The effect of 2,4-D on gene expression in cultured cells

Abstract

The cytotoxic effects of exposure to low concentrations of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) that are typically found in groundwater were investigated, in vitro. Most 2,4-D toxicology studies use high concentrations of the herbicide that are above those typically found in groundwater and measure overt biological endpoints. In contrast, this thesis examines the effects of low concentrations of 2,4-D and measures more subtle and sensitive endpoints such as gene expression and the generation of reactive oxygen species. This work derives from recent cDNA microarray analysis conducted in our laboratory that revealed significant alterations in the expression of 238 genes in cells exposed to nanomolar (nM) concentrations of a commercial formulation of 2,4-D. These findings are extended in this thesis to include the in vitro cytotoxic effects of low concentrations of both technical and commercial 2,4-D on two cell lines. Cells derived from liver (HepG2) and kidney (HEK293) respectively, were chosen, since liver and kidney are known to metabolize 2,4-D in vivo. Cell viability was measured using the Resazurin assay, reactive oxygen species (ROS) were measured with 2’,7’-dichlorofluorescin diacetate (2’,7’-DCFH-DA), and real time–polymerase chain reaction (RT-PCR) was used to assess changes in mRNA expression while protein expression was examined by Western blot.Cell viability studies revealed that low environmental concentrations (0.1 to 100 nM) of 2,4-D induced small, but statistically significant decreases in cell viability. No concentration or time-dependent decreases in cell viability were observed in cells exposed to either forms of low environmental 2,4-D concentrations. HEK293 cells were more susceptible than HepG2 cells to the toxic effects of both forms of 2,4-D, having statistically significant lower viability at all exposure concentrations and durations. Both forms of 2,4-D reduced cell viability in both cell lines, suggesting that cytotoxicity was induced directly by 2,4-D, and not by the ‘inert ingredients’ in the commercial formulation.The ROS assays illustrated that 2,4-D induced statistically significant ROS production in HepG2 and HEK293 cell cultures at concentrations greater than 10 µM and 100 nM respectively. This was both a concentration and time-dependent effect in both cell lines. Although HEK293 cells were more susceptible to 2,4-D, they had 50 to 70% less ROS production than HepG2 cells, at all exposure concentrations and times.The RT-PCR and Western blot analyses showed that exposure of HepG2 and HEK293 cells to low 2,4-D concentrations induced (< 2 fold) alterations in mRNA and protein levels of FTL, FTH1 and PCNA however these changes did not consistently vary with concentration.Taken together, cell viability, ROS and gene expression studies show that low environmental 2,4-D concentrations induced subtle in vitro cytotoxic effects. However we have no evidence that these subtle changes pose a serious health threat to exposed humans.