ABSTRACT
Residues of the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) are ubiquitously found in aquatic biota but potential adverse effects in fish are fairly unknown. To identify molecular effects and modes of action of EHMC we applied a gene expression profiling in zebrafish using whole genome microarrays. Transcriptome analysis and validation of targeted genes were performed after 14 days of exposure of male zebrafish. Concentrations of 2.2 µg/L and 890 µg/L EHMC lead to alteration of 1096 and 1137 transcripts, respectively, belonging to many pathways. Genes involved in lipid metabolism and estrogenic pathway (vtg1), lipid biosynthesis (ptgds), vitamin A metabolic process (rbp2a), DNA damage and apoptosis (gadd45b), and regulation of cell growth (igfbp1a) were investigated by qRT-PCR analysis in whole body, liver, brain and testis. The analysis showed tissue-specific gene profiles and revealed that EHMC slightly affects the transcription of genes involved in hormonal pathways including vtg1, esr1, esr2b, ar, cyp19b and hsd17ß3.
Subject(s)
Cinnamates/toxicity , Gene Expression/drug effects , Sunscreening Agents/toxicity , Water Pollutants, Chemical/toxicity , Zebrafish Proteins/genetics , Animals , Male , Ultraviolet Rays , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
To identify molecular effects of the antineoplastic agent protein kinase C inhibitor 412 (PKC412) (midostaurin), we applied gene expression profiling in zebrafish using whole-genome microarrays. Behavioral, developmental, and physiological effects were investigated in order to analyze for correlations between altered gene expression profiles with effects on development and physiology. Zebrafish blastula-stage embryos were exposed for 6 days postfertilization to nominal levels of 2 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 further investigated. Alteration of swimming behavior was not observed. Pathways of interest affected by PKC412 were angiogenesis, apoptosis, DNA damage response, and response to oxidative stress. Angiogenesis was analyzed in double-transgenic zebrafish embryos Tg(fli1a:EGFP)y1;Tg(gata1:dsRed)sd2; no major defects were induced 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.
Subject(s)
Antineoplastic Agents/toxicity , Embryo, Nonmammalian/drug effects , Gene Expression/drug effects , Protein Kinase Inhibitors/toxicity , Staurosporine/analogs & derivatives , Zebrafish , Animals , Apoptosis/drug effects , Circadian Rhythm/drug effects , Circadian Rhythm/genetics , Circadian Rhythm/physiology , Comet Assay , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/physiology , Gene Expression Profiling , In Situ Nick-End Labeling , Motor Activity/drug effects , Neovascularization, Physiologic/drug effects , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Staurosporine/toxicity , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/physiologyABSTRACT
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.
Subject(s)
Diazepam/pharmacology , Gene Expression Regulation, Developmental/drug effects , Zebrafish/growth & development , Animals , Circadian Rhythm , Gene Expression Profiling , Life Cycle Stages , Male , Nucleic Acid Hybridization , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Zebrafish/genetics , Zebrafish/physiologyABSTRACT
Enzymes belonging to the cytochrome P450 3A (CYP3A) subfamily play an important role in the metabolism of endogenous substances and xenobiotics, including pharmaceuticals. Xenobiotics can alter CYP3A expression and activity, and therefore, changes in CYP3A activity may serve as a biomarker of xenobiotic exposure. To determine changes in CYP3A enzyme activity for environmental risk assessment of xenobiotics including pharmaceuticals, high-throughput assays are needed, but these are missing for fish cells to date. Here, we report on the development of a fluorescent-based CYP3A high-throughput assay for four fish cell lines cultivated in 96-well plates based on 7-benzyloxy-4-trifluoromethylcoumarin as a CYP3A substrate. We show that human CYP3A substrate BFC is catalyzed by fish CYP3A enzymes to a fluorescent product. Its formation is dependent on cell numbers and incubation time. Furthermore, we demonstrate that with this new CYP3A assay induction and inhibition of enzyme activity by pharmaceuticals can be determined. This new cell-based assay is suitable for detection of alteration in CYP3A enzyme activity in large-scale experiments for screening of pharmaceuticals occurring in the environment.
