Analysis of Hepatic Gene Expression Patterns of Fisher 344 Rats Exposed to Pb2+ Using Affymetrix Microarray and Taqman RT-qPCR

Lead contamination of soil and drinking water represent a significant risk to human health, especially for developing infants. Methods employed in determining lead toxic effects on humans using animal models include; histopathological analysis, biochemical analysis such as glycogen levels, aminolevulinic acid (ALA) and DNA and RNA contents and more recently toxicogenomics analysis (study of gene and protein activity response to toxicants. In this study Affymetrix DNA Microarray analyses were used to assess transcriptional profiles due to dose levels and characterize changes in hepatic gene expression in response to Male Fisher 344 rats exposed to 0 ppm, 50 ppm and 500 ppm lead (as acetate) for up to 90 days. To validate gene expression data acquired by Affymetrix Microarray, Taqman RT-qPCR was used to quantify the relative expression levels of selected genes already known to play im-portant roles in mediating Pb2+ toxicity and others identified in our study that might be equally important in this process. Using Taqman RT reagents and Assay-On-Demand offered by Applied Biosystems, the expression of calm1, calm2, Alas1, Atp5o, Ppp3ca, Cyp3a13 and Mapk1 were measured relative to β-Actin. Raw data was exported for normalized gene expression (NGE) analysis using data analysis for real-time PCR (DART-PCR) and relative expression software tool (REST©). Most of the transcripts in the 30d exposure group were up-regulated while most genes in the 90d treatment were down-regulated, relative to controls. Calm1, Calm2, Atp5o, Ppp3ca and Mapk1 were all upregulated in the short treatment regime. For the chronic exposure group, Calm1, Calm2, Atp5o, Ppp3ca, Mapk1 were all down-regulated except Calm2 in the 50 ppm treatment group, relative to controls. Similarly, Alas1 was negatively regulated in all treatment groups except the 90d 50 ppm group. Cyp3a13 was negatively regulated in all treatment groups during all time points. Confirming microarray results are the down-regulation of Calm2 -3.886, Alas1 -1.616, Atp5o -2.706 and Cyp3a13 -1.79 genes in the long term exposure group.