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

Relatively few reported attempts have been made to substitute HPLC for the thin-layer ion-exchange chromatography (TLC) conventionally used in the 32P-postlabelling assay. Using a reverse-phase phenyl-modified silica gel column and a gradient of methanol in 0.5 M sodium phosphate buffer (pH 2.0), we were able to improve the resolution of very similar adducts. Combined with on-line detection of Cerenkov radiation, this method allows separation of sub-femtomole quantities of 32P-labelled nucleoside 3',5'-bisphosphates modified by bulky carcinogens. Using this method, we were able to separate nine of the ten major adducts formed by reaction of the diol-epoxides of ten polycyclic aromatic hydrocarbons with DNA, and resolve different adducts formed by a single carcinogen. The major adducts formed by benzo[b]fluoranthene (BbF) or dibenz[a,h]anthracene in mouse skin in vivo have been shown to be distinct from the adducts formed directly by the bay-region diol-epoxides. The heterocyclic amines IQ and MeIQ have each been shown to form one major DNA adduct in several in vitro and in vivo systems; using HPLC we were able to resolve the two adducts formed by these food mutagens. HPLC is especially useful for the identification of adducts by means of chromatographic comparisons and in the analysis of the multiple adducts formed by complex mixtures of environmental carcinogens. The major adducts formed by benzo[a]pyrene (BaP) and BbF in mouse skin in vivo that were not resolved on TLC were well separated by HPLC and thus a major DNA adduct formed in the skin of mice treated topically with coal tar was found to be derived from BaP rather than BbF.

Topics: Animals; Benz(a)Anthracenes; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Coal Tar; DNA; DNA Damage; Evaluation Studies as Topic; Female; Fluorenes; Humans; In Vitro Techniques; Male; Mice; Phosphorus Radioisotopes; Quinolines; Rats

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

Male and female CDF1 mice were administered a single oral dose of 3 mumol of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and killed 24 h later. DNA was isolated from the livers, lungs, kidneys, colon and forestomach and analysed by 32P-postlabelling for the presence of IQ and MeIQ adducts. Several adduct-enrichment procedures were investigated, including ATP-deficient labelling conditions, butanol extraction and nuclease P1 digestion, and only the ATP-deficient procedure was found to produce the same adduct pattern on polyethyleneimine--cellulose TLC as the standard procedure. Up to nine adduct spots were detected in liver DNA from IQ-treated mice, two of which were not detected in other tissues. The levels of binding in both male and female mice were in the order liver greater than kidney greater than colon greater than forestomach greater than lung. Analysis of DNA from MeIQ-treated mice revealed the presence of up to seven adducts, one of which was detected in liver but not in other tissues. The relative order of DNA binding was kidney greater than liver greater than or equal to colon greater than forestomach greater than lung. As dietary feeding of IQ induces liver, lung and forestomach tumours, and MeIQ induces liver and forestomach tumours in this mouse strain, these binding levels do not correlate with the susceptibility of the organs to carcinogenesis induced by these compounds; the results may indicate the importance of additional factors in determining organ specificity of carcinogenicity.

Topics: Animals; Autoradiography; Carcinogens; Chromatography, Thin Layer; DNA; Female; Male; Mice; Mice, Inbred Strains; Phosphorus Radioisotopes; Quinolines; Tissue Distribution