Effects of Pyriproxyfen on Viability and Increase of Intracellular Lipids in HepG2 Cell Line

Abstract

Abstract Introduction: Pyriproxyfen, (2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine) (PPF) is an insecticidal used in household, agricultural, and horticultural applications to control many insect species. We tested its hepatic toxicity in hepatoma HepG2 cell line, we also evaluate if PPF could induce nonalcoholic fatty liver disease. Materials and methods: The hepatoma HepG2 cell line was exposed for 24-48 hrs with serum-free DMEM to the active principles at different concentrations. The cell viability was assessed by measuring reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). For the evaluation of in vitro steatosis, the cells were rinsed with cold phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde. Images of cell were captured using an optic microscope and stained lipid droplets were then extracted with isopropanol (60%) for quantification by measuring its absorbance at 510 nm. Results: The MTT-test showed that PPF is cytotoxic at all concentrations tested both at 24 h and 48 h. Cell viability is below 50% for concentrations 1-10 ppm while the viability is less than 10% for the concentration 100 ppm. PPF induces the increasing intracellular lipids from 1 ppm concentration. The maximum effect is observed at 100 ppm. Discussion: In our in vitro study we found a loss of cell viability of about 50% for concentrations from 1-10 ppm by the MTT-Test that measures mitochondrial enzyme activity. Because the mitochondrial enzyme activity affected major changes at the starting/beginning of the apoptotic this condition suggested that PPF is strongly cytotoxic to human hepatocytes in the presented assays. Already at 1 ppm concentration PPF induces the increasing intracellular lipids, in HepG2 in vitro culture.