Subcytotoxic inorganic arsenic effects on mitochondria in a human proximal tubule cell line: Implications on mechanism of cell death

Arsenic is an environmental toxicant and a human carcinogen. The kidney is a known target organ for arsenic (As) and is critical for both arsenic biotransformation and elimination. This study investigates the potential of an immortalized human proximal tubular epithelial cell line, HK-2, to serve as a model for low level exposures of the human kidney to arsenic. Subcytotoxic concentrations of arsenite (≤ 10 μM) and arsenate (< 100 μM) were determined by leakage of LDH from cells exposed for 24 hours. Threshold concentrations of arsenite (1-10 μM) and arsenate (10-25 μM) were found to affect mitochondrial MTT processing. Biotransformation of arsenite or arsenate was determined using HPLC-ICP-MS to detect metabolites in cell culture media and lysates. Analysis of media revealed that arsenite was minimally oxidized to arsenate and arsenate was reduced to arsenite. Only arsenite was detected in cell lysates. Pentavalent methylated arsenicals were not detected following exposure to either inorganic arsenical. This demonstrates that the HK-2 cell line is capable of biotransforming inorganic arsenic, primarily reducing arsenate to arsenite. In addition, the mitochondria are a primary target for low-level arsenic toxicity. Previous studies show that at high doses (ppm) inorganic arsenic is toxic to mitochondria primarily by affecting cellular respiration. Mitochondrial injury was further assessed in HK-2 cells by examining mitochondrial membrane potential. Subcytotoxic arsenite caused mitochondrial depolarization, which could subsequently lead to permeability transition and apoptosis. Arsenite also induced translocation of phosphatidylserine to the outer layer of the plasma membrane, indicative of early apoptosis. To confirm whether subcytotoxic arsenite induces cellular and/or mitochondrial morphological alterations consistent with apoptosis, HK-2 cells were evaluated with both light and transmission electron microscopy. Classic morphological changes indicative of apoptosis were not observed at either the light microscopic nor the electron microscopic level; however, evidence of necrotic changes in cytoplasmic structure and morphology--particularly in the mitochondria--were apparent. Therefore, HK-2 cells appear to initiate apoptosis following subcytotoxic arsenite insult, but fail to complete apoptosis and undergo necrosis instead. Subcytotoxic arsenite can be sufficiently toxic to mitochondria that they lose their ability to keep the cell on course for apoptosis