phosphorus-radioisotopes and chrysene

32P-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ([+/-]-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-1 0t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with 32P-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts: efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antibodies, Monoclonal; Benzo(a)pyrene; Carcinogens; Chromatography, Affinity; Chrysenes; DNA Adducts; Fluorescent Antibody Technique; Isotope Labeling; Mice; Mutagenicity Tests; Mutagens; Phosphorus Radioisotopes; Polycyclic Aromatic Hydrocarbons; Single-Strand Specific DNA and RNA Endonucleases; Skin

A 32P-postlabeling procedure for identifying and quantifying hydrophobic DNA adducts was developed (by modifying the method of Randerath and co-workers) in which labeled adducts are separated by high-performance liquid chromatography (HPLC) and quantified by liquid scintillation counting. This method was first developed for fluoranthene-DNA adducts, and methods for optimal detection and quantification of DNA adducts with diol epoxide metabolites of benzo[a]pyrene (BPDE), chrysene (CHDE), and benz[a]anthracene (BADE) have now been established. Analytical conditions slightly different from those adopted for fluoranthene-DNA adducts are required for accurate quantification of BPDE-, CHDE-, and BADE-DNA adducts. In particular, HPLC analysis requires generation of nucleotide 5'-[32P]monophosphate adducts by treatment with nuclease P1, and polycyclic aromatic hydrocarbon adducts demonstrate variable sensitivity to nuclease P1, mediated dephosphorylation. Thus, multiple adducts can be detected in one sample as long as the recovery of adducts under the applied conditions has been determined and chromatographic separation of labeled adducts is achieved. A battery of postlabeling assays can thus make it possible to detect optimally multiple adducts in one DNA sample. Results from these studies indicate that the HPLC- 32P-postlabeling assay is complementary to immunoassays in which related polycyclic aromatic hydrocarbon diol epoxide adducts cross-react for the quantification of adducts.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Animals; Benz(a)Anthracenes; Cattle; Chromatography, High Pressure Liquid; Chrysenes; DNA; DNA Damage; In Vitro Techniques; Phosphorus Radioisotopes; Polycyclic Compounds

32P-Postlabelling analysis is a sensitive method of detecting covalent modification of DNA by chemical carcinogens. We demonstrate that tetrol derivatives of the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (BP) and chrysene become 32P-labelled in the assay in the absence of nucleic acids. The transfer of 32P from [gamma-32P]ATP to the PAH derivatives requires T4 polynucleotide kinase. Phosphorylated dihydrodiols, phenols, triols and parent hydrocarbons were not detected under standard TLC conditions. Labelling of the non-nucleotide substrates was at least 2000-fold less efficient than labelling of a synthetic BP - DNA adduct. Using 75 microCi[gamma-32P]ATP, the detection limit for BP tetrols was 100-200 pg. Labelling of non-adduct substrates is unlikely to interfere with the analysis of DNA isolated from mammalian tissues, but DNA modified by electrophiles in vitro may, if inadequately purified, give rise to spurious radioactive products.

Topics: Adenosine Triphosphate; Animals; Autoradiography; Benzo(a)pyrene; Chromatography, Thin Layer; Chrysenes; DNA; Phenanthrenes; Phosphorus Radioisotopes; Radioisotope Dilution Technique

Incubation of r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-1,2-diol 3,4-oxide), the bay-region diol-epoxide of chrysene, with rat liver microsomes in the presence of NADP+ and DNA, followed by 32P-postlabelling analysis of the DNA, revealed the presence of at least two adducts not detected when anti-chrysene-1,2-diol 3,4-oxide was incubated with DNA alone. The formation of these adducts was not blocked by the epoxide hydrolase inhibitor 1,1,1-trichloropropane-2,3-oxide. One of the adducts cochromatographed with the adduct spot obtained when authentic 9-hydroxy-r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-9-OH-chrysene-1,2-diol 3,4-oxide) was reacted with DNA. Evidence suggested that a second adduct could also be formed by further metabolism of anti-9-OH-chrysene-1,2-diol 3,4-oxide. In addition, evidence was obtained for the further metabolism of the syn-isomer of chrysene 1,2-diol 3,4-oxide and the anti-isomer of a non-bay-region diol-epoxide of dibenz[a,c]anthracene to DNA binding species, but not for that of either the anti- or syn-isomers of the bay-region diol-epoxide of benzo[a]pyrene, the anti-isomers of the bay-region or a non-bay-region diol-epoxide of benz[a]anthracene, or the anti-isomer of the bay-region diol-epoxide of benzo[b]fluoranthene.

Topics: Animals; Benz(a)Anthracenes; Chrysenes; DNA; Male; Microsomes, Liver; Phenanthrenes; Phosphorus Radioisotopes; Rats; Rats, Inbred Strains