Cytotoxicity and Transcriptional Activation of Stress Genes in Human Liver Carcinoma (HepG₂) Cells Exposed to Iprodione

Iprodione (C₁₃H₁₃Cl₂N₃O₃) is a broad spectrum dicarboximide fungicide used on a wide variety of crop diseases. It is used on vegetables, ornamentals, pome and stone fruit, root crops, cotton and sunflowers, to control a variety of fungal pests. Iprodione inhibits the germination of spores and the growth of the fungal mycelium. Experimental studies with mice have indicated that exposure to iprodione at dose levels 5 to 15 folds greater than the LOAEL for liver injury, induces microsomal enzyme activities, hepatocyte proliferation, hepatomegaly, centrilobular hypertrophy, diffuse hypertrophy, and an increase in lauric acid hydroxylation. Currently, there is no toxicological data available on human health effects associated with exposure to iprodione. In this research, we performed the MTT Assay for cell viability to assess the cytotoxicity of iprodione, and the CAT-Tox (L) assay to measure the induction of stress genes in thirteen recombinant cell lines generated from human liver carcinoma cells (HepG₂). The cytotoxicity data indicated a strong concentration - response relationship with regard to iprodione toxicity. The percentages of cell viability were 100 ± 0%, 128.0 ± 41.4%, 97.5 ± 37.7%, 70.1 ± 35.4%, 33.5 ± 16.1%, and 5.1 ± 3.7% in 0, 31.3, 62.5, 125, 250, and 500 μg/mL, respectively. The LC₅₀ was 208.3±83.3 μg/mL. Data obtained from the CAT-Tox (L) assay showed that iprodione is able to induce a significant number of stress genes in HepG₂ cells. At 250 ug/mL exposure, the induction levels were 1.2 ± 0.4, 50.1 ± 17.8, 3.9 ± 1.2, 16.8 ± 7.2, 10.7 ± 0.7, 1.8 ± 0, 26.3 ± 10.0, 7.2 ± 2.4, 1.8 ± 0.0, 6.8 ± 1.3, 6.7 ± 0.5, and 4.3 ± 1.8 for CYP1A1, GSTYa, XRE, HMTIIA, c-fos, NF-kBRE, HSP70, CRE, RARE, GADD153, GADD45, and GRP78, respectively. These results indicate that the metabolism of iprodione involves Phase II biotransformation in the liver (XRE, GSTYa), and that this chemical has the potential to cause cell proliferation and/or inflammatory reactions (c-fos, NF-kB), proteotoxic effects (HSP70, GRP78), metabolic disruption (CRE), and DNA damage (GADD45, GADD153).