Alterations in mitochondrial DNA copy number and oxidative stress markers in blood and muscle tissues of mice following snake envenomation

Snake envenomation represents a significant toxicological and public health concern, causing systemic effects mediated by oxidative stress and cellular injury. Mitochondria are especially susceptible to stress generated by venom, however the initial mitochondrial responses after envenomation are not fully understood. This study investigated time- and tissue-dependent changes in relative mitochondrial DNA (mtDNA) abundance and oxidative stress markers in mice exposed to neurotoxic and hematotoxic snake venoms. Neurotoxic venoms included Naja kaouthia (monocled cobra), Ophiophagus hannah (king cobra), Bungarus fasciatus (banded krait), and Bungarus candidus (Malayan krait), while hematotoxic venoms comprised Calloselasma rhodostoma (Malayan pit viper), Daboia siamensis (Siamese Russell's viper), and Trimeresurus albolabris (white-lipped pit viper). Venoms were administered intramuscularly at empirically determined sublethal doses. Whole blood, tibialis anterior muscle tissue, and mononuclear cells (MNCs) were collected at 1, 6, and 24 hours post-exposure. Relative mtDNA copy number was quantified using SYBR-based real-time quantitative PCR targeting mtDNA-encoded genes (16S rRNA and ND1) normalized to a nuclear reference gene, with additional assessment of cytochrome b (Cytb) and cytochrome c oxidase subunit III (COXIII) as indicators of mitochondrial perturbation. Oxidative stress responses were evaluated by measuring malondialdehyde (MDA) and reduced glutathione (GSH) levels. Venom exposure induced distinct temporal and tissue-specific patterns of mtDNA-associated changes, accompanied by alterations in lipid peroxidation and antioxidant status. Whole blood consistently exhibited earlier and more robust responses than muscle tissue or MNCs. Although mtDNA-related parameters and oxidative stress markers are not specific to envenomation, their combined temporal profiles reflect venom-induced mitochondrial and redox stress. These findings support the potential utility of blood-based mtDNA and oxidative indicators as sensitive, venom-independent tools for evaluating systemic responses to snake envenomation.