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Reactive oxygen species (ROS) are necessary for various cellular processes. However, excess ROS cause damage to many biological molecules and therefore must be tightly regulated in time and space. Hydrogen peroxide (H2O2) is the most commonly used ROS as second messenger in the cell. It is a relatively long‐lived freely diffusible signaling molecule during early events of injury. In the Cnidarian hydra, injury‐induced ROS production is essential for regeneration to proceed. In the present study, we have examined influence of varying exposure to H2O2 on head and foot regeneration in the middlepieces of trisected hydra. We find that longer (4 hours) exposure to 1 mM H2O2 inhibits both head and foot regeneration while shorter exposure (2 hours) does not. Longer exposure to H2O2 resulted in extensive damage to DNA that could not be repaired, probably due to suboptimal induction of APE1, an enzyme necessary for base excision repair (BER). Concomitantly, genes involved in activation of Wnt pathway, necessary for head regeneration, were significantly downregulated. This appeared to be due to failure of both stabilization and transient nuclear localization of β‐catenin. Similarly, genes involved in foot regeneration were also downregulated on longer exposure to H2O2. Thus, exposure to excess ROS inhibits regenerative processes in hydra through reduced expression of genes involved in regeneration and diminished DNA repair.

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Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site CGPC and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH 7.0 and 25 °C respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in E. coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.

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