Enantioselective cytotoxicity profile of o,p'-DDT in PC 12 cells

Abstract

Background: The continued uses of dichlordiphenyltrichloroethane (DDT) for indoor vector control in some developing countries have recently fueled intensive debates toward the global ban of this persistent legacy contaminant. Current approaches for ecological and health risk assessment has ignored the chiral nature of DDT. In this study by employing an array of cytotoxicity related endpoints, we investigated the enantioselective cytotoxicity of o,p'-DDT.

Principal findings: we demonstrated for the first time that R-(-)-o,p'-DDT caused more neuron cell death by inducing more severe oxidative stress, which selectively imbalanced the transcription of stress-related genes (SOD1, SOD2, HSP70) and enzyme (superoxide dismutase and lactate dehydrogenase) activities, and greater cellular apoptosis compared to its enantiomer S-(+)-o,p'-DDT at the level comparable to malaria area exposure (parts per million). We further elucidated enantioselective modes of action using microarray combined with enzyme-linked immunosorbent assay. The enantioselective apoptosis might involve three signaling pathways via caspase 3, tumor protein 53 (p53) and NF(k)B.

Conclusions: Based on DDT stereochemistry and results reported for other chiral pesticides, our results pointed to the same directional enantioselectivity of chiral DDT toward mammalian cells. We proposed that risk assessment on DDT should consider the enantiomer ratio and enantioselectivities.

Figure 1. Oxidative stress induced by racemate and enantiomers of o,p’-DDT.

Effect of the racemate of o,p’-DDT and individual stereoisomers on extracellular lactate dehydrogenase (LDH) release, intracellular superoxide dismutase (SOD) and malondialdehyde (MDA) production at concentration of 3.5×10⁻⁵ mol/L. Data are presented as the relative value of control. PC12 cells were exposed to different compounds for 24 h. The asterisk above each bar indicates a significant difference compared to a negative control (p<0.05, n = 3). Different letters above adjacent bars indicate a significant difference (p<0.05) between the two enantiomers, whereas the same letter indicates no significant difference.

Figure 2. Oxidative stress related gene induction by racemate and enantiomers of o,p’-DDT.

The relative expression of anti-oxidative related genes encoding superoxide dismutase (SOD1 and SOD2) and heat shock protein (HSP) genes in response to racemate and enantiomers of o,p’-DDT. The asterisk denoted p<0.05 relative to the negative control. Different letters above adjacent bars indicate a significant difference (p<0.05) between individual enantiomers or between an enantiomer and racemate, while the same letter indicates no significant difference.

Figure 3. Enantioselectivity in DDT-inducted apoptosis in PC12 cell.

The percent of early apoptotic cells following treatment with individual enantiomers or racemate of DDT was measured with flow cytometry quantified by Annexin V (AV)-PI staining.

Figure 4. Heatmap of 84 apoptotic genes.

The hierarchical clustering was performed for microarray data. Green indicates relative low expression, and red indicates relative high expression.

Figure 5. Apoptotic related proteins induced by racemate and enantiomers of o,p’-DDT.

The protein expression of (A) p53, (B) caspase3 and (C) NF_kB after exposured to rac-o,p’-DDT, R-(+)-o,p’-DDT, and S-(−)-o,p’-DDT. The asterisk indicates p<0.05 relative to a negative control. Different capitalized letters above adjacent bars indicate a significant difference (p<0.05) between individual enantiomers or between an enantiomer and racemate, while the same letter indicates no significant difference.

Figure 6. A speculated signaling pathway mediated by o,p’-DDT for the induction of cells apoptosis.

The red arrowheads indicate the more significant upregulation of apoptosis-related molecules induced by R-form than that of S-form. Reactive oxygen species (ROS), Heat shock proteins (HSPs), Lactate dehydrogenase (LDH), Superoxide dismultase (SOD), Malondialdehyde (MDA).