Superfund Research Program
Effects Related Biomarkers of Toxic Exposures
Project Leader: Terrance J. Kavanagh
Grant Number: P42ES004696
Funding Period: 1995 - 2006
Glutathione (GSH) is a low molecular weight intracellular thiol that is important in free radical scavenging, detoxification of xenobiotics, protein synthesis, DNA synthesis and repair, and cell proliferation. One consequence of exposure to a wide variety of xenobiotics is the induction of glutathione biosynthetic enzymes, especially glutamylcysteine synthetase (GCS), the rate limiting enzyme in GSH biosynthesis. This project is investigating whether changes in the content of GSH, coupled with changes in the expression of mRNA and activity for GCS in accessible body tissues can be utilized as biomarkers of oxidative stress induced by chemical exposures, as well as biomarkers of susceptibility in exposed individuals.
Over the past year, work has been focused on the cloning and amplification of cDNAs and genes for glutamylcysteinyl ligase (GLCL) from mouse kidney mRNA. A high degree of homology for the regulatory (GLCLR) and catalytic subunits (GLCLC) between rat, mouse and human cDNAs was found. The 3' untranslated portion of the mouse GLCLC cDNA was also successfully amplified and a high degree of homology to the rat sequence was discovered. Using an in vitro transcription and translation system, the proteins for GLCLC and GLCLR were expressed and an increase in GCS activity was found when the catalytic subunit cDNA was included alone or in combination with the regulatory subunits. No GCS activity was noted with the regulatory subunit alone.
In related research, a collaborative effort between this project and Bristol-Meyer Squibb is examining the role of GSH status in oxidative stress-induced transmembrane signaling, tyrosine phosphorylation and apoptosis in human peripheral blood lymphocytes. This research has led to the discovery of an interesting marker of oxidative stress (and possibly apoptosis): an increase in red autofluorescence. Another achievement from this collaboration is the development of anti-GLCLR and anti-GLCLC antisera for use in Western analyses and potentially for immunocytochemical (ICC) analyses of animal and human tissues.
The significance of this work is related to the need to develop sensitive and rational biomarkers of exposure, effect and susceptibility in human populations. To that endcharacterizing the sequence of GLCL genes and their expression will provide useful tools for the assessment of chemical exposures, especially to those agents which harm the body by causing oxidative stress.