Research Grants 1998-1999

Fluorescent Probing of Oxidative Stress and Antioxidant Efficacy in a Cell Culture Model

Valerian E. Kagan, PhD
University of Pittsburgh, Pittsburgh, Pennsylvania

Currently, both cell systems and whole animal models used for testing toxic agents thought to act through oxidative stress suffer from a major drawback: metabolic turnover and repair mechanisms for lipids ( a major target of oxidative stress) are extremely efficient. Hence, it is difficult to determine whether oxidative stress has actually occurred. A further drawback in both animal and cell systems is the lack of sensitive measurement techniques. The combination of these two deficiencies means that, at present, extremely large quantities of cells or many animals must be used or, alternatively, extremely large, unphysiological doses of oxidant must be applied.

The model perfected by Kagan and his colleagues eliminates both these drawbacks, thereby providing a system for testing oxidants and antioxidants that is superior both to current animal testing and to current cell culture techniques. Moreoever, the model permits study of site-specific oxidative stress and antioxidant protection in different classes of membrane phospholipids of living cells. Previously, Kagan demonstrated that antioxidants can be successfully evaluated using his model.

Now, the investigator plans to:

Validate the model as a predictor of site-specific oxidative stress in membrane phospholipids of living cells. Kagan's model may be useful as a testing ground for site-specific peroxidation of phospholipids resulting in the lipid-signaling function of phospholipids. The team will use murine leukemia 32 D cells as well as human leukemia HL-60 cells in which they will induce oxidative stress by two toxic chemicals: paraquat and phenol (a major metabolite of benzene). Based on preliminary results they predict that their model system will identify site-specific oxidative stress. They will test whether specific phospholipid peroxidation products may function in signaling pathways in apoptotic cells.
Validate the model as a predictor of site-specific antioxidant protection in membrane phospholipids of living cells. In particular, they plan to test whether the model may provide proof of site-specific antioxidant protection that regulates signaling role(s) of phospholipids in cells. In these experiments, they will use muring leukemia 32 D cells and human leukemia HL-60 cells. In both of these cell lines, they will test whether different isomers and homologues of vitamin E, the major lipid-soluble antioxidant of membranes, protect against site-specific oxidation of membrane phospholipids.
Perfect quantitation of site-specific phospholipid peroxidation through use of high-performance thin-layer chromatographic (HPTLC) separation of phospholipids and laser-induced detection and quantitation of fluorescently-labeled phospholipids. Kagan's fluorescence-HPTLC technique proved to be a powerful tool for quantitating oxidative stress in membrane phospholipids of living cells. This group is working to perfect methodology for separation and analysis of fluorescently labeled phospholipids, especially minor phospholipid classes.

This model has achieved its major goal to date: accurately quantifying the actual degree of lipid peroxidation (without being hidden by repair systems) in cells treated with sub-toxic levels of oxidants as well as the protection afforded by antioxidants. Their developed model may be able to replace much animal testing of prooxidants and antioxidants.