1,N6-Etheno-2'-deoxyadenosine (epsilon dAdo) and 3,N4-etheno-2'-deoxycytidine (epsilon dCyd) are formed in vitro by reaction of DNA with the electrophilic metabolites of vinyl chloride (VC), chloroethylene oxide and chloroacetaldehyde. To detect and quantitate these DNA adducts in vivo, we have raised a series of specific monoclonal antibodies (Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively, were used for detection of epsilon dAdo and epsilon dCyd by competitive radioimmunoassay (RIA), following pre-separation of the etheno adducts from DNA hydrolysates by high performance liquid chromatography. At 50% inhibition of tracer-antibody binding, both Mab had a detection limit of 187 fmol and antibody affinity constants (K) of 2 x 10(9) l/mol. The levels of epsilon dAdo and epsilon dCyd were quantitated in the DNA of lung and liver tissue of young Sprague-Dawley rats exposed to 2000 p.p.m. of VC for 10 days. The epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios were 1.3 x 10(-7) and 3.3 x 10(-7), respectively, in lung DNA, and 5.0 x 10(-8) and 1.6 x 10(-7) in liver DNA. When hydrolysates of 3 mg of DNA were analyzed by RIA at 25% inhibition of tracer-antibody binding, epsilon dAdo and epsilon dCyd were not detected in liver DNA from untreated rats above the limiting epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios of 2.2 x 10(-8) and 3.1 x 10(-8), respectively.
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.
ABSTRACT0'-Ethylguanine (O'-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-Nnitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of 06-EtGua as a determinant for the probability of malignant conversion. Eleven 06-EtGua-repair-proficient (R+) variant subclones were isolated from the 06-EtGua-repair-deficient (1-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, =10-S). Contrary to the 208F wild-type cells, all variants expressed 06-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products 02-and 04-ethylthymine. After exposure to EtNU (S500 jug/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R' variants were as low as 1/28th and 1/56th of those in the R-wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies ofpiled-up foci were found between wild-type and variant cells after exposure to the miyjor reactive metabolite of benzo[a]pyrene, (+)-7l,8a-dihydroxy-9,10a-epoxy-7,8,9,10a-tetrahydrobenzo[a]pyrene, for which stable binding to guanine o6 in cellular DNA has not been observed. The relative capacity of cells for repair of 06-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.
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.
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