title: 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.
abstract: 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 24h 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.
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland; University of Zürich, Institute of Plant Biology, Limnological Station, Seestrasse 187, 8802 Kilchberg, Switzerland.
authors.forename: Susanne
authors.initials: S
authors.lastname: Faltermann
authors.affiliation: University of Basel, Department of Chemistry, St. Johanns-Ring 19, CH-4056 Basel, Switzerland.
authors.forename: Verena
authors.initials: V
authors.lastname: Grundler
authors.affiliation: University of Basel, Department of Chemistry, St. Johanns-Ring 19, CH-4056 Basel, Switzerland.
authors.forename: Karl
authors.initials: K
authors.lastname: Gademann
authors.affiliation: University of Zürich, Institute of Plant Biology, Limnological Station, Seestrasse 187, 8802 Kilchberg, Switzerland.
authors.forename: Jakob
authors.initials: J
authors.lastname: Pernthaler
authors.affiliation: University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz, Switzerland; Swiss Federal Institute of Technology Zurich (ETHZ), Department of Environmental Systems Science, Institute of Biogeochemistry and Pollution Dynamics, Universitätsstrasse 16, 8092 Zürich, Switzerland. Electronic address: karl.fent@fhnw.ch.
authors.forename: Karl
authors.initials: K
authors.lastname: Fent
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000187:drug effects*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D008099:Liver / Q000187:drug effects*; D008387:Marine Toxins; D052998:Microcystins / Q000378:metabolism / Q000633:toxicity; D010456:Peptides, Cyclic / Q000378:metabolism / Q000633:toxicity; D014874:Water Pollutants, Chemical / Q000378:metabolism / Q000633:toxicity; D015027:Zebrafish / Q000196:embryology / Q000502:physiology*
chemical_list: D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D014874:Water Pollutants, Chemical; C063998:nodularin; C057862:cyanoginosin LR
keywords: Cyanobacterial toxin; Endoplasmic reticulum stress; Liver organ culture; Microcystin; Nodularin; Zebrafish