A highly sensitive and specific method for quantitation of O-alkylated DNA adducts and its application to the analysis of human tissue DNA.

Formation and accumulation of O6-alkylguanine and O4-alkylthymine in human tissues is possibly the most relevant marker for cancer risk. Because humans are chronically exposed to diverse kinds of chemicals and eventual DNA structural modifications are supposed to be a complex mixture of adducts at very low levels, it is essential to use an assay with extremely high sensitivity and specificity. We have established a quantitation method, called PREPI, for O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of prefractionation by HPLC, 32P-postlabeling, and immunoprecipitation. The detection limit was about 1 fmole for all three adducts, enabling us to analyze about 1 x 10(-8) levels as a molar ratio to normal counterpart using 100 micrograms of DNA. In a pilot experiment, we analyzed 11 peripheral blood samples from healthy volunteers. O6-Methylguanine was detected in all the cases with a mean value of 2.0 +/- 1.3 x 10(-8) (range, 0.78-4.6 x 10(-8)). Neither O4-methylthymine nor O4-ethylthymine was above the detection limit of 0.8 x 10(-8) as a ratio to thymine.

Introduction 06-Alkylguanine and 04-allkylthymine have been demonstrated to be the most relevant lesions among DNA modifications for induction of cancer by N-nitroso compounds in diverse experimental systems (1)(2)(3). Detection of such DNA lesions in human tissues, therefore, is one of the best markers for the biologically relevant exposure to alkylating agents and may eventuallly lead to a better risk assessment (4)(5)(6). Because DNA modifications in human tissues have been previously demonstrated to be at extremely low levels, the use of an assay with high sensitivity as well as specificity is needed for molecular epidemiological studies on cancer in man. To address this problem, we have established a quantitation method for 0-alkyl DNA adducts and examined its validity for application to human samples in a pilot experiment.
Address reprint requests to N-h. Huh, Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan. by a reverse-phase HPLC system under essentially the same conditions as previously described (5). The alkylated 3 '-MP was then phosphorylated by polynucleotide kinase and [32P]ATP according to Reddy et al. (7), followed by 3 '-dephosphorylation by decreasing the pH to 6.0 (8) for better recognition by antibodies. After purification by HPLC, alkylated 5 '-[32-P]MP was immunoprecipitated with corresponding monoclonal antibodies [ER-6 for 06-methylguanine, EM-3-33 for 04-methylthymine, and ER-01 for 04-ethylthymine (9,10)], and the radioactivity was determined by liquid scintillation counting. Human DNA samples were isolated from peripheral leukocytes of 11 healthy volunteers who had no apparent history ofexposure to alkylating agents.

Results and Discussion
Procedure of PREPI Conditions of each reaction step were optimized using standard DNA treated with N-methyl-N-nitrosourea or N-ethyl-Nnitrosourea. The content of 06-methylguanine (06-MetG), 04-methylthymine (04-MetT), and 04-ethylthymine (04-EtT) in the standard DNA was determined by competitive radioimmunoassay using tritiated tracers and antibodies as described (5). Completion of DNA hydrolysis was monitored by injecting aliquots onto HPLC. 06-Methylguanosine-3 '-monophosphate, 04-methylthymidine-3 '-monophosphate, 06-ethylguanosine-3 '-monophosphate, and 04-ethylthymidine-3 '-monophosphate could be separated from each other and also from normal 3 '-MP under the present conditions, which facilitated independent analysis of each adduct from a given amount of DNA and reduced the amount of [32P]ATP necessary in comparison with the original postlabeling method. After postlabeling, 3 '-phosphate was removed to enhance the recognition by antibodies because the monoclonal antibodies used were originally raised against alkylated nucleosides (9,10). The second HPLC fractionated was used to remove [32P]ATP and nonspecific radioactive byproducts before final immunoprecipitation. The precise amount of alkylated DNA adducts was determined by comparing a curve obtained by serial dilution of samples with a standard curve.

Sensitivity of the Method
A representative standard curve for 04-EtT is shown in Figure  1. Standard curves for 06-MetG and 04-MetT were essentially similar to that for 04-EtT (data not shown). One femtomole of each alkylation product could be precisely determined. Therefore, the relative sensitivity (11), i.e., the lowest relative modification level to be determined is about 1 x 10-8 as a molar ratio to normal counterpart when 100 yg ofDNA is available for analysis (Table 1).

Specificity
Combining several analytical methods with different physicochemical bases is preferable for attaining the high specificity needed, particularly in the case ofhuman samples. The present Abbreviations: ISB, immuno-slot-blot; HPLC, high-pressure liquid chromatography; RIA, radioimmunoassay; PREPI, prefractionation by HPLC, 32P-postlabeling, and immumoprecipitation. method is highly specific because each step, i.e., reverse-phase HPLC fractionation, enzymatic reactions including postlabeling, and immunoprecipitation, contribute to increasing the specificity through different physicochemical or molecular mechanisms.

Relevance versus Feasibility
The validity of any analytical methods for use in molecular epidemiological studies with respect to cancer may be assessed by two independent parameters: relevance to cancer development and feasibility or ease ofuse for mass screening. Quantitation of O-alkylated DNA adducts is considered to have higher relevance to carcinogenesis than hemoglobin adducts or N7-alkylguanine, for example. In our previous studies, to detect 04-EtT in human liver DNA by radioimmunoassay using a tritiated tracer, we had to use more than 20 mg ofDNA (corresponding to 20-30 g liver tissue) because of the limited sensitivity. We attempted to improve the feasibility by increasing the sensitivity ofthe quantitation method without sacrifying the relevance. In the present assay, we need only 100 ytg DNA (corresponding to 10 mL of peripheral blood or several hundred milligrams ofbiopsy tissue specimens) for the analysis of similar relative modification levels.

Analysis of Human Leukocyte DNA
To examine the validity ofthe present method, we quantitated 06-MetG, 04-MetT, and 04-EtT in peripheral leukocyte DNA obtained from 11 healthy volunteers. 06-MetG was detected in all 11 cases, with a mean value of 2.0 ± 1.3 x 10 8 (range 0.78-4.6 x 10 8) as a molar ratio to guanine. 04-MetT and O4--EtT were below the detection limit of 0.8 x 10 8 as a ratio to thymine. The result indicates that humans are exogenously and/or endogenously exposed to methylating agents, and the present method can be practically applied to the analysis of human samples. We are now undertaking studies on a possible correlation between levels ofalkylated DNA adducts and the incidence of cancer using the method described above.