Retention Time (min)

Fig. 1. Detection of HNE-dG adducts as 3',5'-bisphosphates in rat and human tissues. These adducts are formed as two pairs of diastereomers 1,2 and 3,4 and isomers 1 and 2 at the 3',5-bisphosphate level are not separated under the HPLC conditions used. The identities of the radioactive peaks obtained by 32P-postlabeling are indicated by their HPLC comigration with the synthetic UV markers, (a) A blank sample of poly(dA-dC) : poly(dG-dT) is used to ensure that the assay is free of contamination; (b) external standards of HNE-dG adducts are used for quality assurance and quantitation; (c) rat tissues; (d) human tissues. [Reproduced with permission from F.-L. Chung, R. G. Nath, J. E. Ocando, A. Nishikawa, and L. Zhang, Cancer Res. 60, 1507 (2000).]

the addition of standard adduct bisphosphates as UV markers. Figure 1 shows the final HPLC chromatograms demonstrating comigration of the HNE-dG adducts in rat and human liver and colon DNA with the UV markers. To confirm their identities, the adducts were converted to the 5'-monophosphates of HNE-dG with T4 polynucleotide kinase (T4 PNK) and to the ring-opened derivatives by sodium borohydride are then analyzed by HPLC as shown in Fig. 2.

50 60 70 80 Retention Time (min)

Fig. 2. Confirmation of HNE-dG adducts detected in rat colonic DNA. The radioactive peaks detected in tissue DNA, which comigrate with HNE-dG adducts in Fig. 1, are collected from the HPLC together with the UV standards. The concentrated fractions are then treated with NaBHj at pH 11, which opens the propano ring, and subsequently are reduced to N2-[l-(hydroxyethyl)-2-hydroxyheptyl]-dG 3',5'-bisphosphates (a) and with T4 PNK to yield the corresponding 5'-monophosphates (b). In both cases the products comigrated with their UV standards. HNE-dG adduct standards are added to the reaction mixture as references before analysis. Similar results are obtained with rat liver DNA and with human liver and colon DNA. [Reproduced with permission from F.-L. Chung, R. G. Nath, J. E. Ocando, A. Nishikawa, and L. Zhang, Cancer Res. 60,1507 (2000).]

HNE-dG 3',5'-bisphosphates

HNE-dG 5'-monophosphates

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