Biomarkers of Oxidative Stress Study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

doi:10.1016/j.freeradbiomed.2004.09.017

Free Radical Biology and Medicine

Volume 38, Issue 6, 15 March 2005, Pages 698-710

https://doi.org/10.1016/j.freeradbiomed.2004.09.017 Get rights and content

Abstract

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl₄ poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg ip) effects of CCl₄ on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2′-deoxyguanosine (8-OHdG), leukocyte DNA–MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl₄ would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl₄-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl₄ or showed fewer significant effects.

Section snippets

Chemicals and reagents

Carbon tetrachloride and all other chemicals and reagents used in the study were obtained from Sigma–Aldrich Corp. (St. Louis, MO, USA).

Animals and treatment protocol

Male Fisher 344 rats (260–280 g) obtained from Charles River Laboratories (Raleigh, NC, USA) were used in all experiments. The animals were housed three to a cage. Autoclaved hardwood bedding was used in solid-bottom polycarbonate cages with filter tops. Animal rooms were maintained at 20–25°C with 35–70% relative humidity with alternating 12-h light and dark

Plasma

In plasma, the candidates for markers of free radical-induced oxidation were lipid hydroperoxides, MDA, and F₂-isoprostanes. Two assays for lipid hydroperoxides were tested: a lipid peroxidation assay kit and the iodometric assay. When measurements were performed with the kit, statistically significant increases in the levels of lipid hydroperoxides were found for both doses of CCl₄ 2 h after the injection, with the high dose showing a threefold increase compared to controls (Table 1). When

Discussion

The central focus of this multilaboratory study is the identification of the most sensitive and specific biomarkers to assess oxidative damage in in vivo animal studies. The only definitive way to demonstrate excessive free radical activity in vivo is by electron spin resonance, but clearly this is currently inapplicable in clinical practice because of the spin traps that must be administered. Instead, investigators must rely upon the measurement of the oxidation products of free radical

Conclusions

This multilaboratory study to compare different markers of oxidative stress in plasma and urine in a rat model of oxidative stress reveals the following:

1.
Measurements of MDA and 8-iso-PGF_2α concentrations (both by GC/NICI-MS) in plasma are promising candidates for general biomarkers of oxidative stress. Both assays measure oxidative damage to lipids. Due to their GC-MS characteristics, these assays are innovative methodologies that apply advanced technologies. Both assays are highly

Acknowledgments

The authors thank Jean Corbett, John Seely, Ralph Slade, Robert McConnaughey, Kay Crissman, Judy Richards, Linda Quong, and Qu Feng for excellent technical support and acknowledge the contributions of all members of the Committee on Biomarkers of Oxidative Stress Study (BOSS) at NIEHS (Research Triangle Park, NC) for their helpful suggestions, comments, and discussion. The authors also thank Dr. Ann Motten and Ms. Mary J. Mason for editorial assistance. The research described in this article

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Original ContributionBiomarkers of Oxidative Stress Study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Abstract

Section snippets

Chemicals and reagents

Animals and treatment protocol

Plasma

Discussion

Conclusions

Acknowledgments

Ann. N.Y. Acad. Sci.

Free Radic. Biol. Med.

Chem. Phys. Lipids

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Toxicol. Appl. Pharmacol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Anal. Biochem.

Free Radic. Biol. Med.

Biochim. Biophys. Acta

Anal. Biochem.

Methods Enzymol.

Mech. Ageing Dev.

Methods Enzymol.

Anal. Biochem.

Methods Enzymol.

Life Sci.

Pharmacol. Ther.

Toxicol. Appl. Pharmacol.

Int. J. Biochem.

Free Radic. Biol. Med.

Methods Enzymol.

Anal. Biochem.

Anal. Biochem.

Am. J. Clin. Nutr.

J. Lipid Res.

Toxicol. Lett.

J. Biol. Chem.

Chem. Phys. Lipids

J. Biol. Chem.

J. Am. Coll. Cardiol.

Anal. Biochem.

Prostaglandins

J. Biol. Chem.

J. Biol. Chem.

Original Contribution
Biomarkers of Oxidative Stress Study II: Are oxidation products of lipids, proteins, and DNA markers of CCl₄ poisoning?