ABSTRACT
As part of a program to develop methods for dosimetry of exposure to sulfur mustard, we developed immunochemical methods for the detection of the major adduct, N7-[2-[(hydroxyethyl)thio]ethyl]guanine (N7-HETE-Gua), formed after alkylation of DNA with sulfur mustard. After immunization of rabbits with calf thymus DNA treated with sulfur mustard, we obtained the antiserum W7/10 with a high specificity for DNA adducts of sulfur mustard. With this serum, a competitive enzyme-linked immunosorbent assay was developed in which sulfur mustard adducts to DNA could be detected with a minimum detectable amount of 1-5 fmol per well and a selectivity that allows detection of one N7-HETE-Gua among 5 x 10(6) unmodified nucleotides in single-stranded DNA. The complications that arise to isolate double-stranded DNA from biological samples and to make the DNA single-stranded without destruction of the sulfur mustard adducts result in about a 20-fold higher limit for adduct detection in DNA from human blood than in single-stranded DNA. Presently, adducts in white blood cells can be detected after exposure of human blood to sulfur mustard concentrations > or = 2 microM. We synthesized N7-HETE-GMP for use as a hapten to generate monoclonal antibodies against this adduct. After immunization of mice with this adduct coupled to the carrier protein keyhole limpet hemocyanin we obtained several hybridomas producing monoclonal antibodies that recognize N7-HETE-Gua, containing an intact imidazolium ring. The sensitivity of the competitive ELISA with the monoclonal antibodies was comparable to that of the assays performed with the rabbit antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
DNA/chemistry , Leukocytes/metabolism , Mustard Gas/chemistry , Thymus Gland/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Cattle , DNA/immunology , Enzyme-Linked Immunosorbent Assay , Female , Guanine/analogs & derivatives , Guanine/immunology , Guanine/metabolism , Haptens/chemistry , Haptens/immunology , Hemocyanins , Humans , Immunochemistry , In Vitro Techniques , Leukocytes/chemistry , Leukocytes/drug effects , Mice , Mice, Inbred BALB C/immunology , Mustard Gas/pharmacology , Thymus Gland/chemistry , Thymus Gland/drug effectsABSTRACT
As part of a program to develop methods for verification of alleged exposure to sulfur mustard, we synthesized and characterized the adducts most likely formed by alkylation of DNA with sulfur mustard: N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine (1), bis[2-(guanin-7-yl)ethyl] sulfide (2), N3-[2-[(2-hydroxyethyl)thio]ethyl]adenine (3), and O6-[2-[(2-hydroxyethyl)thio]ethyl]-guanine and its 2'-deoxyguanosine derivative. Incubation of double-stranded calf thymus DNA and human blood with [35S]sulfur mustard in vitro followed by enzymatic degradation of the DNA and mild depurination afforded three major radioactive peaks upon HPLC analysis. These peaks were identified as 1-3 by coelution with the synthetic markers and mass spectrometric and electronic spectra. Compound 1 appeared to be the most abundant adduct, which is in agreement with previous investigations on DNA alkylation with sulfur mustard.
