phosphorus-radioisotopes and quinone

Our interest in benzene-DNA adduct formation and their consequence has led us to develop a number of sensitive methods for their analysis. A HPLC method for the analysis of 32P-postlabelled benzene-DNA adducts was developed and used to detect adducts formed from the reaction of DNA or individual deoxynucleotides with the metabolites para-benzoquinone (p-BQ) and hydroquinone (HQ). Reaction of DNA with BQ yielded four adducts, the major product being a deoxycytidine adduct. HQ formed a single detectable deoxyguanosine DNA adduct, which was a minor product of the reaction of DNA with p-BQ. The supF forward mutation assay was used to assess the mutagenicity of p-BQ and HQ after transfection of treated plasmid in the human kidney cell line, Ad293. Single base substitution mutations at GC base pairs (bp) predominated for each treatment. However, when the mutation spectra achieved for each treatment were compared they were shown to be significantly different (p=0.004). These results may suggest either a possible role for the minor benzene-deoxyguanosine adducts in benzene genotoxicity or that HQ is causing DNA modification via a different mechanism, such as oxidative damage.

Topics: Benzene; Benzoquinones; Cell Line; Chromatography, High Pressure Liquid; DNA Adducts; Genes, Suppressor; Humans; Hydroquinones; Mutagenicity Tests; Phosphorus Radioisotopes; RNA, Transfer

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DN

Topics: Autoradiography; Base Sequence; Benzoquinones; Cell-Free System; Chromatography, High Pressure Liquid; Deoxyadenosines; Deoxycytidine; DNA Adducts; DNA Repair; HeLa Cells; Humans; Kinetics; Molecular Sequence Data; N-Glycosyl Hydrolases; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Protein Binding

The suitability of C18 reversed-phase thin-layer chromatography (TLC) for enrichment of adducts in the 32P-postlabeling assay was investigated for structurally diverse classes of DNA adducts derived from benzo[a]pyrene, 2-acetylaminofluorene, benzoquinone, safrole, and mitomycin C. The TLC enrichment involved retention of adducts to the C18 phase followed by elution with organic solvent-water. Adduct patterns obtained by the C18 purification were qualitatively similar to those obtained by the nuclease P1 and butanol procedures, the two commonly used enrichment methods. Adduct recoveries by the C18 method varied for different adducts and were significantly lower than those obtained by the other two techniques.

Topics: 2-Acetylaminofluorene; Animals; Benzo(a)pyrene; Benzoquinones; Butanols; Carcinogens; Chromatography, Thin Layer; DNA; Female; Isotope Labeling; Liver; Lung; Mice; Mitomycin; Phosphorus Radioisotopes; Rats; Safrole; Single-Strand Specific DNA and RNA Endonucleases

Using P1 nuclease enhanced 32P postlabeling, we investigated DNA adduct formation in HL-60 promyelocytic leukemia cells treated with the benzene metabolites hydroquinone, catechol, and 1,2,4-benzenetriol. Comparison of the slopes of the dose-response curves showed that hydroquinone was 7-9 times more effective than 1,2,4,-benzenetriol and catechol at inducing DNA adducts. Comparison of hydroquinone with catechol showed a good correlation between adduct formation and cytotoxicity. Similar comparisons of hydroquinone and 1,2,4,-benzenetriol suggest that cellular processes in addition to DNA adduct formation contributed to cytotoxicity. In cells treated with the combination of hydroquinone and either catechol or 1,2,4,-benzenetriol, DNA adduct formation was 3-6 times greater than the sum of adduct formation produced by single-agent treatments. Treatment with hydroquinone and 1,2,4,-benzenetriol produced DNA adducts not detected after treatment with either metabolite alone. The synergistic interaction of benzene metabolites in the production of DNA adducts may play an important role in the genotoxic effects of benzene in vivo.

Topics: Autoradiography; Benzene; Benzoquinones; Catechols; Cell Line; Cell Survival; DNA, Neoplasm; Humans; Hydroquinones; Kinetics; Leukemia, Promyelocytic, Acute; Phosphorus Radioisotopes; Radioisotope Dilution Technique

Treatment of DNA digests with nuclease P1 prior to 32P-labeling of adducts has previously been shown to enhance the sensitivity of the 32P-postlabeling assay for the detection of aromatic carcinogen-DNA adducts. The enhancement was based on the ability of nuclease P1 to remove the 3'-phosphate from normal nucleotides but not the corresponding phosphate from most aromatic adducted nucleotides. We investigated the utility of another 3'-dephosphorylating enzyme, nuclease S1, for this purpose, and found it to be as effective as nuclease P1. The recovery of DNA adducts derived from benzo[a]-pyrene (B[a]P), benzoquinone (BQ) and 2-acetylaminofluorene (AAF) was comparable after enhancement with either enzyme. Some differences were, however, observed. Recovery of a minor B[a]P adduct was 1.5 times higher by the S1 procedure. Among minor adducts of BQ, two showed higher values (2.8- and 6.1-fold) by the S1 procedure and one by the P1 procedure (2.4-fold). The major AAF adduct, deoxyguanosine-C8-AF, exhibited poorer recovery (1-11%) by either procedure, while the minor adducts, deoxyguanosine-N2-AAF and deoxyguanosine-C8-AAF, showed better recovery (2-3 times) than by the enhancement procedure involving extraction of adducts into butanol. Our results show that the nuclease S1 assay can complement the nuclease P1 assay, with improved recoveries for some adducts. Considering the complexity of the postlabeling assay, this additional variant may prove useful in unequivocal detection of DNA adducts.

Topics: 2-Acetylaminofluorene; Autoradiography; Benzo(a)pyrene; Benzoquinones; Carcinogens; Chromatography, Thin Layer; DNA; DNA Damage; Phosphorus Radioisotopes; Single-Strand Specific DNA and RNA Endonucleases

We have examined DNA adduct formation and cytotoxicity in HL-60 cells treated with either hydroquinone (HQ) or p-benzoquinone (p-BQ). Treatment of HL-60 cells with either HQ or p-BQ produced the same DNA adduct. The DNA adduct level varied from 0.05 to 10 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. To achieve the same DNA adduct level required higher concentrations and longer treatment times with HQ compared to p-BQ. p-BQ was also more cytotoxic to HL-60 cells than HQ. Reaction of calf thymus DNA with a p-BQ/HQ mixture produced five adducts as detected by 32P-postlabeling. Two isomers of (hydroxy)-1,N2-benzetheno-2'- deoxyguanosine-3'-phosphate were isolated from the reaction of 2'-deoxyguanosine-3'-phosphate with a p-BQ/HQ mixture and one of the isomers was identified as adduct no. 1 of the DNA reaction. The DNA adduct formed in HL-60 cells treated with HQ or p-BQ did not correspond to any of the principal adducts formed in DNA reacted with p-BQ/HQ. This result suggests that cellular mechanisms modify DNA adduct formation by HQ and p-BQ.

Topics: Benzoquinones; Cell Survival; DNA; Humans; Hydroquinones; Leukemia, Promyelocytic, Acute; Phosphorus Radioisotopes; Tumor Cells, Cultured

Cytidine-3'-phosphate was reacted with p-benzoquinone under neutral aqueous conditions, and the fluorescent product formed was isolated and characterized. The structure of the covalent adduct was identified as (3'-hydroxy)-3,N4-benzetheno-cytidine-3'-phosphate by high-resolution MS and 1H NMR spectroscopy. A similar product was isolated from the reaction of 2'-deoxycytidine-3'-phosphate with a hydroquinone-p-benzoquinone mixture. 32P-Postlabeling of calf thymus DNA reacted with p-benzoquinone detected several adducts, the principal adduct being (3'-hydroxy)-3,N4-benzetheno-2'-deoxycytidine-3'-phosphate. Our studies demonstrate that the reaction of DNA with p-benzoquinone in vitro leads to multiple DNA adducts. 32P-Postlabeling may allow detection of benzene-DNA adducts in vivo.

Topics: Benzoquinones; Carcinogens; Cytidine Monophosphate; Cytosine Nucleotides; Deoxycytidine Monophosphate; Deoxycytosine Nucleotides; DNA; Phosphorus Radioisotopes; Quinones; Radioisotope Dilution Technique; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

The carcinogenicity of benzene has been considered to be in part mediated by its chemically reactive metabolic product benzoquinone (BQ), which is formed from the intermediary metabolites phenol and hydroquinone (HQ). We have evaluated the DNA-binding capability of these chemicals in vitro and in vivo by postlabeling. Treatment of rat Zymbal glands in culture with phenol and HQ or direct reaction of BQ with DNA produced DNA adducts, which were detectable by the nuclease P1-enhanced 32P-postlabeling assay as 5'-32P-labeled 3',5'-bisphosphate products. The enhancement of sensitivity in this assay is based on the previous finding that nuclease P1 hydrolyzes the phosphate attached to the 3' side of normal nucleotides but not the corresponding phosphate of most aromatic/bulky adducted nucleotides. Also based on this hydrolytic property of nuclease P1, we developed an additional sensitive procedure that permitted the detection of DNA lesions as 5'-32P-labeled products of dinucleotides, pXpN, or of nucleoside monophosphates, pX, where X and N indicate an adducted nucleoside and a normal nucleoside respectively. In the latter assay, adducted DNA was first digested with nuclease P1 and acid phosphatase to yield XpN and N. The latter were then 32P-labeled to yield [5'-32P] pXpN or 32P-labeled and treated with venom phosphodiesterase to obtain [5'-32P]pX. After optimization of enzymatic conditions, the modified nuclease P1 assay yielded adduct recoveries similar to those obtained by the bisphosphate assay for in vitro phenol-, HQ- and BQ-DNA adducts. Neither of the nuclease P1-enhanced postlabeling procedures showed exposure-specific adducts in vivo in the bone marrow, Zymbal gland, liver and spleen of female Sprague-Dawley rats at 24 h after the last of four single, daily p.o. doses of 75 mg/kg phenol or 150 mg/kg phenol/HQ (1:1). Our results show that phenol, HQ and BQ produce adducts in vitro, but corresponding adducts are not detected in vivo with phenol and phenol/HQ, even when measured by the standard and modified nuclease P1 postlabeling methods capable of detecting 1 adduct in 10(9-10) DNA bases.

Topics: Acid Phosphatase; Adenosine Triphosphate; Animals; Benzoquinones; DNA; Female; Hydroquinones; Nucleosides; Nucleotides; Phenol; Phenols; Phosphorus Radioisotopes; Pyrenes; Quinones; Rats; Rats, Inbred Strains; Single-Strand Specific DNA and RNA Endonucleases

Formation of DNA-adducts by 3-BHA or its metabolites, i.e., tert-butyl-1,4-benzoquinone (TBQ) and 5-methoxy-3-tert-butyl-1,2-benzoquinone (3-TBOQ), as well as DNA-adduct formation by 4-nitroquinoline-N-oxide (4NQO), in rat forestomach were examined by an enzymatic 32P-postlabeling assay. Four DNA-adducts were clearly detected in the forestomach after treatment of rats with 4NQO. The sensitivity was 1.9 certain adducts per 10(8) normal nucleotides. On the contrary, no DNA adducts were detected in the forestomach of rats given either a single or repeated oral administration (5 days) of 3-BHA, TBQ or 3-TBOQ. The analyses were carried out under conditions which could detect the DNA-adducts produced by reaction of TBQ with calf thymus DNA in vitro. The results suggest that formation of aromatic adducts in vivo by 3-BHA, TBQ or 3-TBOQ in the rat forestomach-DNA is not evident or at least below the detection limits of the current bioassay.

Topics: 4-Nitroquinoline-1-oxide; Animals; Benzoquinones; Butylated Hydroxyanisole; DNA; Gastric Mucosa; Male; Phosphorus Radioisotopes; Quinones; Rats; Rats, Inbred F344; Sulfhydryl Compounds