Global gene expression induced by CP1020 (Cyanopeptolin) in zebrafish eleuthero-embryo (Danio rerio)
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ABSTRACT: Cyanobacteria produce various cyanotoxins, which can cause severe effects to other organisms. Microcystins, one group of such toxins, primarily produced by species of Microcystis, are strong hepatotoxins and inhibit potently protein phosphatases 1 and 2A. Microcystin is the most studied cyanotoxin, however, others are not investigated. Eutrophication of water bodies promotes the occurrence of toxic algal blooms and since a anthropogenic caused increase in eutrophication events can be observed, it is becoming increasingly important to study the consequences and to increase the knowledge on toxins associated with algal blooms. Recently a new cyanobacteria toxin from a Microcystis strain, CP1020, was described. CP1020 belongs to the class of cyanopeptolins and its toxicity was shown to be comparable to that of microcystin (Gademann et al., 2009). It is a strong protease inhibitor inhibiting trypsin in the picomolar range (IC50 = 670 pM) and effects survival of the freshwater crustacean Thamnocephalus platyurus (LC50) 8.8 M-NM-
Project description:Cyanobacteria produce various cyanotoxins, which can cause severe effects to other organisms. Microcystins, one group of such toxins, primarily produced by species of Microcystis, are strong hepatotoxins and inhibit potently protein phosphatases 1 and 2A. Microcystin is the most studied cyanotoxin, however, others are not investigated. Eutrophication of water bodies promotes the occurrence of toxic algal blooms and since a anthropogenic caused increase in eutrophication events can be observed, it is becoming increasingly important to study the consequences and to increase the knowledge on toxins associated with algal blooms. Recently a new cyanobacteria toxin from a Microcystis strain, CP1020, was described. CP1020 belongs to the class of cyanopeptolins and its toxicity was shown to be comparable to that of microcystin (Gademann et al., 2009). It is a strong protease inhibitor inhibiting trypsin in the picomolar range (IC50 = 670 pM) and effects survival of the freshwater crustacean Thamnocephalus platyurus (LC50) 8.8 μM (Gademann et al., 2009). Nothing is known, however, about the toxicity of CP1020 to fish. Furthermore, no information is available on the toxic modes of action, in addition to the proteinase activity. Consequently our study has the aim to elucidate the modes of action of CP1020 on zebrafish eleuthero-embryos. By using a microarray technique, we will analyse alterations of global gene expression by CP1020 at two different concentrations. Thereby, we hope to elucidate the whole array of affected biological pathways to elucidate the mechanisms by which CP1020 affect fish.
Project description:Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained in the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the University of Tennessee Institutional Animal Care and Use Committee (Protocol #1690-1007). Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl. Embryos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (± 15 min.), such that larvae used in experiments were of similar age at the time of exposure. All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27± 1 ºC and 14:10h light:dark photoperiod. Experiment Overall Design: At 72 h post-fertilization, zebrafish larvae were exposed to lyophilized Microcystis and purified MC-LR at concentrations of 100 and 1,000 µg/L. Controls consisted of zebrafish system water (negative control) and zebrafish system water containing 0.05% ethanol (vehicle control). Larvae from both control groups as well as 100 µg/L MC-LR, 1,000 µg/L MC-LR, and lyophilized Microcystis were exposed in groups of 50 with three replicates and were sacrificed after 96 hours for total RNA extraction and subsequent microarray analysis. All larvae were exposed in beakers containing 100 ml of solution. Experiment Overall Design: Water samples for microcystin analysis and water quality measurements were taken during the experiment, and mortality and behavioral observations were recorded at 24-hour intervals. Microcystin analysis was conducted by protein phosphatase inhibition assay. Measured concentrations of microcystin-LR were 140 ± 12 SD (low concentration) and 1,703 ± 71 SD (high concentration). The concentration of microcystin-LR in the lyophilized Microcystis treatment was 4.5 µg/L. Water quality parameters measured included dissolved oxygen (6.7 mg/L), pH (6.9), total alkalinity (36 mg/L as CaCO3), total hardness (18 mg/L as CaCO3), and ammonia (<0.2 mg/L). No significant mortality or behavioral changes in larvae were observed during the exposure.
Project description:To identify molecular effects of the antineoplastic agent PKC412 (Midostaurin), we applied gene expression profiling in zebrafish using whole genome microarrays. Zebrafish eleuthero-embryos were exposed for 6 dpf to nominal levels of 2 μg/L and 40 μg/L PKC412. Among the 259 and 511 altered transcripts at both concentrations, respectively, the expressions of genes involved in the circadian rhythm were of interest. Alteration of swimming behaviour was not noted. Pathways of interest affected by PKC412 were angiogenesis, apoptosis, DNA damage response and response to oxidative stress. Angiogenesis was not altered by PKC412 treatment at both concentrations. Apoptosis occurred in olfactory placodes of embryos exposed to 40 μg/L, and DNA damage was induced at both PKC412 concentrations. However, there were no significant effects on reactive oxygen species formation. This study leads to the conclusion that PKC412-induced alterations of gene transcripts are partly paralleled by physiological effects at high, but not at low PKC412 concentrations expected to be of environmental relevance. Gene expression in zebrafish eleuthero-embryos was measured after exposure for 6dpf to 2 ug/L and 40 ug/L PKC412 or to the respective controls. A total of 12 arrays (Agilent 4 × 44 K Zebrafish microarray) were used, including four for the water control group, four for the solvent control group, four for the 2 μg/L and four for the 40 μg/L PKC412 dose group.
Project description:Microcystis aeruginosa cells were treated with phosphorus repletion, depletion and starvation. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic method was employed to explore to the effects of phosphorus limitation on Microcystis aeruginosa cells at the protein level. This investigation would contribute to the understanding of global cellular responses of Microcystis to phosphorus limitation and provide theoretical basis for deciding whether it is an effective way to control Microcystis blooms by phosphorus reduction.
Project description:We applied zebrafish whole genome microarrays to identify molecular effects of diazepam, a neuropharmaceutical encountered in wastewater-contaminated environments, and to elucidate its neurotoxic mode of action. Behavioral studies were performed to analyze for correlations between altered gene expression with effects on the organism level. Male zebrafish and zebrafish eleuthero-embryos were exposed for 14 d or up to 3 d after hatching, respectively, to nominal levels of 273 ng/L and 273 μg/L (determined water concentrations in the adult experiment 235 ng/L and 291 μg/L). Among the 51 and 103 altered transcripts at both concentrations, respectively, the expression of genes involved in the circadian rhythm in adult zebrafish and eleuthero-embryos were of particular significance, as revealed both by microarrays and quantitative PCR. The swimming behavior of eleuthero-embryos was significantly altered at 273 μg/L. The study leads to the conclusion that diazepam-induced alterations of genes involved in circadian rhythm are paralleled by effects in neurobehavior at high, but not at low diazepam concentrations that may occur in polluted environments. Gene expression in male zebrafish brain was measured after exposure for 14 d to 273 ng/L and 273 ug/L diazepam or to the water control. A total of 11 arrays (Agilent 4 × 44 K Zebrafish microarray) were used, including three for the water control group, four for the 273 ng/L and four for the 273 μg/L diazepam dose group.
Project description:Elevated levels of adsorbable organic bromine compounds (AOBr) have been detected in German lakes, and cyanobacteria like Microcystis, which are known for the synthesis of microcystins, are one of the main producers of natural organobromines. However, very little is known about how environmental realistic concentrations of organobromines impact invertebrates. Here, the nematode C. elegans was exposed to AOBr-containing surface water samples and to a Microcystis aeruginosa enriched batch culture (MC-BA) and compared to single organobromines and microcystin-LR exposures. Stimulatory effects were observed in certain life trait variables, which were particularly pronounced in nematodes exposed to MC-BA. A whole genome DNA-microarray revealed that MC-BA led to the differential expression of more than 2000 genes, many of which are known to be involved in metabolic, neurologic, and morphologic processes. Moreover, the up-regulation of cyp- and the down-regulation of abu-genes suggested the presence of chronic stress. However, the nematodes were not marked by negative phenotypic responses. The observed difference in MC-BA and microcystin-LR (which impacted lifespan, growth and reproduction) exposed nematodes was hypothesized to be likely due to other compounds within the batch culture. Most likely the exposure to low concentrations of organobromines appears to buffer the effects of toxic substances, like microcystin-LR.
Project description:As an essential primary producer, cyanobacteria play an important role in the global cycle for both carbon and nitrogen in the ecosystems. Though the influence of nanoplastics on the carbon metabolism of cyanobacteria, especial Microcystis aeruginosa, a dominant species causing cyanobacterial blooms, is well studied, little is known about nanoplastics affecting the nitrogen metabolism.
Project description:Microcystins are produced by the cyanobacteria, most commonly Microcystis aerµginosa. Upon ingestion, toxic microcystins are actively absorbed by fish, birds and mammals where they are primarily liver toxins. Groups of 4-6 rats were exposed to 0, 1, 10, 50 or 100 µg/kg microcystin-LR for 0.5, 1, 3 or 6 hours and gene expression analysis performed on liver with samples hybridized to whole rat genome RG230_2.0 GeneChip arrays (Affymetrix, CA).
Project description:Elevated levels of adsorbable organic bromine compounds (AOBr) have been detected in German lakes, and cyanobacteria like Microcystis, which are known for the synthesis of microcystins, are one of the main producers of natural organobromines. However, very little is known about how environmental realistic concentrations of organobromines impact invertebrates. Here, the nematode C. elegans was exposed to AOBr-containing surface water samples and to a Microcystis aeruginosa enriched batch culture (MC-BA) and compared to single organobromines and microcystin-LR exposures. Stimulatory effects were observed in certain life trait variables, which were particularly pronounced in nematodes exposed to MC-BA. A whole genome DNA-microarray revealed that MC-BA led to the differential expression of more than 2000 genes, many of which are known to be involved in metabolic, neurologic, and morphologic processes. Moreover, the up-regulation of cyp- and the down-regulation of abu-genes suggested the presence of chronic stress. However, the nematodes were not marked by negative phenotypic responses. The observed difference in MC-BA and microcystin-LR (which impacted lifespan, growth and reproduction) exposed nematodes was hypothesized to be likely due to other compounds within the batch culture. Most likely the exposure to low concentrations of organobromines appears to buffer the effects of toxic substances, like microcystin-LR. Nematodes were exposed to filtrated samples of Lake Stößensee Berlin (August and October) and compared to the control-group (exposure to water). Furthermore, nematodes were exposed to filtrated M. aeruinosa batch culture samples and compared to another control-group (exposure to Z-Medium). Three samples (biological replicates) were prepared for each group.