Identification of copper-responsive genes in an early life stage of the fathead minnow Pimephales promelas

Abstract

While physiological changes associated with copper toxicity have been studied in adult fathead minnow, Pimephales promelas, little is known about the effect of copper on newly hatched larvae. As a result we initiated an investigation on the mechanism of copper toxicity in 24 h post-hatch larvae using gene expression changes to identify responsive genes. Fish were exposed to copper concentrations of 0, 50, 125 and 200 mug/L in a 48 h toxicity test. Total RNA from survivors was used in a differential display assay to screen for differentially expressed gene products. Altogether, 654 copper-responsive differentially expressed bands were collected. Database searches found homology for 261 sequences. One hundred and sixty-one bands were homologous to NCBI genes of known function, of which 69 were individual genes. The most abundant categories of functional genes responding to copper were involved in protein synthesis/translational machinery and contractile proteins. Twenty-one dose-responsive genes were measured for expression changes using real-time quantitative PCR. Differential gene expression was validated for 11 of 13 genes, when a 1.2 times qPCR difference between the copper and control samples was observed. Transcripts identified as titin, cytochrome b, fast muscle specific heavy myosin chain 4, fast muscle troponin I, proteasome 26S subunit and troponin T3a were induced over twofold. Differential display bands identified as 60S ribosomal proteins L27 and L12 were repressed approximately threefold. We conclude that copper exposure affects several cellular pathways in larval fathead minnows with protein synthesis, ribosome structure, and muscle contractile proteins being the most sensitive to this stress.

Linking molecular biomarkers with higher level condition indicators to identify effects of copper exposures on the endangered delta smelt (Hypomesus transpacificus)

“…SER Ca ATPase was a particularly informative gene, because the measured downregulation supports indications of interference of Cu with Ca 2þ homeostasis, neurological signaling, and muscle activity. The strong downregulation of a-actin, at all concentrations, sustains reported effects of Cu on contractile muscle proteins [28], further supported by increases in m-calpain expression, involved in muscle regeneration [25].…”

“…It was downregulated, significantly correlating (r ¼ 0.978) with increasing Cu concentration. Downregulation of a-amylase transcription has previously been associated with Cu exposure [28]. Chitinase, an enzyme required in the digestion of chitin structures in the exoskeletons of crustaceans and many insects, was significantly upregulated on exposure to Cu.…”

Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review

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Influence of copper on early development: Prenatal and postnatal considerations

“…There were several genes that were downregulated following exposure to high Cu concentrations and the majority of these genes were associated with the regulation of growth and longevity. Other pathways disturbed by excessive Cu exposure to fathead minnows included the oxidative stress response pathway, protein folding, proteolysis, PI3K pathway, and the MAP‐kinase pathway [196].…”