Analysis Of carcinogen-DNA Adducts Research Articles

Beyond monotherapy: Integrating immunotherapy into current treatment regimens

While we have shown that these urinary metabolites are risk biomarkers for lung cancer in these studies, they are not fully predictive. This is because these biomarkers are mainly measuring exposure but do not take into account the critical metabolic activation step leading to the formation of DNA adducts which cause the multiple mutations observed in lung cancer. Quantitation of DNA adducts is challenging because of their low levels, typically less than 1 adduct per 10 normal bases, and the small amounts of DNA available for studies in living humans. To address this question, we are developing high resolution mass spectrometric methods for quantifying oral cell DNA adducts as a surrogate for DNA adduct formation in the lung. In one recent study, we found remarkably high levels of DNA adducts of tobaccospecific compounds in in the oral mucosa cells of smokers compared to non-smokers. We are also able to quantify formaldehyde-DNA adducts in oral mucosa cells. As mass spectrometric methods for analysis of carcinogen-DNA adducts become increasingly more sensitive and specific, it appears likely that quantitation of a panel of tobacco carcinogen DNA adducts in smokers, leading to identification of susceptible individuals, may be feasible.

Carcinogen biomonitoring in human exposures and laboratory research: validation and application to human occupational exposures

A multiple biomarker approach is required to integrate for metabolism, temporal response and exposure-response kinetics, biological relevance, and positive predictive value. Carcinogen DNA adduct analysis can be used in animal and in vitro studies to detect absorption permutations caused by mixture interactions, and to control metabolic variation when specific CYP450 genes (1A1 or 1A2) are knocked out. These enzymes are not critical to the metabolic activation of model Polycyclic Aromatic Compounds (PAC) and aromatic amines, respectively, as suggested by in vitro analysis. Several human studies have been carried out where multiple biomarkers have been measured. In a study of benzidine workers, the similarities in elimination kinetics between urinary metabolites and mutagenicity is likely responsible for a better correlation between these markers than to BZ-DNA adducts in exfoliated cells. In a study of rubber workers, the relationship between specific departments, urinary 1 HP and DNA adducts in exfoliated cells coincided with the historical urinary bladder cancer risk in these departments; the same relationship did not hold for urinary mutagenicity. In a study of automotive mechanics, biomarkers were used to monitor the effectiveness of exposure interventions. These data reinforce the notion that carcinogen biomarkers are useful to monitor exposure, but that a complementary approaches involving effect and perhaps susceptibility biomarkers is necessary to obtain the necessary information.

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by32P-postlabeling

32P-Postlabeling is a widely applied assay for the analysis of carcinogen-DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 microM) for 18 hr and from liver of rats i.p.-treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol-extraction and a salting-out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol-enriched 32P-postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting-out procedure (63.2 +/- 13.7 adducts per 10(8) nucleotides, n = 9) as compared with the phenol-extraction (14.3 +/- 0.8). RNA was approximately 4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched 32P-postlabeling in the liver of B[a]P-exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE-DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P-derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by 32P-postlabeling.

Development and application of non-invasive biomarkers for carcinogen-DNA adduct analysis in occupationally exposed populations

Biological monitoring of exposures to carcinogenic compounds in the workplace can be a valuable adjunct to environmental sampling and occupational medicine. Carcinogen-DNA adduct analysis has promise as a biomarker of effective dose if target organ samples can be obtained non-invasively. We have developed non-invasive techniques using exfoliated urothelial and bronchial cells collected in urine and sputum, respectively. First morning urine samples were collected from 33 workers exposed to benzidine or benzidine-based dyes and controls matched for age, education, and smoking status. Sufficient DNA for 32P-postlabelling analysis was obtained from every sample. Mean levels of a specific DNA adduct (which co-chromatographed with standard characterized by MS) were elevated significantly in the benzidine-exposed workers relative to controls. In addition, workers exposed to benzidine had higher adduct levels than those exposed to benzidine-based dyes. This study demonstrates the usefulness of these non-invasive techniques for exposure/effect assessment. To be useful in occupational studies, biomarkers must also be sensitive to exposure interventions. We have conducted topical application studies of used gasoline engine oils in mice and found that the levels of carcinogen-DNA adducts in skin and lung can be significantly lowered if skin cleaning is conducted in a timely manner. The combination of useful, non-invasive techniques to monitor exposure and effect and industrial hygiene interventions can be used to detect and prevent exposures to a wide range of carcinogens including those found in used gasoline engine oils and jet exhausts.

Analysis of 4-aminobiphenyl-DNA adducts in human urinary bladder and lung by alkaline hydrolysis and negative ion gas chromatography-mass spectrometry.

Analysis of carcinogen-DNA adducts has been regarded as a useful means of assessing human exposure to chemical carcinogens. We have established a method for quantitation of 4-aminobiphenyl (4-ABP)-DNA adducts by alkaline hydrolysis and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Aliquots of DNA (typically 100 micrograms/ml) were spiked with an internal standard, d9-4-ABP, and were hydrolyzed in 0.05 N NaOH at 130 degrees C overnight. The liberated 4-ABP was extracted with hexane and derivatized using pentafluoropropionic anhydride in trimethylamine for 30 min at room temperature prior to GC-NICI-MS. With in vitro [3H]N-hydroxy-4-ABP modified DNA standards, we observed 59 +/- 7% (n = 9) recovery of the 4-ABP and a linear correlation between hydrolyzed 4-ABP and the adduct levels ranging from about 1 in 10(8) to 1 in 10(4) nucleotides (r = 0.999, n = 9). The method was further validated by comparison of the results with that obtained by the 32P-postlabeling method. There was excellent agreement (r = 0.994, p < 0.001) between the two methods for quantitation of the adduct in eight samples of Salmonella typhimurium DNA treated with 4-ABP and rat liver S9, although the 32P-postlabeling method gave slightly higher values. The DNA adducts in 11 human lung and 8 urinary bladder mucosa specimens were then determined by our GC-NICI-MS method. The adduct levels were found to be < 0.32 to 49.5 adducts per 10(8) nucleotides in the lungs and < 0.32 to 3.94 adducts per 10(8) nucleotides in the bladder samples.(ABSTRACT TRUNCATED AT 250 WORDS)

32P-postlabeling of bile components: bulky adduct-like behavior in polyethyleneimine-cellulose thin layer chromatography.

The 32P-postlabeling assay has been used widely in carcinogen-DNA adduct analysis because of its sensitivity and reproducibility. Cloned T4 polynucleotide kinase (PNK), routinely used in this assay, phosphorylates the 5'-OH groups of adducted nucleotides in the presence of [gamma-32P]ATP. However, as an exception to this property, PNK has been reported to phosphorylate non-adducted carcinogen metabolites, such as tetrol derivatives of benzo[a]pyrene and chrysene. Also, PNK phosphorylates both 5'-OH and 3'-OH groups of safrole-adducted deoxydinucleoside monophosphates having an unmodified purine in the 3'-position. In the present study we show that T4 PNK catalyzed the transfer of [32P]phosphate from [gamma-32P]ATP to rat bile components or purified bile acids (derivatives of 3 alpha-hydroxy-5 beta-cholanic acid) in the absence of nucleic acids or nucleases. However, labeling of the bile acids appeared over 100,000-fold less efficient than labeling of 2'-deoxyadenosine-5'-monophosphate. There was no reaction in the absence of bile components or PNK. Dehydrocholic acid, which lacks hydroxyl groups, was resistant to phosphorylation. On polyethyleneimine-cellulose TLC maps, 32P-labeled rat bile extract gave an array of non-polar radioactive spots which resembled carcinogen-DNA adducts, while 32P-labeled purified bile acids each gave a single spot. These 32P-labeled products liberated 32Pi upon incubation with prostatic acid phosphatases. Two of the radioactive spots obtained from rat bile were identified as phosphorylated taurocholic and taurodeoxycholic acids by co-chromatography with 32P-labeled standards. These findings demonstrate for the first time that PNK is able to phosphorylate natural products other than nucleotides and further emphasize the need to rule out contamination with bile acids and possibly other bulky/hydrophobic alcohols when analyzing DNA samples by 32P-postlabeling.

Improved reversed-phase high-performance liquid chromatographic separation of 32P-labelled nucleoside 3',5'-bisphosphate adducts of polycyclic aromatic hydrocarbons

32P-Postlabelling is a sensitive technique for the detection and analysis of carcinogen-DNA adducts. In this paper we describe the development of an improved high-performance liquid chromatography (HPLC) method for the separation of 32P-labelled 3',5'-bisphosphates of nucleosides modified by reactive derivatives of carcinogenic polycyclic aromatic hydrocarbons (PAH). Optimal resolution of the major 32P-postlabelled DNA adducts formed by the anti-diol-epoxides of ten PAH was achieved using a phenyl-modified silica gel column with a gradient of methanol in phosphate buffer at low pH and high ionic strength. Use of a radioactivity flow detector coupled to the HPLC apparatus allowed detection of subfemtomole quantities of labelled adducts.

3.4 Immunocytochemical analysis of carcinogen-DNA adducts in normal and preneoplastic tissues

3.4 Immunocytochemical analysis of carcinogen-DNA adducts in normal and preneoplastic tissues

Preparation of microgram quantities of BaP-DNA adducts using isolated rat hepatocytes in vitro

The analysis of carcinogen-DNA adducts generally requires the preparation (by chemical or biological means) of DNA adduct standards, in amounts sufficient for chemical characterization. We have established conditions for the in vitro biological preparation of microgram quantities of DNA adducts derived from benzo[a]pyrene (BaP), fluoranthene and 7,12-dimethylbenzanthracene, using isolated rat hepatocytes. The metabolic activation of 180 microM BaP by isolated rat hepatocytes in a calf-thymus-DNA (CT-DNA)-supplemented medium resulted in the formation of 2.9 micrograms of BaP adducted to 56.7 mg of DNA. The average level of binding in this experiment was 148 +/- 8 pmol BaP bound/1 mg DNA, which compares favorably to the 10-30 pmol BAP/1 mg DNA which is typical of mouse skin adducts in vivo. In another experiment, BaP-DNA adduct formation in calf-thymus DNA added to hepatocyte incubations was further increased to 327 +/- 27 pmol/mg DNA, by physical shearing of the DNA prior to the incubation. The HPLC profile of the BaP adducts produced using hepatocytes plus CT-DNA is virtually indistinguishable from that produced by tumor-initiating doses of BaP applied to mouse skin in vivo, and the major DNA adduct formed by the hepatocytes co-elutes with the (+)-anti-diol-epoxide adduct of deoxyguanosine. Similar experiments using fluoranthene and 7,12-dimethylbenzanthracene also resulted in substantial DNA adduct formation; however, incubations using dibenz[a,h]anthracene did not. These results indicate that isolated rat hepatocytes in vitro can be useful for the preparation of DNA adducts of a number of polycyclic aromatic hydrocarbons, in quantities sufficient for chemical characterization.

Postlabeling analysis of carcinogen-DNA adducts in mussel, Mytilus galloprovincialis

The 32P-postlabeling analysis of DNA adducts formed in mussel digestive gland homogenates incubated with 2-aminofluorene (32 μ m) showed one major and one minor adduct in the range of 1 adduct per 1–4 × 10 8 nucleotides. The predominant adduct was found to be chromatographically similar to the known C8-aminofluorene-substituted guanine adduct. The metabolic activation of 2-aminofluorene was blocked in the presence of methimazole (470 μ m), a competitive inhibitor of FAD-containing monooxygenase. In contrast to 2-aminofluorene, benzo( a)pyrene-modified DNA either did not show any adduct or a very weak adduct spot (1 adduct per 1–4 × 10 9 nucleotides). The samples of DNA from mussel, carp and bream, which had no obvious exposure to pollutants, showed one to several weak ‘natural adducts’. The origin of these adducts is as yet undetermined. Since postlabeling assay enables measurement of carcinogen-DNA adducts formed with any, even unknown, chemicals from the composite environmental mixture of genotoxic pollutants, it may become a valuable tool for risk assessment also in marine environments.

Postlabeling methods for carcinogen-DNA adduct analysis.

Radioactive carcinogens have provided most of our present knowledge about the chemistry of interactions between carcinogens and biological systems. The requirement of radioactive carcinogens has restricted carcinogen-DNA binding studies to chemicals that are readily available in isotopically labeled form, i.e., a minute fraction of all potentially mutagenic or carcinogenic chemicals. To extend the scope of carcinogen-DNA binding studies, an alternative method, which does not require radioactive test chemicals, has been developed. In this approach, radioactivity (32P) is being incorporated into DNA constituents by polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP after exposure of the DNA in vitro or in vivo to a nonradioactive, covalently binding chemical, and evidence for the alteration of DNA nucleotides is provided by the appearance of extra spots on autoradiograms of thin-layer chromatograms of digests of the chemically modified DNA. Quantitation of adduct levels is accomplished by scintillation counting. The sensitivity of the technique depends on the experimental conditions for 32P-labeling and on the chemical structure of the adducts. Greater sensitivity may be achieved if adducts can be separated as a class from the normal nucleotides. This is the case for an estimated 80% of all carcinogens, giving rise to bulky and/or aromatic substituents in DNA. Under the present conditions, one such adduct in 10(9) to 10(10) normal nucleotides can be detected. A total of approximately 80 compounds has been studied thus far Binding to DNA of rodent tissues was readily detected by the 32P-postlabeling assay for all known carcinogens among these compounds, and adducts were detected in DNA from human placenta of smokers.

Biochemical studies of the tracheobronchial epithelium.

Tracheobronchial epithelium has been a focus of intense investigation in the field of chemical carcinogenesis. We have reviewed some biochemical investigations that have evolved through linkage with carcinogenesis research. These areas of investigation have included kinetics of carcinogen metabolism, identification of carcinogen metabolites, levels of carcinogen binding to DNA, and analysis of carcinogen-DNA adducts. Such studies appear to have provided a reasonable explanation for the susceptibilities of the respiratory tracts of rats and hamsters to carcinogenesis by benzo(a)pyrene. Coinciding with the attempts to understand the initiation of carcinogenesis in the respiratory tract has also been a major thrust aimed at effecting its prevention both in humans and in animal models for human bronchogenic carcinoma. These studies have concerned the effects of derivatives of vitamin A (retinoids) and their influence on normal cell biology and biochemistry of this tissue. Recent investigations have included the effects of retinoid deficiency on the synthesis of RNA and the identification of RNA species associated with this biological state, and also have included the effects of retinoids on the synthesis of mucus-related glycoproteins. Tracheal organ cultures from retinoid-deficient hamsters have been used successfully to indicate the potency of synthetic retinoids by monitoring the reversal of squamous metaplasia. Techniques applied to this tissue have also served to elucidate features of the metabolism of retinoic acid using high pressure liquid chromatography. In brief, formidable strides have been made in biochemistry specific to this important target tissue, despite the inability to acquire tracheobronchial epithelium in large quantities.

Highly persistent polycyclic aromatic hydrocarbon-DNA adducts in mouse skin: Detection by 32P-postlabeling analysis

A 32P-postlabeling method for carcinogen-DNA adduct analysis recently developed in our laboratory was applied to skin DNA from mice treated topically with polycyclic aromatic hydrocarbons (PAHs). After application of 4 doses of 1.2 μmol each of benzo[ a]pyrene (BP), 3-methylcholanthrene (MC) and 7,12-dimethylbenz[ a]anthracene (DMBA), respectively, total covalent adduct binding in mouse skin DNA initially amounted to 1 adduct in 6.0 × 10 4–1.3 × 10 5 nucleotides. Four weeks after treatment, these levels had declined to 1 adduct in 1.4 × 10 6–2.7 × 10 6 nucleotides. Substantial removal of DNA adducts occurred during the first 2 weeks after carcinogen application while adducts remaining thereafter underwent little or no repair between 2 and 4 weeks after treatment. These results raise the possibility that the persistent adducts occupy specific genomic sites in quiescent cells where they may not be amenable to repair because of localized conformational alterations of DNA or shielding by associated proteins.

Isolation and quantitation of DNA-bound benzo(a)pyrene metabolites: Comparison of hydroxyapatite and precipitation procedures

The hydroxyapatite and precipitation procedures are two commonly used methods for isolating DNA, and both have been employed in studies on chemical carcinogenesis. A comparison was made of the specific activity (pmol/mg DNA) of benzo(a)pyrene metabolite-DNA adducts in DNA isolated by the two procedures from the lung, liver, and forestomach of A/HeJ mice given oral benzo(a)pyrene. Total DNA-associated radioactivity per milligram DNA was higher with the precipitation procedure than with the hydroxyapatite method. This was partly due to large amounts of early eluting peaks in the high-pressure liquid chromatography analyses of digests of DNA isolated by the precipitation procedure. However, the specific activities obtained for binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide isomers to DNA isolated by the two methods were in close agreement. The hydroxyapatite procedure resulted in the isolation of purer DNA and required less time and sample handling than the precipitation method. For isolation of DNA from whole tissue for carcinogen-DNA adduct analysis, these properties make the hydroxyapatite procedure superior to the precipitation method. Our findings have important implications when comparing the results of DNA-binding studies which employ different DNA isolation procedures.