Heat-induced oxidative stress and inflammation involve in cadmium pollution history in the spleen of zebrafish.

Zebrafish were exposed to 0, 2.5 and 5 µg/L cadmium (Cd) for 10 weeks, and then each group was exposed to 26 °C(control) and 32 °C (high temperature) for 7 days. 22 indicators were compared between 26 °C and 32 °C in the spleen, including body weight, LPO and NO levels, activity levels of Cu/Zn-SOD, CAT and iNOS, MTs protein levels, and mRNA levels of Nrf2, Cu/Zn-SOD, CAT, HSF1, HSF2, HSP70, MTF-1, MTs, IL-6, IL-10, IL-1ß, TNF-α, iNOS and NF-κB. Most indicators were not significantly affected by heat in fish from no Cd pollution. However, almost all of indicators were responsive to heat in fish pre-exposed to Cd. Several indicators were sensitive to heat in fish pre-exposed to 2.5 µg/L Cd such as iNOS activities, and mRNA levels of iNOS and IL-10. Most other indicators were sensitive to heat in fish pre-exposed to 5 µg/L. The mRNA levels of HSP70 and MTF-1 were up-regulated by heat in fish pre-exposed to 0, 2.5 and 5 µg/L Cd. However, the magnitude of increase was the greatest in fish pre-exposed to 5 µg/L Cd. These differences between control and high temperature would serve as biomarkers to distinguish healthy from Cd-polluted group. The findings imply that metal pollution history should be carefully considered when screening heat biomarkers in fish.

Liver X Receptor Gene Expression is Enhanced in Children with Obstructive Sleep Apnea-Hyperpnoea Syndrome and Cyclooxygenase-2 (COX-2) is Correlated with Severity of Obstructive Sleep Apnea-Hypopnea Syndrome (OSAHS).

BACKGROUND Liver X receptor (LXR) is a nuclear receptor presenting in macrophages; it works indispensably in lipid metabolism control and also negatively regulates the expression of inflammatory genes in macrophages. There are many LXR-related studies in adults with metabolic syndrome but rare reports in obese children with obstructive sleep apnea-hypopnea syndrome (OSAHS). The aim of this study was to investigate the expression of LXR, cholesterol ester transfer protein (CETP), and cyclooxygenase-2 (COX-2) genes in obese children with OSAHS compared with obese children without OSAHS and non-obese children. MATERIAL AND METHODS Sleep monitoring was conducted in 80 obese children with sleep disorders. Fasting morning blood samples from the 80 obese children and 51 normal children were collected and separated, so that macrophages were obtained after culture. Fluorescence quantitative real-time PCR (RT-PCR) was used to detect expression levels of the LXR, CETP, and COX-2 genes. RESULTS LXR, COX-2, and CETP levels in the OSAHS group were higher than those in the other two groups (P<0.05), and the LXR levels in the group of obese children without OSAHS were higher than those in control group (P<0.05). COX-2 expression in the group with moderate to severe OSAHS was higher than that in the group with mild OSAHS (P<0.05). Meanwhile, there were no significant differences in the LXR and CETP levels between the moderate to severe OSAHS group and the mild OSAHS group (P>0.05). CONCLUSIONS LXR gene expression was significantly increased in obese children with OSAHS. The severity of OSAHS was positively correlated with COX-2 levels.

[Establishment of a rat model of oral food allergy].

OBJECTIVE: To establish a food allergy model in Brown Norway (BN) rats by gavage of ovalbumin (OVA) without any adjuvant, and to evaluate this model. METHODS: A total of 20 male BN rats aged 3 weeks were randomly divided into allergy group and control group (n=10 each). BN rats in the allergy group were given OVA 1 mg per day by gavage, and all the rats were treated for 41 days continuously. On day 42, the rats in the allergy group were given OVA 100 mg by gavage for challenge. The rats in the control group were given normal saline of the same volume by gavage. Differences in body length, body weight, and food intake were compared between the two groups on days 7, 14, 21, 28, 35, and 42. ELISA was used to measure the serum OVA-IgE level and plasma histamine level after challenge on day 42, and the changes in rats' appearance and fecal properties were observed. The model of food allergy was considered successful when the serum OVA-IgE level in the allergy group was no less than the mean serum OVA-IgE level + 3 standard deviation in the control group. RESULTS: There were no significant differences in body length, body weight or food intake between the allergy and control groups at all time points (P>0.05). On day 21, the control group had a significantly higher food intake than the allergy group (P<0.05). On day 42 after challenge, the allergy group showed significantly higher serum OVA-IgE and plasma histamine levels than the control group (P<0.05). The sensitization rate (rate of successful modeling) was 90%. The fecal properties showed no significant differences between the two groups. CONCLUSIONS: OVA by gavage without any adjuvant can successfully establish the model of food allergy in BN rats and has a high success rate. Food allergy induced by OVA may reduce food intake within a short period of time, but no influence on rats' body length or body weight has been observed.

Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro.

Peroxisome proliferator-activated receptor gamma (PPARγ) is ligand-inducible transcription factor and has important roles in lipid metabolism, cell proliferation and inflammation. In the present study, yellow catfish Pelteobagrus fulvidraco PPARγ cDNA was isolated from liver by RT-PCR and RACE, and its molecular characterization and transcriptional regulation by insulin in vivo and in vitro were determined. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene. PPARγ1 and PPARγ2 mRNA covered 2426 bp and 2537 bp, respectively, with an open reading frame (ORF) of 1584 bp encoding 527 amino acid residues. Yellow catfish PPARγ gene was organized in a manner similar to that of their mammalian homologs, implying a modular organization of the protein's domains. A comparison between the yellow catfish PPARγ amino acid sequence and the correspondent sequences of several other species revealed the identity of 55-76.2%. Two PPARγ transcripts (PPARγ1 and PPARγ2) mRNAs were expressed in a wide range of tissues, but the abundance of each PPARγ mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection of insulin in vivo significantly stimulated the mRNA expression of total PPARγ and PPARγ1, but not PPARγ2 in the liver of yellow catfish. In contrast, incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1, PPARγ2 and total PPARγ. To our knowledge, for the first time, the present study provides evidence that PPARγ1 and PPARγ2 are differentially expressed with and among tissues during different developmental stages and also regulated by insulin both in vivo and in vitro, which serves to increase our understanding on PPARγ physiological function in fish.

Hormone-sensitive lipase in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA tissue expression and transcriptional regulation by leptin in vivo and in vitro.

Hormone-sensitive lipase (hsl) plays a pivotal role in regulation of lipolysis in mammals, but information is very scarce about its gene structure and function in fish. In this study, two distinct hsl cDNAs, designated hsl1 and hsl2, were firstly isolated and characterized from yellow catfish Pelteobagrus fulvidraco. The validated cDNAs encoding for hsl1 and hsl2 were 2739 and 2629bp in length, encoding peptides of 679 and 813 amino acid residues, respectively, and shared 57.7% amino acid identity. The phylogenetic analysis revealed that hsl1 and hsl2 derived from paralogous genes that might have arisen during a teleost-specific genome duplication event. Both hsl mRNAs were expressed in a wide range of tissues, but the abundance of each hsl mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin (rb-hLEP) stimulated the mRNA expression of hsl2, but not hsl1, in the liver and hepatocytes of P. fulvidraco, respectively, suggesting that two hsl isoforms might serve different roles in lipid metabolism. To our knowledge, for the first time, the present study provides evidence that two hsl mRNAs are differentially expressed with and among tissues during different developmental stages and also differentially regulated by leptin both in vivo and in vitro, which serves to increase our understanding on hsl physiological function in fish.

Molecular characterization, tissue distribution and kinetic analysis of carnitine palmitoyltransferase I in juvenile yellow catfish Pelteobagrus fulvidraco.

Up to date, only limited information is available on genetically and functionally different isoforms of CPT I enzyme in fish. In the study, molecular characterization and their tissue expression profile of three CPT Iα isoforms (CPT Iα1a, CPT Iα1b and CPT Iα2a) and a CPT Iß isoform from yellow catfish Pelteobagrus fulvidraco is determined. The activities and kinetic features of CPT I from several tissues have also been analyzed. The four CPT I isoforms in yellow catfish present distinct differences in amino acid sequences and structure. They are widely expressed in liver, heart, white muscle, spleen, intestine and mesenteric adipose tissue of yellow catfish at the mRNA level, but with the varying levels. CPT I activity and kinetics show tissue-specific differences stemming from co-expression of different isoforms, indicating more complex pathways of lipid utilization in fish than in mammals, allowing for precise control of lipid oxidation in individual tissue.

In vitro effects of selenium on copper-induced changes in lipid metabolism of grass carp (Ctenopharyngodon idellus) hepatocytes.

The present study was performed to evaluate the in vitro effects of selenium (Se) supplementation to prevent copper (Cu)-induced changes in lipid metabolism of hepatocytes from grass carp (Ctenopharyngodon idellus). Four groups (control and 100 µM Cu in combination with 0, 5, and 10 µM Se, respectively) were chosen. Compared with the control, activities of glucose 6-phosphatedehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme, and carnitine palmitoyltransferase I (CPT I) of all three Cu-exposed groups at 24 and 48 h were significantly greater. However, among three Cu-exposed groups, increasing Se concentration tended to increase activities of G6PD and ME at 24 h and 6PGD activity at 24 and 48 h but decreased CPT I activity at 24 h. Compared with the control, Cu exposure alone, or in combination with Se, downregulated mRNA levels of sterol regulatory element-binding protein-1 (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase, peroxisome proliferator activated receptor alpha (PPARα), CPT I, and hormone-sensitive lipase (HSL) at 24 h as well as SREBP-1c, FAS, and ACC mRNA levels at 48 h. However, upregulated mRNA levels of PPARα, CPT I, and HSL, as well as decreased triglyceride content, were recorded at 48 h. Thus, although toxic at greater levels, lower levels of Se provided significant protection against Cu-induced changes in lipid metabolism. For the first time, our study indicates the dose- and time-dependent effects of Se addition on changes in lipid metabolism induced by Cu in fish hepatocytes and provides new insights into Se-Cu interaction at both enzymatic and molecular levels.

Differential effects of acute and chronic zinc exposure on lipid metabolism in three extrahepatic tissues of juvenile yellow catfish Pelteobagrus fulvidraco.

The aim of this study was to determine the potential mechanisms of exposure to waterborne zinc (Zn) on lipid metabolism in three extrahepatic tissues (ovary, muscle and mesenteric adipose tissue) of female yellow catfish Pelteobagrus fulvidraco. Female yellow catfish were chronically exposed to Zn (0.05, 0.35 or 0.86 mg Zn/l; duration of treatment 8 weeks) or acutely exposed to a high level of Zn (4.71 mg Zn/l for 96 h). Following the respective treatment, lipid deposition and mRNA levels of 11 genes (CPT IA, CPT IB, PPARα, PPARγ, SREBP-1, G6PD, 6PGD, FAS, ACCa, ACCb and LPL) involved in lipid metabolism were determined. Waterborne Zn exposure significantly reduced growth performance and lipid content in muscle but had no significant effect on lipid content in ovary and mesenteric adipose tissue. The change in the levels of the mRNA genes under study was Zn concentration-dependent and tissue-dependent. Pearson correlations between the mRNA levels of three transcriptional factors and enzymes in these tissues revealed that variations in gene expression as a result of the different Zn treatments underlay the patterns of lipid metabolism, which in turn affected fat storage and mobilization. To our knowledge, this is the first study to demonstrate the effect of waterborne Zn exposure on lipid metabolism in extrahepatic tissues at the molecular level. These results therefore contribute to our understanding of Zn-induced toxicity in fish.

In vitro exposure to copper influences lipid metabolism in hepatocytes from grass carp (Ctenopharyngodon idellus).

In the present study, three different copper (Cu) concentrations (control, 10 and 100 lM, respectively) and three incubation times (24, 48 and 96 h) were chosen to assess in vitro effect of Cu on lipid metabolism in hepatocytes of grass carp Ctenopharyngodon idellus. Increased glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carnitine palmitoyltransferase I activities were observed in hepatocytes with increasing Cu concentration and exposure duration. Cu decreased mRNA levels of several lipogenic and lipolytic genes at 24 h. However, at 48 h, Cu down-regulated the process of lipogenesis but up-regulated that of lipolysis. The Cudriven up-regulation of lipolytic genes was maintained after 96 h and accompanied by a decreased intracellular triglyceride accumulation, while no effect on lipogenic genes was shown. Thus, 96-h Cu exposure induced lipid depletion, possibly due to the upregulation of lipolysis. Although in this process, lipogenesis might be up-regulated, it was not enough to compensate lipid consumption. Our study represents the first approach to concentration- and time-dependent in vitro effects of Cu on lipid metabolism of fish hepatocytes and provides new insights into Cu toxicity in fish at both enzymatic and molecular levels.

Characterization and tissue distribution of leptin, leptin receptor and leptin receptor overlapping transcript genes in yellow catfish Pelteobagrus fulvidraco.

In the present study, full-length cDNA sequences of leptin (LEP), leptin receptor (LEPR) and leptin receptor overlapping transcript (LEPROT) were cloned from yellow catfish Pelteobagrus fulvidraco, and their tissue distribution profiles were determined. The validated cDNA of yellow catfish leptin (ycLEP), leptin receptor (ycLEPR) and LEPROT were 1119, 4195 and 827bp in length, encoding the peptide of 172, 1086 and 130 amino acid residues, respectively. The phylogenetic analysis revealed that fish LEP, LEPR and LEPROT were separated from tetrapod, and also ycLEPS were separated from other fish species. The ycLEP mRNA expression levels were highest in liver, followed by ovary, mesenteric fat and spleen, and lowest in intestine, heart, muscle, pituitary and testis. The ycLEPR mRNA levels were highest in pituitary, intermediate in mesenteric fat, liver, ovary, muscle and spleen, and lowest in heart, intestine and testis. The ycLEPROT mRNA levels were highest in pituitary, followed by spleen, mesenteric fat, heart, ovary, liver, muscle, testis and intestine. Identification and tissue distribution of yellow catfish LEP, LEPR and LEPROT genes provided initial step towards understanding their biological roles in yellow catfish.

Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro.

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2',5'-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K(m) values for G6-P and NADP(+) were determined to be 0.026, 0.0068 mM, respectively. The V(max) values for G6-P and NADP(+) were 2.20 and 2.27 µM min(-1) mg protein(-1), respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10(-6) min(-1) mg protein(-1), respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC50 values of Zn(+2), Mn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn(+2), Al(+3), and Cd(+2) showed competitive inhibition, while Cu(+2) inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.

Zinc pre-exposure improves Zn resistance by demethylation of metallothionein 2 and transcription regulation of zinc-regulatory genes in zebrafish ZF4 cells.

Mild zinc (Zn) pre-exposure can promote Zn resistance of organism, but the underlying molecular mechanisms are largely unknown. Two experiments were performed using zebrafish ZF4 cells, including short-term and long-term Zn pre-exposure experiments. In the short-term test, the cells were pre-exposed to 100 µM Zn for 24 h, transferred into fresh medium with 4.4 µM Zn for 24 h, and then re-exposed to 250 µM Zn. In the long-term test, the cells were pre-exposed to 100 µM Zn intermittently for 10 passages (3 days per passage), transferred into fresh medium with 4.4 µM Zn for 5 passages, and then re-exposed to 250 µM Zn. Both pretreatments resulted in higher resistance to 250 µM Zn. Exposure to 250 µM Zn caused a more than 2-fold increase in Zn content without Zn pretreatment but did not affect Zn content in the Zn pretreated cells. The Zn pretreated cells had low methylation levels of the metal-response element (MRE) at locus -87 in the promoter of mt2 (metallothionein 2). The up-regulated mRNA expression of Zn-regulatory genes (mtf-1, mt2, slc30a1a, slc30a4, slc30a5, slc30a6 and slc30a7) in the long-term Zn pretreated cells and mt2, slc30a4, slc30a6 and slc30a7 in the short-term Zn pretreated cells were observed. Exposure to 250 µM Zn in combination with the Zn pretreatments up-regulated mRNA expression of these genes and reduced methylation levels of the MRE compared with 250 µM Zn alone and the control. Taken together, the data suggested that demethylation of MRE in the promoter of mt2 and transcriptional induction of mt2 and Zn exporter genes offered Zn resistance in fish ZF4 cells. The traditional toxicological evaluation based on continuous exposure may overestimate the risk of fluctuating concentrations of Zn in the environment.

Early life stage exposure to cadmium and zinc within hour affected GH/IGF axis, Nrf2 signaling and HPI axis in unexposed offspring of marine medaka Oryzias melastigma.

Information on transgenerational effects of cadmium (Cd) and zinc (Zn) within hour of exposure is scarce. To the end, larvae of marine medaka Oryzias melastigma at 0 day-post-hatching (dph) were subjected to LC50 for 96-h of Cd or Zn for 0.5 and 6 h, and then transferred into clear water for 95 days until the generation of offspring larvae at 25 dph. Growth, antioxidant capacity and stress response in offspring larvae were examined. Exposure to Zn for 0.5 h or Cd for 0.5 h and 6 h promoted growth performance and reduced total antioxidant capacity (TAC) and activities of superoxide dismutase (SOD) and catalase (CAT). Malondialdehyde (MDA) and cortisol levels declined in larvae following Zn exposure for 0.5 h, whereas Cd exposure increased MDA content and did not affect cortisol levels. These physiological changes could be partially explained by transcription of genes in the hormone/insulin-like growth factor-I (GH/IGF) axis, NF-E2-related factor 2 (Nrf2) signaling, and hypothalamus-pituitary-interrenal (HPI) axis. For example, Zn exposure for 0.5 h up-regulated genes encoding growth hormone (gh) and insulin-like growth factor binding protein (igfbp1) and down-regulated mRNA levels of nrf2, Kelch-like-ECH-associated protein 1 gene (keap1a), keap1b, sod1, mineralocorticoid receptor (mr), corticotropin-releasing hormone receptor (crhr1), corticotropin-releasing hormone binding protein (crhbp), cytochrome P450 (cyp11a1, cyp17a1) and hydroxysteroid dehydrogenase (hsd3b1). Cd exposure for 0.5 and 6 h up-regulated growth hormone release hormone (ghrh) and igfbp1, down-regulated nrf2 and keap1a, and did not affect mRNA levels of HPI axis genes. Taken together, this study demonstrated that short-term metal exposure during larvae phase had positive and negative effects on offspring even after a long recovery.

Cadmium induced oxidative stress, endoplasmic reticulum (ER) stress and apoptosis with compensative responses towards the up-regulation of ribosome, protein processing in the ER, and protein export pathways in the liver of zebrafish.

The present study identified that exposure to 5, 10, and 20 µg/L Cd for 48 days reduced growth, increased Cd accumulation and levels of reactive oxygen species (ROS) and lipid peroxidation, and induced ER stress and cellular apoptosis in the liver in a dose-dependent manner. However, the survival rate was not affected by Cd. The increased production of ROS might result from reduced catalase (CAT) and copper/zinc-superoxide dismutase (Cu/Zn-SOD) activities, which might trigger ER stress pathways and subsequently induce apoptotic responses, ultimately leading to growth inhibition. Transcriptomic analyses indicated that the differentially expressed genes (DEGs) involved in metabolic pathways were significantly enriched and dysregulated by Cd, suggesting that metabolic disturbances may contribute to Cd toxicity. However, there were increases in glutathione peroxidase (GPX) activity, protein levels of metallothioneins (MTs) and heat shock protein 70 (HSP70), and mRNA levels of sod1, cat, gpx, mt2, and hsp70. Furthermore, DEGs related to ribosome, protein processing in the ER, and protein export pathways were significantly enriched and up-regulated by Cd. These increases may be compensatory responses following oxidative stress, ER stress, and apoptosis to resist negative effects. Taken together, we demonstrated that environmentally relevant levels of Cd induced adaptive responses with compensatory mechanisms in fish, which may help to maintain fish survival at the cost of growth.

Transgenerational effects of zinc in zebrafish following early life stage exposure.

Although toxic effects of zinc (Zn) have been well established in the different developmental stages in fish, long-lasting effects of Zn exposure during embryonic development have not been explored. Exposure to an environmentally relevant Zn concentration of 10 µM (650 µg/L) during the first five days after fertilization did not affect survival, body weight, malformations or overall hatching success of F0 and F1 larvae. Zn exposure did, however, result in delayed hatching in both the F0 and F1 generations and caused significant changes in homeostasis of Zn and selenium (Se) in F0 and F1 fish. This was especially pronounced when F1 embryos from Zn-exposed parents were treated with 30 µM (2000 µg/L) Zn. In the F0 generation, skewed sex ratio towards males and changes in homeostasis of Zn, Se and manganese (Mn) in the brain, gill, liver and gonad of adult fish were also observed. These changes were associated with altered expression of Zn- and Mn-regulatory genes and sex differentiation genes in F0 and F1 fish. The present study suggests that fish may carry memory from embryo-larval Zn exposure into adulthood and further to the next generation. The present study shows that ecotoxicological risk of an exposure to Zn during embryo-larval development may persist long after recovery and may also manifest in the F1 generation.

Combined effects of polystyrene microplastics and cadmium on oxidative stress, apoptosis, and GH/IGF axis in zebrafish early life stages.

The toxicological interactions of microplastics (MPs) and heavy metals have been paid much attention in aquatic organism. The mechanisms are not fully clear, particularly in fish early life stages. To the end, zebrafish embryos were exposed to 500 µg/L MPs, 5 µg/L cadmium (Cd), and their combination for 30 days. Body weight, adsorption characteristics of Cd onto MPs, Cd accumulation, oxidative stress, apoptosis, and growth hormone/insulin-like growth factor-I (GH/IGF) axis were examined. Exposure to MPs and Cd alone reduced body weight, which was aggravated by co-exposure. An increase in reactive oxygen species (ROS) levels was observed in larvae exposed to Cd or MPs + Cd, suggesting an induction of oxidative stress. Lipid peroxidation levels were not affected by exposure to MPs and Cd alone but dramatically enhanced by co-exposure, which may be explained by the reduction of total antioxidant capacity (TAOC) and activity levels of Mn-superoxide dismutase (Mn-SOD) and catalase (CAT) after co-exposure. Increased apoptotic cells were observed in the vertebral body of larvae exposed to Cd, the esophagus of larvae exposed to MPs, and both organs of larvae exposed to MPs + Cd, which was further confirmed by changes in the activities of Caspase-3, Caspase-8 and Caspase-9. PCR array on the transcription of genes related to growth, oxidative stress and apoptosis was examined, showing that the combined exposure resulted in greater magnitude of changes than MPs and Cd alone. The results indicate that MPs can enhance the negative effects of Cd on growth, oxidative damage and apoptosis in early life stages of zebrafish. However, the adsorption of Cd onto MPs was not observed and the combined exposure did not increase the Cd content in larvae compared to the single Cd exposure, implying that vector role of MPs in Cd uptake is negligible.

Nutritional-status dependent effects of microplastics on activity and expression of alkaline phosphatase and alpha-amylase in Brachionus rotundiformis.

Tissue-nonspecific alkaline phosphatase (ALPL) and alpha-amylase (AMY) are essential in the immune and digestive systems, respectively. Microplastics (MPs) pose a risk to zooplankton which may be in a state of feeding, starvation, or subsequent refeeding. However, molecular characterization of both enzymes and the regulated mechanisms affected by nutritional statuses and MPs remain unclear in zooplankton. In the present study, four full-length genes encoding ALPL and two genes encoding AMY were cloned and characterized from an isolated marine rotifer, Brachionus rotundiformis, including alplA, alplB, alplC, alplD, amy2a, and amy2al. AMY activity and expression of amy2a and amy2al were reduced by starvation and recovered after refeeding compared with feeding. ALPL activity remained unchanged among different statuses, while alplA, alplB and alplD were down-regulated by starvation and refeeding compared with feeding. ALPL activity was not affected by exposure to 10, 100 and 1000 µg/L MPs in rotifers subjected to feeding, starvation and refeeding, whereas AMY activity was significantly enhanced by 1000 µg/L MPs in rotifers subjected to refeeding. Gene expression of the tested genes, except amy2a, was significantly responsive to MPs, especially in the feeding rotifers, depending on MPs concentrations and nutritional statuses. Two-way ANOVA confirmed that these changes were strongly associated with the interaction between MPs concentrations and nutritional statuses. The present study is the first to demonstrate a nutritional status-dependent impact of MPs on immune and digestive responses, and provides more sensitive molecular biomarkers for assessing MPs toxicity using the species as model animals.

Particles rather than released Zn2+ from ZnO nanoparticles aggravate microplastics toxicity in early stages of exposed zebrafish and their unexposed offspring.

Knowledge on the interaction between microplastics (MPs) and zinc oxide nanoparticles (ZnO NPs) is limited. Here, we investigated effects of embryo-larvae exposure to 500 µg/L polystyrene MPs (5 µm), 1200 µg/L ZnO NPs (< 100 nm), 500 µg/L dissolved Zn2+ from ZnSO4, and the mixtures of MPs and ZnO NPs or ZnSO4 on exposed F0 larvae and unexposed F1 larvae. Consequently, ZnO particles adhered to MPs surfaces rather than Zn2+, and increased Zn transport into larvae. Growth inhibition, oxidative stress, apoptosis, and disturbance of growth hormone and insulin-like growth factor (GH/IGF) axis were induced by MPs and ZnO NPs alone, which were further aggravated by their co-exposure in F0 larvae. MPs + ZnO increased apoptotic cells in the gill and esophagus compared with MPs and ZnO NPs alone. Reduced growth and antioxidant capacity and down-regulated GH/IGF axis were merely observed in F1 larvae from F0 parents exposed to MPs + ZnO. Contrary to ZnO NPs, dissolved Zn2+ reversed MPs toxicity, suggesting the protective role of Zn2+ may be not enough to ameliorate thfie negative effects of ZnO particles. To summarize, we found that particles rather than released Zn2+ from ZnO nanoparticles amplified MPs toxicity in early stages of exposed zebrafish and their unexposed offspring.

Genome-wide identification of seven superoxide dismutase genes in the marine rotifer Brachionus rotundiformis and modulated expression and enzymatic activity in response to microplastics and nutritional status.

Microplastics (MPs) pollution has attracted worldwide attention. Superoxide dismutase (SOD) is a sensitive indicator for assessing the toxic effects of MPs in aquatic organisms. However, few studies have been performed to identify all genes encoding SOD in aquatic invertebrates. Especially, effects of MPs on SOD activity and expression in aquatic organisms under starvation or a subsequent refeeding status are unclear. In the present study, all full-length genes encoding SOD were cloned and characterized from the marine rotifer Brachionus rotundiformis, including CuZnSOD1, CuZnSOD2, CuZnSOD3, CuZnSOD4, CuZnSOD5, MnSOD1, and MnSOD2. The CuZnSOD1, CuZnSOD2 and MnSOD2 are homologous to SODs from vertebrates and the other SOD proteins are rotifer-specific according to the results from the phylogenetic tree. The conserved signature sequences and binding sites of Cu2+, Zn2+and Mn2+ were also identified in the seven SOD proteins. Compared with feeding, starvation down-regulated SOD activity and mRNA expression of CuZnSOD2, CuZnSOD4, CuZnSOD5, MnSOD1 and MnSOD2 while refeeding maintained SOD activity comparable to the feeding level and up-regulated CuZnSOD5 and MnSOD2. Intake of MPs by B. rotundiformis was observed by examining fluorescence signals from the fluorescently-labeled microplastics under different nutritional status. Exposure to MPs reduced rotifer density and increased malondialdehyde (MDA) content and SOD activity in the rotifers under the refeeding condition, but did not affect these indicators under the feeding and starvation conditions. However, mRNA expression of some tested genes was responsive to MPs in the fed, starved and refed rotifers. The present study for the first time demonstrated a nutritional status-dependent effect of MPs on oxidative stress response, and provided more sensitive molecular biomarkers for assessing the toxicity of MPs using B. rotundiformis as a model animal.

Effects of continuous and intermittent cadmium exposure on HPGL axis, GH/IGF axis and circadian rhythm signaling and their consequences on reproduction in female zebrafish: Biomarkers independent of exposure regimes.

Typical biomarkers of cadmium (Cd) pollution have well been confirmed in fish from continuous exposure pattern. However, in a natural environment, fish may be exposed to Cd intermittently. In this study, juvenile female zebrafish were exposed for 48 days to 10 µg/L Cd continuously, 20 µg/L for 1 day in every 2 days or 30 µg/L for 1 day in every 3 days. The toxic effects were evaluated using 8 various physiological and biochemical endpoints like specific growth rate (SGR), 17ß-estradiol (E2) and vitellogenin (VTG) concentrations in plasma, reproductive parameters (gonadosomatic index (GSI), egg-laying amount, spawning percentage, and hatching and mortality rate of embryos). Transcription of 59 genes related to hypothalamic-pituitary-gonadal-liver (HPGL) axis, circadian rhythm signaling and insulin-like growth factor (IGF) system was examined. SGR, spawning percentage, E2 and VTG levels declined in fish exposed to 10 and 20 µg/L Cd but remained relatively stable in fish exposed to 30 µg/L Cd. Exposure to 10, 20 and 30 µg/L Cd significantly reduced GSI, hatching rate and mortality rate. Similarly, mRNA expression of 27 genes were sensitive to both continuous and intermittent Cd exposure. Among these genes, expression levels of 10 genes had more than 5-fold increase or decrease, including mRNA levels of vtg1, vtg2, vtg3, esr1, igf2a, igf2b, igfbp5b, nr1d1, gnrh3 and gnrhr4. The most sensitive molecular biomarker was vtg3 expression with 1500-3100 fold increase in the liver. The present study, for the first time, provides effective candidate biomarkers for Cd, which are independent of exposure regimes.