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

Abstract

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.

Bypass of Mutagenic O6-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

“…The highest adduct levels were measured in the sigmoid colon and rectum. [11][12][13] Moreover, levels of O 6 -CMG from exfoliated colon cells have been shown to increase with increased meat consumption. 12-14 O 6 alkylguanine (O 6 -alkylG) adducts, especially O 6 -MeG, are primarily repaired by O 6 -methylguanine DNA methyltransferase (MGMT) via direct damage reversal.…”

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

“…The analysis of O 6 CMdG in blood was also investigated in volunteers that had consumed high levels of processed (nitrite-preserved red) meat compared to a vegetarian diet in a small scale pilot study. Previous research had identified the presence of the O 6 CMdG adduct in volunteers consuming a high red meat diet (20) and established that processed meat leads to higher levels of DNA damage than red meat (23). Thus, the present study sought to identify differences in O 6 CMdG adducts between a diet high in processed meat compared to a vegetarian diet.…”

“…The ISB has since been used to detect the M 1 dG adducts in mice livers (17) and human colorectal mucosa (18) and more recently we applied this methodology to the measurement of O 6 -carboxymethyl-deoxyguanosine adducts (O 6 CMdG, Fig. 1), using a polyclonal antibody (19), derived from nitrosated amines, for in vitro studies and in human blood samples of volunteers consuming a high red meat diet (20). The latter was confirmed by alternative qualitative methods of measuring O 6 CMdG (21).…”

Synthesis of deuterium and C‐13‐labelled ethyl glycolate and their subsequent use in the synthesis of labelled analogues of the DNA adduct O6‐carboxymethyl‐2′‐deoxyguanosine

“…Glycine ethyl ester.HCl (0.99 g, 7.09 mmol) was dissolved in water (1.8 cm 3 ) and dichloromethane (4 cm 3 ), stirred and cooled to -10° C. Aq. sodium nitrite (4.43 M, 1.8 cm 3 ) was added to the cooled solution, then 5% H 2 SO 4 (0.65 cm 3 ) was added dropwise and the solution stirred for 10 mins.…”

“…One such adduct is O 6 -carboxymethyl-2'-deoxyguanosine (O 6 CMdG) that has been linked to nitrosated amines from red meat diets [2] . Previous research has examined this adduct using an immunoslot blot assay which is very sensitive but only a single adduct can be analysed at a time and difficulties can occur due to cross-reactivity of the antibodies [3,4] . A limited supply of a polyclonal antibody does exist for O 6 CMdG but attempts to produce a monoclonal antibody have been unsuccessful and a different technique will therefore be needed in the future.…”