Superfund Research Program
Effects Related Biomarkers of Toxic Exposures
Project Leader: Terrance J. Kavanagh
Grant Number: P42ES004696
Funding Period: 1995 - 2006
Oxidative stress plays an important role in a number of diseases including cardiovascular disease, pulmonary fibrosis, diabetes, Parkinson's disease and Alzheimer's disease. Because the antioxidant glutathione has been shown to be altered in a number of these disease, Dr. Kavanagh has begun to examine the role of the rate limiting enzyme in glutathione synthesis, glutamate-cysteine ligase, in their pathophysiology. The researchers have now investigated the role of a GAG trinucleotide repeat polymorphism in GCLC a number of diseases including cystic fibrosis, Type 1 diabetes mellitus (T1DM) and Parkinson's disease (PD). In CF and T1DM, they have found associations between the numbers of repeats and the severity of disease (CF) or the age of onset (T1DM - females only). A manuscript describing the results in CF patients has been submitted for publication. T1DM data have been presented at several national meetings and a manuscript detailing these studies will be submitted shortly. Initial results with the PD patient population failed to show an association between this polymorphism and risk for PD. Dr. Kavanagh's team plans to expand this analysis to a larger number of patients in the near future.
Using homologous recombination and embryonic stem cell technology, project investigators have generated transgenic mice which are null for the Gclm gene. Researchers from the University of Cincinnati reported that Gclm mice are viable, whereas GCLC mice are not. Dr. Kavanagh has also found that the Gclm null mice are viable. The University of Washington model differs in a significant feature from the model the University of Cincinnati has created. The UC researchers used a neomycin resistance gene oriented in reverse orientation to disrupt the first coding exon of the Gclm gene. Dr. Kavanagh's team targets the same exon using a b-galactodosidase-neomycin (b-GEO) cDNA gene in the forward orientation, which can thus be used as a convenient in vivo reporter of the native Gclm promoter activity. This activity is easily ascertained using either colorometric (Xgal) or fluorescence (fluorescein digalactoside) staining. These mice will be used to assess the effects of oxidative stress inducing agents (MeHg, cadmium, CCl4, diquat) on Gclm expression in vivo and to determine the role of GCLM in protecting mice from oxidative stress.
In collaboration with Dr. Hooper (Project 7), these researchers have begun to assess whether Gcl genes are appropriate biomarkers of heavy metal exposure in small mammals captured at a Superfund site. Using quantitative real-time PCR, they have examined Gclc, Gclm, metallothionein (MT) and glyceraldehyde phosphate dehydrogenase (Gapdh) mRNA expression in the livers of deer mice captured at the Anaconda Smelter site in Montana. Dr. Hooper and colleagues have already made measurements on the extent of heavy metal contamination (arsenic, cadmium, lead, zinc, and copper) in these animals. Preliminary analyses of RT-PCR data indicate significant correlation between transcriptional gene expression for Gclc and cadmium liver concentrations in both males and females; and between both Gcl subunits and copper concentrations in females only. However, no correlation was found for MT and liver metal concentrations. Interestingly, a correlation has also been found between Gclc and Gclm mRNA expression for males and females suggesting possible co-regulation. Also, GAPDH was negatively correlated with cadmium exposure. This latter finding is in agreement with other reports that this housekeeping gene may be responsive to oxidative stress. This team is following up these observations by examining the expression of GCL and MT protein expression by Western analyses and also subjecting the mRNA to cDNA microarray analysis.