The adduct O6‐carboxymethyl‐2′‐deoxyguanosine (O6CMdG) is of importance as it has been previously linked to high red meat diet in humans, and as yet, a liquid chromatography‐mass spectrometry (LC‐MS) method has not been developed due to lack of appropriate standards. The synthesis of the deuterated and C‐13 analogues required the use of [2H2]‐ and [13C2]ethyl glycolate to label the carboxymethyl moiety of O6CMdG. [2H2]Ethyl glycolate was synthesised via acid hydrolysis of ethyl diazoacetate using deuterated solvents (59% yield), whilst [13C2]ethyl glycolate was synthesised from [13C2]glycine in a three‐step procedure (35% yield). The labelled ethyl glycolates were then used to synthesise [2H2]‐ and [13C2]O6CMdG for future use as internal standards in the LC‐MS analysis of biological samples. Copyright © 2011 John Wiley & Sons, Ltd.

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 (M1dG) adduct and the O6-carboxymethyl–deoxyguanosine (O6CMdG) 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 M1dG adducts, and the data showed no correlation with adduct levels in other tissues, indicating that the EPIC blood samples were not useful for studying M1dG adducts. Blood samples from a processed meat versus vegetarian diet intervention (n=6) were analyzed for O6CMdG, 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 107 nucleotides for the analysis of O6CMdG. Subsequent reanalysis for O6CMdG showed a weakly significant increase in O6CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted.

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.

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 mu g to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled (C-14- and H-3-) binding, P-32-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 similar to 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.

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.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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 15 N‐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. Copyright © 2007 John Wiley & Sons, Ltd.

AIMS: GHB is produced naturally in the human body and is also a Class C controlled substance under the Misuse of Drugs Act 1971. It is notorious because of its association with drug facilitated sexual assaults (DFSA). Studies have indicated that large variations in urinary and blood concentrations of endogenous GHB occur across population groups (1). At present the recognised cut off values of 10 µg/mL in urine and 5 µg/mL in blood may only provide reliable evidence if the sample was taken <12 hours after ingestion because GHB is rapidly metabolised and excreted from the body (2). A recent study, using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), found a significant difference (> 13.5%) in the values of ∂13C found in endogenous GHB in five postmortem blood samples (range: 13.8 - 86.3 µg/mL) compared to synthetically produced GHB with the implication that stable isotope measurements could significantly increase time frames of detection in reported DFSA (3). The aim of this study is to determine the ∂13C values of GHB at concentrations below the recognised cut off values in urine samples. METHODS: GHB can be derivatised or converted to gamma-butyrolactone (GBL) for GC analysis. We have derivatised GHB using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) (linear response: 0.05 - 25 µg/mL), and converted GHB to GBL (linear response: 0.5 - 50 µg/mL) based upon on a method developed ‘in-house’ by the Forensic Science Service Ltd. RESULTS AND CONCLUSIONS: Preliminary ∂13C (%) values for synthetic GBL at 50 µg/mL (mean -26.5%; σn-1 0.06; n = 3) and GHB-TMS derivatives at 200 µg/mL (mean -34.2%; σn-1 0.21; n = 3) have been obtained using a Thermo Finnigan MAT 253 IR-MS coupled to a Trace Ultra GC/Combustion III Interface. However, we found that extraction of GHB from urine using solid phase extraction (CLEAN SCREEN® GHB and Oasis® MAX cartridges) did not remove interfering compounds sufficiently to be able to precisely determine ∂13C values at levels less than 10 µg/mL. Therefore, we propose synthesising an immunising antigen to make an antibody to specifically target GHB using immunoaffinity column extraction. (1) A.A. Elian (2002).Forensic Science International 128, 120-122. (2) P.V. Kavanagh, P. Kenny and J. Feely (2001). Journal of Pharmacy and Pharmacology 53, 399-402. (3) C. Saudan, M. Augsburger, P. Kintz, M. Saugy, and P. Mangin (2005). Journal of Analytical Toxicology 29, 777-781.

Rats were fed from weaning on 1 of 3 diets. Those fed a cafeteria diet had livers that were enlarged and abnormal by visual inspection. The rats themselves appeared healthy, had a normal growth rate, and were not significantly different in weight from control animals. Histologic examination revealed the livers of these rats to be rich in lipids and glycogen. Liver function tests showed a depressed level of alanine transaminase and an abnormal high-density lipoprotein/low-density lipoprotein. Dietary lipids generate free radicals that can interact with, and damage, DNA. However, when DNA was extracted from the livers and examined for the presence of the adduct M1-dG, there were no significant differences in adduct levels in livers from animals fed any of the diets. We conclude that the cafeteria diet can have long-term adverse effects on liver function even though overt measures of health may be unimpaired, body mass is maintained within normal limits, and liver DNA is not adversely affected.

5234 Intrroduction: O6-Carboxymethylguanine (O6-CMG) is a modified DNA base derived from nitrosation of glycine (Cupid et al, ChemRes Toxicol 2004, 17, 294-300). As O6-Methylguanine (O6-MeG) is inevitably formed concurrently we have inserted synthesized O6-CMG into defined sites of the p53 cDNA sequence using nucleic acid chemistry and used the modified seqeunces to explore the consequences of mispairing. Methods: O6-CMG was synthesised as a suitably pro\. tected 2’-deoxynucleotide and incorporated into 25- or 26-mers which were then inserted into defined sites on the coding and non-coding strands of the human p53 cDNA sequence iwhich was then ligated into a double-stranded circular plasmid. The p53 seqeunce was amplified using PCR. Single-nucleotide changes that occured in the adduct-containing codons were detected using ARMS-PCR. Unmodified- and O6-MeG-containing sequences were used as negative and positive controls. Results: PCR amplification and the ARMS-PCR assay did not introduce any detectable artefactual mutations in the negative control. O6-MeG resulted in the expected G to T mutations. However, O6-CMG gave not only G to T mutations but also G to A. Conclusions: The pattern of mutations induced by O6-CMG in this system were identical to those produced in a yeast-based functional assay which was in turn similar to the pattern of mutations in p53 from human GI tract cancers. O6-CMG produces a pattern of mutations which is quite distinct from the well-studied O6-MeG. These results underline the possible role of glycine nitrosation in the aetiology of human GI tract cancers. Current studies are aimed at establishing a precursor-product relationship between dietary glycine and O6-CMG in human DNA using stable isotope methodology. We gratefully acknowledge support from the World Cancer Research Fund

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.

This report covers some highlights in chemical genetics published during 2006. Significant advances have been made in the development of diversity-oriented synthesis to make new molecules with natural product-like skeletal diversity. The use of small molecules to interact with specific pathways in order to understand biological mechanisms and to identify new drug targets is also a main focus of this report.

1971 Aim: Westernisation and the consumption of a high fat/high red meat diet, have been associated with an increased risk for colon cancer. Malondialdehyde is the mutagenic and carcinogenic end product of lipid peroxidation associated with polyunsaturated fat intake capable of reacting with DNA to form adducts such as the malondialdehyde-deoxyguanosine adduct M1dG. High red meat diets have been associated with colorectal cancer risk due to the increased formation of alkylating agents such as N-nitroso compounds (NOC’s), some of which are known to be potent carcinogens. The aim of this study was to investigate the reliability of exfoliated cells as a source of biomarkers of diet related DNA damage within the colon. Methods: Exfoliated colonocytes from stools from individuals consuming a normal Western diet were isolated using a non-invasive immunomagnetic bead technique (Bandaletova et al., 2002). These exfoliated colonocytes were then stained using the avidin-biotin method for the presence of the adducts M1dG and 06-carboxymethylguanine (O6-CoMedG). The colon carcinoma cell line HT-29 exposed to 1,1,3,3 tetraethoxypropane to induce the M1dG adduct, and potassium diazoacetate to induce the O6-CoMedG adduct in vitro was used as a positive control. Results: HT-29 cells were shown to stain for M1dG and O6-CoMedG adducts after treatment with 20mM 1,1,3,3-tetraethoxypropane and 1mM potassium diazoacetate respectively. Exfoliated cells were consistently extracted from faecal samples and were shown to stain positively for M1dG and O6-CoMedG adducts in stool samples from individuals consuming a normal Western diet. Conclusion: In this study, we have shown that exfoliated colonocytes can be reliably extracted from stool and that the M1dG and O6-CoMedG adducts can be visually detected in exfoliated colonocytes from humans consuming a normal Western diet. HT-29 cells treated with the adduct inducing compounds 1,1,3,3 tetraethoxypropane and potassium diazoacetate provided reliable positive controls. The presence of the M1dG and O6-CoMedG adducts in exfoliated colonocytes are indicators of DNA damage as a result of diet related genotoxic compounds. Bandaletova,T, Bailey N, Bingham S, Loktionov A. (2002). Isolation of exfoliated colonocytes from the human stool as a new technique for colonic cytology. APIMS 110 239-46.

We note with some concern the content of articles in the International Journal of Occupational and Environmental Health,1Tomatis L The IARC monographs programme: changing attitudes towards public health.Int J Occup Environ Health. 2002; 8: 144-152Crossref PubMed Scopus (49) Google Scholar, 2Huff J IARC monographs, industry influence, and upgrading, downgrading, and under-grading chemicals.Int J Occup Environ Health. 2002; 8: 149-270Crossref Scopus (51) Google Scholar and Commentaries in The Lancet Oncology3Burton A Is industry influencing IARC to downgrade chemicals?.Lancet Oncol. 2003; 4: 4Summary Full Text Full Text PDF PubMed Scopus (6) Google Scholar and The Lancet.4EditorialTransparency at IARC.Lancet. 2003; 361: 189Summary Full Text Full Text PDF PubMed Scopus (8) Google Scholar We are aware of the specific criticisms of both a perceived change in focus of the International Agency for Research on Cancer (IARC) monographs programme with respect to public health and the apparent absence of independence of the membership of working groups. These criticisms are not valid. With respect to the commitment to public health, we consider that the IARC monographs have assigned high priority to assessment of exposures that are widespread, such as those entailing biological agents and lifestyle factors. Understanding of the relevant hazards in these complex areas has the potential to contribute to reduced cancer risk for many of the world's population. Working groups for such assessments represent a wide range of expertise. However, in its traditional heartland of assessing occupational and environmental agents, the IARC has sometimes encountered difficulties in finding members with sufficient expertise who do not have real or perceived interests in the agents being assessed. We believe that measures the IARC has in place to ensure that members with inappropriate interests do not exert undue influence on evaluations are effective. It is noteworthy that every published IARC monograph contains all evidence that is deemed pertinent to make an informed assessment of carcinogenic hazard, including mechanistic data that increasingly contribute to evaluations that lie above or below the default criteria. We are convinced that the IARC endeavours to bring together working groups with the best expertise available, and that these groups strive to make assessment on the best scientific evidence that is openly available. On behalf of the many scientists who serve, or who have served, as members of IARC monograph working groups, we unreservedly repudiate the implied slur on the integrity and ability of all concerned. This correspondence letter is a result of a statement unanimously adopted by members of the sixth advisory group of the IARC monographs programme (Lyon, France; Feb 11–14, 2003). Our action is taken in the absence of any wish, comment, or otherwise by the director, IARC, or IARC staff. BWS is chair, HG is vice-chair, and DS and TK are subgroup chairs of the advisory group of the IARC monographs programme.