Superfund Research Program
Effects Related Biomarkers of Toxic Exposures
Project Leader: Terrance J. Kavanagh
Grant Number: P42ES004696
Funding Period: 1995 - 2006
The researchers identified 5' cis acting regions of the mouse GLCLr gene that participate in both basal and inducible expression (Hudson and Kavanagh, 2000). In a collaboration with Dr. Jeff Chan (UC San Francisco) Nrf-1 deficient cells fail to support basal and inducible expression of GLCLr in mouse embryonic fibroblasts deficient for this transcription factor. The investigators have also subcloned approximately 6 kb of the GLCLc mouse promoter sequence into a reporter plasmid, and preliminary data indicates that a putative antioxidant response element located approximately 3.4 kb 5' to the transcription start site is important for basal transcription. A study which examined tissue and time dependant expression of GLCLc and GLCLr at the mRNA and protein levels in adult mice has been completed. Both subunits show some tissue specific diurnal variation which is consistent with previously reported diurnal variation in GLCL activity by others. It has also been found that the expression of the two subunits is not always coordinated. As a follow-on to previously published work, the researchers examined the expression of GLCL subunits during embryonic development, and found similar discrepancies in the expression of the two subunits. GLCL expression is high during early phases of organogenesis (day 10-12), then subsides, and then is increased again shortly before birth. Continuing previous studies which investigated the induction of GLCL by methylmercury, project investigators examined the induction of GLCL subunits in mouse embryos exposed to MeHg in utero. It appears that there is a significant increase in the expression of both subunits in the yolk sac at gestation day 14 in mice exposed to1 and 4 ppm MeHg. However, no change was apparent at either day 10 or 18. An increase was also observed in the expression of both subunits at day 18 in the placenta. No differences have been noted at any day in the expression of GLCL subunits in the embryo proper. This underscores the importance of the extra-embryonic tissues in defense against MeHg toxicity.
GLCL transgene mRNA expression has now been characterized in 3 GLCLc and 7 GLCLr transgenic mouse lines. Researchers found that 2 GLCLc and 3 GLCLr founder lines are inducible with the mifepristone "switch" such that mRNA levels equal or exceed that of the endogenous mRNA in GLCL/GLVP bigenic mice administered 5 mg/kg mifepristone i.p. An increase in GLCLr protein was also found in two of the GLCLr lines, as well as a correlation between GLCLr protein and the level of GLCLc protein and GLCL activity. In collaboration with Drs. Jun Yu, Gang Wang and Maynard Olson from the UW Genome Center and Department of Medical Genetics, project investigators determined that there are no common exonic polymorphisms in the GLCLc or GLCLr genes in humans. This resequencing effort was carried out on DNA from a population of more than 50 ethnically diverse people. Therefore, the researchers focused on the role of the GAG trinucleotide repeat in GLCLc 5' untranslated region in fibrotic lung disease. In collaboration with Dr. Ganesh Raghu of the UW Division of Pulmonary Medicine, it was shown that there is an under-representation of the 9- repeat allele in patients with idiopathic pulmonary fibrosis (IPF).