The Effects of Lead and Cadmium on Erythroid Differentiation and Gata-1 Regulated Gene Expression.

Lead (Pb) and Cadmium (Cd) are among the most toxic of industrial pollutants. Anemia is one of the pathologic effects caused by exposure to these heavy metals. While previous investigations have identified the inhibition of heme synthetic enzymes by Pb and the inhibition of erythropoietin production by Cd as likely causes for these toxin-induced anemias, we were intrigued by recent reports that heavy metals, including Pb and Cd are able to replace coordinating zinc (Zn) atoms in Zn finger transcription factors (e.g., P53, TFIIIA and Sp1), disrupting their function. These reports lead us to consider the hypothesis that another cause of the anemias associated with Pb and Cd poisoning might be the displacement of Zn atoms from the two Zn fingers of the erythroid-specific transcription factor GATA-1. GATA-1 is required for normal erythropoiesis and binding sites for GATA-1 are found in the regulatory elements of erythroid-specific genes. While most of the previous studies had been performed in cell-free systems, addressing our hypothesis in a cell-based system, could add weight to pathophysiologic relevance of Zn replacement by heavy metals. To test our hypothesis, we used the model of murine erythroleukemia (MEL) cell differentiation in response to dimethylsulfoxide (DMSO). We first treated MEL cells with increasing concentrations of Pb acetate (0, 0.1, 1, 4 10 and 100 uM) or Cd acetate at the same concentrations and then measured cell viability by trypan blue staining. Decreased cell viability was seen with Pb concentrations above 4 uM by 4 days in culture. Decreased viability was not seen at any Cd concentration through 7 days. We next examined the effect of the same concentrations of Pb and Cd on differentiation of MEL cells as measured by benzidine staining. Control cells treated only with DMSO showed 0% positive staining on day 0 and 90% staining by day 7 of the experiment. Pb treatment at all concentrations had no effect on differentiation. Cd, at concentrations of 10 and 100uM, appeared to slow the kinetics of differentiation but the proportion of benzidine positive cells by day 7 was only slightly reduced. This lack of effect on our model of differentiation lead us ask whether Pb or Cd could be affecting the expression of individual genes which require GATA-1 for proper expression. We used quantitative real-time PCR to measure the levels of mRNA for β-globin, α-globin, erythropoietin receptor and GATA-1 genes daily over the 7 day course of the differentiation experiment. The β-actin gene served as a control for these experiments. As expected, levels of β- and α-globin mRNAs increased 5–10 fold during the experiment. Levels of the other genes, including β-actin, decreased over the time course. No dose-dependent changes in the levels of gene expression were observed with either Pb or Cd treatments. Given these findings, we wanted to determine whether Pb and Cd were capable of inhibiting the ability of GATA-1 to bind DNA. Following pre-incubation with the Pb or Cd, gel mobility shift experiments were conducted using MEL nuclear extract and an oligonucleotide known to bind GATA-1 from the β-globin LCR. No changes in binding were seen at the concentrations used in our experiments. Only at concentrations of 1mM or higher of either metal was DNA binding inhibited. These findings suggest that our hypothesis was incorrect and that at concentrations associated with Pb and Cd toxicity in humans there apear to be no significant effects on erythroid differentiation or GATA-1 function.