Final Reports (1999)

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

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

Quantitative assays of lipid peroxidation are essential for evaluating oxidative damage from various sources, and in testing the efficacy of antioxidant interventions. At present such assays are often performed in chemical systems, then transferred to animal models. However, the results of chemical testing, both of oxidative stressors and of antioxidants, are often not applicable in vivo. This is due to the effects of cell metabolism on potential oxidants and antioxidants and the effects of lipid turnover and repair mechanisms on the products of lipid oxidation. As a result of the present situation, a great deal of confusion is generated, and much needless animal testing is performed, in an attempt to extrapolate chemical data to the in vivo situation. An obvious solution is to use cell systems as an intermediate testing model. However, present cell systems have several disadvantages, which they share with animal models, that often dissuade researchers from their use. The major problem was the lack of a sensitive probe for lipid peroxidation that is unaffected by cell repair systems. We solved this problem by developing our cell model for quantitating site-specific oxidative stress in membrane phospholipids.

Our

Specific Aims were:

Specific Aim 1. Validate the model as a predictor of protective effects of exogenous antioxidant compounds. This Specific Aim included three components:
1. Studies of site-specific action of vitamin E and its homologues against uniform oxidation of phospholipids in human erythroleukemia K562 cells and rat aortic smooth muscle cells induced by lipid-soluble azo-initiator of peroxyl radicals (AMVN).
2. Studies of vitamin E antioxidant protection against site-specific oxidation of phospholipids by tert-butyl hydroperoxide (t-BuOOH) in K562 cells.
3. Studies of antioxidant effects of nitric oxide (NO) against AMVN-induced phospholipid peroxidation in human erythroleukemia K562 cells and rat aortic smooth muscle cells.
Specific Aim 2. Validate the model as a test for endogenous antioxidant mechanisms. This Specific Aim included two components:
1. Studies of antioxidant properties of Bcl-2 gene product in PC12 and 32D cells.
2. Studies of antioxidant properties of metallothioneins in murine lung microvascular endothelial cells.

Both Specific Aims were successfully studied and completed during the funding period. Novel methodology for investigating site-specific oxidative stress in membrane phospholipids has been developed and successfully used in studies of oxidative stress mechanisms in live cells. The power of our newly developed approach and different applications of the technique are illustrated by 20 papers published during this period of time in prestigious peer-reviewed journals as follows:

Ritov, V.B., Banni, S., Yalowich, J.C., Day, B.W., Claycamp, H.G., Corongiu, F.P., Kagan, V.E. (1996). Non-random peroxidation of different classes of membrane phospholipids in live cells detected by metabolically integrated cis-parinaric acid. Biochim. Biophys. Acta 1283: 127-140.
Ritov, V.B., Menshikova, E.V., Goldman, R., Kagan, V.E. (1996). Direct oxidation of polyunsaturated cis-parinaric fatty acid by phenoxyl radicals generated by peroxidase/ H2O2 in model systems and in HL-60 cells. Toxicol. Lett. 87: 121-129.
Fabisiak, J.P., Kagan, V.E., Ritov, V.B., Johnson, D.E., Lazo, J.S. (1997). Bc1-2 inhibits selective oxidation and extemalization of phosphatidylserine during paraquat-induced apoptosis. Am. J. Physiol.: Cell Physiol. 41(2): 675-684.
Tyurin, V.A., Carta, G., Tyurina, Y.Y., Banni, S., Day, B.W., Corongiu, F.P., Kagan, V.E. (1997). Peroxidase-catalyzed oxidation of Carotene in HL-60 cells and in model systems: involvement of phenoxyl radicals. Lipids 32(2):131-142.
Osaka, K. , Ritov, V.B., Bernardo, J. F., Branch, R.A., Kagan, V.E. (1997). Amphotericin B protects cis-parinaric acid against peroxyl radical-induced oxidation: amphotericin B as an antioxidant. Antimicrob. Agents and Chemother. 41(4): 743-747.
Gorbunov, N.V., Yalowich, J.C., Gaddam, AS., Thampatty, P., Kisin, E.R., Elsayed, N.M., Kagan, V.E. (1999). Nitric oxide prevents oxidative damage produced by tert-butyl hydroperoxide in erythroleukemia cells via nitrosylation of heme and non-heme iron: electron paramagnetic resonance evidence. J. Biol. Chem. 272: 12328-12341.
Tyurina, Y.Y., Tyurin, V.A., Carta, G., Quinn, P.J., Schor, N.F., Kagan, V.E. (1997). Direct evidence for antioxidant effect of Bcl-2 in PC-12 rat pheochromocytoma cells. Arch. Biochem. Biophys. 344: 413-423.
Osaka, K., Tyurina, Y.Y., Dubey, R.K., Tyurin, V.A., Ritov, V.B., Quinn, P.J., Branch, R.A., Kagan, V.E. (1997). Amphotericin B acts as an intracellular antioxidant: protection against 2,2'-azobis(2,4-dimethylvaleronitrile)-induced peroxidation of membrane phospho-lipids in rat aortic smooth muscle cells. Biochem. Pharmacol. 54: 937-945.
Pitt, B.R., Schwarz, M., Woo, E.S., Yee, E., Wasserloos, K., Tran, S., Weng, W., Mannix, R.J., Watkins, S.A., Tyurina, YY., Tyurin, V.A., Kagan, V.E., Lazo, J.S. (1997). Overexpression of metallothionein decreases the sensitivity of pulmonary endothelial cells to oxidant injury. Am. J. Physiol.: Lung Cell. Molec. Physiol. 17(4): 856-865.
Tyurin, V.A., Tyurina, Y.Y., Quinn, P.J., Schor, N.F., Balachandran, R., Day, B.W., Kagan, V.E. (1998). Glutamate-induced cytotoxicity in PC12 pheochromocytoma cells: role of oxidation of phospholipids, glutathione and protein sulfhydryls revealed by bcl-2 transfection. Mol. Brain Res. 60: 270-281.
Gorbunov, N.V., Tyurina, Y.Y., Salama, G., Day, B.W., Claycamp, H G., Argyros, S., Elsayed, N.M., Kagan, V.E. (1998). Nitric oxide protects cardiomyocytes against tert-butyl hydroperoxide-induced formation of alkoxyl and peroxyl radicals and peroxidation of phosphatodylserine. Biochem. Biophys. Res. Commun. 244: 647-651.
Fabisciak, J.P., Kagan, V.E., Tyurina, Y.Y., Tyurin, V.A., Lazo, J.S. (1998). Paraquat-induced phosphatidylserine oxidation and apoptosis is independent of activation of phospholipase A2. Am. J. Physiol.: Lung Cell. Molec. Physiol. 18(5): 793-802.
Fabisciak, J.P., Tyurina, Y.Y., Tyurin, V.A., Lazo, J.S., Kagan, V.E. (1998). Random versus selective membrane phospholipid oxidation in apoptosis: role of phosphatidylserine. Biochemistry 37: 13781-13790.
Shvedova, A. A., Tyurina, Y.Y., Gorbunov, N.V., Tyurin, V.A., Castranova, V., Ojimba, J., McLaughlin, M.K., Kagan, V.E. (1999). tert-Butyl hydroperoxide/hemoglobin-induced oxidative stress and damage to mesenteric smooth muscle cells: different effects of nitric oxide and nitrosothiols. Biochem. Pharmacol. 57: 989-1001.
Goldman, R., Claycamp, H.G., Sweetland, M.A., Sedlov, A.V., Tyurin, V.A., Kisin, E.R., Tyurina, Y.Y., Ritov, V.B., Wenger, S.L., Grant, S.G., Kagan, V.E. (1999). Myeloperoxidasecatalyzed redox-cycling of phenol promotes lipid peroxidation and thiol oxidation in HL60 cells. Free Radical Biol. Med. 27: 1050-1063.
Day, B.W., Tyurin, V.A., Tyurina, Y.Y., Liu, M., Facey, J.A., Carta, G., Kisin, E.R., Dubey, R.K., Kagan, V.E. (1999). Peroxidase-catalyzed pro- versus anti-oxidant effects of 4-hydroxytamoxifen-enzyme specificity and biochemical sequelae. Chem. Res. Toxicol. 12(1): 28-37.
Fabisiak, J.P., Tyurin, V.A., Tyurina, Y.Y., Borisenko, G.G.. Korotaeva, A., Pitt, B.R., Lazo, J.S., Kagan, V.E. (1999). Redox regulation of copper-metallothionein. Arch. Biochem. Biophys. 363: 171-181.
Kagan, V.E., Yalowich, J.C., Borisenko, G.G., Tyurina, Y.Y., Tyurin, V.A., Thampatty, P., Fabisiak, J.P. (1999). Mechanism-based chemopreventive strategies against etoposideinduced acute myeloid leukemia: free radical/antioxidant approach. Mol. Pharmacol. 56: 494-506.
Fabisiak, J.P., Tyurin, V.A., Tyurina, Y.Y., Sedlov, A., Lazo, J.S., Kagan, V.E. (2000). Nitric oxide dissociates lipid oxidation from apoptosis and phosphatidylserine extemalization during oxidative stress. Biochemistry 39: 127-138.
Shvedova, A.A., Kommineni, C., Jeffries, B.A., Castranova, V., Tyurina, Y.Y., Tyurin, V.A., Serbinova, E.A., Fabisiak, J.P., Kagan, V.E. (2000) Redox-cycling of phenol induces oxidative stress in human epidermal keratinocytes. J. Invest. Dermatol. (in press).

In addition, our newly developed methodology has been also published as an invited chapter in a prestigious series Methods in Molecular Biology:

Kagan, V.E., Ritov, V.B., Tyurina, Y.Y., Tyurin, V.A. (1998). Sensitive and specific fluorescent probing of oxidative stress in different classes of membrane phospholipids in live cells using metabolically integrated fcis-parinaric acid. Methods Mol. Biol. 108: 71-87.