Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach.

Immunoslot blot assays have been used for the analysis of many DNA adducts, but problems are frequently encountered in achieving reproducible results. Each step of the assay was examined systematically, and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimization was performed on both the malondialdehyde-deoxyguanosine (M(1)dG) adduct and the O(6)-carboxymethyl-deoxyguanosine (O(6)CMdG) adduct to demonstrate the applicability to other DNA adducts. Blood samples from the European Prospective Investigation on Cancer (EPIC) study (n = 162) were analyzed for M(1)dG adducts, and the data showed no correlation with adduct levels in other tissues, indicating that the EPIC blood samples were not useful for studying M(1)dG adducts. Blood samples from a processed meat versus vegetarian diet intervention (n = 6) were analyzed for O(6)CMdG, and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 10(7) nucleotides for the analysis of O(6)CMdG. Subsequent reanalysis for O(6)CMdG showed a weakly significant increase in O(6)CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted.

Quantification of radiation-induced single-strand breaks in plasmid DNA using a TUNEL/ELISA-based assay.

To accurately quantify the number of single-strand breaks (SSBs) induced in plasmid DNA molecules after irradiation, a new type of assay methodology has been explored. The new method is based on the TUNEL (terminal deoxynucleotide transferase dUTP nick end-labeling) assay that was adopted for use under ELISA (enzyme-linked immunosorbent assay) conditions. The assay was found to both improve the quantification and reduce the uncertainties in measurement of SSBs compared with the commonly used agarose gel electrophoresis (AGE) method. Together with AGE, the new method can provide the additional data necessary for an accurate analysis of both SSB and double-strand break (DSB) formation in DNA molecules after irradiation. Furthermore, since only small amounts of DNA are required, the ELISA method can be used to quantify the damage in samples of DNA that are smaller than those required for AGE analysis. As an example of the data obtainable using the new method, plasmid DNA samples were irradiated with vacuum-ultraviolet (VUV) light in an aqueous solution at 170 nm and subsequently analyzed by ELISA. The results were compared directly with those from AGE analysis. The ELISA gave results for SSBs that were an order of magnitude higher than those from AGE and suggested that DSBs are more likely to be the result of two SSBs rather than a single event and that a damaged molecule is more likely to be susceptible to VUV light than an undamaged one.

Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage.

The formation of deoxyribonucleic acid (DNA) adducts can have important and adverse consequences for cellular and whole organism function. Available methods for identification of DNA damage and quantification of adducts are reviewed. Analyses can be performed on various samples including tissues, isolated cells, and intact or hydrolyzed (digested) DNA from a variety of biological samples of interest for monitoring in humans. Sensitivity and specificity are considered key factors for selecting the type of method for assessing DNA perturbation. The amount of DNA needed for analysis is dependent upon the method and ranges widely, from <1 microg to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled ((14)C- and (3)H-) binding, (32)P-postlabeling, and methods dependent on gas chromatography (GC) or high-performance liquid chromatography (HPLC) with detection by electron capture, electrochemical detection, single or tandem mass spectrometry, or accelerator mass spectrometry. Sensitivity is ranked, and ranges from approximately 1 adduct in 10(4) to 10(12) nucleotides. A brief overview of oxidatively generated DNA damage is also presented. Assay limitations are discussed along with issues that may have impact on the reliability of results, such as sample collection, processing, and storage. Although certain methodologies are mature, improving technology will continue to enhance the specificity and sensitivity of adduct analysis. Because limited guidance and recommendations exist for adduct analysis, this effort supports the HESI Committee goal of developing a framework for use of DNA adduct data in risk assessment.

Unambiguous structural elucidation of base-modified purine nucleosides using NMR.

A general and unambiguous approach has been developed for structural elucidation of modified purine nucleosides using NMR spectroscopy. Systematic assignment of proton and carbon signals of modified nucleosides was firmly established by COSY and the anomerism of the glycosidic linkage of synthetic nucleosides clearly elucidated by NOESY experiments. Characteristic properties of 15N-isotopic labelling at specific positions of nucleosides were also employed for structural studies. The reported approach is applicable to other modified nucleosides and nucleotides, as well as nucleobases.

Potassium diazoacetate-induced p53 mutations in vitro in relation to formation of O6-carboxymethyl- and O6-methyl-2'-deoxyguanosine DNA adducts: relevance for gastrointestinal cancer.

Nitrosated glycine derivatives react with DNA to form O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG) and O6-methyl-2'-deoxyguanosine (O6-MedG) adducts concurrently. O6-CMdG is not repaired by O6-alkylguanine alkyltransferases and might be expected to lead to mutations via a similar mechanism to O6-MedG. Potassium diazoacetate (KDA) is a stable form of nitrosated glycine and its ability to induce mutations in the p53 gene in a functional yeast assay was studied. Treatment of a plasmid containing the human p53 cDNA sequence with KDA afforded readily detectable levels of O6-CMdG and O6-MedG. The treated plasmid was used to transform yeast cells and coloured colonies harbouring a p53 sequence with functional mutations were detected. Recovery of the mutated plasmids followed by DNA sequencing enabled the mutation spectrum of KDA to be characterised. The most common mutations induced by KDA were substitutions with >50% occurring at GC base pairs. In contrast to the methylating agent methylnitrosourea which gives predominantly (>80%) GC-->AT transitions, KDA produced almost equal amounts of transitions (GC-->AT) and transversions (GC-->TA and AT-->TA). This difference is probably due to a different mode of base mispairing for O6-CMdG compared with O6-MedG. The pattern of mutations induced by KDA was very similar to the patterns observed in mutated p53 in human gastrointestinal tract tumours. These results are consistent with the hypothesis that nitrosation of glycine (or glycine derivatives) may contribute to characteristic human p53 mutation profiles. This conclusion is borne out by recent observations that O6-CMdG is present in human DNA both from blood and exfoliated colorectal cells and is consistent with recent epidemiological studies that have concluded that endogenous nitrosation arising from red meat consumption is related to an increased risk of colorectal cancer.

Red meat enhances the colonic formation of the DNA adduct O6-carboxymethyl guanine: implications for colorectal cancer risk.

Red meat is associated with increased risk of colorectal cancer and increases the endogenous formation of N-nitrosocompounds (NOC). To investigate the genotoxic effects of NOC arising from red meat consumption, human volunteers were fed high (420 g) red meat, vegetarian, and high red meat, high-fiber diets for 15 days in a randomized crossover design while living in a volunteer suite, where food was carefully controlled and all specimens were collected. In 21 volunteers, there was a consistent and significant (P < 0.0001) increase in endogenous formation of NOC with the red meat diet compared with the vegetarian diet as measured by apparent total NOC (ATNC) in feces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O(6)-carboxymethyl guanine (O(6)CMG) was significantly (P < 0.001) higher on the high red meat diet. In 13 volunteers, levels were intermediate on the high-fiber, high red meat diet. Fecal ATNC were positively correlated with the percentage of cells staining positive for O(6)CMG (r(2) = 0.56, P = 0.011). The presence of O(6)CMG was also shown in intact small intestine from rats treated with the N-nitrosopeptide N-acetyl-N'-prolyl-N'-nitrosoglycine and in HT-29 cells treated with diazoacetate. This study has shown that fecal NOC arising from red meat include direct acting diazopeptides or N-nitrosopeptides able to form alkylating DNA adducts in the colon. As these O(6)CMG adducts are not repaired, and if other related adducts are formed and not repaired, this may explain the association of red meat with colorectal cancer.

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies.

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2'-deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2'-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2'-deoxyinosine 9 was prepared from 2'-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-15N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-beta-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.

Detection of O6-carboxymethyl-2'-deoxyguanosine in DNA following reaction of nitric oxide with glycine and in human blood DNA using a quantitative immunoslot blot assay.

Previous research has shown that a range of nitrosated glycine derivatives react with DNA to form O6-carboxymethylguanine and O6-methylguanine DNA adducts [Harrison et al. (1999) Chem. Res. Toxicol. 12, 106-111). Nitrosated glycine derivatives may be formed in the gastrointestinal tract from the reaction of dietary glycine with nitrosating agents. The aim of this study was to further investigate the role of dietary glycine in the formation of O6-guanine adducts at physiologically relevant concentrations. In vitro studies were performed by reacting 10 microM to 50 mM glycine with nitric oxide in the presence of oxygen. An HPLC assay was developed to measure the resulting nitrosated glycine derivative, diazoacetate anion. The amount of nitrosating agent present in the reaction mixture was determined by colorimetric measurement of nitrite, the hydrolysis product of N2O3. Diazoacetate anion formation depended linearly on glycine concentration. Solutions of nitrosated glycine reacted with 2'-deoxyguanosine and calf thymus DNA to give O6-carboxymethyl-2'-deoxyguanosine and, at high concentrations of glycine and nitric oxide, O6-methyl-2'-deoxyguanosine. At physiological concentrations of glycine and nitric oxide, diazoacetate anion was not detectable. Studies with synthetic diazoacetate anion showed that concentrations < 14 microM did not give detectable O6-carboxyethylguanine in DNA, even when a sensitive immunoslot blot assay was used. However, O6-carboxymethylguanine was detected in human blood DNA samples obtained from three volunteers consuming a standardized high meat diet, using the immunoslot blot assay. O6-Carboxymethylguanine levels ranged from 35 to 80 (detection limit = 15) O6-carboxymethylguanine per 10(8) bases. These studies provide further evidence that nitrosated amino acids may be risk factors for gastrointestinal tract cancers.

The enemy at the gates? DNA adducts as biomarkers of exposure to exogenous and endogenous genotoxic agents.

Genomic DNA is under continuous assault by various chemical species produced by normal cellular metabolism. In addition, exposure to exogenous agents adds further insult. Modification of DNA by chemical carcinogens has long been recognized as an early event in carcinogenesis and many DNA adducts have been characterized. There appears to be great value in using DNA adducts as markers of exposure to genotoxic (i.e. DNA-damaging) agents and some may be even more useful as indicators of risk of disease. Studies of the relationship between aflatoxin exposure and liver cancer have illustrated particularly well the advantages of using specific DNA adducts and other biomarkers, not only to better characteristic the risk factors, but also as endpoints in intervention studies. DNA adducts of endogenous genotoxins such as malondialdehyde and nitrosated glycine are particularly informative in studies of the effects of diet on cancer risk. DNA adducts may also be useful in identifying no-exposure levels in risk assessment of low-level environmental exposures such as 1,3-butadiene (BD).

Detection of malondialdehyde DNA adducts in human colorectal mucosa: relationship with diet and the presence of adenomas.

Colorectal biopsies from normal mucosa of participants in the United Kingdom Flexible Sigmoidoscopy Trial and European Prospective Investigation on Cancer (EPIC; n = 162) were analyzed for the presence of malondialdehyde-deoxyguanosine (M(1)-dG), a DNA adduct derived from lipid peroxidation. The aim was to investigate whether dietary factors can modulate M(1)-dG levels and whether M(1)-dG in normal mucosa is a risk factor for colorectal adenomas. Samples were analyzed using a sensitive immunoblot blot assay. This study has shown for the first time that M(1)-dG is present in human colorectal tissue. M(1)-dG levels ranged from undetectable (n = 13) to 12.23 per 10(7) total bases. Mean levels were 4.3 +/- 3 and 4.6 +/- 2.9 per 10(7) total bases in men and women, respectively. In men, there were positive associations of adduct levels with height and age, and inverse associations with body mass index. Legumes, fruit, salad, and whole meal bread were inversely associated with M(1)-dG adducts, whereas consumption of offal, white meat, beer, and alcohol were positively associated with elevated levels. In women, there was an inverse association of the adduct with the ratio of polyunsaturated:saturated fatty acids (P = 0.019) and a weak positive correlation with saturated fat (P < 0.061). When levels of adducts were compared in individuals with and without adenomas, there was a trend for higher levels in individuals presenting with adenomas especially in the highest category of M(1)-dG adducts (P < 0.005).