Alkylpurines are liberated from alkylated DNA by glycosylase repair enzymes and, in most cases, excreted in urine without further metabolism. This phenomenon forms the basis of noninvasive methods to ...measure DNA alkylation in vivo. In the case of methyl adducts, such as 7-methylguanine (7-MeGua), natural backgrounds exist due to RNA turnover. However, deuterated ( d3) methylating agents or precursors give rise to d3-7- MeGua and d3-3-methyladenine (3-MeAde), which can be readily quantitated using gas chromatography - mass spectrometry (GC-MS). A deuterated probe drug, such as d6-aminopyrine, can be used to measure endogenous nitrosation levels in experimental animals. In contrast, for higher alkyl homologues of alkylpurines, natural backgrounds are low or nonexistent and can be directly measured by GC-MS using stable isotope labeled internal standards. For example, increased levels of urinary 3-ethyladenine were observed in cigarette smokers. Due to recent advances in analytical methodology, notably immunoaffinity cleanup of urine, measurements of excreted DNA adducts can be used in studies in human populations exposed to low levels of alkylating carcinogens.
Helicobacter pylori infection is associated with elevated gastric mucosal concentrations of the lipid peroxidation product malondialdehyde and
reduced gastric juice vitamin C concentrations. ...Malondialdehyde can react with DNA bases to form the mutagenic adduct malondialdehyde-deoxyguanosine
(M 1 -dG). We aimed to determine gastric mucosal levels of M 1 -dG in relation to H. pylori infection and malondialdehyde and vitamin C concentrations. Patients ( n = 124) attending for endoscopy were studied. Levels of antral mucosal M 1 -dG were determined using a sensitive immunoslot-blot technique; antral mucosal malondialdehyde was determined by thiobarbituric
acid extraction, and gastric juice and antral mucosal ascorbic acid and total vitamin C were determined by high-performance
liquid chromatography. Sixty-four H. pylori -positive patients received eradication therapy, and endoscopy was repeated at 6 and 12 months. Levels of M 1 -dG did not differ between subjects with H. pylori gastritis ( n = 85) and those with normal mucosa without H. pylori infection ( n = 39; 56.6 versus 60.1 adducts/10 8 bases) and were unaffected by age or smoking habits. Malondialdehyde levels were higher (123.7 versus 82.5 pmol/g; P < 0.001), gastric juice ascorbic acid was lower (5.7 versus 15.0 μmol/ml; P < 0.001), and antral mucosal ascorbic acid was unchanged (48.0 versus 42.7 μmol/g) in H. pylori gastritis compared with normal mucosa. Multiple regression analysis revealed that M 1 -dG increased significantly with increasing levels of malondialdehyde, antral ascorbic acid, and total antral vitamin C. M 1 -dG levels were unchanged 6 months (63.3 versus 87.0 adducts/10 8 bases; P = 0.24; n = 38) and 12 months (66.7 versus 77.5 adducts/10 8 bases; P = 0.8; n = 13) after successful eradication of H. pylori. M 1 -dG thus is detectable in gastric mucosa, but is not affected directly by H. pylori .
The endogenous formation of nitrosoproline (NPRO) following administration of nitrate and proline is reported in ten healthy young adults. There was a relatively constant basal excretion of NPRO, 26 ...+/- 10 (SD) nmol/day, in excess of amounts found in the diet. This basal synthesis of NPRO was not reduced by ascorbic acid (2 g/day) or alpha-tocopherol (400 mg/day). A significant rise in the excretion of NPRO was observed following the administration of nitrate and proline, ranging from 29 to 318 nmol/24 h with a mean of 100 nmol/24 h. 15NNitrate was used as a tracer to study the observed excess excretion of NPRO in urine. The data revealed that urinary NPRO excretion as a result of endogenous synthesis is not totally derived from ingested nitrate as its precursor. The ingestion of ascorbic acid and alpha-tocopherol inhibited the incorporation of 15Nnitrate into NPRO by 81 and 59%, respectively. An additional nitrosamino acid, N-nitrosothiazolidine-4-carboxylic acid, was present in the urine. It was found that N-nitrosothiazolidine-4-carboxylic acid increased 6-fold upon ingestion of nitrate. Ascorbic acid and alpha-tocopherol blocked this nitrate induced synthesis.
A gas chromatographic-mass spectrometric method has been developed for the determination of N-7-2H3methyl 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-7-2H3methyl guanine is then measured by gas chromatography-mass spectrometry, enabling the amount of deuterated base to be determined. Preliminary experiments with 2H3methyl methanesulfonate in rats showed measurable excretion of N-7-2H3methyl guanine. 4-(Di2H3methylamino)antipyrine alone gave no detectable amount of alkylated base, but coadministration of nitrite resulted in excretion of deuterated N-7-methyl guanine.
Alkoxy acids are the active metabolites of teratogenic glycol ethers. To examine the relationship of chemical structure to embryotoxicity, the effects of 6 acids on the development of 9.5-day rat ...embryos over 48 h in culture were studied. Methoxyacetic acid (MAA) and ethoxyacetic acid (EAA) (5 mM) were growth-retarding and induced gross structural defects, with MAA being more effective. n-Propoxyacetic acid (n-PAA) and n-butoxyacetic acid (n-BAA) (5 mM) were markedly less embryotoxic and produced only minor anomalies. Thus, the activities of these substituted acetic acids decreased with the increase in the length of the alkoxy chain. 3-Methoxypropionic acid (3-MPA) and 4-methoxybutyric acid (4-MBA) (5 mM) were much less active than MAA and induced only minor defects. Therefore in this series: RO(CH2)nCOOH, an increase in the value of n caused a greater reduction in embryotoxicity than did an increase in chain length of the alkyl group R.
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