Assessment of oxidative damage to DNA, transcriptional expression of key genes, lipid peroxidation and histopathological changes in carp Cyprinus carpio L. following exposure to chronic hypoxic and subsequent recovery in normoxic conditions

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Abstract

In fish, a complex set of mechanisms deal with environmental stresses including hypoxia. In order to probe the hypothesis that hypoxia-induced stress could be manifested in varieties of pathways, a model species, mirror carp (Cyprinus carpio), were chronically exposed to hypoxic condition (dissolved oxygen level: 1.80±0.6mg/l) for 21 days and subsequently allowed to recover under normoxic condition (dissolved oxygen level: 8.2±0.5mg/l) for 7 days. At the end of these exposure periods, an integrated approach was applied to evaluate several endpoints at different levels of biological organisation. These included determination of (i) oxidative damage to DNA in erythrocytes (using modified comet assay), (ii) lipid peroxidation in liver samples by measuring the malondialdehyde production using the 2-thiobarbituric acid [i.e. thiobarbituric acid reactive substances (TBARS) assay] and (iii) histopathological changes in gills. In addition, transcriptional expression of hypoxia-inducible factor 1 α (HIF-1α) and genes involved in the repair of oxidative damage to DNA (i.e. ogg1) and base excision repair (i.e. xrcc1) using reverse transcription polymerase chain reaction in liver samples were also determined. The results suggested significantly enhanced expression of these genes in response to hypoxia compared to concurrent normoxic controls. While the expression of HIF-1α reverted to control values within 7 days exposure to normoxic condition (P < 0.05), the transcriptional expression of the two genes involved in DNA repair process remained significantly high under the recovery period, which complemented the induction of oxidative damage to DNA. Hypoxic groups showed significantly increased values for TBARS level (~2-fold) and histopathological changes in gill tissues compared to both normoxic and recovery groups. Overall, oxidative damage to DNA determined by modified comet assay reflected the observed biological responses in other tissues of the fish. Along with other parameters, this integrated experimental design further strengthens the applications of the comet assay as an important technique to assess stress-induced DNA damage in ecotoxicological studies.

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