Radiolabeled Cu-ATSM as a novel indicator of overreduced intracellular state due to mitochondrial dysfunction: studies with mitochondrial DNA-less ρ0 cells and cybrids carrying MELAS mitochondrial DNA mutation

ObjectivesRadiolabeled Cu-diacetyl-bis (N4-methylthiosemicarbazone) (⁎Cu-ATSM), including 60/62/64Cu-ATSM, is a potential imaging agent of hypoxic tumors for positron emission tomography (PET). We have reported that ⁎Cu-ATSM is trapped in tumor cells under intracellular overreduced states, e.g., hypoxia. Here we evaluated ⁎Cu-ATSM as an indicator of intracellular overreduced states in mitochondrial disorders using cell lines with mitochondrial dysfunction.MethodsMitochondrial DNA-less ρ0206 cells; the parental 143B human osteosarcoma cells; the cybrids carrying mutated mitochondria from a patient of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (2SD); and that carrying wild-type one (2SA) were used. Cells were treated under normoxia or hypoxia, and 64Cu-ATSM uptake was examined to compare it with levels of biological reductant NADH and NADPH.Resultsρ0206 cells showed higher 64Cu-ATSM uptake than control 143B cells under normoxia, whereas 64Cu-ATSM uptake was not significantly increased under hypoxia in ρ0206 cells. Additionally, 64Cu-ATSM uptake showed correlate change to the NADH and NADPH levels, but not oxygenic conditions. 2SD cells showed increased 64Cu-ATSM uptake under normoxia as compared with the control 2SA, and 64Cu-ATSM uptake followed NADH and NADPH levels, but not oxygenic conditions.Conclusions64Cu-ATSM accumulated in cells with overreduced states due to mitochondrial dysfunction, even under normoxia. We recently reported that 62Cu-ATSM-PET can visualize stroke-like episodes maintaining oxygen supply in MELAS patients. Taken together, our data indicate that ⁎Cu-ATSM uptake reflects overreduced intracellular states, despite oxygenic conditions; thus, ⁎Cu-ATSM would be a promising marker of intracellular overreduced states for disorders with mitochondrial dysfunction, such as MELAS, Parkinson's disease and Alzheimer's disease.