Comparison of the Cytotoxicity of the Nitroaromatic Drug Flutamide to Its Cyano Analogue in the Hepatocyte Cell Line TAMH: Evidence for Complex I Inhibition and Mitochondrial Dysfunction Using Toxicogenomic Screening

Abstract

Flutamide (FLU) is an antiandrogen primarily used in the treatment of metastatic prostate cancer. It is an idiosyncratic hepatotoxicant that sometimes results in severe liver toxicity. FLU possesses a nitroaromatic group, which may be a contributor to its mechanism of toxicity. A nitro to cyano analogue of FLU (CYA) was synthesized and used to test this hypothesis in the TGFalpha-transfected mouse hepatocyte cell line (TAMH). MTT cell viability assays and confocal microscopy showed that hepatocytes are more sensitive to cytotoxicity caused by FLU than CYA (LD 50 75 vs 150 microM, respectively). Despite the structural modification, the antiandrogen activity of CYA is comparable to that of FLU. Comparisons of transcriptomic changes caused by FLU with those caused by a panel of known cytotoxicants [acetaminophen, tetrafluoroethylcysteine, diquat, and rotenone (ROT)] indicated that FLU results in a temporal gene expression pattern similar to ROT, a known inhibitor of complex I of the electron transport chain. A subsequent microarray analysis comparing FLU to CYA and ROT revealed many similarities among these three compounds; however, FLU and ROT result in more substantial changes than CYA in the expression of genes associated with oxidative phosphorylation, fatty acid beta-oxidation, antioxidant defense, and cell death pathways. Electron microscopy confirmed that FLU leads to mitochondrial toxicity that has some similarities to the mitochondrial effects of ROT, but the morphologic changes caused by FLU were greater in scope with both intra- and intercellular manifestations. Biochemical studies confirmed that both ROT and FLU deplete cellular ATP levels and inhibit complex I of the electron transport chain to a greater extent than CYA. Thus, as compared to CYA, the nitroaromatic group of FLU enhances cytotoxicity to hepatocytes, likely through mechanisms involving mitochondrial dysfunction and ATP depletion that include complex I inhibition.