Chromium(III)-induced apoptosis of lymphocytes: death decision by ROS and Src-family tyrosine kinases

Apoptosis is an active process induced by a variety of physiological and external stimuli, in which elimination of damaged cells are effected through a genetically controlled process. In this study, we have examined the mechanism of chromium(III) [Cr(III)]-induced cytotoxicity with respect to its relationship to oxidative stress. Morphology, flow cytometry, and DNA fragmentation studies show that tris-(1,10-phenanthroline)chromium(III) [Cr(III)-phen], tris-(2,2'-bipyridyl)chromium(III) [Cr(III)-bpy], trans-diaqua[1,2-bis(salicylideneamino)ethanechromium(III)] [Cr(III)-salen], and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] [Cr(III)-salprn] induced apoptosis of lymphocytes. Pentaammineaquachromium(III) [Cr(III)-hpa] does not induce apoptosis. Apoptosis induced by these complexes involves the generation of reactive oxygen species (ROS) as seen by increased fluorescence of dichloroflourescein (DCF) observed through flow cytometry. Pretreatment of lymphocytes with antioxidants completely abrogate apoptosis. Cr(III) treatment also increased the expression and activation of Src-family tyrosine kinases viz. p56lck, p59fyn, and p53/56lyn, as seen by immunoblotting and immune complex kinase assay. PP2, a selective Src-family tyrosine kinase inhibitor, abolishes apoptosis, indicating that Src-family tyrosine kinases are directly involved in eliciting apoptosis. Interestingly, a one-to-one correlation between the expression of Src-family tyrosine kinases and ROS is observed, since antioxidants pretreatment inhibits the expression and the activation of these kinases. These results further indicate that Cr(III)-induced apoptosis is mediated through production of ROS, which in turn activates the Src-family tyrosine kinases. The increased activation of Src-family tyrosine kinases may be a mechanism involved in apoptosis of lymphocytes elicited by various other physiological stimuli that exploit ROS as a second messenger