Copper Toxicity and Calcium Supplementation on nkx2.5 and mt2 Gene Expression in Zebrafish Embryos

Sahib Mohammad bakir ^1*, Theodore B. Henry ², Richard D. Handy ³

This study determined how copper toxicity and calcium supplementation affect gene expression in early zebrafish embryos to inform aquatic environmental health and developmental biology.

Abstract

Background: Heavy metals are pervasive pollutants in aquatic ecosystems, posing significant threats to the survival and development of aquatic organisms, particularly during their early life stages. While copper is necessary for fish growth and maturation, elevated levels of dissolved copper in water can lead to adverse effects on early developmental stages. The aim of this study was to evaluate the expression pattern of the nkx2.5 gene and the potential of Mt2 expression as a biomarker for metal toxicity. Methods: The study assessed the expression levels of the nkx2.5 gene at different time points during embryonic development, focusing particularly on its peak expression time. Zebrafish embryos were exposed to dissolved copper, either alone or in combination with calcium (Cu+Ca), to examine the effects on nkx2.5 expression. The expression of Mt2, a potential marker for metal toxicity, was also analyzed under copper exposure conditions. Results: The results revealed that nkx2.5 gene expression peaked at 16 hours post-fertilization (hpf), a critical time period for heart formation and development in zebrafish embryos. Exposure to both copper and calcium (Cu+Ca) significantly increased nkx2.5 expression by nearly tenfold compared to control conditions or exposure to copper alone. Furthermore, the expression of Mt2 showed a substantial sixfold increase under copper exposure conditions in the absence of additional calcium (Ca2+), indicating metal-induced stress. Conclusion: The findings of this study determined the susceptibility of zebrafish embryos to metal exposure during early developmental stages, leading to disruptions in cellular biology and heart development.

Keywords: Zebrafish, Heavy metals, Embryonic development, nkx2.5 gene expression, Copper toxicity, Mt2 biomarker, Calcium.

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