Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes.
To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants--a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids-phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol-in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.
Shvedova
A A
AA
Health Effects Laboratory Division, NIOSH, Morgantown, WV 26505, USA.
Tyurina
Y Y
YY
Tyurin
V A
VA
Kikuchi
Y
Y
Kagan
V E
VE
Quinn
P J
PJ
D000328:Adult; D000975:Antioxidants / Q000494:pharmacology*; D001391:Azo Compounds / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005231:Fatty Acids, Unsaturated / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D015227:Lipid Peroxidation / Q000187:drug effects / Q000502:physiology*; D009570:Nitriles / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010743:Phospholipids / Q000378:metabolism*; D012867:Skin / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D014810:Vitamin E / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology
D000975:Antioxidants; D001391:Azo Compounds; D005231:Fatty Acids, Unsaturated; D005456:Fluorescent Dyes; D009570:Nitriles; D016877:Oxidants; D010743:Phospholipids; C055792:2,2'-azobis(2,4-dimethylvaleronitrile); D014810:Vitamin E; D014969:Xanthine Oxidase; C013641:parinaric acid