3-ethyladenine and 3-methyladenine

Methylating and ethylating agents are used in the chemical industry and produced during tobacco smoking. They generate DNA base damage whose role in cancer induction has been documented. Alkylated bases are repaired by the base excision repair pathway. We have established the repair efficiency of methylated and ethylated bases by various Escherichia coli repair proteins, namely 3-methyladenine-DNA-glycosylase I (TagA protein), which excises 3-methyladenine and 3-methylguanine, 3-methyladenine-DNA-glycosylase II (AlkA protein), which has a broad substrate specificity including 3- and 7-alkylated purines and the formamidopyrimidine(Fapy)-DNA-glycosylase (Fpg protein) repairing imidazole ring-opened 7-methylguanine. The comparison of the Km values of these various enzymes showed that methylated bases were excised more efficiently than ethylated bases. Several 3-alkyladenine derivatives have been synthesized and examined for their ability to inhibit the activity of the various repair proteins. We have shown that 3-ethyl-, 3-propyl-, 3-butyl- and 3-benzyladenine were much more efficient inhibitors of TagA protein than 3-methyladenine. The inhibitory effect was increased with the increase of the size of alkyl-group and IC50 for 3-benzyladenine was 0.4 +/- 0.1 microM as compared to 1.5 +/- 0.3 mM for 3-methyladenine. These compounds inhibited neither the AlkA protein nor human 3-methyladenine-DNA-glycosylase (ANPG protein). Moreover, 3-hydroxyethyladenine did not affect the activity of any of these enzymes. Taken together, these results suggest that hydrophobic interactions are involved in the mechanism of inhibition and/or recognition and excision of alkylated purines by TagA protein.

Topics: Adenine; Alkylating Agents; Bacterial Proteins; DNA; DNA Damage; DNA Glycosylases; DNA Methylation; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Escherichia coli; Escherichia coli Proteins; Kinetics; Lipoproteins; N-Glycosyl Hydrolases

The urinary excretion of the DNA alkylation products 3-methyladenine (3-MeAde) and 3-ethyladenine (3-EtAde) after controlled exposure to cigarette smoke over a period of 4 days was determined by competitive radioimmunoassay after separation by HPLC. Twenty-four hour urine samples were collected from five smokers and five non-smokers. Days 1 and 3 (control days) were without smoking, on days 2 and 4 smokers consumed 24 cigarettes each within 8 h in an unventilated room (45 m3) in the presence of non-smokers. Average levels of carbon monoxide during exposure were 15-20 p.p.m., 2.8-3.5 mg/m3 of respirable suspended particles and 75-86 micrograms/m3 of nicotine. Carboxyhemoglobin levels increased by 9.0 and 1.8% in smokers and passive smokers respectively. On control days, urinary excretion of 3-MeAde was similar in smokers and non-smokers (4.7-6.2 micrograms/24 h). Smoking resulted in a significant increase (P < 0.01) in 3-MeAde excretion (13.6-14.8 micrograms/24 h); no change was observed in the average excretion of 3-MeAde by passive smokers (4.8-4.9 micrograms of 3-MeAde/24h). Baseline 3-EtAde excretion on control days was similar in smokers and passive smokers (13.7-32.8 ng/24 h). In smokers, the amount of urinary 3-EtAde was increased > 5-fold (119.3-138.5 ng/24 h) on smoking days; no effect on 3-EtAde excretion was observed on average in passive smokers (18.0-25.2 ng/24 h). The nature of the DNA-reactive agent(s) responsible for the increased urinary excretion of 3-alkyladenines, in particular of the sensitive indicator 3-EtAde, remains to be determined.

Topics: Adenine; Adult; Environmental Exposure; Humans; Male; Smoking; Tobacco Smoke Pollution

Immunoaffinity gels were prepared by coupling monoclonal antibody (Mab) EM-6-47 to protein A-Sepharose, and were used to make small columns retaining 3-alkyladenines (3-alkAde) of diverse structure. An analytical procedure for determination of 3-methyladenine (3-MeAde), 3-ethyladenine (3-EtAde), 3-(2-hydroxyethyl)adenine (3-HOEtAde) and 3-benzyladenine (3-BzAde) was developed. Deuterated internal standards (d3-3-MeAde, d5-3-EtAde, d4-3-HOEtAde and d7-3-BzAde) were synthesized and added to urine samples prior to immunoaffinity purification. 3-alkAde were separated and quantitated as tert-butyl-dimethylsilyl (TBDMS) derivatives by capillary gas chromatography-low resolution mass spectrometry (GC-MS). Detection limits for 3-MeAde, 3-EtAde and 3-HOEtAde were 0.2 pmol/ml urine and for 3-BzAde, 1 pmol/ml urine. Studies in two volunteers showed that 3-MeAde and 3-HOEtAde were excreted almost quantitatively (> 90%) within 24 h, that 3-EtAde was less well excreted (67-74%) and that 3-BzAde was poorly excreted (21-25%). Studies on basal levels of 3-alkAde urinary excretion in three volunteers showed that 3-MeAde was > 90% derived from the diet as the preformed product. 3-HOEtAde was present at approximately 10 nmol/day and was reduced to approximately 1 nmol/day when the diet was standardized suggesting that it is also dietary in origin. 3-BzAde was not detected in human urine. 3-EtAde was not only excreted at low levels (< 1 nmol/day) but was also only very slightly affected by diet. This general and sensitive method will be useful in biomonitoring studies in subjects exposed to alkylating agents of diverse structure.

Topics: Adenine; Adult; Antibodies, Monoclonal; Deuterium; Diet; Female; Gas Chromatography-Mass Spectrometry; Humans; Immunosorbent Techniques; Male; Sepharose; Staphylococcal Protein A

We describe an immunoanalytical procedure for the detection and quantitation of 3-alkyladenines in biological samples with the use of anti-(3-alkyladenine) monoclonal antibodies (Mab). A new hapten-protein conjugate, 3-ethyl-8-(3-carboxypropyl)-adenine, was used for immunization of BALB/c mice after conjugation to carrier proteins via the carboxyl group. Eighty-nine hybridomas were established which secrete anti-(3-alkyladenine) Mab with antibody affinity constants ranging from 1 x 10(7) to 5 x 10(9) l/mol for 3-ethyladenine (3-EtAde). One of these Mab (EM-6-47) had detection limits of 30 fmol for 3-EtAde, 17 fmol for 3-n-butyladenine (3-BuAde) and 475 fmol for 3-methyladenine (3-MeAde) respectively, at 25% inhibition of tracer-antibody binding. The binding pattern of Mab EM-6-47 revealed high specificity for adenine substituted at N-3 with different alkyl residues and no, or very low, cross-reactivity with other alkylated or unmodified nucleic acid components or structurally related compounds. 3-MeAde and 3-EtAde can be well separated from nucleic acids, and from rat and human urine samples, using HPLC with two successive stationary phases. Using Mab EM-6-47 in conjunction with a competitive RIA, both 3-MeAde and 3-EtAde were detected in the range of 100-300 ng (3-MeAde) and 2-10 ng (3-EtAde) in urine samples (10 +/- 2 ml) of untreated rats collected over a 24 h period. Only 3-MeAde (range 1.3-24.20 micrograms) was found in human urine samples. The concentration of 3-EtAde in rat urine increased significantly during the 24 h following a single i.v. application of N-ethyl-N-nitrosourea. After i.p. application of known amounts of 3-MeAde and 3-EtAde, greater than 90% of 3-MeAde and greater than 70% of 3-EtAde were excreted in rat urine within the subsequent 24 h. The concentration of 3-alkyladenines in body fluids (urine) may thus provide a useful indicator of environmental exposure to nucleic acid-reactive agents, and the immunoanalytical procedure described here permits the sensitive determination of adenines carrying different substituents at N-3.

Topics: Adenine; Animals; Antibodies, Monoclonal; Body Fluids; Cross Reactions; DNA; Enzyme-Linked Immunosorbent Assay; Female; Humans; Mice; Mice, Inbred BALB C; Models, Molecular; Molecular Conformation; Radioimmunoassay; Rats; Rats, Inbred Strains