Negative effect of chronic cadmium exposure on growth, histology, ultrastructure, antioxidant and innate immune responses in the liver of zebrafish: Preventive role of blue light emitting diodes.

The present study explored the possible preventive effects of blue light emitting diodes (LEDs) on cadmium (Cd)-induced oxidative stress and immunotoxicity in zebrafish. To this end, zebrafish were exposed to a white fluorescent bulb or blue LEDs (LDB, peak at 450nm, at an irradiance of 0.9W/m2), and 0 or 30µgL-1 waterborne Cd for 5 weeks. Growth performance, survival rate, and hepatic histology, ultrastructure, antioxidant and innate immune responses were determined in zebrafish. Cd exposure alone reduced growth and survival rate, and induced oxidative damage and changes in histology and ultrastructure. However, Cd exposure in combination with LDB apparently relieved these negative effects. The alleviation of adverse effects might result from the up-regulation of antioxidant and innate immune genes at transcriptional, translational, or post-translational levels. Cd exposure alone dramatically enhanced mRNA levels of nuclear transcription factor κB (NF-κB) and E2-related factor (Nrf2). However, compared to Cd exposure alone, Cd exposure in combination with LDB apparently down-regulated both genes. Taken together, our results suggest that chronic Cd exposure induced a negative effect on zebrafish, possibly involved in NF-κB-induced immunotoxicity and Nrf2-induced oxidative stress. Finally, for the first time, our data demonstrated that LDB could protect fish against Cd toxicity.

Preheating mitigates cadmium toxicity in zebrafish livers: Evidence from promoter demethylation, gene transcription to biochemical levels.

The working hypothesis for this study was that moderate heat stress would alleviate the deleterious effects of subsequent cadmium (Cd) exposure on fish. Thus, zebrafish (Danio rerio) were subjected to water maintained at 26°C and 34°C for 4days, and then exposed to 0 or 200µg/L Cd for 1 week at 26°C. Multiple indicators were measured from livers of zebrafish at different levels, including DNA, RNA, protein and enzymatic activity associated with oxidative stress, inflammation and metal transport. The ameliorative effect of preheatinging on Cd toxicity was demonstrated. In the Cd-exposed groups, preheating decreased mortality and lipid peroxidation, increased activity levels of catalase (CAT) and copper/zinc-superoxide dismutase (Cu/Zn-SOD), and up-regulated mRNA levels of heat shock protein 70 (HSP70) and heat shock factor 2 (HSF2). Preheating also mitigated Cd-induced increases in protein and mRNA levels of metallothioneins (MTs), and mRNA levels of several inflammation-related genes. Furthermore, preheating alone dramatically up-regulated mRNA levels of genes related to antioxidant and immune defenses, zinc and copper transporters, protein folding, and reduced methylation levels in the HSF binding motif of the HSP70 promoter. Overall, preheating-induced accumulation of transcripts via demethylation might support the rapid defense responses at post-transcriptional levels caused by subsequent Cd exposure, indicating an adaptive mechanism for organisms exposed to one mild stressor followed by another.

Circadian time-dependent antioxidant and inflammatory responses to acute cadmium exposure in the brain of zebrafish.

Up to date, little information is available on effects of circadian rhythm on metal-induced toxicity in fish. In this study, zebrafish were acutely exposed to 0.97mgL-1 cadmium for 12h either at ZT0 (the light intensity began to reached maximum) or at ZT12 (light intensity began to reached minimum) to evaluate the temporal sensitivity of oxidative stress and inflammatory responses in the brain of zebrafish. Profiles of responses of some genes at mRNA, protein and activity levels were different between ZT0 and ZT12 in the normal water. Exposure to Cd induced contrary antioxidant responses and similar inflammatory responses between ZT0 and ZT12. However, the number of inflammatory genes which were up-regulated was significantly greater at ZT12 than at ZT0. And, the up-regulated inflammatory genes were more responsive at ZT12 than at ZT0. At ZT12, antioxidant genes were down-regulated at mRNA, protein and activity levels. Contrarily, antioxidant genes were not affected at mRNA levels but activated at the protein and/or activity levels at ZT0. Reactive oxygen species (ROS) sharply increased and remained relatively stable when fish were exposed to Cd at ZT12 and ZT0, respectively. Positive correlations between ROS levels and mRNA levels of nuclear transcription factor κB (NF-κB) and between mRNA levels of NF-κB and its target genes were observed, suggesting that ROS may play an essential role in regulating the magnitude of inflammatory responses. Taken together, oxidative stress and immunotoxicity in the brain were more serious when fish were exposed to Cd in the evening than in the morning, highlighting the importance of circadian rhythm in Cd-induced neurotoxicity in fish.

Heat indicators of oxidative stress, inflammation and metal transport show dependence of cadmium pollution history in the liver of female zebrafish.

Environmental stressors such as high temperature and metal exposure may occur sequentially, simultaneously, previously in aquatic ecosystems. However, information about whether responses to high temperature depend on Cd exposure history is still unknown in fish. Zebrafish were exposed to 0 (group 1), 2.5 (group 2) and 5µg/L (group 3) cadmium (Cd) for 10 weeks, and then each group was subjected to Cd-free water maintained at 26°C and 32°C for 7days respectively. 26 indicators were used to compare differences between 26°C and 32°C in the liver of female zebrafish, including 5 biochemical indicators (activity of Cu/Zn-SOD, CAT and iNOS; LPO; MT protein), 8 molecular indicators of oxidative stress (mRNA levels of Nrf2, Cu/Zn-SOD, CAT, HSF1, HSF2, HSP70, MTF-1 and MT), 5 molecular indicators of inflammation (mRNA levels of IL-6, IL-1ß, TNF-α, iNOS and NF-κB), 8 molecular indicators of metal transport (mRNA levels of, ZnT1, ZnT5, ZIP8, ZIP10, ATP7A, ATP7B and CTR1). All biochemical indicators were unchanged in group 1 and changed in group 2 and 3. Contrarily, differences were observed in almost all of molecular indicators of inflammation and metal transport in group 1, about half in group 2, and few in group 3. We also found that all molecular indicators of oxidative stress in group 2 and fewer in group 1 and 3 were significantly affected by heat. Our data indicated that heat indicators of oxidative stress, inflammation and metal transport showed dependence of previous cadmium exposure in the liver of zebrafish, emphasizing metal pollution history should be carefully considered when evaluating heat stress in fish.

Acute exposure to waterborne cadmium induced oxidative stress and immunotoxicity in the brain, ovary and liver of zebrafish (Danio rerio).

Cadmium (Cd) is an environmental contaminant that poses serious risks to aquatic organisms and their associated ecosystem. The mechanisms underlying Cd-induced oxidative stress and immunotoxicity in fish remain largely unknown. In this study, adult female zebrafish were exposed to 0 (control), 1mgL-1 Cd for 24h and 96h, and the oxidative stress and inflammatory responses induced by Cd were evaluated in the brain, liver and ovary. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) increased in a time-dependent manner after treatment with Cd in the brain and liver. The increase may result from the disturbance of genes including copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), inducible nitric oxide synthase (iNOS), and ciclooxigenase-2 (COX-2) at mRNA, protein and activity levels. Although ROS, NO and MDA were not significantly affected by Cd in the ovary, the up-regulation of Cu/Zn-SOD, CAT, iNOS, and COX-2 was observed. Exposure to Cd induced a sharp increase in the protein levels of tumor necrosis factor alpha (TNF-α) in the brain, liver and ovary, possibly contributing to activate inflammatory responses. Furthermore, we also found a dramatic increase in mRNA levels of NF-E2-related factor 2 (Nrf2) and nuclear transcription factor κB (NF-κB) at 24h in the liver and ovary. The corresponding changes in the mRNA levels of Kelch-like-ECH-associated protein 1 (Keap1a and Keap1b) and the inhibitor of κBα (IκBαa and IκBαb) may contribute to regulate the transcriptional activity of Nrf2 and NF-κB, respectively. Contrarily, mRNA levels of Nrf2, NF-κB, Keap1, Keap1b, IκBαa and IκBαb remained stable at 24 and 96h in the brain. Taken together, we demonstrated Cd-induced oxidative stress and immunotoxicity in fish, possibly through transcriptional regulation of Nrf2 and NF-κB and gene modifications at transcriptional, translational, post-translational levels, which would greatly extend our understanding on the Cd toxicity.

Molecular cloning, expression analysis and cellular localization of an LFRFamide gene in the cuttlefish Sepiella japonica.

Neuropeptides are important regulators of physiological processes in metazoans, such as feeding, reproduction, and heart activities. In this study, an LFRFamide gene was identified from the cuttlefish Sepiella japonica (designated as SjLFRFamide). The full-length sequence of SjLFRFamide cDNA has 841bp, and the open reading frame contains 567bp encoding 188 amino acids, which shared high similarity with precursor SOFaRP2 from Sepia officinalis. The deduced SjLFRFamdie precursor protein contains a signal peptide and four different FLPs (FMRFamide-like peptides): one pentapeptide (TIFRFamide), two hexapeptides (NSLFRFamide and GNLFRFamide) and one heptapeptide (PHTPFRFamide). Multiple sequence alignment showed that SjLFRFamide contains rather conserved mature peptides, which all ended in FRF. The phylogenetic analysis suggests that SjLFRFamide belongs to the LFRFamide subfamily. The tissue distribution analysis through quantitative real-time PCR method showed that SjLFRFamide mRNA is significantly expressed in the brain, and slight trace are detected in female nidamental gland and accessory nidamental gland. In situ hybridization assay of the brain indicated that SjLFRFamide is transcribed in several different functional lobes, suggesting SjLFRFamide might associate with multiple physiological regulations, such as feeding, chromatophore regulation and reproduction. This is the first study describing LFRFamide in S. japonica, which might have great importance for cuttlefish artificial breeding.

Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd(2.).

The 70-kDa heat shock proteins (HSP70) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. The full-length HSP70 cDNA of Sepiella maindroni (designated as SmHSP70, GenBank accession no. KJ739788) was 2109 bp, including an ORF of 1950 bp encoding a polypeptide of 649 amino acids with predicted pI/MW 5.24/71.30 kDa, a 62 bp-5'-UTR and a 97 bp-3'-UTR. BLASTp analysis and phylogenetic relationship strongly suggested that the amino acid sequence was a member of HSP70 family. Multiple sequence alignment revealed that SmHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTP-binding site motif and 'EEVD' motif. Time-dependent mRNA expression of SmHSP70 in the liver was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmHSP70 played a significant role in mediating the environmental stress and immune response against pathogens.