Quantitative Detection of Benzo[a]pyrene Diolepoxide-DNA Adducts by Cryogenic Laser Induced Fluorescence
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- 作者：Wen Luo ; Rajan Gurjuar ; Can Ozbal ; Koli Taghizadeh ; Arthur Lafleur ; Ramachandra R. Dasari ; Helmut Zarbl ; and William G. Thilly
- 刊名：Chemical Research in Toxicology
- 出版年：2003
- 出版时间：January 2003
- 年：2003
- 卷：16
- 期：1
- 页码：74 - 80
- 全文大小：125K
- 年卷期：v.16,no.1(January 2003)
- ISSN：1520-5010
文摘
In the present report, we describe a fluorescence-based method capable of measuring benzo[a]pyrene diolepoxide (BPDE) adducts in intact genomic DNA, with a sensitivity of a fewhundreds copies per cell. The assay is based on cryogenic laser-induced fluorescence technologyat liquid nitrogen temperatures, coupled with an intensified charge-coupled device camera,and incorporates several enhancements to existing methodologies. One important modificationwas the incorporation of terbium(III) nitrate pentahydrate, Tb(NO3)3, as an internal fluorescencestandard to correct for differences in light scattering and fluctuations in instrument parameters.Since the fluorescence spectrum of Tb(NO3)3 does not overlap with those of BPDE-DNAadducts, use of this lanthanide salt markedly improved the sensitivity of cryogenic laser-inducedfluorescence. The limit of quantification of the assay is 6.4 BPDE-DNA adducts/108 nucleotides,or 776 adducts/cell, using 22.5 
g of genomic DNA. This assay is rapid, highly sensitive, andeconomical and has been applied to monitor DNA adduct levels as a function of time afterexposure to BPDE in repair-competent human lymphoblastoid AHH-1 and TK6 cells.
g of genomic DNA. This assay is rapid, highly sensitive, andeconomical and has been applied to monitor DNA adduct levels as a function of time afterexposure to BPDE in repair-competent human lymphoblastoid AHH-1 and TK6 cells.