Characterization of human serum albumin modified by hair dye component, 4-chloro-1,2-phenylenediamine: Role in protein aggregation, redox biology and cytotoxicity

Abstract

Hair dyes and their components have been described as mutagenic and carcinogenic. They penetrate the skin, undergo auto-oxidation, yield protein reactive derivatives and modify proteins in an irreversible manner. Investigations into the impact of aromatic diamines on protein structures are expected to have a significant clinical value. This study documents the strong potential of 4-chloro-1, 2-phenylenediamine (4-Cl-o-PDA), a hair dye component, in the modification of human serum albumin (HSA). HSA was modified in vitro by 4-Cl-o-PDA and changes in tertiary and secondary structural characteristics were analyzed by UV–visible, fluorescence and far UV circular dichroism, fourier transform infrared spectroscopical techniques and ANS binding assay. Interaction of 4-Cl-o-PDA and HSA was studied by molecular docking and drug displacement assay. Formation of aggregates and inhibitory effect of antioxidant curcumin was analyzed by ThT binding assay, ThT fluorescence and electron microscopy. Changes in oxidation status of HSA were studied by thiol estimation and NBT assay. Effect of 4-Cl-o-PDA on cellular redox status, DNA integrity and cytotoxicity was analyzed in lymphocytes using dichlorofluorescein method, comet assay and MTT assay. We observed gross structural damage in HSA modified by 4-Cl-o-PDA. Modification led to unmasking of hydrophobic clusters, loss of its alpha helix and an increase in beta strand and unordered structure in HSA. 4-Cl-o-PDA strongly binds at sub-domain IB of HSA. HSA modified by 4-Cl-o-PDA formed amyloid fibrillar aggregates, whose formation was inhibited by antioxidant treatment. Modified HSA showed reduction of free sulfydryl content. 4-Cl-o-PDA-HSA led to the generation of reactive oxygen species in human lymphocytes. Modified HSA caused an increase in the tail length of DNA indicating enhanced single strand DNA breaks in cells. Modified protein was found to be genotoxic and cytotoxic. This works offers insights in the role of hair dye components in the biological world.