David E. G. Shuker

Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization.

The assessment of human cancer risk from chemical exposure requires the integration of diverse types of data. Such data involve effects at the cell and tissue levels. This report focuses on the specific utility of one type of data, namely DNA adducts. Emphasis is placed on the appreciation that such DNA adduct data cannot be used in isolation in the risk assessment process but must be used in an integrated fashion with other information. As emerging technologies provide even more sensitive quantitative measurements of DNA adducts, integration that establishes links between DNA adducts and accepted outcome measures becomes critical for risk assessment. The present report proposes an organizational approach for the assessment of DNA adduct data (e.g., type of adduct, frequency, persistence, type of repair process) in concert with other relevant data, such as dosimetry, toxicity, mutagenicity, genotoxicity, and tumor incidence, to inform characterization of the mode of action. DNA adducts are considered biomarkers of exposure, whereas gene mutations and chromosomal alterations are often biomarkers of early biological effects and also can be bioindicators of the carcinogenic process.

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 μg to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled (14C- and 3H-) binding, 32P-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 ∼1 adduct in 104 to 1012 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.

The contribution of epidemiology.

Epidemiologic studies directly contribute data on risk (or benefit) in humans as the investigated species, and in the full food intake range normally encountered by humans. This paper starts with introducing the epidemiologic approach, followed by a discussion of perceived differences between toxicological and epidemiologic risk assessment. Areas of contribution of epidemiology to the risk assessment process are identified, and ideas for tailoring epidemiologic studies to the risk assessment procedures are suggested, dealing with data collection, analyses and reporting of both existing and new epidemiologic studies. The dietary habits and subsequent disease occurrence of over three million people are currently under observation worldwide in cohort studies, offering great potential for use in risk assessment. The use of biomarkers and data on genetic susceptibility are discussed. The paper describes a scheme to classify epidemiologic studies for use in risk assessment, and deals with combining evidence from multiple studies. Using a matrix approach, the potential contribution to each of the steps in the risk assessment process is evaluated for categories of food substances. The contribution to risk assessment of specific food substances depends on the quality of the exposure information. Strengths and weaknesses are summarized. It is concluded that epidemiology can contribute significantly to hazard identification, hazard characterisation and exposure assessment.

Determination of N-7-[2H3]methyl guanine in rat urine by gas chromatography-mass spectrometry following administration of trideuteromethylating agents or precursors

A gas chromatographic-mass spectrometric method has been developed for the determination of N-7-[2H3]methyl guanine in urine in the presence of large natural levels of N-7-methyl guanine. Urine is fractionated on heptanesulfonic acid-treated C-18 Sep-pak cartridges, followed by derivatization to give a volatile N-heptafluorobutyryl-O6-2,3,4,5, 6-pentafluorobenzyl derivative which is separated on an SE52 fused silica capillary column. Using N-7-ethyl guanine as an internal standard, the total amount of N-7-methyl guanine is determined by gas chromatography-flame ionization detection. The percentage of N-7-[2H3]methyl guanine is then measured by gas chromatography-mass spectrometry, enabling the amount of deuterated base to be determined. Preliminary experiments with [2H3]methyl methanesulfonate in rats showed measurable excretion of N-7-[2H3]methyl guanine. 4-(Di[2H3]methylamino)antipyrine alone gave no detectable amount of alkylated base, but coadministration of nitrite resulted in excretion of deuterated N-7-methyl guanine.

Lack of correlation between the observed stability and pharmacological properties of S-nitroso derivatives of glutathione and cysteine-related peptides.

S-Nitrosothiols (RSNOs) have been widely studied as donors of nitric oxide. In general, RSNOs are considered to be somewhat unstable; however, they are both potent vasodilators and inhibitors of platelet aggregation. In order to improve our understanding of the factors that determine the biological activity of RSNOs, the chemical stability and pharmacological activity of a series of RSNOs was determined. Results show that millimolar solutions of S-nitrosocysteine (SNOCys) and S-nitroso-L-cysteinylglycine (SNOCysGly) were the least stable, whereas S-nitroso-3-mercaptopropionic acid (SNOPROPA) and S-nitroso-N-acetyl-L-cysteine (SNONAC) were the most stable of the compounds tested. Recent evidence suggests that RSNOs, such as SNONAC, are as unstable as SNOCys at micromolar concentrations. The decomposition of certain RSNOs is catalysed by trace amounts of copper (II) ions, with this phenomenon being particularly evident for SNOCys and SNOCysGly. The decomposition of the more stable RSNOs, including S-nitroso-L-glutathione (SNOGSH) and L-gamma-glutamyl-L-cysteine (SNOGluCys), were not as sensitive to copper ions. The decomposition of the stable RSNO, SNOGSH, was more rapid in the presence of excess thiol, whereas the decay of the unstable RSNO, SNOCys, was reduced with added thiol. All RSNOs tested inhibited platelet aggregation, relaxed vascular smooth muscle, and inhibited cell growth in the nanomolar range, but their order of potency did not correlate with their chemical stability of millimolar solutions. It is apparent that the potency of an RSNO in a physiological situation will depend on the concentration of the compound present, the presence of trace metal ions such as copper, and the occurrence of transnitrosation reactions.

Characterization of Azo Coupling Adducts of Benzenediazonium Ions with Aromatic Amino Acids in Peptides and Proteins

A synthetic peptide, VLSPADKTNWGHEYRMF(cmC)QIG, was reacted with 4-chlorobenzenediazonium hexafluorophosphate as a model for reactions of aromatic diazonium ions with proteins. At a ratio of diazonium ion to peptide of 0.8:1, three products could be seen by reversed-phase HPLC. Electrospray mass spectrometric analysis of the isolated products revealed that two of the products had the same mass of 2648 Da, being 138 Da higher than the parent peptide and corresponding to the addition of a 4-chlorobenzenediazo group. The third isolated product had a mass of 2787 Da which corresponded to the addition of two 4-chlorobenzenediazo groups (276 Da). Digestion of the monoadducted intact peptides with trypsin or endoproteinase Glu-C and HPLC separation of adduct oligopeptides followed by sequencing with electrospray ionization tandem mass spectrometry showed unambiguously that histidine and tyrosine residues were the major sites of modification. Incubation of human serum albumin with 4-chlorobenzenediazonium hexafluorophosphate at molar ratios of 1:1, 1:2, and 1:10 resulted in adduct formation as detected by shifts in the HPLC retention time of the protein and also by an increase in mass as determined by electrospray mass spectrometry.

Quantification of Radiation-Induced Single-Strand Breaks in Plasmid DNA using a TUNEL/ELISA-Based Assay

Abstract 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.

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).

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-β-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.

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.