Abstract
Lead compounds are widely distributed environmental toxicants. Long-term lead exposure causes kidney, brain, and lung cancer in animals. Epidemiological studies indicate that lead is a possible human carcinogen. Previous studies have shown that sustained ERK1/2 activation elicited by lead acetate (Pb(II)) enhances nucleotide excision repair synthesis and confers anti-cytotoxicity and anti-mutagenicity. Moreover, Pb(II) genotoxicity is enhanced when cells have low level of apurinic/apyridimic endonuclease/redox factor-1 (APE/Ref-1), an enzyme functioning in the DNA base excision repair pathway and the redox activation of transcription factors such as AP-1, NF-κB and p53. In this study, we investigate the roles of ERK1/2 signal and APE/Ref-1 in gene expression profiling caused by lead acetate in human non-small adenocarcinoma CL3 cells. The cellular ERK1/2 activation was modulated using U0126, an inhibitor of MKK1/2. The involvement of APE/Ref-1 in gene expression was examined using APEas-CL3 cell line whose APE/Ref-1 level was 50% as comparison with its counterpart Babe-CL3. By using a home-made cDNA microarray with systemic quality control, we have screened a list of Pb(II)-induced genes whose expression were associated with ERK1/2 signal or APE/Ref-1 levels. Among them, PEA15 that can enhance ERK1/2 localization in the cytosol was selected for further investigation. We next adopted reverse transcription coupled with polymerase chain reaction (PCR) or real-time PCR and Western blotting to examine the gene expression and protein levels of PEA15. The results confirmed that Pb(II) can induce PEA15 gene expression and protein levels via ERK1/2 signal. This finding suggests that a negative feedback regulation of ERK1/2 is achieved by induction of PEA15 expression. On the other hand, the PEA15 gene expression and protein levels in APEas-CL3 cells were markedly lower than those in Babe-CL3 cells, suggesting APE/Ref-1 mediates in inducing PEA15 gene expression. Pb(II) could induce PEA15 gene expression and protein levels in both APEas-CL3 and Babe-CL3 cells. Taken together, we have established a cDNA microarray system and obtained several candidate genes for further investigation of the roles of ERK1/2 signal and APE/Ref-1 in Pb(II) toxicology.