Carcinogenesis, Teratogenesis & Mutagenesis

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Establishment and application for an oxidative stress biomarker quantitative analysis system

YIN Jian1,WANG Ai-ping2,LI Wan-fang2,JIN Hong-tao2,WEI Jin-feng2,*   

  1. Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050
  • Received:2014-05-21 Revised:2014-09-03 Online:2014-09-30 Published:2014-09-30

Abstract:

OBJECTIVE: To develop a detection method for free radical scavenging capacity in biological samples,based on the oxygen radical absorbance capacity (ORAC) method,which was applied to evaluate the biological antioxidant capacity and to classify the extent of environmental pollution and toxic effects. METHODS:Zebrafish was chosen as the experimental animal,to test the hydroperoxyl free radical (ROO·). The azo compound 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was chosen as the oxygen free radical source. Fluorescein (FL) as the fluorescence indicator and Trolox as the quantitative standard. Then fluorescence intensity decay process was recorded after free radicals reacted with FL,and the ability of antioxidant delay the fluorescence intensity recession was assessed to evaluate the effect of chemicals on ORAC. The established method was applied to study the effect of different concentrations of four heavy metals (copper,cadmium,chromium and lead) on ORAC of zebrafish. RESULTS:The established method showed high specificity and good accuracy (between 97% and 108%) and precision (CV less than 5%);this method also had good reproducibility (CV is 2.92%). The limit of quantitation (LOQ) and the limit of detection (LOD) were 3.03 and 1.00 μmol Trolox/mg protein,respectively,the correlation coefficient(R2) was ≥ 0.99. According to the new established quantitative analysis methods,we determined the physiological normal range (0-50 μmol Trolox/mg protein) and classified the heavy metal contamination degree and toxic effects based on the effect of metals on ORAC of zebrafish. CONCLUSION:The established method may be suitable for evaluating biological antioxidant capacity and classifying environmental pollution and toxic effects. It could be effective in enhancing the inter laboratory data recognition and promoting the application of biomarkers for antioxidant protection in environmental pollution monitor and environmental risk assessment.

Key words: oxygen radical absorbance capacity, environmental pollution, zebrafish, heavy metal, environmental risk assessment