Optimization of the 32P-postlabeling Assay for Detecting Benzo(a)pyrene-induced DNA Adduct Formation in Zacco platypus
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Objectives: 32P-postlabeling assay is the most sensitive method of detecting DNA adduct formation. However,it is limited by a low sample throughput and use of radioisotopes (RI). In this study, we modified it to minimizethese limitations and applied it to Z. platypus exposed to Benzo(a)pyrene (BaP) in order to investigate DNAadduct formation (effect biomarker for pollutants) in Z. platypus for assessing risk of waterborne BaP exposure. Methods: DNA hydrolysis was performed only with Micrococcal nuclease (MNase), RI reduction test was performedand the overlapping steps between thin layer chromatography (TLC) and radioisotope high-performanceliquid chromatography (RI-HPLC) were omitted. The application of a modified method to Z. platypus exposedto BaP was performed. Results: The results revealed that the amount of RIs used can be reduced roughly 10-fold. Because the analysistime was shortened by 8.5 hours, the sample throughput per hour was increased compared with the previousmethod. The results of applying modified 32P-postlabeling assay to Z. platypus, DNA adduct formation in Z. platypus showed dose-dependency with the BaP concentration. Only BPDE-dGMP was detected as a DNAadduct. Conclusion: These results demonstrate that the modified 32P-postlabeling assay is a suitable method for detectingDNA adduct formation in Z. platypus exposed to waterborne BaP and will be useful in risk assessment of carcinogeniceffect in aquatic environment due to BaP.
Abstract
Objectives: 32P-postlabeling assay is the most sensitive method of detecting DNA adduct formation. However,it is limited by a low sample throughput and use of radioisotopes (RI). In this study, we modified it to minimizethese limitations and applied it to Z. platypus exposed to Benzo(a)pyrene (BaP) in order to investigate DNAadduct formation (effect biomarker for pollutants) in Z. platypus for assessing risk of waterborne BaP exposure. Methods: DNA hydrolysis was performed only with Micrococcal nuclease (MNase), RI reduction test was performedand the overlapping steps between thin layer chromatography (TLC) and radioisotope high-performanceliquid chromatography (RI-HPLC) were omitted. The application of a modified method to Z. platypus exposedto BaP was performed. Results: The results revealed that the amount of RIs used can be reduced roughly 10-fold. Because the analysistime was shortened by 8.5 hours, the sample throughput per hour was increased compared with the previousmethod. The results of applying modified 32P-postlabeling assay to Z. platypus, DNA adduct formation in Z. platypus showed dose-dependency with the BaP concentration. Only BPDE-dGMP was detected as a DNAadduct. Conclusion: These results demonstrate that the modified 32P-postlabeling assay is a suitable method for detectingDNA adduct formation in Z. platypus exposed to waterborne BaP and will be useful in risk assessment of carcinogeniceffect in aquatic environment due to BaP.
참고문헌 (28)
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