Abstract
Cadmium compounds are ubiquitous environmental toxicants that can cause genotoxicity and cytotoxicity, and have been categorized as group 1 carcinogen by International Agency for Research on Cancer. Previous studies have indicated that cadmium-elicited activation of p38 MAPK triggers cell cycle arrest or apoptosis. Cadmium cytotoxicity is also associated with the generation of free radicals. In this thesis, I investigated the role of nitric oxide (NO) elicited by cadmium chloride (Cd) in regulating p38 MAPK signaling pathway and cytotoxicity in H157 lung cancer cells. By using NO fluorescence capture dye in combined with flow cytometry analysis, I found that Cd could induce NO levels in H157 cells. Co-treatment of a NO scavenger PTIO or a NO donor GSNO, respectively, increased or decreased Cd-induced cytotoxicity as determined by the MTT assay. Immunoblotting showed that PTIO co-treatment further enhanced the capability of Cd to elicit p38 phosphorylation. The results thus suggest that NO may down-regulate p38 activation and thereby prevents Cd-induced cell death. S-nitrosylation on p38 was observed following in vitro and in vivo GSNO treatments as analyzed by SNO-RAC technique in combined with immunoblotting. Cd also markedly induced S-nitrosylation on endogenous p38 in H157 cells. Furthermore, specific single, double, triple cysteine (Cys) residues on p38α were changed to serine/alanine residues using Flag-p38α expression vector as a temple and site-directed mutagenesis procedure. These p38α mutant plasmids were transfected into H293 cells and allowed overexpression to generate Flag-p38α mutant proteins for in vitro NO modification and S-nitrosylation assay. The results showed that Cys39 and Cys162 of p38α maybe the major S-nitrosylation sites. These Flag-p38α expression vectors containing Cys mutations could provide further studies to elucidate the importance of S-nitrosylation on p38 signaling. Together, results obtained in this study suggest that NO generation following Cd stimulation promotes S-nitrosylation on p38 that may lessen Cd-induced cytotoxicity.