Abstract:
Para-Phenylenediamine (PPD) has long been used in two-thirds of permanent oxidative hair dye formulations. Epidemiological studies and several in vitro as well as in vivo studies have showed equivocal results in potential carcinogenicity of PPD however, the underlying mechanisms still remains elusive. The present study therefore aims to investigate the cytotoxicity and the mechanism of action, namely the signal transduction pathways involved upon PPD exposure on normal rat kidney (NRK-52E) cells and human urothelial (UROtsa) cells. Cell viability assay was carried using the MTT calorimetric method and cell cycle analysis was detected by flow cytometry. The mode of cell death was determined using annexin V binding assay. To further confirm apoptosis mitochondrial membrane potential and caspase-3 assays were performed. Reactive oxygen species generation was detected by 2′,7′- dichlorofluorescin diacetate staining and protein expression was studied using western blot analysis. Our results showed that PPD decreased cell viability of NRK52E cell significantly and induced reactive oxygen species (ROS) and apoptosis in a dose dependant manner. PPD treated NRK-52E cells showed up-regulation of phosphorylated-SAPK-JNK protein expression and down-regulation of Ras and Raf protein expression; however, Akt, Bcl-2, Bcl-xL and Bad protein expression levels were not significantly changed as compared to control. On the other hand, PPD treatment on UROtsa cells decreased cell viability and increased sub-G1 hypodiploid cells in a dose dependant manner. Cell death due to apoptosis was detected using annexin V binding assay. Further analysis showed PPD induced mitochondrial dysfunction through the loss of mitochondrial membrane potential (MMP) and increased caspase-3 levels in UROtsa cells. PPD treated UROtsa cells generated ROS in a time dependant manner. Western blot analysis of PPD treated UROtsa cells down-regulated phosphoylated proteins from all three pathways the NF-κB, Wnt and mTOR pathways. In conclusion oxidative stress and apoptosis induced by PPD is correlated with the activation of PTK-Ras-Raf-JNK and inhibition of PI3K/Akt pathways in NRK52E cells whereas PPD induced oxidative stress and apoptosis in UROtsa cells were correlated with the inhibition of NF-κB, Wnt and mTOR pathways.
