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Doctoral Dissertation Announcement
Candidate:Worlanyo Eric Gato
Doctor of Philosophy
Title: Gene Expression Analysis of Fisher 344 Male Rats Exposed to 0PPM, 50PPM and 500PPM Lead Acetate in Drinking Water
Dr. Jay Means, Chair
Dr. Michael Barcelona
Dr. David Huffman
Dr. Robert Eversole
Date: Wednesday, February 21, 2007 2:00 - 4:00 p.m.
2734 Wood Hall
Lead contamination of soil and drinking water represent a significant risk to human health, especially for developing infants. Various methods are employed in determining lead intoxication effects on humans using rats as model organisms. Histopathological analysis, multi-elemental analyses, biochemical analyses such as glycogen levels, aminolevulinic acid (ALA) and DNA and RNA contents and recently toxico-genomic analysis are some of the approaches used. As a way of laying the foundation for research in analyzing global hepatic differential gene expression patterns and profiles in Fisher 344 rat liver, the effects of lead toxicity on Fisher 344 rats were assessed by histology and tissue residues were measured by inductively-coupled plasma mass spectrometry (ICP-MS).
ICP-MS results showed a significant accumulation of lead in blood, liver, kidney and bone marrow in lead exposed groups. With the exception of kidney, the 90-days treatment groups showed markedly high levels of lead in blood, liver and marrow than the 30-days exposed groups. Potential interactions of calcium, iron, cobalt, copper, zinc and nickel and lead examined showed positive and negative correlation for 30 and 90 days treatment period respectively. Hepatic histopathology produced no evidence of necrosis or changes in architecture of hepatocytes in the 0ppm and 50ppm for the 30-day duration of exposure in the case of the liver. In contrast, necrosis and alterations in the structure and disposition of the liver and kidney tissues were observed for the 500ppm treatment group.
The scatterplot data suggest a greater number of genes were differentially expressed in the 90 days 500ppm Pb2+ treatment than the other dose groups. Using a 2-fold expression difference threshold, genes either up/down-regulated appear to be the same for both treatments during the 30 days exposure period. Interestingly, 90 days 50ppm treatment showed less than half the number of genes expressed compared to the 30 days treatment. In contrast, the 90 days 500ppm group had over one thousand genes differentially expressed at 2-fold and more than twice the number of genes of the other treatment groups at 3-, 5-, and 10-fold levels. Differentially expressed genes included genes cited in the literature and those not reported to be affected by lead toxicity.
Expression profiles analyzed by clustering and gene ontology (GO) showed clustering patterns that appear to be similar for both time points and dose levels. However, the short term exposure group (30d) showed far fewer genes being affected by ±2-fold than in the sub-chronic treatment group (90d). This was confirmed by multidimensional scaling plot which showed that a majority of arrays congregate around the origin. Gene ontology analysis revealed 15 GO categories affected by chronic (90d) lead exposure, whiles three GO categories were observed to be significantly affected for short exposure (30d) periods. The following pathways; Regulation of eIF4e and p70 S6 Kinase, Control of skeletal myogenesis by HDAC and calcium/calmodulin-dependent kinase (CAMK), Role of MEF2D in T-cell Apoptosis are significantly perturbed by lead poison in vivo.
Affymetrix Microarray expression data was validated by Taqman quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Using Taqman RT reagents the expression of calm1, calm2, Alas1, Atp5o, Ppp3ca, Cyp3a13 and Mapk1 were measured relative to β-Actin. Most of the transcripts in the 30d exposure group were up-regulated while most transcripts in the 90d treatment were down-regulated, relative to controls. Confirming microarray results are the down-regulation of Calm2: -3.886, Alas1: -1.616, Atp5o: -2.706 and Cyp3a were13: -1.79 genes in the long term exposure group.