Direct analysis of mitochondrial toxicity of antiretroviral drugs

Abstract

Mitochondrial toxicity is a serious side-effect of antiretroviral drugs, especially nucleoside reverse transcriptase inhibitors (NRTI). An in vitro assay to predict mithocondrial toxicity of in-use and developmental NRTI would be invaluable. To test the ability of a cytofluorimetric technique to predict the mitochondrial-dependent pancreatic and hepatic toxicity we used didanosine (ddI) alone or in combination with hydroxyurea (HU). The technique is based on the ability of the lipophilic cation JC-1 to enter selectively into mitochondria and change its colour as the membrane potential changes due to toxicity. Mitochondrial toxicity by HU and ddI was evaluated in pancreatic and hepatic human cell lines. The results were expressed as mitochondrial toxicity index (MTI), ranging from 0 to 100: the negative control was 0, and 100 indicating maximal toxicity. Dose-dependent pancreatic toxicity of ddI was evident after 14 days of culture (MTI 34 ± 4 at 100 μM, 10 ± 4 at 10 μM, 2 ± 3 at 1 μM ddI). HU alone was not toxic (MTI 7 ± 10 at 100 μM, 2 ± 2 at 50 μM and 2 ± 4 at 10 μM HU); however, HU increased the toxicity of high, but not low, concentrations of ddI. For example, the MTI of 10 μM ddI plus 50 μM HU was 54 ± 9. Negligible mitochondrial toxicity was observed in the hepatic cell line exposed to ddI alone or in combination with HU. This in vitro assay might have in vivo relevance. First, ddI-related pancreatitis is dose dependent, and is reported more frequently than hepatic failure, consistent with our in vitro results. Second, patients who developed pancreatitis during randomized, controlled trials were treated with HU in combination with 400 mg ddI once daily (high peak concentration of ddI in the blood). In contrast, no pancreatitis was observed when HU was combined with 200 mg ddI twice daily (low peak concentration of ddI). These in vivo results are consistent with our in vitro observation that HU increases pancreatic cell toxicity in the presence of high concentrations of ddI. The in vitro assay described here might be used to predict the mitochondrial toxicity of other NRTI, alone or in combination.