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

Objectives: Radiolabeled Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) (*Cu-ATSM), including (60/62/64)Cu-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.

Methods: Mitochondrial DNA-less ρ(0)206 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 (64)Cu-ATSM uptake was examined to compare it with levels of biological reductant NADH and NADPH.

Results: ρ(0)206 cells showed higher (64)Cu-ATSM uptake than control 143B cells under normoxia, whereas (64)Cu-ATSM uptake was not significantly increased under hypoxia in ρ(0)206 cells. Additionally, (64)Cu-ATSM uptake showed correlate change to the NADH and NADPH levels, but not oxygenic conditions. 2SD cells showed increased (64)Cu-ATSM uptake under normoxia as compared with the control 2SA, and (64)Cu-ATSM uptake followed NADH and NADPH levels, but not oxygenic conditions.

Conclusions: (64)Cu-ATSM accumulated in cells with overreduced states due to mitochondrial dysfunction, even under normoxia. We recently reported that (62)Cu-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.