Mitochondrial toxicity assays

HPNE and AsPC-1 cell lines were plated at a final density of 20,000-50,000 cells/well and allowed to adhere for at least 24 h prior to the assay. The initiation of toxicant exposure begins by removing the growth media and replacing with DMEM supplemented with either 25 mM glucose or 10 mM galactose. This method of determining mitochondrial toxicity is similar to that described in the study by Sanuki et al (55) and Dott et al (56). The treatment groups consisted of cells exposed to Cd (0.1-100 μM) or control (media only) for 4 h followed by the addition of 12 mM MTT dye (1.2 mM final concentration). The absorbance was measured at 565 nm, and the data were then expressed as the means ± SEM of n=12 in duplicate.

To establish the association between cell death and the loss of ATP, we used a luciferin-based detection system (Mitochondrial ToxGlo™; Promega) (57). The principle of the mitochondrial toxicity tests is that substituting 10 mM galactose for 25 mM glucose will increase susceptibility to mitochondrial toxins (58-60). The cells were exposed to CdCl₂ (1 or 50 μM) for 48 h prior to the initiation of the assay. These two concentrations were selected based on 50 μM approximating 6 ppm, and 1 μM was significantly lower than our threshold of 10 μM. Following 30 min of incubation at 37°C in a CO₂ incubator, viability was determined by measuring the fluorescence (485 nm_ex/525 nm_em) emitted. The second step in the multiplex was to quantify the amount of ATP present by directly adding the luciferin-based detection system into the wells that have already been quantified for cell viability with the amount of luminescence being directly proportional to the amount of ATP present. Collectively, by comparing the cellular responses in the two datasets, a better understanding of CdCl₂ can be obtained as a cellular toxicant that results in mitochondrial dysfunction or non-mitochondrial related cytotoxic mechanisms.