Parental Transfer of Microcystin-LR-Induced Innate Immune Dysfunction of Zebrafish: A Cross-Generational Study.

Transgenerational effects of microcystin-LR (MC-LR) released by cyanobacterial blooms have become a hot topic. In the present study, adult zebrafish pairs were exposed to 0, 0.4, 2, and 10 µg/L MC-LR for 60 days and the embryos (F1 generation) were hatched without or with continued MC-LR exposures at the same concentrations until 5 days postfertilization (dpf). The results showed the existence of MC-LR both in F0 gonads and in F1 embryos and indicated that MC-LR could be transferred directly from the F0 adult fish to F1 offspring. The adverse effects on sex hormone levels, sexual development, and fecundity in F0 generation along with abnormal development in F1 offspring were observed. Furthermore, downregulation of antioxidant genes (cat, mn-sod, gpx1a) and upregulation of innate immune-related genes (tlr4a, myd88, tnfα, il1ß) as well as increased proinflammation cytokine contents (TNF-α, IL-1ß, IL-6) were noticed in F1 offspring without/with continued MC-LR exposures. In addition, significant differences between the two F1 embryo treatments demonstrated that continuous MC-LR exposure could result in a higher degree of inflammatory response compared to those without MC-LR exposure. Our findings revealed that MC-LR could exert cross-generational effects of immunotoxicity by inhibiting the antioxidant system and activating an inflammatory response.

AMPKα1 overexpression improves postoperative cognitive dysfunction in aged rats through AMPK-Sirt1 and autophagy signaling.

Postoperative cognitive dysfunction (POCD) is a common complication in elderly patients who undergo surgery involving anesthesia. Its underlying mechanisms remain unclear. Autophagy plays an important role in the damage and repair of the nervous system and is associated with the development of POCD. Using a rat model, adenosine monophosphate-activated protein kinase α1 (AMPKα1), an important autophagy regulator, was found to be significantly downregulated in rats with POCD that was induced by sevoflurane anesthesia or by appendectomy. Overexpression of AMPKα1-ameliorated POCD, as indicated by decreased escape latencies and increased target quadrant swimming times, swimming distances, and platform crossing times during Morris water maze tests. AMPKα1 overexpression activated autophagy signals by increasing the expression of light chain 3 II (LC3-II) and Beclin1 and decreasing the expression of p62 in the hippocampus of rats with POCD. Moreover, blocking autophagy by 3-methyladenine partly attenuated AMPKα1-mediated POCD improvement. Furthermore, overexpression of AMPKα1 could upregulate the expression of p-AMPK and Sirt1 in the hippocampus of rats with POCD. Intriguingly, inhibiting AMPK signals via Compound C effectively attenuated AMPKα1-mediated POCD improvement, concomitant with the downregulation of p-AMPK, Sirt1, LC3-II, and Beclin1 and the upregulation of p62. We thus concluded that overexpression of AMPKα1 can improve POCD via the AMPK-Sirt1 and autophagy signaling pathway.

Efficient U(VI) adsorption on iron/carbon composites derived from the coupling of cellulose with iron oxides: Performance and mechanism.

Novel iron/carbon composites were successfully prepared via coupling of cellulose with iron oxides (e.g. α-FeOOH, Fe2O3 and Fe(NO3)3·9H2O) at different temperatures under nitrogen atmosphere. Characterization by various techniques implied that chemical interaction between cellulose and Fe3O4/Fe0 existed in the as-prepared iron/carbon composites. The site of interaction between cellulose and iron precursors was illustrated (mainly combined with COO-). The self-reduction of Fe3+ to Fe2+ or even Fe0 and the interaction between carbon and Fe3O4/Fe0 in the calcination process realized the strong magnetism of the composites. Batch experiments and spectroscopic techniques indicated that the maximum adsorption capacity of MHC-7 for U(VI) (105.3 mg/g) was significantly higher than that of MGC-7 (86.0 mg/g) and MFC-7 (79.0 mg/g), indicating that Fe2O3 can be regarded as the remarkable iron resource for the iron/carbon composites. XPS results revealed that the oxygen-containing groups were responsible for the adsorption process of U(VI) on iron/carbon composites, and the adsorption of carbon and reduction of Fe0/Fe3O4 toward U(VI) were synergistic during the reaction process. In addition, the iron/carbon composites exhibited a good recyclability, recoverability and stability for U(VI) adsorption in the regeneration experiments. These findings demonstrated that the iron/carbon composites can be considered as valuable adsorbents in environmental cleanup and the Fe2O3 was a promising iron resource for the preparation of iron/carbon composites.

Survival strategies of Wuchang bream (Megalobrama amblycephala) juveniles for chronic ammonia exposure: Antioxidant defense and the synthesis of urea and glutamine.

This study aimed to explore how Wuchang bream (Megalobrama amblycephala) survive and defend against the toxicity of ambient total ammonia nitrogen (0, 5, 10, 20 and 30 mg/L TA-N) during 30-day exposure. As a result, hepatic malondialdehyde and protein carbonylation as well as histopathological alterations increased with increasing TA-N level, which suggested that chronic ammonia exposure caused oxidative stress and damage in the liver of fish. Meanwhile, the activities of hepatic total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glucose 6-phosphate dehydrogenase (G6PD) as well as the mRNA expression of Cu/Zn sod, cat, gpx and g6pd were elevated significantly along with significant reduction of glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH) (P < 0.05). These results indicated that hepatic antioxidant responses were activated to alleviate oxidative damages induced by ammonia, in which lower-concentration ammonia only initiate SOD-CAT-GR-G6PDH defense and higher ammonia activated the SOD-CAT-GPx-GSH-GR-G6PDH antioxidant response. In addition, significant increases of serum urea and hepatic ammonia, urea, glutamine, arginase as well as glutamine synthetase were detected with the increase of TA-N (P < 0.05), while serum ammonia levels kept stable (P > 0.05). The present findings further revealed that ammonia could be detoxified directly into glutamine and urea in Wuchang bream to cope with ammonia exposure. In conclusion, under chronic ammonia exposure, enhanced hepatic antioxidant responses as well as increased urea and glutamine synthesis worked in combination to allow Megalobrama amblycephala to defend against environmental ammonia toxicity.

Waterborne microcystin-LR exposure induced chronic inflammatory response via MyD88-dependent toll-like receptor signaling pathway in male zebrafish.

Waterborne microcystin-LR (MC-LR) released by cyanobacterial blooms in eutrophic water bodies have caused serious risk to aquatic animal and human health. In the present study, we for the first time conducted a comprehensive in vivo investigation on chronic inflammatory responses and its molecular pathways of different environmental relevant levels of MC-LR (0, 0.4, 2 and 10 µg/L) in male zebrafish (Danio rerio). The results showed that chronic MC-LR exposure caused splenic inflammatory changes including the formation of melano-macrophage centers, remarkable elevation of serum tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL1ß) levels as well as significant upregulated expression of MyD88-dependent toll-like receptor (TLR/MyD88) signaling pathway genes (tlr4a, myd88, erk2, p38a, il1ß and tnfα). The immunohistochemical and western blot results further validated that higher MC-LR concentrations tended to enhance the MyD88 signal. Moreover, significant decreases of serum C3 levels along with splenic c3b expression in the 10 µg/L exposure group proved that chronic MC-LR exposure could ultimately decrease the innate immunity of fish. Our findings revealed that chronic exposure of MC-LR could cause chronic inflammation through TLR/MyD88 signaling pathway and subsequently induce immune disorders in male zebrafish, which also urge us to pay more attention on the potential immunotoxicity of long-term exposure to low concentration of MC-LR.

Chronic Microcystin-LR Exposure Induces Abnormal Lipid Metabolism via Endoplasmic Reticulum Stress in Male Zebrafish.

In order to explore effects of low levels of continuous microcystin-LR (MC-LR) (a cyanotoxin) exposure on hepatic lipid metabolism on the basis of the endoplasmic reticulum stress (ERS) pathway, we exposed adult male zebrafish to MC-LR (0, 1, 5, and 25 µg/L) for 60 days, and hepatic histopathology as well as lipid metabolic parameters were determined with mRNA levels of ERS signal molecules and downstream factors, along with genes associated with lipid metabolism in zebrafish liver. The results revealed that prolonged exposure to MC-LR remarkably altered the levels of hepatic total cholesterol and triglyceride and led to hepatic steatosis, which was also confirmed by hepatic cytoplasmic vacuolization in Hematoxylin/eosin (H&E) stain and lipid droplet accumulation in Oil Red O stain. The severity of hepatic damage and lipidation was increased in a dose-related manner. MC-LR exposure significantly upregulated transcriptional levels of ERS markers including hspa5, mapk8, and chop, indicating the occurrence of ERS in the liver of zebrafish. Concurrently, MC-LR significantly improved mRNA expression of unfolded protein response (UPR) pathway-related genes including atf6, eif2ak3, ern1, and xbp1s, suggesting that all of the three UPR branches were activated by MC-LR. MC-LR also induced significant upregulation of downstream lipid metabolism-related factors and genes including srebf1, srebf2, fatty acid synthase (fasn), acetyl-CoA carboxylase (acaca), stearoyl-CoA desaturase (scd), HMG CoA reductase (hmgcra), and HMG CoA synthase (hmgcs1), and downregulation of genes associated with lipolysis such as triglyceride hydrolase gene (atgl), hormone-sensitive enzyme gene (hsla), and carnitine palmitoyltransferase gene (cpt1aa). Our present results indicated that the cause of hepatic lipid accumulation by MC-LR was mainly by upregulating lipogenic and cholesterol genes but downregulating the expression of lipolytic genes through the induction of srebf1 and srebf2, which were involved in the activation of ERS signal pathways.

Persistent Exposure to Environmental Levels of Microcystin-LR Disturbs Cortisol Production via Hypothalamic-Pituitary-Interrenal (HPI) Axis and Subsequently Liver Glucose Metabolism in Adult Male Zebrafish (Danio rerio).

There is growing evidence that microcystin-LR (MC-LR) is a new endocrine disruptor, whereas the impacts of persistent exposure to MC-LR on the hypothalamic-pituitary-interrenal (HPI) axis and health hazards thereafter have not been investigated. In this work, adult male zebrafish (Danio rerio) were immersed into MC-LR solutions at concentrations of 0, 1, 5 and 25 µg/L for 30 d, respectively. The results showed that persistent MC-LR exposure caused an extensive upregulation of HPI-axis genes but an inhibition of brain nuclear receptors (gr and mr), which finally increased serum cortisol levels. Furthermore, the decreased expression of hepatic gr might partly be responsible for the strong inhibition on the expression of downstream genes involved in glucose metabolic enzymes, including gluconeogenesis-related genes (pepck, fbp1a, g6pca), glycogenolysis-related gene (pyg), glycolysis-related genes (gk, pfk1b, pk) and glycogenesis-related gene (gys2). These findings are in accordance with the decline in serum glucose, indicating that long-term MC-LR exposure caused a lower production of glucose relative to glucose lysis. Our above results firstly establish the link between persistent MC-LR exposure and impaired glucose metabolism, suggesting that long-term MC-LR-mediated stress might threaten fish's health.

Full-Length Transcriptome from Camellia oleifera Seed Provides Insight into the Transcript Variants Involved in Oil Biosynthesis.

Camellia oleifera Abel., belonging to the genus Camellia of Theaceae, has been widely used as a cooking oil, lubricant, and in cosmetics. Because of complicated polyploidization and large genomes, reference genome information is still lacking. Systematic characterization of gene models based on transcriptome data is a fast and economical approach for C. oleifera. Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) and Illumina RNA-Seq combined with gas chromatography were performed for exploration of oil biosynthesis, accumulation, and comprehensive transcriptome analysis in C. oleifera seeds at five different developmental stages. We report the first full-length transcriptome data set of C. oleifera seeds comprising 40,143 deredundant high-quality isoforms. Among these isoforms, 37,982 were functionally annotated, and 271 (2.43%) belonged to fatty acid metabolism. A total of 8,344 full-length unique transcript models were obtained, and 8,151 (97.69%) of them produced more than two isoforms, suggesting a high degree of transcriptome complexity in C. oleifera seeds. A total of 783 alternative splicing (AS) events were identified, among which the retained intron was the most abundant. We also obtained 1,910 long noncoding RNAs (lncRNAs) and found that AS events occurred in these lncRNAs. Potential transcript variants of genes involved in oil biosynthesis were also investigated. After performing weighted correlation network analysis, we found seven "gene modules" and hub genes for each module showing a significant association with oil content. The series test of clusters classified these modules into four significant profiles based on gene expression patterns. Protein-protein interaction network analysis showed that upregulated WRI1 interacted with 17 genes encoding the enzymes playing key roles in oil synthesis. MYB and ZIP transcriptional factors also showed significant interactions with key genes involved in oil synthesis. Collectively, our data advance the knowledge of RNA isoform diversity in seeds at different developmental stages and provide a rich resource for functional studies on oil synthesis in C. oleifera.

The Protective Roles of Dietary Selenium Yeast and Tea Polyphenols on Growth Performance and Ammonia Tolerance of Juvenile Wuchang Bream (Megalobrama amblycephala).

In order to investigate protective roles of dietary selenium yeast (SY) and tea polyphenols (TPs) on growth of juvenile Wuchang bream (Megalobrama amblycephala) and its resistance under ammonia stress, juvenile Wuchang bream were randomly assigned into four groups: a control group fed basal diets and three treatment groups fed basal diets supplemented with 0.50 mg/kg SY, 50 mg/kg TPs and a combination of 0.50 mg/kg SY and 50 mg/kg TPs, respectively. After 60 days of feeding, growth parameters of Wuchang bream were measured along with serum hormones and the transcription of growth axis-related genes. Then fish were exposed to ammonia stress of 22.5 mg/L total ammonia nitrogen. Hepatic oxidative damage parameters, antioxidant responses and ultrastructure were evaluated before ammonia exposure (0 h) and at 3, 6, 12, 24, and 48 h after ammonia exposure. Results show that before ammonia exposure, the growth parameters, serum GH and IGF-1 levels as well as the growth axis-related gene expression (gh, ghr2 and igf-1) for the SY and combination groups were higher than those determined for the fish on the control diet. In contrast, the administration of TP alone didn't have significant effects on the growth parameters and growth-related hormones. After ammonia exposure, compared with the control, remarkable increases in the activity and mRNA expression of hepatic antioxidant enzymes (glutathione peroxidase and catalase superoxide dismutase) in three treatment groups were observed along with decreases of hepatic malondialdehyde and protein carbonylation levels, indicating that the single and combined supplementation of SY and TPs could enhance antioxidant capacity to alleviate oxidative stress and damage by ammonia. Consistent with this finding, alterations of the liver ultrastructure in three treatment groups were less severe and faster recovery than in the control group after ammonia exposure. In conclusion, a basal diet supplemented with the combination of 0.50 mg/kg SY and 50 mg/kg TPs could has very beneficial effects on the whole aspects of the growth and ammonia resistance in Wuchang bream juveniles.

Nitrite Enhances MC-LR-Induced Changes on Splenic Oxidation Resistance and Innate Immunity in Male Zebrafish.

Hazardous contaminants, such as nitrite and microcystin-leucine arginine (MC-LR), are released into water bodies during cyanobacterial blooms and may adversely influence the normal physiological function of hydrobiontes. The combined effects of nitrite and MC-LR on the antioxidant defense and innate immunity were evaluated through an orthogonal experimental design (nitrite: 0, 29, 290 µM; MC-LR: 0, 3, 30 nM). Remarkable increases in malondialdehyde (MDA) levels have suggested that nitrite and/or MC-LR exposures induce oxidative stress in fish spleen, which were indirectly confirmed by significant downregulations of total antioxidant capacity (T-AOC), glutathione (GSH) contents, as well as transcriptional levels of antioxidant enzyme genes cat1, sod1 and gpx1a. Simultaneously, nitrite and MC-LR significantly decreased serum complement C3 levels as well as the transcriptional levels of splenic c3b, lyz, il1ß, ifnγ and tnfα, and indicated that they could jointly impact the innate immunity of fish. The severity and extent of splenic lesions were aggravated by increased concentration of nitrite or MC-LR and became more serious in combined groups. The damages of mitochondria and pseudopodia in splenic macrophages suggest that oxidative stress exerted by nitrite and MC-LR aimed at the membrane structure of immune cells and ultimately disrupted immune function. Our results clearly demonstrate that nitrite and MC-LR exert synergistic suppressive effects on fish innate immunity via interfering antioxidant responses, and their joint toxicity should not be underestimated in eutrophic lakes.

The synergistic effects of waterborne microcystin-LR and nitrite on hepatic pathological damage, lipid peroxidation and antioxidant responses of male zebrafish.

Hazardous materials from decaying cyanobacterial blooms, such as microcystin-LR (MC-LR) and nitrite pose serious challenges to aquatic organisms. To assess combined toxic effects of MC-LR and nitrite on hepatic pathology, lipid peroxidation and antioxidant responses of fish, adult male zebrafish (Danio rerio) were exposed to solutions with different combined concentrations of MC-LR (0, 3, 30 µg/L) and nitrite (0, 2, 20 mg/L) for 30 d. The results showed that hepatic pathological lesions progressed in severity and extent with increasing concentration of single factor MC-LR or nitrite and became more severe in co-exposure groups. Concurrently, significant increases in malondialdehyde (MDA) revealed the occurrence of oxidative stress caused by MC-LR, nitrite and both of them, which was indirectly verified by remarkable decreases in the total antioxidant capacity (T-AOC) as well as the transcription and activity of antioxidant enzymes (CAT and GPx). Hepatic mitochondria were damaged as the common action site of MC-LR and nitrite, suggesting that oxidative stress played a significant role in the mechanisms of the hepatotoxicity of MC-LR and nitrite. The depletion of hepatic glutathione (GSH) indicated the importance of GSH/glutathione-S-transferases (GST) system in these two chemicals detoxification. These results clearly illustrated that MC-LR and nitrite have synergistic effects on the histostructure, antioxidant capacity and detoxification capability in the liver of zebrafish. Therefore, the combined pollution of MC-LR and nitrite in eutrophic lakes can reduce the defense mechanism of the fish and accelerate the consumption of GSH, which compromise the survival of the fish during prolonged cyanobacterial blooms episodes.

Sirt3-Mediated Autophagy Contributes to Resveratrol-Induced Protection against ER Stress in HT22 Cells.

Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in neuronal survival and death. Resveratrol is a non-flavonoid polyphenol that has neuroprotective effects against many neurological disorders. Here, we investigated the potential protective effects of resveratrol in an in vitro ER stress model mimicked by tunicamycin (TM) treatment in neuronal HT22 cells. We found that TM dose-dependently decreased cell viability and increased apoptosis, which were both significantly attenuated by resveratrol treatment. Resveratrol markedly reduced the expression or activation of ER stress-associated factors, including GRP78, CHOP, and caspase-12. The results of immunocytochemistry and western blot showed that resveratrol promoted autophagy in TM-treated cells, as evidenced by increased LC3II puncta number, bcelin1 expression and LC3II/LC3I ratio. Pretreatment with the autophagy inhibitor chloroquine could reduce the protective effects of resveratrol. In addition, the expression of Sirt3 protein and its downstream enzyme activities were significantly increased in resveratrol-treated HT22 cells. To confirm the involvement of Sirt3-mediated mechanisms, siRNA transfection was used to knockdown Sirt3 expression in vitro. The results showed that downregulation of Sirt3 could partially prevented the autophagy and protection induced by resveratrol after TM treatment. Our study demonstrates a pivotal role of Sirt3-mediated autophagy in mediating resveratrol-induced protection against ER stress in vitro, and suggests the therapeutic values of resveratrol in ER stress-associated neuronal injury conditions.

Single and combined exposure of microcystin-LR and nitrite results in reproductive endocrine disruption via hypothalamic-pituitary-gonadal-liver axis.

Microcystin-LR (MC-LR) released by Microcystis blooms degradation usually co-exists with a chemical called nitrite, posing a serious harm to aquatic organisms. To assess the single and combined effects of MC-LR and nitrite on the reproductive endocrine system, a fully factorial experiment was designed and adult male zebrafish (Danio rerio) were exposed to 9 treatment combinations of MC-LR (0, 3, 30 µg/L) and nitrite (0, 2, 20 mg/L) for 30 d. The results showed that both MC-LR and nitrite caused concentration-dependent effects including the growth inhibition, decreased gonad index as well as testicular injuries with widen intercellular spaces and seminiferous epithelium deteriorations. And testicular pathological changes in the co-exposure groups of MC-LR and nitrite were similar but more serious than those in single-factor exposure groups. Concurrently, exposure to MC-LR or nitrite alone could significantly decrease T levels by downregulating gene expressions (gnrh2, lhß, ar, lhr) in the hypothalamic-pituitary-gonadal-liver-axis (HPGL-axis), and there were significant interactions between MC-LR and nitrite on them. In contrast, E2 levels as well as transcriptional levels of cyp19a1b, cyp19a1a and vtg1 showed significant inductions with increasing MC-LR concentrations, indicating an estrogen-like effect of MC-LR. Our findings illustrated that co-exposure of MC-LR and nitrite synergistically cause reproductive dysfunction by interfering with the HPGL axis in male fish, which prompt us to focus more on the potential risks in fish reproduction and even population dynamics due to the wide occurrence of toxic cyanobacterial blooms.