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Effect of administration of the carcinogen DMN on urinary 7-MeG

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Abstract

1. Evidence is presented for the excretion of 7-methylguanine in normal rat urine at a rate of approx. 65mug./day. Experiments with animals in which the nucleic acids had been prelabelled by treatment of the neonatal rats with [(14)C]-formate gave evidence that the methylated base originated in the nucleic acids of the rat. 2. Injection of [(14)C]dimethylnitrosamine leads to an increased excretion of 7-methylguanine, and the base becomes labelled in the methyl group. The disappearance of labelled 7-methylguanine formed in nucleic acids of rats treated with the carcinogen therefore does not take place by an N-demethylation reaction, but by liberation of the intact methylated base.

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... Previous animal studies on urinary N7-alkylating guanine demonstrated that its level correlated with the degree of DNA alkylation in target organs. 33 Most of the urinary N7methylguanine was found in the liver when rats were treated with 3 H-labeled dimethylnitrosamine. 44 The excretion of N7methylguanine has also been shown to correlate well with liver DNA methylation. 45 A similar study also showed that the urinary excretion of N7-aflatoxin B-guanine adducts correlated well with the levels of the adducts in the liver. ...
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... The fractions containing e7Gua from a Sephadex G-10 chromatography were pooled and lyophilized ; the residue was taken up in 2 ml water brought to pH 2.5 with H,S04 and the ethylated purine precipitated with silver nitrate as described by Craddock and Magee [14]. The silver salt was dissolved in 1 M HC1, the AgCl discarded by centrifugation and the supernatant rechromatographed on Sephadex G-10 (with NaN, in the elution buffer). ...
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Noninvasive methodologies for measuring carcinogen exposure in humans, based on the use of urinary markers, are being developed and validated for use in molecular epidemiological studies. A range of 3-alkyladenines can be determined in urine samples by an immunoaffinity purification-GC/MS approach [3-methyladenine, 3-ethyladenine, 3-(2-hydroxyethyl)adenine, and 3-benzyladenine]. Using this method, recent results in human subjects suggest that urinary 3-alkyladenines are potentially useful markers of alkylating agent exposure, particularly where the backgrounds of such adducts are much lower than 3-methyladenine. Urinary excretion of S-benzylmercapturic acid has been studied in experimental animals as a marker of exposure to benzylating agents such as N-nitroso-methylbenzylamine. 3-Nitrotyrosine (NTyr) is formed in vivo in tissue or blood proteins after exposure to nitrosating and/or nitrating agents such as tetranitromethane. After turnover of proteins, NTyr is released and excreted in urine as metabolites 3-nitro-4-hydroxy-phenylacetic acid and 3-nitro-4-hydroxyphenylacetic acid, which are determined by GC with a thermal energy analyzer. The sensitivity and specificity, combined with ease of use, of these noninvasive biomonitoring approaches means that they may be readily incorporated into molecular epidemiological studies in which exposure to nitrosating and alkylating agents may be important risk factors.
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Chapter
The mechanism of action of many carcinogens is considered to involve the formation of electrophilic alkylating agents which react with nucleophilic sites in DNA giving rise to a range of adducts (Miller, 1978). All of the nitrogen and oxygen atoms in purines and pyrimidines are known to form adducts with various carcinogens, although the relative amounts of each adduct vary greatly depending on the reactivity of the alkylating species (Singer and Kusmierek, 1982). O6-alkylguanine adducts are promutagenic lesions which can be repaired by alkyltransferases which remove the alkyl group. In contrast, N7- and N3-purine adducts are repaired by glycosylases which excise the alkylated base (Karran and Lindahl, 1985). Excised alkylpurines are known to be resistant to catabolism and are excreted in urine. These observations are the basis of methods for the non-invasive measurement of human exposure to alkylating carcinogens using excreted DNA adducts (Shuker, 1989). This approach has been particularly successful in studies on human exposure to aflatoxin B1 (Groopman et al., 1991).
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Chapter
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The urinary 7-methyl guanine excretion of Sprague-Dawley rats was determined up to 21 days after a single, relatively high dose of the carcinogens dimethyl nitrosamine, diethyl nitrosamine N-nitroso morpholine (intraperitoneal injection), trans-dimethylamino stilbene and 9,10-dimethyl-benzanthracene (oral application). This was achieved by use of column chromatography on Dowex IX 8 as well as isotope dilution and optical density measurement (the mean deviation was 5,7%). The normal amount excreted by male rats (weight 230–250 g) was 210–255 ? per day. A perceptible increase in 7-methyl guanine excretion was found only after application of dimethyl nitrosamine and then only in the case of those animals which died three days after injection. All of the other carcinogens investigated caused an immediate decrease which lasted 6–9 days, the minimum occuring between the first and fifth day.
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A metabonomic approach based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC/MS) was used to study the nephrotoxicity of rhizoma alismatis (RA) in rats. Potential biomarkers of RA toxicity were identified and the toxicological mechanism is discussed. Urine samples were collected from control and treated rats at various stages and analyzed by UPLC/MS in positive ionization mode. Histopathological analysis was used to evaluate renal function. The differences in the metabolic profiles of the control and treated rats were clearly distinguishable with principal components analysis (PCA) of the chromatographic data, and significant changes in 13 metabolite biomarkers were detected in the urine. This metabonomic method combined with PCA could discriminate the treated rats from the control rats on days 60, 120, and 180 after treatment, before serious organic renal damage was apparent on day 180 with histopathology. This research indicates that UPLC/MS-based metabonomic analysis of urine samples can be used to predict the chronic nephrotoxicity induced by rhizoma alismatis. Copyright © 2011 John Wiley & Sons, Ltd.
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Human exposure to carcinogenic alkylating agents can lead to the formation of covalently bound adducts in DNA, some of which are excreted in urine as alkylated purines following DNA degradation and repair. Tandem mass spectrometric methods have been developed for the qualitative and quantitative determination of such alkylpurines in human urine. Short-chain alkyl- and hydroxyalkylguanines have been synthesized with the substituents at the N-7-, O6- and N2-positions of guanine. Examination of the product ion scans of their molecular ions (electron impact (EI) ionization) revealed that the ion at m/z 151, [guanine]+, was common to all of the alkylguanines studied, with the exception of the methylated analogues. Precursor ion scans of this ion on partially purified human urine extracts showed the presence of several ions (e.g. m/z 179, 195) which were consistent with molecular ions for alkylguanines. The presence of these and other constituents was confirmed by product ion spectra of molecular ions (EI and fast atom bombardment), and by high-performance liquid chromatographic separation prior to tandem mass spectrometry (MS/MS). Evidence was obtained for the presence of N-7-methyl-, N2-dimethyl-, N2-dimethyl-, N2-ethyl- and N-7-(2-hydroxyethyl)guanine. Quantitative methods were established for these five alkyl guanines using gas chromatography mass spectrometry (GC/MS) and GC/MS/MS. Deuterated internal standards were synthesized and added to the urine prior to extraction of alkylpurines by Sep-Pak cartridge chromatography. The products were converted into their tert-butyldimethylsilyl derivatives and analysed by selected ion monitoring (SIM) of [M – 57]+ or by multiple reaction monitoring (MRM) of the fragmentation M+˙ → [M – 57]+. The MRM method yielded values for N-7-methylguanine of 2.57 ± S.D. 1.32 mg day−1 (n = 6), N2-methylguanine of 0.31 ± 0.10 mg day−1 (n = 10) and N2-dimethylguanine of 0.21 ± 0.23 mg day−1 (n = 10). N2-Ethyl- and N-7-(2-hydroxyethyl)guanine could only be detected by SIM at levels of ∼0.5 and 2 μg day−1, respectively. The MRM analyses, although inherently less sensitive than the SIM analyses, exhibit greater selectivity and consequently fewer contaminant ions.
Article
The degree of alkylation of proteins was used to determined tissue doses Dt i.e. the concentration of free alkylating agent, integrated over time, in resting male mice exposed for 1–2 h to air containing 1–35 ppm ethylene oxide (EO). The exposure doses were thus 0.03–2% of LD50. The results agree with an absorption of all EO in alveolar ventilation, a rapid distribution to all organs, and a rapid detoxication and excertion (biological half-life about 9 min).Dt is proportional to the exposure dose within the range studied. In most organs, including the testes, the Dt is about 0.5 μM · h per ppm · h of expsoure. The degree of alkylation of DNA agreed with expectation from the doses determined.Expressing genetic risks of environmental chemicals in the frame of reference of radiation hazard will facilitate comparison and summations of risks of various origins. On the basis of dose-effect curves of EO and X-rays in barely, a tissue dose of EO in man of · h may be provisionally set equal to 80 rad of low LET radiation. Allowing for the difference in alveolar ventilation between mouse and man, this would mean that epoxide operators working at 5 ppm EO 40 h/week receive a weeky gonad dose of EO amounting to about 4 “rad-equivalenst”. Various data show that this risk estimate is realistic.
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Rats treated with di(2-chloroethyl)methylamine (HN2), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine 0-24 h after treatment. Ethyl methanesulphonate (EMS) and dimethylnitrosamine (DMN) gave negative results in this respect but all five alkylating agents increased the excretion of 1-methyl-nicotinamide (1-meNmd). In addition, a larger quantity of 7-methylguanine (7MG) and uric acid was excreted after DMN treatment. 1,4-Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5-fluorouracil (5FU), cytosine arabinoside (araC), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz-[a]anthracene (7-BrMBA) gave negative results.
The methylation of transfer RNA (tRNA) and of ribosomal RNA (rRNA) was studied in normal animals and in animals fed diets containing the carcinogens dimethylnitrosamine, aflatoxin or ethionine. The rats were injected with [14C]methionine, and the nucleic acids were isolated, hydrolysed with HClO4 and analysed by column chromatography on Dowex-50.The level of labelling of each methylated base in tRNA was increased by the carcinogenic treatment. The increase probably does not reflect merely an increased rate of synthesis of tRNA, as each methylated base increases to a different extent in precancerous liver, in contrast to the situation in intestine, where the labelling of the different methylated bases is higher than in liver, but each is increased to a similar extent. Also, in precancerous liver, the increase in protein synthesis, judged by the incorporation of [14C]methionine into protein, is in most cases much less than the increased methylation of tRNA, while in intestine the incorporation of [14C]-methionine into protein and the methylation of tRNA exceed the values in liver in equal proportions. There also appears to be a change in composition of the major bases of tRNA during carcinogenesis produced by dimethylnitrosamine and by aflatoxin. These results add evidence to the view that there is a change in the relative abundance of different species of tRNA synthesised during carcinogenesis, the change being towards more highly methylated species.In the case of rRNA there is an increase in labelling of each methylated base, but the increase may correlate with an increased synthesis of rRNA. There is evidence for a change in the kinetics of the reactions leading to the formation of rRNA precursor.
Article
Alkylation of nucleic acids occurs both physiologically within living cells and after the administration of compounds that are either themselves direct chemical alkylating agents or are converted into alkylating agents by metabolic activation. Some of these compounds are highly potent carcinogens. Carcinogenicity of these agents is due to the alkylation of certain cellular components because no other degradation product nor is the compound itself oncogenic. This chapter deals with the formation and metabolism of alkylated purines in nucleic acids. It briefly discusses other alkylation reactions leading to the alkylphosphate triester production and alkylated pyrimidines. It also presents evidences favoring particular critical targets for the action of alkylating carcinogens. The attack on nucleic acids by carcinogenic alkylating agents is not entirely random and generally leads to the formation of alkylated nucleosides at many different sites distributed throughout the cellular nucleic acids. Carcinogenesis is not necessarily mediated through mutagenesis in somatic cells. However, it is observed that carcinogenic action could be mediated through a distinct action of the electrophilic reactant.
Article
The chemical structure, reactivity and metabolic fate of the insecticide dichlorvos (2,2-dichlorovinyl dimethyl phosphate) are discussed in relation to the possible genotoxicity of this and other methyl phosphate triesters. Recent attempts to demonstrate the methylation of DNA following exposure of bacteria and animals to dichlorvos are reviewed. On the basis of comparative data relating mutagenesis to methylation reactions, it seems entirely appropriate to conclude that the mutagenicity of dichlorvos to bacteria is due solely to methylation of the bacterial DNA under the conditions of these tests. However, the methylation of mammalian DNA could not be demonstrated under realistic exposure conditions (when the alkylating mutagen methyl methanesulphonate afforded clearly measurable methylation). The failure to detect methylation by dichlorvos in vivo is attributed to the operation of highly efficient enzyme-catalysed biotransformations which rely largely on the phosphorylating reactivity of dichlorvos. The biotransformation pathways, characterised mostly in the rat, appear to be common also to pig, mouse, hamster, and man.
Article
The covalent binding of chemical carcinogens to DNA of mammalian organs is expressed per unit dose, and a 'Covalent-Binding Index', CBI, is defined. CBI for various carcinogens span over 6 orders of magnitude. A similar range is observed for the carcinogenic potency in long-term bioassays on carcinogenicity. For the assessment of a risk from exposure to a carcinogen, the total DNA dmaage can be estimated if the actual dose is also accounted for. A detailed description is given for planning and performing a DNA-binding assay. A complete literature survey on DNA binding in vivo (83 compounds) is given with a calculation of CBI, where possible, 153 compounds are listed where a covalent binding to any biological macromolecule has been shown in vivo or in vitro. Recent, so far unpublished findings with aflatoxin M1, macromolecule-bound aflatoxin B1, diethylstilbestrol, and 1,2-epithiobutyronitrile are included. A comparison of CBI for rat-liver DNA with hepatocarcinogenic potency reveals a surprisingly good quantitative correlation. Refinements for a DNA-binding assay are proposed. Possibilites and limitations in the use of DNA binding in chemical carcinogenesis are discussed extensively.
Article
The degree of alkylation of haemoglobin was determined at different times after treatment of mice with one directly active alkylating agent, ethylene oxide, and one agent that requires metabolic activation, dimethylnitrosamine. Because of the random alkylation of red blood cells of various ages and the stability of alkylated haemoglobin, the amount of alkylated amino acids in haemoglobin decreases linearly with time, reaching the value zero after about 40 days, the life-span of erythrocytes in the mouse. This provides a basis for the use of haemoglobin as a monitor for integral doses of genotoxic environmental chemicals.
Article
Structural and functional analysis of T cell receptor (TcR)-ligand binding would be greatly advanced by the availability of an intact, assembled TcR in soluble form. We have produced such a molecule, by splicing the extracellular domains of a TcR to the glycosyl phosphatidylinositol membrane anchor sequences of Thy-1. The molecule is expressed in the absence of CD3 on the cell surface, and can be cleaved from the membrane by treatment with phosphatidylinositol-specific phospholipase C. The alpha and beta chains of the soluble molecule are paired in the native conformation as judged by reactivity with the anti-V beta 8 monoclonal antibody F23.1, and with the anti-clonotypic monoclonal antibody 1B2; it is a disulfide-linked dimer with a mol. mass of 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, and 47 kDa after reduction. We conclude that we have generated an alpha/beta TcR in soluble form.
Article
Humans are exposed to many chemical carcinogens (Doll & Peto 1981), including a large number of alkylating agents. Estimates have been made of the relative contributions of various chemicals to the total burden of carcinogens, but these are very uncertain due to the difficulty of quantitatively assessing human exposure. In addition, there are likely to be substantial differences between individuals in metabolism, excretion and other parameters for a given chemical. These problems have led to a search for biomonitoring methods which permit an assessment of individual human exposure to specific carcinogens.
Article
Following i.p. injection of [methyl-14C]methyl methanesulphonate (MMS) into rats (100 mg/kg) 3-[14C]methyladenine was identified as a urinary product excreted mainly up to 24 h after treatment, the amount over this period being about 0.02 mumol 3-methyladenine. When [14C]MMS and L-[methyl-3H]methionine were injected together no methyl-3H-label was detected in 3-methyladenine, nor was this product detected following injection of [methyl-14C]methionine alone or of [14C]formate. Isotopically labelled 1-methylnicotinamide (1-meNmd) was detected following all the treatments listed, and as previously found by Chu and Lawley, 1-meNmd excretion was enhanced by MMS treatment as judged by increased excretion of 1-[3H]meNmd when [14C]MMS and [3H]methionine were given together. The extent of labelling of 1-meNmd was much lower following injection of [14C] formate, than that from methionine or MMS. The results showed that 3-methyladenine derived only from direct chemical methylation by MMS. They also support the previous suggestion that [methyl-14C]meNmd can result from direct methylation, with a maximal amount of about 3% of excreted meNmd deriving from this route. The possible utility of the methods described for monitoring in vivo alkylation is discussed.
Article
Investigation of urinary markers as indices of endogenous nitrosation and of gastric cancer etiology has been a major focus of our work. As part of this effort, studies have been carried out on a Colombian population at high risk for gastric cancer. In this group, nitrosoproline excretion was highly correlated with nitrate excretion in the subpopulation with advanced gastric pathology, but not in control subpopulations with more normal stomachs. Neither urinary 7-methylguanine nor 3-methyladenine was strongly related to gastric pathology or to urinary nitrate or nitrosoproline levels. More recently, as evidence has accumulated concerning the importance of nitric oxide as a cellular messenger, we have begun research toward developing markers for the presence of nitric oxide and for endogenous nitrosation via this compound. Nitric oxide is formed from arginine by activated endothelial cells as a messenger for vasodilation. We have shown that prolonged exercise leads to increased urinary nitrate and that when 15N-arginine is ingested by humans, 15N-nitrate levels increase in 24-hr urine collections. Nitrosohydroxyethylglycine and 3-nitrotyrosine were evaluated as indices for the formation of N-nitrosomorpholine and for the nitration of protein, respectively, under experimental conditions (e.g., immunostimulation) expected to enhance nitric oxide formation. Nitrotyrosine has not proved useful as a biomarker for nitration/nitrosation reactions in immunostimulated rats. Immunostimulation of rats following administration of morpholine led to increases in urinary nitrate and nitrosohydroxyethylglycine. This procedure, however, would not be appropriate for humans due to the toxicity of morpholine and the carcinogenicity of N-nitrosomorpholine.
Article
The reaction of β-propiolactone with guanosine and related compounds was shown to give the previously reported product 7-(2-carboxyethyl)guanine, and a second product which was characterized as 7,9-di-(2-carboxyethyl)guanine. Evidence for this structure included an elementary analysis of the compound, comparison of its u.v. spectra and pKa with those for analogous structures, and comparison of its spectra and RF with those for the compound prepared by an alternative method. A mechanism for the reaction of β-propiolactone with guanosine and related compounds is suggested.
Article
The metabolic relationship between urinary 7-methylguanine and 8-hydroxy-7-methylguanine has been studied. 7-[7-15N]Methylguanine was administered to a normal human subject and the presence of isotope in urinary purine bases was followed for 7 days. Most of the 15N given was recovered in the 7-methylguanine and 8-hydroxy-7-methylguanine excreted on the first day. The isotope concentration in these two compounds was quite similar. No excess isotope was detected in xanthine or uric acid. Since only negligible isotope was recovered after the first day, it may be concluded that no appreciable pool of 7-methylguanine exists. A substantial proportion of this compound is rapidly oxidized to 8-hydroxy-7-methylguanine in man by an unknown process. The isotopic 7-methylguanine required was prepared by methylation of guanine in alkaline solution with methyl chloride. 9-Methylguanine and 3-methylguanine were major by-products, and small amounts of 1-methylguanine and N2-methylguanine were isolable.