Levels Of carcinogen-DNA Adducts Research Articles

Ellagic Acid Inhibits DNA Binding of Benzo(a)pyrene Activated by Different Modes.

The possible mechanisms of inhibition of benzo(a)pyrene (BaP) binding to DNA in vivo and in vitro by ellagic acid (EA) were investigated. In the in vitro studies BaP was activated by cytochrome P-450-dependent mixed function oxidase system, ascorbate-dependent lipid peroxidation, and peroxidation of linoleic acid. Reactions were performed in the dark at 37°C for 20 to 30min in buffered aqueous solutions with 2mg DNA and 40nmol tritiated BaP. The levels of BaP-DNA adducts were determined by assay of the radioactivity. BaP-DNA adduct levels ranged from 146 to 170fmol/mg DNA in ascorbate-and NADPH-dependent reactions and the level was 8.25±0.425pmol/mg DNA in the hematin-mediated linoleic acid reaction. Addition of EA to the reaction mixtures resulted in a significant inhibition in BaP-DNA adduct formation, depending upon the concentration of EA; e.g., 500μM EA resulted in 42 to 53% inhibition of binding in the three systems of carcinogen activation. Similarly, EA feeding to male NMRI mice at a concentration of 12μg/ml drinking water significantly decreased the levels of carcinogen-DNA adducts in the lungs. Reactions performed in vitro, in which DNA was preincubated overnight at 4°C with EA, revealed a decrease of 20% in adduct formation. MDA formation during the process of lipid peroxidation, stimulated by NADPH and ascorbate in the liver microsomes of mouse and by hematin in linoleic acid, was taken as an index for the free radical reactions. Addition of EA to in vitro systems inhibited the MDA formation with an IC50 (concentration for 50% inhibition) of 480, 400, and 400μM for NADPH, ascorbate, and hematin-mediated free radical reactions, respectively.

No increase in carcinogen-DNA adducts in the lungs of monkeys exposed chronically to marijuana smoke

Rhesus monkeys exposed to marijuana smoke either 7 or 2 days weeks (HI and LO groups, respectively), or ethanol-extracted marijuana smoke for 7 days week (EM) or sham treatment (SH) for 1 year were sacrificed 7 months following the last exposure. Pulmonary levels of carcinogen-DNA adducts were determined. Although mean or median adduct levels were not statistically different, 15 of 22 adduct measures were highest in the EM group and lowest 12 of 22 times in the SH group. The levels of aromatic carcinogen-DNA adducts seem no higher in the lungs of animals exposed to marijuana smoke than in untreated animals. Ethanol-extracted marijuana may have effects greater than marijuana itself.

32P-postlabelling analysis of DNA from human tissues

DNA extracted from human lung, bladder, liver, pancreas, cervix and breast tissue samples taken at autopsy (37 sample sets) was analysed by the nuclease P1 enhancement modification of the 32P-postlabelling assay for levels of aromatic carcinogen DNA adducts. Results were combined with those from a previous study for statistical analysis of 56 sample set (32 male + 24 female). A strong trend was seen for increased adduct levels in the lung DNA of smokers and a weak association for the bladder DNA of smokers compared to non-smokers. Aromatic adducts were also detected in other tissues.

Smoking related carcinogen-DNA adducts in biopsy samples of human urinary bladder: identification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl as a major adduct.

The prevalence of covalent modifications to DNA (carcinogen-DNA adducts) in 42 human urinary bladder biopsy samples was investigated by 32P-postlabeling methods, with enhancement by both nuclease P1 treatment and 1-butanol extraction. Total mean carcinogen-DNA adduct levels and the mean levels of several specific adducts were significantly elevated in DNA samples of 13 current smokers, as opposed to 9 never smokers or 20 ex-smokers (5 years abstinence). There was no significant difference between the latter two groups. Several DNA adducts enhanced by nuclease P1 treatment were chromatographically similar to putative hydrocarbon DNA adducts reported earlier for placenta and lung DNA samples obtained from cigarette smokers. Putative aromatic amine adducts were detected by 1-butanol extraction that were not present when the samples were treated with nuclease P1. One of these displayed chromatographic behavior identical to the predominant adduct induced by the human urinary bladder carcinogen, 4-aminobiphenyl, which is present in cigarette smoke. This adduct comigrated in several thin-layer chromatographic systems with a synthetic N-(deoxyguanosin-8-yl)-4-amino[2,2'-3H]biphenyl-3',5'-bisphosphate marker. Moreover, when this adduct was eluted from the thin-layer chromatograms of several individuals and injected onto an HPLC system, the 32P from the human bladder DNA samples coeluted in the same fraction as the tritiated synthetic N-(deoxyguanosin-8-yl)-4-aminobiphenyl marker. These data reinforce an association between cigarette smoking and DNA damage and suggest a molecular basis for the initiation of human urinary bladder cancer by cigarette smoke.

The role of intestinal microflora in the metabolic activation of 6-nitrochrysene to DNA-binding derivatives in mice

6-Nitrochrysene has previously been shown to be a potent lung and liver carcinogen following i.p. administration to newborn mice and to be metabolically activated to DNA-binding derivatives by nitro-reduction or a combination of nitro-reduction and ring oxidation. In this study, we have examined fecal metabolites and DNA-carcinogen adducts in 5-week-old conventional and germfree Balb/c mice treated with [ 3H]6-nitrochrysene in order to determine if the metabolic activation pathway(s) for this compound in these mice differs from that observed in preweanling mice. We further evaluated the role of the intestinal microflora on the metabolism and generation of DNA-reactive metabolites in this system. The amount of 6-aminochrysene excreted in the feces of germfree mice within 48 h after treatment with a single i.p. dose of [ 3H]6-nitorchrysene (0.03 μmol/5 μl/g body wt) was approximately 25% of that excreted in identically treated conventional mice. However, the levels of carcinogen-DNA adducts in the lungs and livers of conventional and germfree Balb/c mice were similar at the 24 and 48 h time points examined. HPLC analysis of hydrolysates of liver and lung DNA indicated that adducts derived from both N-hydroxy-6-aminochrysene and trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene metabolites were formed in the liver whereas only the latter adduct was detected in the lung. This contrasts with previous findings in preweanling mice where the adduct derived from the trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene metabolite was the single major adduct detected in both liver and lung DNA. The proportion of adducts derived from N-hydroxy-6-aminochrysene was significantly greater in the liver DNA of germfree mice than in the liver DNA of conventional mice.

Detection and characterization of carcinogen—DNA adducts in exfoliated urothelial cells from 4-aminobiphenyl-treated dogs by 32P-postlabelling and subsequent thin-layer and high-pressure liquid chromatography

The recent development of sensitive methods to detect carcinogen-DNA adducts offers a useful biochemical approach to human risk assessment. However, a major obstacle to developing a human biomonitoring method for carcinogen-DNA adducts has been the problem of obtaining target tissue DNA samples by non-invasive means. This work describes a method for the isolation of nanogram quantities of DNA from urothelial cells exfoliated into urine and the detection of carcinogen-DNA adducts from that DNA by 32P-postlabelling methods. Urine samples were collected on ice from dogs treated with 4-aminobiphenyl (ABP) over a 2-week period and pooled according to an experimental plan that involved analysis of cumulative 48- or 72-h samples. The pooled samples were sieved and then washed repeatedly with a sucrose buffer to dissolve contaminating triple phosphate (MgNH4PO4), calcium oxalate and uric acid crystals. DNA was isolated using an enzyme-solvent extraction method with the DNA being co-precipitated from ethanol with glycogen. The DNA was hydrolysed and postlabelled with 32P under conditions of excess ATP so that nucleotides were labelled quantitatively. Adducts observed on the resulting thin-layer chromatograms were identical to those obtained from DNA modified in vitro with N-hydroxy-4-aminobiphenyl and from dog bladder urothelial DNA isolated from the ABP-dosed animals at termination of the experiment. Furthermore, a dose-related increase in ABP-DNA adduct formation was demonstrated. Thus, it appears that the carcinogen-DNA adduct levels in the exfoliated bladder cells are reflective of the levels in the intact urothelium once steady-state levels have been achieved. To establish the identity of the major ABP-urothelial DNA adduct in chronically-treated dogs, the predominant 32P-postlabelled adduct was eluted from the thin-layer chromatograms and co-injected on an HPLC system with a synthetic [3H]N-(deoxyguanosin-8-yl)-4-aminobiphenyl-3',5'-bisphosphate standard. Dual-label analysis of 3H and 32P indicated that both eluted from the column in the same fraction, which coincided with the UV absorbance peak of the synthetic marker. Preliminary experiments with exfoliated urothelial cells from human urines indicate that these methods should have general utility for monitoring humans exposed to urinary bladder carcinogens and for investigations of the biochemical mechanisms by which such adducts are formed in the urothelium.

Specific BPDE I modification of replicating and parental DNA from early S phase human foreskin fibroblasts.

Replicating DNA was modified by BPDE I to a greater extent than parental DNA when human fibroblast cells were treated with the carcinogen for 30 min in early S phase. Synchronized cells were exposed to 5-bromodeoxyuridine and treated with non-radioactive BPDE I and [methyl-3H]thymidine in early S phase. The density- and tritium-labeled, replicated DNA was separated from parental DNA in a CsCl gradient. The individual carcinogen-DNA adduct levels in both samples were quantitated by using the 32P-postlabeling method. The total modification of replicated DNA was 1.4-2.4 times greater than parental DNA. This difference was mainly reflected by differences in the main adducts, identified as the 3', [5'-32P]bisphosphates of 7R and 7S-BPDE I-dG. Confirmation of the identity of these two specific carcinogen-DNA adducts was accomplished by co-chromatography on t.l.c. with 3H-labeled 3',5'-bisphosphate adducts. The two 3H- and 32P-labeled adducts were isolated and dephosphorylated. The resultant 3H-labeled deoxyribonucleoside adducts were analyzed on h.p.l.c. and identified by co-chromatography with authentic standards. These results suggest that preferential modification of replicating DNA occurs when human cells are treated with BPDE I in early S phase. The ultimate result of this specific modification is the expression of a transformed phenotype.

Benzo(a)pyrene diolepoxide-DNA adducts detected by synchronous fluorescence spectrophotometry.

Using benzo(a)pyrene (BP) as a model carcinogen we are currently applying a fluorescence technique to detect the very low levels of carcinogen-DNA adducts in human populations due to environmental exposure. In synchronous fluorescence spectrophotometry for detection of BP-diol epoxide-DNA, excitation and emission wavelengths are scanned simultaneously with a fixed wavelength difference (delta lambda) of 34 nm. Compared to conventional fluorescence methods only one peak emerges because excitation and emission peaks have to match delta lambda to show. Because of the quenching effect of DNA, samples are hydrolyzed by acid. After this, BP-diol epoxide (BPDE)- -modified DNA gives a peak at the same wavelength and of the same fluorescence yield as BP-tetrols. When DNA from peripheral blood lymphocytes of 44 coke oven workers were analyzed, 10 had a sharp peak at 379. Among 36 coke oven workers from another factory, 4 had detectable levels of adducts. A much smaller percentage of samples was positive in a group of aluminum plant workers. We have also found BPDE-DNA adducts in DNA from pulmonary alveolar macrophages and peripheral blood lymphocytes from tobacco smokers and some of the nonsmokers.

DNA Adduct Formation by 7, 12-Dimethylbenz[<italic>a</italic>]anthracene and Its Noncarcinogenic 2-Fluoro Analogue in Female Sprague-Dawley Rats<xref ref-type="fn" rid="FN2">2</xref>

The potent polycyclic aromatic hydrocarbon 7,12 dimethylbenz[a]anthracene (DMBA) bound to the DNA of numerous organs of the female outbred Sprague-Dawley rat after iv administration under a regimen known to produce a high yield of mammary adenocarcinomas. The maximum DNA binding levels observed following iv administration of 5 mg (20 mumol) DMBA range from approximately 12 mumol hydrocarbon/mol deoxyribonucleic for the liver to approximately 5 mumol hydrocarbon/mol deoxyribonucleotide for the mammary gland, the target tissue. Administered under identical conditions, the noncarcinogenic analogue 2-fluoro-7,12-dimethylbenz[a]anthracene (2F-DMBA) bound to the DNA at levels of about 5-10% that of DMBA (i.e., 0.3-1.6 mumol/mol deoxyribonucleotide). Chromatographic analysis of the hydrocarbon-deoxyribonucleoside adducts produced showed that for DMBA at least two major types of identifiable adducts were observed in all tissues examined, the major one being that resulting from the reaction of a DMBA bay-region diol-epoxide. The other adduct type resulted from the binding of an analogous diol-epoxide of a DMBA metabolite, 7-hydroxymethyl-12-methylbenz[a]anthracene. Adducts from 2F-DMBA were observed on high-pressure liquid chromatography but were in quantities insufficient for characterization. The finding of higher levels of chromatographically identical DMBA-DNA adducts in the nontarget (liver) tissue than in the target tissue indicated that adduct formation per se was not a sufficient stimulus for the cancer induction. However, the failure of the structurally similar 2F-DMBA, which produced only very low levels of DNA adducts, to induce mammary cancer implies that certain levels of carcinogen-DNA adducts may be necessary for carcinogenesis.