Effects of the glucocorticoid clobetasol propionate and its mixture with cortisol and different class steroids in adult female zebrafish

Ecotoxicological effects of glucocorticoids and steroid mixtures in the environment are not sufficiently known. Here we investigate effects of 11–14 days exposure of female zebrafish to the glucocorticoid clobetasol propionate (Clo), cortisol (Cs), their mixture and mixtures with five different class steroids (Clo + triamcinolone + estradiol + androstenedione + progesterone) in liver, brain and gonads. Cs showed little activity, while Clo reduced the condition factor at 0.57 and 6.35 μg/L. Clo induced differential expression of genes in the liver at 0.07–6.35 μg/L, which were related to circadian rhythm (per1, nr1d2), glucose metabolism (g6pca, pepck1), immune system response (fkbp 5, socs3, gilz), nuclear steroid receptors (pgr and pxr), steroidogeneses and steroid metabolism (hsd11b2, cyp2k22). Clo caused strong transcriptional down-regulation of vtg. Similar upregulations occurred in the brain for pepck1, fkbp5, socs3, gilz, hsd11b2, and nr1d2a, while cyp19b was down-regulated. Effects of Clo + Cs mixtures were similar to Clo alone. Transcriptional alterations were different in mixtures of five steroids with no alteration of vtg in the liver due to counteraction of Clo and estradiol. Induction of fkbp5 (brain) and sult2st3 (liver) and downregulation of cyp19a (gonads) occurred at 1 μg/L. Histological effects of the five steroids mixture in gonads were characterized by a decrease of mature oocytes. Our data indicate that effects of steroids of different classes sum up to an overall joint effect driven by the most potent steroid Clo.ISSN:0166-445XISSN:1879-151

Comparative effects of nodularin and microcystin-LR in zebrafish: 2. Uptake and molecular effects in eleuthero-embryos and adult liver with focus on endoplasmic reticulum stress

Microcystin (MC) and nodularin are structurally similar cyanobacterial toxins that inhibit protein phosphatases. Additional modes of action are poorly known, in particular for nodularin. In our associated work, we showed that active cellular uptake is mediated by the organic anion transporting polypeptide drOatp1d1 in zebrafish (Faltermann et al., 2016). Here, we assessed the transcriptional expression of three genes encoding three uptake transporters during embryonic development from 24 h post fertilization (hpf) to 168 hpf. Transcripts of drOatp1d1 and drOatp2b1 are present at 24 hpf. The abundance increased after hatching and remained about constant up to 168 hpf. Transcripts of drOatp2b1 were most abundant, while drOapt1f transcripts showed very low relative abundance compared to drOatp1d1 and drOatp2b1. We further demonstrated the uptake of fluorescent labeled MC-LR in eleuthero-embryos and its accumulation in the glomerulus of the pronephros. An important molecular effect of MC-LR in human liver cells is the induction of endoplasmic reticulum (ER)-stress. Here, we investigated, whether MC-LR and nodularin similarly lead to induction of ER-stress in zebrafish by analyzing changes of mRNA levels of genes indicative of ER-stress. In zebrafish liver organ cultures short- and long-term exposures to 0.15 and 0.3 μmol L−1 MC-LR, and 0.5 and 1 μM L−1 nodularin led to significant transcriptional induction of several ER-stress marker genes, including the chaperone glucose regulated protein 78 (bip), the spliced form of x-box binding protein (xbp-1s), the CCAAT-enhancer-binding protein homologous protein (chop) and activating transcription factor 4 (atf4). Furthermore, strong transcriptional changes occurred for tumor necrosis factor alpha (tnfa) and dual specificity phosphatase 5 (dusp5), associated with mitogen activated protein kinase (MAPK) pathway. However, no alterations in transcript levels of pro-apoptotic genes Bcl-2 like protein 4 (bax) and p53 occurred. In contrast to adult liver, MC-LR and nodularin did not result in detectable changes of mRNA levels of selected target genes involved in ER-stress in zebrafish eleuthero-embryos, nor was the abundance of transcripts belonging to the MAPK and pro-apoptosis pathways altered. In conclusion, our data indicate that MC-LR and nodularin have similar transcriptional effects. They lead to changes in mRNA levels of genes that suggest induction of ER-stress, and furthermore, lead to increased level of tnfα mRNA in the adult liver, which suggests a novel (transcriptional) mode of action in fish. However, although taken up by eleuthero-embryos, no transcriptional changes induced by these cyanobacterial toxins were detected. This is probably due to action to specific organs such as liver and kidneys that could not be identified by whole-embryo sampling

Comparative effects of nodularin and microcystin-LR in zebrafish: 2. Uptake and molecular effects in eleuthero-embryos and adult liver with focus on endoplasmic reticulum stress

Microcystin (MC) and nodularin are structurally similar cyanobacterial toxins that inhibit protein phosphatases. Additional modes of action are poorly known, in particular for nodularin. In our associated work, we showed that active cellular uptake is mediated by the organic anion transporting polypeptide drOatp1d1 in zebrafish (Faltermann et al., 2016). Here, we assessed the transcriptional expression of three genes encoding three uptake transporters during embryonic development from 24 h post fertilization (hpf) to 168 hpf. Transcripts of drOatp1d1 and drOatp2b1 are present at 24 hpf. The abundance increased after hatching and remained about constant up to 168 hpf. Transcripts of drOatp2b1 were most abundant, while drOapt1f transcripts showed very low relative abundance compared to drOatp1d1 and drOatp2b1. We further demonstrated the uptake of fluorescent labeled MC-LR in eleuthero-embryos and its accumulation in the glomerulus of the pronephros. An important molecular effect of MC-LR in human liver cells is the induction of endoplasmic reticulum (ER)-stress. Here, we investigated, whether MC-LR and nodularin similarly lead to induction of ER-stress in zebrafish by analyzing changes of mRNA levels of genes indicative of ER-stress. In zebrafish liver organ cultures short- and long-term exposures to 0.15 and 0.3 μmol L−1 MC-LR, and 0.5 and 1 μM L−1 nodularin led to significant transcriptional induction of several ER-stress marker genes, including the chaperone glucose regulated protein 78 (bip), the spliced form of x-box binding protein (xbp-1s), the CCAAT-enhancer-binding protein homologous protein (chop) and activating transcription factor 4 (atf4). Furthermore, strong transcriptional changes occurred for tumor necrosis factor alpha (tnfa) and dual specificity phosphatase 5 (dusp5), associated with mitogen activated protein kinase (MAPK) pathway. However, no alterations in transcript levels of pro-apoptotic genes Bcl-2 like protein 4 (bax) and p53 occurred. In contrast to adult liver, MC-LR and nodularin did not result in detectable changes of mRNA levels of selected target genes involved in ER-stress in zebrafish eleuthero-embryos, nor was the abundance of transcripts belonging to the MAPK and pro-apoptosis pathways altered. In conclusion, our data indicate that MC-LR and nodularin have similar transcriptional effects. They lead to changes in mRNA levels of genes that suggest induction of ER-stress, and furthermore, lead to increased level of tnfα mRNA in the adult liver, which suggests a novel (transcriptional) mode of action in fish. However, although taken up by eleuthero-embryos, no transcriptional changes induced by these cyanobacterial toxins were detected. This is probably due to action to specific organs such as liver and kidneys that could not be identified by whole-embryo sampling

Anti-Inflammatory Activity of Cyanobacterial Serine Protease Inhibitors Aeruginosin 828A and Cyanopeptolin 1020 in Human Hepatoma Cell Line Huh7 and Effects in Zebrafish (Danio rerio)

Intensive growth of cyanobacteria in freshwater promoted by eutrophication can lead to release of toxic secondary metabolites that may harm aquatic organisms and humans. The serine protease inhibitor aeruginosin 828A was isolated from a microcystin-deficient Planktothrix strain. We assessed potential molecular effects of aeruginosin 828A in comparison to another cyanobacterial serine protease inhibitor, cyanopeptolin 1020, in human hepatoma cell line Huh7, in zebrafish embryos and liver organ cultures. Aeruginosin 828A and cyanopeptolin 1020 promoted anti-inflammatory activity, as indicated by transcriptional down-regulation of interleukin 8 and tumor necrosis factor α in stimulated cells at concentrations of 50 and 100 µmol·L−1 aeruginosin 828A, and 100 µmol·L−1 cyanopeptolin 1020. Aeruginosin 828A induced the expression of CYP1A in Huh7 cells but did not affect enzyme activity. Furthermore, hatched zebrafish embryos and zebrafish liver organ cultures were exposed to aeruginosin 828A. The transcriptional responses were compared to those of cyanopeptolin 1020 and microcystin-LR. Aeruginosin 828A had only minimal effects on endoplasmic reticulum stress. In comparison to cyanopeptolin 1020 our data indicate that transcriptional effects of aeruginosin 828A in zebrafish are very minor. The data further demonstrate that pathways that are influenced by microcystin-LR are not affected by aeruginosin 828A

Molecular effects of the cyanobacterial toxin cyanopeptolin (CP1020) occurring in algal blooms: Global transcriptome analysis in zebrafish embryos

Higher water temperatures due to climate change combined with eutrophication of inland waters promote cyanobacterial blooms. Some of the cyanobacteria produce toxins leading to drinking water contamination and fish poisoning on a global scale. Here, we focused on the molecular effects of the cyanobacterial oligopeptide cyanopeptolin CP1020, produced by Microcystis and Planktothrix strains, by means of whole-genome transcriptomics. Exposure of 72 hpf old zebrafish embryos for 96 h to 100 and 1,000 μg/L CP1020 resulted in differential transcriptional alteration of 396 and 490 transcripts (fold change ≥ 2), respectively, of which 68 gene transcripts were common. These belong to genes related to various important biological and physiological pathways. Most clearly affected were pathways related to DNA damage recognition and repair, circadian rhythm and response to light. Validation by RT-qPCR showed dose-dependent transcriptional alterations of genes belonging to DNA damage and repair and regulation of circadian rhythm. This leads to the hypothesis that CP1020 acts on DNA and has neurotoxic activity. This transcriptome analysis leads to the identification of novel and unknown molecular effects of this cyanobacterial toxin, including neurotoxicity, which may have important consequences for humans consuming contaminated drinking water

Active Glucocorticoids Have a Range of Important Adverse Developmental and Physiological Effects on Developing Zebrafish Embryos

Glucocorticoids in aquatic systems originating from natural excretion and medical use may pose a risk to fish. Here, we analyzed physiological and transcriptional effects of clobetasol propionate (CLO), cortisol and cortisone in zebrafish embryos as single compounds and binary mixtures. CLO and cortisol, but not cortisone showed a concentration-dependent decrease in muscle contraction, increase in heart rate, and accelerated hatching. CLO also induced immobilization and edema at high concentrations. Transcription analysis covering up to 26 genes showed that mostly genes related to glucose metabolism, immune system and development were differentially expressed at 91 ng/L and higher. CLO showed stronger effects on immune system genes than cortisol, which was characterized by upregulation of <i>fkbp5</i>, <i>irg1l</i>, <i>gilz</i>, and <i>socs3</i>, and development genes, matrix metalloproteinases <i>mmp-9</i> and <i>mmp-13</i>, while cortisol led to stronger upregulation of the gluconeogenesis genes <i>g6pca</i> and <i>pepck1.</i> CLO also induced genes regulating the circadian rhythm, <i>nr1d1</i> and <i>per1a</i>. In contrast, cortisone led to down-regulation of <i>vitellogenin</i>. Binary mixtures of cortisol and CLO mostly showed a similar activity as CLO alone on physiological and transcriptional end points but additive effects in heart rate and <i>pepck1</i> upregulation, which indicates that mixtures of glucocorticoids may be of concern for developing fish