lactobacillus rhamnosus bacteria were administered via the exposure media. After the singular or combined exposure, interaction between PFBS and probiotics on the growth of zebrafish larvae was measured. PFBS exposure significantly decreased the larval body weight, weight gain and specific growth rate, while probiotic supplementation efficiently inhibited the growth retardation caused by PFBS. Furthermore, PFBS and probiotic combinations remarkably activated the antioxidant capacity to timely scavenge the reactive oxidative species and protect the larvae from lipid peroxidation. Biochemical assay and fluorescent staining verified that PFBS exposure significantly promoted the production of bile acids, which were further enhanced by the probiotics. In coexposed zebrafish larvae, up-regulation of peroxisome proliferator-activated receptor (PPARb) would enhance the ß-oxidation of fatty acids to meet the energy demand from larval growth, subsequently decreasing fatty acid concentrations. In addition, probiotic supplements masked the dysbiosis of PFBS and potently shaped the gut microbiota, which closely modulated the production of bile acids. Overall, the present findings underline the beneficial effects of probiotics to protect the developing larvae from the aquatic toxicities of PFBS, thus highlighting the potential application values of probiotic recipe in aquaculture and ecological reservation.

Fingerprinting fecal DNA and mRNA as a non-invasive strategy to assess the impact of polychlorinated biphenyl 126 exposure on zebrafish.

In toxicological studies, experimental animals are generally subjected to dissection to obtain the tissues of concern, which causes great harm to the animals. In this regard, it is necessary to test and develop a non-invasive strategy to prevent the animals from anthropic injury when achieving scientific objectives. Therefore, zebrafish fecal DNA and mRNA pools were assessed by using metagenomic and transcriptomic analyses based on their potential to diagnose toxicological impairment of polychlorinated biphenyl (PCB) 126, a model persistent organic pollutant. The results showed that there was abundant zebrafish DNA and mRNA in the feces, which were, however, associated with contrasting profiles of physiological activities. As compared to DNA fragments, fecal mRNA provided a better representation of zebrafish physiological status. PCB126 exposure dramatically shifted the composition of fecal zebrafish DNA and mRNA as a function of sex. The differential mRNA caused by PCB126 clearly identified the toxicological fingerprint of PCB126. In summary, this study provides preliminary clues about the potential of fecal genes (mRNA in particular) in the development of non-invasive toxicological approaches. In the future, it is expected that more works will be conducted to screen sensitive diagnostic biomarkers from feces to increase the rate and reduce the cost of ecological risk assessment.

Antagonistic interaction between perfluorobutanesulfonate and probiotic on lipid and glucose metabolisms in the liver of zebrafish.

Perfluorobutanesulfonate (PFBS) and probiotic bacteria can interact to induce hepatic hypertrophy. However, the molecular events occurring in the hypertrophic liver are still unknown. Therefore, we performed this follow-up study using adult zebrafish that were exposed for 40 days to 0 and 10 µg/L PFBS, with or without dietary supplementation of probiotic Lactobacillus rhamnosus. After PFBS or/and probiotic exposures, proteome perturbation, histological pathogenesis and glucose metabolism were investigated in the livers. Proteomic analysis showed potent intervention of PFBS or/and probiotic with hepatic functions. PFBS single exposure caused marked disturbances in lipid metabolisms, which may underlie the severe vacuolization in male liver. The addition of probiotic alleviated the lipid metabolic disorders of PFBS. Furthermore, probiotic supplementation enhanced ATP energy production using glucose in mitochondrial respiratory chain of male fish. However, PFBS alone caused remarkable increase in blood glucose level (by 2.5-fold relative to the control), underlining the onset of hyperglycemia symptom. In contrast, the liver of male fish from the coexposure group functioned appropriately, which immediately increased insulin levels by 2.2-fold to reduce the glucose accumulation in blood. In female liver, PFBS alone significantly decreased the blood glucagon concentration by 2.9-fold. The deficiency of glucagon hormone consequently contributed to the accumulation of glycogen (3.2-fold) therein. Vigorous antagonistic interaction between PFBS and probiotic was noted with respect to glucose metabolism, which restored ATP, glucose, glycogen and glucagon to the control levels. Overall, the present study finds that probiotic L. rhamnosus is efficient to mitigate the metabolic disorders of PFBS on lipid and glucose, highlighting the potential values of probiotic bacteria to protect the aquatic ecosystem.

Shift in skin microbiota and immune functions of zebrafish after combined exposure to perfluorobutanesulfonate and probiotic Lactobacillus rhamnosus.

Dysbiosis of fish skin microbiome and immunity by environmental pollutants are rarely studied in toxicological research in spite of their importance for fish health. In the present study, adult zebrafish were exposed to 0 and 10 µg/L of perfluorobutanesulfonate (PFBS) for 40 days, with or without the supplementation of probiotic Lactobacillus rhamnosus, with objectives to explore the interaction between PFBS pollutant and probiotic bacteria on skin mucosal microbiota and immune response. Amplicon sequencing analysis found that PFBS alone significantly disturbed the microbial community composition and abundance on the skin, favoring the growth of stress-tolerant bacteria (e.g., Deinococcus and Enhydrobacter genera). However, the administration of probiotic inhibited the dysbiosis of PFBS and shaped the skin microbiome in the combined exposure group. PFBS single exposure also promoted the production of mucus on the skin of male zebrafish, which may be related to the growth of Limnobacter bacteria. In contrast, probiotic supplements remarkably improved the immune functions in male skin mucus from the combined group, as evidenced by the consistent increases in lysozyme activity, immunoglobulin concentrations and peroxidase activity. Overall, the present study provides the first clue about the singular and combined effects of PFBS and probiotic on skin microbiota and immunity, highlighting the beneficial action of probiotic L. rhamnosus against PFBS stress.

Interaction between probiotic additive and perfluorobutanesulfonate pollutant on offspring growth and health after parental exposure using zebrafish.

Perfluorobutanesulfonate (PFBS) pollutant and probiotic bacteria can interact to affect the reproductive outcomes of zebrafish. However, it is still unexplored how the growth and health of offspring are modulated by the combination of PFBS and probiotic. In the present study, adult zebrafish were exposed to 0 and 10 µg/L PFBS for 40 days, with or without dietary supplementation of probiotic Lactobacillus rhamnosus. After parental exposure, the development, growth and viability of offspring larvae were examined, with the integration of molecular clues across proteome fingerprint, growth hormone/insulin-like growth factor (GH/IGF) axis, calcium homeostasis, hypothalamic-pituitary-adrenal (HPA) axis and nutrient metabolism. Parental probiotic supplementation significantly increased the body weight and body length of offspring larvae. Despite the spiking of PFBS, larvae from the combined exposure group still had longer body length. RNA processing and ribosomal assembly pathways may underlie the enhancement of offspring growth by probiotic bacteria. However, the presence of PFBS remarkably increased the concentrations of cortisol hormone in offspring larvae as means to cope with the xenobiotic stress, which required more energy production. As evidenced by the proteomic analysis, the addition of probiotic bacteria likely alleviated the energy metabolism disorders of PFBS, thus allocating more energy for the larval offspring growth from the combined group. It was noteworthy that multiple molecular disturbances caused by PFBS were antagonized by probiotic additive. Overall, the present study elucidated the intergenerational interaction between PFBS and probiotic on offspring growth and health after parental exposure.

Probiotic Lactobacillus rhamnosus modulates the impacts of perfluorobutanesulfonate on oocyte developmental rhythm of zebrafish.

Potent interaction between probiotic bacteria and perfluorobutanesulfonate (PFBS), an aquatic pollutant of emerging concern, was previously reported on reproduction of zebrafish. However, the underlying mechanism is largely unexplored. In this regard, the present study continued to focus on the interactive modes between probiotics and PFBS. Adult zebrafish were exposed for 28 days to 0 and 10 µg/L PFBS with or without dietary supplementation of probiotic Lactobacillus rhamnosus. With the relevance to fecundity outcome, a suite of reproductive indices at transcriptional, hormonal, proteomic and histological levels of biological organization were measured herein. The fecundity monitoring results showed that probiotic additive shifted the impacts of PFBS on egg spawn, gradually approaching the control level. Based on ovary histological observation, oocyte growth was significantly promoted by probiotics or/and PFBS exposures, while the presence of probiotic bacteria partially antagonized the effects of PFBS on oocyte growth. The combination of probiotics and PFBS increased the concentration of maturation inducing hormones in ovary. Despite the enhanced hormonal signals, gene transcriptions of ovarian local auto/paracrine factors were consistently decreased in all exposure groups, suggesting the blocked transition from oocyte growth phase toward oocyte maturation phase. Ovary proteomic analysis found that PFBS exposure with or without probiotic bacteria mainly affected the RNA metabolic processes, although the addition of probiotics exerted extra influences on amino acid metabolism. Overall, the present study provided more mechanistic evidence about the interactive behavior between probiotic bacteria and PFBS pollutant. Feed additive of probiotic bacteria modulated the impacts of PFBS on egg production rhythm through oocyte growth and maturation phases.