Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a "'C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations (10Be, 14C, 26Al, 41Ca, and 129I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of the technology. In this study, the relationship between exposure to low levels of 2-amino-3, 8-dimethyl[2-14C~imidazo[4,5-f]quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Instrument reproducibility in this study is 2%; sensitivity is 1 adduct per 10"1 nucleotides. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, we demonstrate at least 1 order of magnitude Improvement over the best adduct detection sensitivity reported to date and 3-5 orders of magnitude improvement over other methods used for adduct measurement. An additional improvement of 2 orders of magnitude in sensitivity is suggested by preliminary experiments to develop bacterial hosts depleted in radiocarbon. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.Carcinogens covalently bound to any of the deoxynucleotide bases present in DNA (DNA adducts) have been proclaimed as markers of carcinogen exposure. The relationship between adduct formation and exposure, however, has been primarily established at high carcinogen doses and not at lower, more environmentally relevant, levels because of limitations in assay sensitivity. As a consequence, the significance of using adducts as a measure of carcinogen exposure in the human population is unknown. Currently, the most sensitive technique for adduct detection is the 32p postlabeling assay. The 32p postlabeling assay has permitted measurement of 1 adduct in 1010 nucleotides and has been used to detect carcinogen-DNA binding in occupationally exposed humans and smokers, but accurate quantitative measurement at levels <1 adduct per 107-101 nucleotides is difficult because of variability in adduct recovery (1-3). The ability of accelerator mass spectrometry (AMS) to measure concentrations of rare isotopes in 20-Asg to 1-mg samples suggested to us that its extension to the biomedical sciences was a natural and potentially powerful application of the technology (4). The great enhancement in 14C detection sensitivity available with AMS offers the distinct advantage of detecting extremely small amount...

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25–26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by 32P‐postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case‐by‐case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance. Environ. Mol. Mutagen. 35:222–233, 2000 © 2000 Wiley‐Liss, Inc.

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure͞activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiestersugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3 end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.C onsumption of foods containing heterocyclic amines (HA) has been implicated in the etiology of human cancers, including cancer of the colon, lung, and breast (1-4). The mutagen 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is the most mass abundant of the HAs, which are formed in meat and fish by the condensation of amino acids with creat(in)ine during cooking (5, 6). PhIP has been shown to induce tumors in several organs in rodents (7-9) and form DNA adducts, which are considered initiating events in chemical carcinogenesis (10,11). Treatment of cultured mammalian cells with PhIP results in predominantly single-base substitutions (12, 13). A Ϫ1 frame-shift hotspot also has been observed in a 5Ј-GGGA-3Ј sequence in the Apc gene of PhIP-induced rat colon tumors and in the lacI gene of rat mammary glands (14-16).The major DNA adduct formed in vivo, and the only one unequivocally identified to date, is derived from the binding of metabolically activated PhIP to the C8 position of guanine [C8-dG-PhIP {N2-(2Ј-deoxyguanosin-8-yl)-PhIP}; refs. 17 and 18]. The reactive form of PhIP, a nitrenium ion, arises as a consequence of N-hydroxylation, which is catalyzed primarily by cytochrome CYP1A2 in humans (19,20), followed by N:Osulfation or N:O-acetylation (21). Analysis of phage vectors containing a site-specific C8-dG-PhIP adduct replicated in mammalian cells...

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