Use of molecular and genomic approaches to characterize molecular and cellular effects of lead toxicity

The toxic metal lead is a widespread environmental health hazard that can adversely affect human health. Lead can substitute for biologically essential metals such as calcium and zinc in protein metal-binding domains. The binding of lead to these domains can cause abnormal protein activity resulting in altered gene expression patterns and perturbed cellular pathways. In an effort to further examine this hypothesis we undertook studies of the molecular and cellular consequences of lead toxicity. We have demonstrated that the normally calcium-dependent binding characteristics of the synaptic vesicle protein synaptotagmin I are altered by lead. Nanomolar concentrations of lead induce the interaction of synaptotagmin I with phospholipid liposomes. The C2A domain of synaptotagmin I is required for lead-mediated phospholipid binding. Lead protects synaptotagmin I from proteolytic cleavage in a manner similar to calcium. However, lead is unable to promote the interaction of synaptotagmin I and syntaxin. Finally, lead is able to directly inhibit the ability of calcium to induce the interaction of synaptotagmin I and syntaxin. We have also employed cDNA microarrays to analyze the effects of lead exposure on gene expression patterns in immortalized rat astrocytes. We have identified novel putative targets of lead-mediated toxicity including members of the family of calcium/phospholipid binding annexins. We have demonstrated the ability to distinguish lead treated samples from control or sodium samples solely on the basis of large-scale gene expression patterns using two complementary clustering methods. Finally, we have shown that the calcium-dependent phospholipid binding protein annexin A5, initially identified as a differentially regulated gene by our microarray analysis, is directly bound and activated by nanomolar concentrations of lead. During the analysis of our microarray data we developed a set of novel bioinformatics tools called “Database Referencing of Array Genes ONline” or “DRAGON”. Based on our findings, we conclude that both synaptotagmin 1 and annexin A5 may be important, physiologically relevant targets of lead. Furthermore, we conclude that microarray technology is an effective tool for the identification of lead-induced patterns of gene expression and molecular targets of lead.