Accelerator mass spectrometry in biomedical dosimetry: relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA.

Abstract

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...

Low-level biological dosimetry of heterocyclic amine carcinogens isolated from cooked food.

“…Adduct formation at higher doses does not seem to correlate with target tissues for carcinogenesis, suggesting that adduct formation alone is not necessarily indicative of tumorigenicity. The linearity of the adduct dose-response curve reported here for PhIP and also earlier for 2-amino-3,8-dimethylimidazo [4,5-JAquinoxaline (6) suggests that no or a very low threshold exists for DNA damage and that DNA adduct levels are indicative of exposure. The levels at which DNA damage and that DNA adduct levels are indictive of exposure.…”

“…AMS isolates and counts specific nuclei as opposed to measuring atomic decay. Thus, AMS methodology results in a 106-fold improvement in isotope detection efficiency compared to scintillation counting for 'C and has allowed us to quantitatively measure DNA adduct levels on the order of 10 -1I to 10 -12 adducts/nucleotide (6). We also have applied the 32p_ postlabeling assay to mechanistic studies of the effects of these carcinogens on DNA.…”

In-Utero and Lactational Exposure of 3,3',4,4',5-Pentachlorobiphenyl Modulate Dimethlbenz[a]anthracene-Induced Rat Mammary Carcinogenesis.

“…However, dioxins are considered non-genotoxic carcinogens, since they do not form DNA adducts and are negative in vitro tests for genetic toxicity 24 . The PCB and TCDD possess high affinity to aryl hydrocarbon receptor (AhR), and their interactions with this cytosolic receptor are the essential first step in the resulting toxic actions 1,5 .…”

Integrated approach for evaluating species and interindividual differences in responsiveness to dioxins and structural analogs.

“…TCDD is ineffective in short-term tests for genetic toxicity (5,6); moreover, it does not bind covalently to DNA either in in vitro or in vivo, even when methods are used that can detect one adduct in 1011 normal nucleotides (7). Therefore, TCDD is generally considered to be a nongenotoxic carcinogen; however, it may exert indirect genotoxic effects, as evidenced by one study in which TCDD increased the frequency of strand breaks in rat liver nuclei, possibly by increasing iron bioavailability and lipid peroxidation (8).…”