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.

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.

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.

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.

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.

High salinity acclimatization alleviated cadmium toxicity in Dunaliella salina: Transcriptomic and physiological evidence.

In the present study, we tested the hypothesis that high salinity acclimatization can mitigate cadmium (Cd) toxicity in the microalga Dunaliella salina. To this end, microalgal cells were subjected to high salinity (60 g/L) for 12 weeks until the growth rate remained stable between generations and were then exposed to 2.5 mg/L of Cd for 4 days. Acute Cd toxicity impaired cell growth by increasing Cd bioaccumulation and lipid peroxidation, which reduced cellular pigment, total protein, and glutathione content. It also significantly weakened photosynthetic efficiency and total antioxidant capacity. However, acclimatization to high salinity alleviated these negative effects under Cd stress. To understand the potential mechanisms behind this phenomenon, 12 cDNA libraries from control, Cd-exposed (Cd), high salinity-acclimated (Salinity), and high salinity-acclimated with Cd exposure (Salinity + Cd) cells were derived using RNA sequencing. A total of 2019, 1799, 2150 and 1256 differentially expressed genes (DEGs) were identified from sample groups Salinity / Control, Cd / Control, Salinity + Cd / Control, and Salinity + Cd / Cd, respectively. Some of these DEGs were significantly enriched in ribosome, photosynthesis, stress defense, and photosynthesis-antenna proteins. Among these genes, 82 ribosomal genes were up-regulated in Salinity / Control (corrected P = 3.8 × 10-28), while 81 were down-regulated in Cd / Control (corrected P = 1.1 × 10-24). Moreover, high salinity acclimatization up-regulated 8 photosynthesis genes and 18 stress defense genes compared with the control. Additionally, 3 photosynthesis genes, 11 stress defense genes and 11 genes encoding light harvesting proteins were up-regulated by high salinity acclimatization under Cd exposure. Overall, high salinity acclimatization mitigated Cd toxicity, possibly by up-regulating the transcription of photosynthesis, stress defense, and ribosomal genes. These results provide new insights on cross-tolerance in microalgae.

Transcription activation of ß-carotene biosynthetic genes at the initial stage of stresses as an indicator of the increased ß-carotene accumulation in isolated Dunaliella salina strain GY-H13.

ß-carotene is an efficient antioxidant and its accumulation is an oxidative response to stressors. Dunaliella salina strain GY-H13 is rich in ß-carotene under environmental stresses, which was selected as material to understand the molecular mechanism underlying ß-carotene biosynthesis. Seven full length cDNA sequences in ß-carotene biosynthesis pathway were cloned, including geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), phytoene desaturase (PDS), 15-cis-zeta-carotene isomerase (ZISO), zeta-carotene desaturase (ZDS), prolycopene isomerase (CRTISO), lycopene beta-cyclase (LCYb). The seven protein sequences from the strain GY-H13 showed the highest similarity with other D. salina strains. Especially, PSY, PDS and LCYb protein sequences shared 100 % identity. Phylogenetic analysis indicated all proteins from GY-H13 firstly clustered with those from other D. salina strains with a bootstrap of 100 %. Multiple alignment indicated several distinct conserved motifs such as aspartate-rich domain (ARD), dinucleotide binding domain (DBD), and carotene binding domain (CBD). These motifs are located near ligand-binding pocket, which may be required for the activity of enzyme. Expression levels of these genes and ß-carotene content were measured over 24-h cycle, showing clear daily dynamics. All genes were dramatically up-regulated in the morning but the highest accumulation of ß-carotene was observed at noon, suggesting a lag-effect between gene transcription and biological response. Furthermore, the accumulation of ß-carotene increased under nitrogen deficiency, Cd exposure and high light and decreased under high salinity in a time-dependent manner. No gene of ß-carotene biosynthesis was up-regulated by high salinity while most genes were activated by the other stresses at the beginning stage of exposure. Growth inhibition and oxidative damage were also observed under high salinity. Overall, transcription activation of ß-carotene biosynthetic genes at the initial stage of stress exposure is a determinant of the increased accumulation of ß-carotene in microalgae, which help their survive under harsh environments. The newly isolated D. salina strain GY-H13 would be a promising microalgae model for investigating the molecular mechanism of stress-induced ß-carotene biosynthesis.

Larimichthys crocea is a suitable bioindicator for monitoring short-term Cd discharge along the coast: An experimental study.

The present study evaluated the feasibility of using a marine cage fish Larimichthys crocea as a model for monitoring short-time Cd discharge near the sewage outlet. Fish were exposed to 0, 20, 100, 500 and 2500 µg/L for 6 h. Cd concentrations in gills, and left and right lobes of hepatopancreas were examined as well as activity levels of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathion-S-transferase (GST), glutathione reductase (GR), lipid peroxidation, glutathione (GSH) and mRNA levels of 19 genes encoding these enzymes. Cd concentrations increased at 100, 500 and 2500 µg/L Cd in gill and at 2500 µg/L Cd in hepatopancreas. Lipid peroxidation increased and GSH levels declined in gills at 2500 µg/L Cd. On the contrary, oxidative damage was not observed in hepatopancreas but GSH levels increased at all tested concentrations of Cd in the left lobe and at 20 µg/L Cd in the right lobe. The enhanced antioxidant response was confirmed in gills due to the increased activity levels of antioxidant enzymes and the up-regulated mRNA levels of most genes. However, disordered antioxidant response was observed in hepatopancreas, showing a dose- and lobe-dependent effect. RNA-seq and q-PCR analyses were performed to investigate differently expressed genes between both lobes under different concentrations of Cd. The most significantly enriched pathway term was pancreatic secretion, where the right lobe showed higher mRNA levels of 18 genes encoding pancreatic digestive enzymes than the left one under Cd stress. Interestingly, both lobes had the same mRNA levels of digestive enzyme genes and antioxidant genes in fish without Cd exposure. Overall, Larimichthys crocea is very sensitive to environmental exposure to cadmium. The present study for the first time investigates Cd-induced antioxidant response in Larimichthys crocea, also is the first to find lobe-dependent effects in fish.

Waterborne zinc induced lobe-dependent effect on oxidative stress and energy metabolism in hepatopancreas of Larimichthys crocea.

The study aimed to compare differences in oxidative stress and energy metabolism between the left and right lobe of hepatopancreas in large yellow croaker Larimichthys crocea exposed to 0 (control), 20, and 100 µM Zn for 96 h. Tipical biomarkers were examined including the proportion of white hepatopancreas, lipid content, malondialdehyde (MDA) level, glutathione (GSH) content, activity levels of enzymes (Cu/Zn-superoxide dismutase, Cu/Zn-SOD; catalase, CAT; glutathione peroxidase, GPx; glutathione reductase, GR; mitochondrial ATP synthase, F-ATPase; malate dehydrogenase, MDH; succinate dehydrogenase, SDH; hepatic lipase, HTGL; lipoprotein lipase, LPL), mRNA levels of genes encoding these enzymes (sod1, cat, gpx1a, gr, atp5b, mdh, sdh, htgl, and lpl), and gene expression of signaling molecules the NF-E2-related nuclear factor 2 (nrf2) and Kelch-like ECH-associated protein 1 (keap1). A whitish color in the left lobe of hepatopancreas was observed in the control and Zn-exposed fish. Contrarily, the right lobe of hepatopancreas tended towards red with increasing Zn levels. The phenomenon was further confirmed by that lipid content was reduced in the right lobe and was not significantly affected in the left lobe by Zn. The right lobe showed higher energy consumption than the left lobe as reflected by the up-regulation of activity levels of HTGL, LPL, F-ATPase, MDH, and SDH. Lipid peroxidation declined by 20 µM Zn and was unchanged by 100 µM Zn in both lobes, which could be explained by increased activity levels of Cu/Zn-SOD and GPx. However, the magnitude of increase in Cu/Zn-SOD activity was greater in the right lobe than that in the left one. The difference in enzyme activity between two lobes may be involved in changes in mRNA levels of sod1, gr, atp5b, sdh, htgl, lpl, and nrf2, which was further confirmed by positive relationships between enzyme activity and gene expression. Our data also showed positive correlations between nrf2 expression and mRNA levels of its target genes, suggesting that Nrf2 was required for the protracted induction of these genes. Our results demonstrated the potential molecular mechanism of Zn-induced differences between lobes of hepatopancreas, suggesting that the sampling part of hepatopancreas should be considered with caution when assessing metal contamination.

Cu pre-exposure alters antioxidant defense and energy metabolism in large yellow croaker Larimichthys crocea in response to severe hypoxia.

The aim of the present study was to evaluate the effects of Cu pre-exposure on antioxidant defense and energy metabolism in the liver of the large yellow croaker exposed to severe hypoxia. Fish were pre-acclimated to 0 and 30 µg Cu L-1 for 96 h, and subsequently exposed to 7.0 and 1.5 mg DO L-1 for another 24 h. Hypoxic stress alone increased reactive oxygen species and hepatic vacuoles. When compared to hypoxic stress alone, hypoxic stress plus Cu pre-exposure increased mortality and ROS production, and worsened histological structure by inhibiting antioxidant defense and aerobic metabolism, and enhancing anaerobic metabolism, suggesting Cu pre-acclimation aggravated hypoxia-induced oxidative damage. NFE2-related nuclear factor 2 and hypoxia-inducible factor-1α might participate in the transcriptional regulation of genes related to antioxidant response and energy metabolism, respectively. In conclusion, Cu pre-acclimation had a synergistic effect on antioxidant response and energy metabolism in fish under severe hypoxia, which contributes to understanding the molecular mechanisms underlying negative effects of Cu pre-acclimation against hypoxic damage in fish.

A functional gene encoding carnitine palmitoyltransferase 1 and its transcriptional and kinetic regulation during fasting in large yellow croaker.

Carnitine palmitoyltransferase 1 (CPT1) plays an essential role in maintaining energy supply via fatty acid oxidation, especially under fasting. In this study, the complete cDNA sequence of cpt1a was cloned from liver of large yellow croaker (Larimichthys crocea), with an open reading frame of 2319 bp encoding a protein of 772 amino acids. Bioinformatics analysis predicted the presence of conserved functional motifs and amino acid residues. The highest mRNA expression of cpt1a was observed in the liver. Phylogenetic tree clearly shows that CPT1A protein is a homologue of mammalian CPT1A. Recombinant protein rCPT1A showed catalytic activity, with Michaelis constant (km) (≈1.38 mM) and maximal reaction rates (Vmax) for carnitine (≈12.66 nmols/min/mg protein). The cpt1a mRNA expression dramatically increased and CPT1 activity remained unchanged after fasting. Fasting did not significantly change Vmax and free carnitine (FC) content in liver. Interestingly, catalytic efficiency (Vmax/Km) and FC/Km increased in fish fasted for 4 days, implying FC contents might be enough to ensure the optimal fatty acid oxidation. Contrarily, both indicators declined when fish fasted for 12 days. The present results demonstrated cpt1a has a biological function and showed that the transcriptional and kinetic regulation of CPT1 during fasting, emphasizing that fasting-induced fatty acid oxidation depends on changes in kinetic properties instead of CPT1 activity and transcription.

Negative effects of acute cadmium on stress defense, immunity, and metal homeostasis in liver of zebrafish: The protective role of environmental zinc dpre-exposure.

In the study, zebrafish were exposed to 0 and 200 µg/L Zn for 8 weeks, and then both groups were transferred to water including 0, 100, and 200 µg/L Cd for 4 days, respectively. Acute Cd exposure caused negative effects on stress defense, immune, and metal transport systems by increasing lipid peroxidation, iNOS activity and mRNA levels of il-6 and inos, and decreasing Cu/Zn-SOD and HSP70 levels, and mRNA levels of sod1, cat, hsp70, p65, mtf-1, znt5, zip7, atp7a, and atp7b. Lipid peroxidation was significantly reduced by Zn pre-exposure under Cd exposure, which may be explained by the enhanced stress defense capacity and the weaken inflammatory response. Firstly, Zn pre-exposure increased MTs and HSP70 levels and CAT activity in Cd-free water, which may facilitate fish quick response to Cd. Secondly, Zn pre-exposure reduced Cd accumulation at 100 and 200 µg/L Cd, down-regulated il-6 and il-1ß at 100 µg/L Cd and p65 at 200 µg/L Cd, and increased Cu/Zn-SOD and CAT activities at 200 µg/L Cd. Thirdly, Zn pre-exposure alone up-regulated transcription factors (hsf1, hsf2, and mtf-1, and nrf2) and their target genes (sod1, cat, hsp70, and mt2) under Cd exposure in a dose-dependent manner. It should be noted that Zn pre-exposure down-regulated several metal transport genes dramatically at 0 and 100 µg/L Cd, which may be an important mechanism for reducing Cd import into livers. Overall, long-term and environmental Zn pre-exposure mitigated Cd toxicity by the enhanced stress defense capacity and the down-regulated metal transport and inflammatory responses.

The lagged effects of environmentally relevant zinc on non-specific immunity in zebrafish.

Responses to zinc (Zn) during exposure have well studied but the effects after the exposure are commonly neglected. In the study, non-specific immune response to zinc in blood and spleen of zebrafish was evaluated after exposure. At first, fish were subjected to 0 (control) and 200 µg/L zinc (Zn) for 6 weeks. Specific growth rate, survival rate, blood albumin level, and the activities of Cu/Zn-SOD and iNOS were not significantly changed by Zn exposure. Conversely, Zn increased the levels of globulin and hemoglobin, CAT activity, and mRNA levels of nrf2, sod1, cat, hsf1, hsp70, p65, il-6, il-1ß, tnf-α and inos. In the second experiment, zebrafish were transferred to a recovery period for 4 and 8 days. The increased activities of Cu/Zn-SOD and CAT and the up-regulated mRNA levels of nrf2, cat, p65, tnf-α, and inos still were observed. In the third experiment, zebrafish from 4 d post-exposure were re-exposed to the high levels of Zn and cadmium (Cd) (600, 1200 µg/L Zn; 100, 200 µg/L Cd) for 4 days. 100 µg/L Cd caused a higher survival rate in the Zn-exposed fish than the control, suggesting Zn pre-exposure might develop the tolerance to Zn and Cd. Although transcriptional levels of sod1, hsf1, hsf2, hsp70, il-6 and il-1ß and activity levels of iNOS recovered to the control levels at 4 and 8 d post-exposure, differences in magnitude of responsiveness were observed between normal fish and Zn-exposed fish. Overall, Zn acclimation persisted when fish recovered, which provides a new perspective about Zn toxicology.

Transcriptional and physiological responses of Dunaliella salina to cadmium reveals time-dependent turnover of ribosome, photosystem, and ROS-scavenging pathways.

Effects on short-term (6 h) and long-term (96 h) exposure to cadmium (Cd) at 0.1, 0.5 and 2.5 mg/L in microalga Dunaliella salina were assessed using both physiological end points and gene expression analysis. Different physiological responses between the short-term and long-term exposures were observed. Upon 6 h after Cd exposure, lipid peroxidation and cell ultrastructure remained unchanged, while contents of chlorophyll a, chlorophyll b, carotenoids were increased at 0.5 and 2.5 mg/L Cd. Contrarily, 96 h after Cd exposure, lipid peroxidation levels were increased, while pigments content was decreased, and damaged cell ultrastructure was apparent at 2.5 mg/L Cd. Activities of antioxidant enzymes (APX, SOD, GST, GPX, and GR) changed differently both at 6 h and 96 h after Cd exposure. Upon 6 h after Cd exposure, SOD and GST activity increased at all three doses, GR and GPX activity increased at 0.5 mg/L Cd while APX activity increased at 0.1 mg/L Cd. Contrarily, 96 h after Cd exposure, activities of all the antioxidant enzymes increased both at 0.1 and 0.5 mg/L Cd; but there was a decrease in SOD and GR activity in D. salina exposed to 2.5 mg/L Cd. RNA-seq and qRT-PCR analyses indicated that genes involved in ROS-scavenge, photosystem, and ribosome functions were differentially expressed. The most significantly enriched function was the ribosome, in which more than 30 ribosome genes were up-regulated at 6 h but down-regulated at 96 h after Cd exposure at 2.5 mg/L. Our study indicated for the first time that genes encoding ribosomal proteins are the primary target for Cd in microalgae, which allowed gaining new insights into temporal dynamics of toxicity and adaptive response pathways in microalgae exposed to metals.

Life-cycle exposure to cadmium induced compensatory responses towards oxidative stress in the liver of female zebrafish.

The current study examined effects of waterborne cadmium (Cd) at environmental relevant concentrations (0, 2.5 and 5 µg/L) on growth, survival, histology, ultrastructure, metal homeostasis, and antioxidant responses in female zebrafish from embryos to sexually maturity for 15 weeks. Growth and survival rate were not significantly affected by Cd exposure. There were no significant changes in ultrastructure of cellular organelles, reactive oxygen species (ROS) levels, lipid peroxidation (LPO) in liver. However, Cd exposure increased Cd and lipid accumulation, reduced contents of zinc, copper and reduced glutathione (GSH), and down-regulated activity of copper/zinc-superoxide dismutase (Cu/Zn-SOD) in liver of zebrafish. Contrarily, the mRNA and activity levels of catalase (CAT), the mRNA levels of Cu/Zn-SOD, and the mRNA and protein levels of metallothioneins (MTs) were up-regulated. The transcriptional regulation of Cu and Zn transporters might be a vital mechanism by which fish slow the Zn and Cu uptake. Taken together, our data demonstrated that long-term and low-dose Cd induced adaptive responses with interlinked compensatory mechanism, which may protect fish against oxidative stress.

Effects of heat and cadmium exposure on stress-related responses in the liver of female zebrafish: Heat increases cadmium toxicity.

In this study, female zebrafish (Danio rerio) were exposed to 26°C or 34°C, 0 or 197µg/L cadmium (Cd), singly or in combination for 7days. Multiple stress-related indicators were evaluated in the liver. Mortality, lipid peroxidation (LPO) and ultrastructural damage increased significantly by Cd exposure alone, and were not affected by heat alone. Interestingly, the combined exposure increased LPO, ultrastructural damage, and mortality compared with Cd exposure alone. The results indicated that elevated temperature increased Cd toxicity, which could be explained by several reasons. Firstly, Cd-exposed fish failed to activate the antioxidant defense system under heat stress. Secondly, expression levels of heat shock protein 70 (HSP70) were not significantly up-regulated by heat in Cd-exposed fish but increased by 117 times in Cd-free fish. Besides, hypermethylation of heat shock factor (HSF) binding motif in HSP70 promoter was observed during the combined exposure, indicating that simultaneous exposure may have partially suppressed the cytoprotective up-regulation of HSP70. Thirdly, heat induced an immunosuppressive effect in Cd-exposed fish, as reflected by the reduced mRNA and activity levels of nitric oxide synthase (iNOS) and interleukin-1ß (IL-1ß) expression levels. Finally, heat down-regulated Zir-, Irt-like protein 8 (ZIP8) and copper transporter 1 (CTR1) and up-regulated metallothioneins (MTs) in Cd-exposed fish, possibly suggesting Cu and Zn depletion and Cd accumulation. Hence, our data provide evidences that warmer temperatures can potentiate Cd toxicity, involved in the regulation of gene transcription, enzymatic activity, and DNA methylation. We found that heat indicators showed varied sensitivity between normal and Cd-exposed fish, emphasizing that the field metal pollution should be carefully considered when evaluating effects of climate change.