phosphorus-radioisotopes and 2-amino-3-8-dimethylimidazo(4-5-f)quinoxaline

Estimating the cancer risk posed by heterocyclic amines depends on measuring how chemical dose influences measurable indicators of cancer progression. This data ideally should encompass the range of actual human exposure, at the low dose end, and laboratory animal studies, at the high dose end. Accelerator mass spectrometry (AMS) has been used to measure the absorption, fate, and DNA adduct dosimetry of the heterocyclic amines PhIP and MeIQx at doses equivalent to human consumption following single-dose administration and chronic daily dosing. AMS is a nuclear physics technique which specifically counts nuclei of cosmogenic isotopes, rather than relying on decay. For tracing 14C, sensitivity is increased 10(6)-fold relative to decay counting. We have found that tissue clearance rates for [2-(14)C]-PhIP are rapid (t1/2 = 1 h) at low dose (41 ng/kg), with most of the radiocarbon distributed to the liver and G.I. tract. MeIQx-DNA adduct levels decrease linearly with dose (5 mg/kg-500 ng/kg) in single dose exposures. Likewise, the biologically available dose of [2-(14)C]-MeIQx decreases linearly with decreasing dose (5 mg/kg-1 ng/kg). On chronic daily dosing, it takes 40 days for adducts to reach steady-state in tissues and adduct levels appear to decrease linearly with decreasing dose, except possibly at very low doses. DNA binding of PhIP involves both sulfation or acetylation of the N-hydroxylated PhIP. Quantitatively, sulfation appears to be an important pathway for PhIp activation in rodent tissue cytosols while acetylation appears quantitatively more important in human tissue cytosols. The greatest activity is in liver and intestinal tissues for both pathways. The specific DNA adducts formed in vivo and in vitro from exposure to PhIP and MeIQx are likely guanine adducts. These data suggest that DNA adduct dosimetry responds linearly with dose but may become sub-linear at very low doses for chronic exposure and that factors other than DNA adduction may be critical to explain these heterocyclic amines' tumorigenicity.

Topics: Animals; Carcinogens; DNA Adducts; Humans; Imidazoles; Mass Spectrometry; Mutagens; Neoplasms; Phosphorus Radioisotopes; Quinoxalines; Radioisotope Dilution Technique; Rodentia

The effect of chlorophyllin on 2-amino-3,8-dimethyimidazo[4,5-f]quinoxaline (MeIQx)-mediated DNA-adduct formation in Drosophila was studied. Third-instar larvae of Drosophila were fed MeIQx at 1 mg/6.5 g-feed/bottle, with or without chlorophyllin (100-300 mg). After a 6 h feeding exposure to MeIQx, the larvae were divided into 2 groups. The first group was examined for covalent DNA adducts by 32P-postlabeling assay. The second group was assayed for DNA damage by allowing the larvae to develop to adults and measuring the male/female ratio (males, DNA repair-deficient; females, DNA repair-proficient). The 32 P-postlabeling results indicated a significant decrease in DNA adduct levels in larvae treated with MeIQx and 300 mg chlorophyllin (1.7 +/- 0.7 adducts/10(7) nucleotides) as compared with MeIQx-treated larvae 6.5 +/- 2.1 adducts/10(7) nucleotides). The results on male/female sex ratios also indicated a chlorophyllin-induced decrease in DNA damage by exposure to MeIQx. The suppressive effect of chlorophyllin on the genotoxic actions of a polycyclic mutagen, MeIQx, may be a result of complex formation between chlorophyllin and the mutagen.

Topics: Animals; Antimutagenic Agents; Autoradiography; Chlorophyllides; DNA; DNA Adducts; DNA Damage; Drosophila; Female; Male; Mutagens; Phosphorus Radioisotopes; Quinoxalines

The carcinogenic heterocyclic amine 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) is widely distributed in cooked foods. The nuclease P1 method increased the sensitivity of the standard 32P-postlabeling analysis about 1000-fold for detection of MeIQx-DNA adducts. The recovery of MeIQx-DNA adducts by the nuclease P1 method was determined to be about 50% using liver DNA of a rat treated with [14C]MeIQx intragastrically. By the nuclease P1 method five adducts were detected in the liver DNA of rats fed MeIQx and two of them, including the most abundant one, were identified as MeIQx-deoxyguanosine adducts by comparison with the adducts formed in in vitro reactions of N-acetoxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline with the four 2'-deoxyribonucleotides. The most abundant adduct in vivo was identified as N2-(deoxyguanosin-8-yl)-MeIQx 3',5'-diphosphate (3',5'-pdGp-C8-MeIQx). MeIQx-DNA adduct levels in human tissues could be determined by the nuclease P1 modification of the 32P-postlabeling method in combination with HPLC, and thus provide information on the roles of MeIQx in human carcinogenesis.

Topics: Animals; Chromatography, Thin Layer; DNA; Guanosine Diphosphate; Isotope Labeling; Liver; Male; Phosphorus Radioisotopes; Quinoxalines; Rats; Rats, Inbred F344; Sensitivity and Specificity; Single-Strand Specific DNA and RNA Endonucleases

The 32P-postlabeling method was used to examine the adducts in DNA, polynucleotides, and mononucleotides reacted in vitro with the N-hydroxy and N-acetoxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Adduct profiles were compared to those found in vivo in liver of cynomolgus monkeys fed IQ, MeIQx or PhIP. The N-acetoxy derivatives of IQ, MeIQx and PhIP (generated in situ from the corresponding N-hydroxylamine in the presence of acetic anhydride) each formed three principal adducts in DNA. Adduct 1 of IQ, MeIQx and PhIP was chromatographically identical to the 32P-labeled bis(phosphate) derivative of N-(deoxyguanosin-8-yl)-IQ, N-(deoxyguanosin-8-yl)-MeIQx, and N-(deoxyguanosin-8-yl)-PhIP respectively, and this adduct comprised approximately 65% of total adduct levels found in DNA in vitro. The C8-guanine adduct and the two minor adducts were also found in poly(dG-dC). poly(dG-dC), suggesting that the two minor adducts of IQ, MeIQx and PhIP are also formed on the guanine base. The N-acetoxy derivatives of IQ, MeIQx, and to a much lesser extent PhIP, also formed adducts with adenine-containing polynucleotides including poly(dA), poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT), but these adenine adducts were chromatographically different from those found in DNA. The three guanine adducts of N-acetoxy-IQ, -MeIQx and -PhIP found in vitro in DNA and in guanine-containing polynucleotides were also found in the liver of monkeys fed IQ, MeIQx or PhIP respectively, indicating that metabolic activation via N-hydroxylation and esterification occurred in vivo in monkeys. With each compound, the C8-guanine adduct was the predominant adduct found in vivo. The results indicate similarities among IQ, MeIQx and PhIP in the DNA adducts formed in vitro and in vivo and substantiate the use of the 32P-postlabeling method for comparative adduct studies.

Topics: Animals; Autoradiography; Chromatography, High Pressure Liquid; DNA; DNA Damage; Haplorhini; Imidazoles; Liver; Mutagens; Phosphorus Radioisotopes; Polynucleotides; Quinolines; Quinoxalines

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) are carcinogens found in cooked meats that form DNA adducts upon metabolic activation. Purified DNA from Chinese hamster ovary (CHO) cells was reacted in vitro with the active metabolites N-acetoxy-IQ or N-acetoxy-MelQx, and the adduct levels in the 5' dihydrofolate reductase (DHFR) gene and downstream region were quantitated by Southern hybridization. Adducted and restricted DNA was treated with Escherichia coli uvrABC excinuclease or alkali (0.1 N NaOH, 37 degrees C, 60 min) to incise DNA at IQ and MelQx adduct sites. The DNA was then denatured with formamide, electrophoresed on a neutral agarose gel, transferred to a support membrane, and hybridized with sequence-specific DNA probes. Both uvrABC and alkali reduced the intensity of Southern hybridization in proportion to the number of IQ or MelQx adducts in DNA, indicating that these adducts are substrates for uvrABC and that they form alkali-labile lesions in DNA. IQ and MelQx adduct levels were the same in the 5' DHFR gene and in the downstream region. Southern hybridization analysis of pBR322 containing known levels of IQ or MelQx adducts showed that the efficiency of cutting IQ or MelQx adducts by uvrABC excinuclease and alkali was approximately 30% and 15%, respectively. 32P-postlabeling studies examining adduct level in bulk DNA further showed that the adduct profiles were identical in pBR322, CHO DNA, and cultured CHO cells exposed to the reactive metabolites of IQ or MelQx. The results indicate that IQ and MelQx adducts can be quantitated in specific genomic sequences and that this method should be directly applicable to studies of gene-specific repair of these adducts in cultured cells.

Topics: Alkalies; Animals; Autoradiography; Blotting, Southern; CHO Cells; Cooking; Cricetinae; Cricetulus; DNA; DNA Damage; DNA Repair; DNA, Bacterial; Electrophoresis; Endodeoxyribonucleases; Escherichia coli; Food Contamination; Intercalating Agents; Mutagens; Phosphorus Radioisotopes; Plasmids; Quinolines; Quinoxalines; Tetrahydrofolate Dehydrogenase; Ultraviolet Rays

Topics: Animals; Binding Sites; Carcinogens; DNA; DNA Damage; Humans; Imidazoles; Mass Spectrometry; Mice; Phosphorus Radioisotopes; Quinoxalines

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 14C-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(11) 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.

Topics: Animals; Carbon Radioisotopes; Carcinogens; Dioxins; DNA; Dose-Response Relationship, Drug; Kinetics; Liver; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Phosphorus Radioisotopes; Polychlorinated Dibenzodioxins; Quinoxalines

We report the effects of several inducers of P450 metabolizing enzymes on DNA adduct formation by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in C57BL/6 mice. We also examined the role of N:O-acetylation and the nitrenium ion in the genotoxicity of MeIQx, since these have been implicated in the activation of other aminoimidazoazaarenes (AIA) to DNA reactive species. Mice were given phenobarbital (PB), Aroclor 1254, beta-naphthoflavone (BNF) or corn oil, i.p., followed 3-5 days later with oral administration of MeIQx. Induction of Aroclor and BNF produced DNA with 8-fold more adducts than either the corn oil-alone or PB-treated animals. Both corn oil-alone and PB-treated animals were similar. Four major adducts were found in all cases with no differences among inducers as judged by co-chromatography. Azido-MeIQx induced calf-thymus-DNA adducts produced identical adduct profiles to those seen for the mouse DNA. Similar adduct profiles were obtained from Salmonella TA98, and the nitroreductase deficient strains (TA98NR and TA98/1,8-DNP6) exposed to MeIQx in the presence of Aroclor-induced-mouse-liver S9. Adduct frequencies in TA98/1,8-DNP6 were significantly lower than in TA98 and TA98NR. The data described in this report demonstrate that induction quantitatively increases adduct numbers but does not affect the types of DNA damage. These data also suggest that the same DNA reactive intermediates are formed in vivo as in vitro and support the hypothesis that the metabolism of MeIQx involves the P450I family of isozymes, N:O-acetyltransferases and possibly a nitrenium ion. The application of radioanalytic scanners for quantitation of 32P-postlabelling adduct maps is described.

Topics: Animals; DNA; DNA, Bacterial; Liver; Male; Mice; Mice, Inbred C57BL; Mutagens; Phosphorus Radioisotopes; Quinoxalines; Radioisotope Dilution Technique; Salmonella typhimurium