Superfund Research Program
Effects Related Biomarkers of Toxic Exposures
Project Leader: Terrance J. Kavanagh
Grant Number: P42ES004696
Funding Period: 1995 - 2006
Dr. Kavanagh previously reported that basal and BHQ-inducible expression of the mouse glutamate-cysteine ligase modifier subunit gene (Gclm) was dependent on regulatory elements located within 787 bp upstream of its translation start codon. This region is highly homologous to the human GCLM promoter and contains a conserved antioxidant response element (ARE). Electromobility shift assays (EMSAs) reveal constitutive and BHQ-inducible binding of mouse Hepa-1 nuclear factors to this ARE, and mutagenesis of the ARE in a luciferase reporter construct containing 1.3 kb of Gclm promoter preferentially diminished inducible expression and affected basal expression as well. EMSAs and site-directed mutagenesis studies reveal constitutive and BHQ-inducible binding of nuclear factors at a putative Nrf/AP1 site upstream of the ARE. The transcription factor Nrf1 mediates Gclm expression as evidenced by a 10-fold reduction in luciferase reporter activity in Nrf1-deficient cells, which can be partially restored upon co-transfection with Nrf1 cDNA. Taken together, these data indicate regulation of Gclm by the transcription factors Nrf1 and Nrf2 via the ARE and upstream Nrf/AP1 binding site.
Project investigators generated transgenic mice designed to conditionally over-express GCLC or GCLM using the mifepristone (RU486) sensitive GeneSwitchTM system, which utilizes a liver specific (transthyretin) promoter to transactivate GCL transgene expression. We have conducted some preliminary experiments to determine the sensitivity of these animals to carbon tetrachloride-induced liver injury. Frozen liver sections were stained with diphenyl-1-pyrenylphosphine (DPPP) for measurement of lipid peroxides. Liver injury was more apparent in control animals receiving CCl4 than in GCL transgenic animals. These data were recently presented at the International Society for Analytical Cytology meeting in San Diego.
In collaboration with Drs. Moira Aitken and Ed McKone, researchers have examined the role of a GAG trinucleotide repeat polymorphism in GCLC in cystic fibrosis (CF). Individuals with the same mutation in CFTR show a great deal of heterogeneity in CF disease progression. Association of this polymorphism with disease severity was highly statistically significant for individuals with milder CFTR genotypes, suggesting an important role for GCL in a subset of CF patients.