Fate of mitochondrial DNA (mtDNA) in zebrafish (Danio rerio)under oxidative stress

Abstract

Stress in various formssuch as extreme drought, temperature, salt intrusion, emergence of new pathogens, industrial chemicals discharge in to the aquatic system causing serious health problem of the fishes, which ultimately affect the expected production level in fisheries sector. All these environmental stress factors increase cellular reactive oxygen species (ROS) levelmainly superoxide(O2•−), hydroxyl radicals (OH•), hydrogen peroxide (H2O2) leading to secondary oxidative stress effects in fish. Under oxidative stress, ROS might affect the cellular macromolecules like protein, lipid, DNA etc. This study focused to examine the secondary effects of the environmental pollutants that is the oxidative stress on mitochondrial DNA (mtDNA) in fish. Tropical freshwater zebrafish (Danio rerio) was used as experimental model in this study and the oxidative stress was induced by H2O2treatment. Firstly we examined the acute toxicity of H2O2on zebrafish by estimating LC50 value which was not reported yet. The fishes were exposed to different concentrations of H2O2 for static acute toxicity test. The concentration-mortality (%) data were analyzed through Probit analysis to estimate the 96 h median lethal concentration (LC50) which was 257.494 µM. Secondly, the oxidative stress effect on mtDNA was examined in both H2O2 treated and non-treated zebrafish during 96 hours period. We have designed two sets of mtDNA specific primers to observe H2O2 stress effect in two specific zone of mtDNA, one is from 1 bp to 8500 bp and the other is 8244 bp to 16585 bp. After polymerase chain reaction we observed a probable nucleotide loss in mtDNA in fish under oxidative stress with the primer set 2. This oxidative damage was observed from 8266 bp to 16563 bp in both brain and egg cell of H2O2treated zebrafish. After in silico analysis, we found that this particular sequence consists of several hot spots which are repeated sequences and prone to oxidative damage. This study finds a potential sequence in mtDNA to find the oxidative damage in fish. Our findings might be the basis to develop a molecular biomarker to study the secondary oxidative stress effect due to any environmental pollutants.