Diagnosis and dosimetry of exposure to sulfur mustard: Development of standard operating procedures. Further exploratory research on protein adducts - Final report

In continuation of previous grants, Standard Operating Procedures (SOPs) have been developed for retrospective detection of exposure to sulfur mustard, i.e., an immunoslotblot assay for DNA adducts in blood and skin, and the modified Edman procedure for determination of adducts to the N-terminal valine in hemoglobin. Both SOPs were validated (day-to-day variability, inter- and intraindividual variation) and
could be properly set up and carried out at another institute (MRICD) within one working day. In vivo persistence studies showed that in hairless guinea pigs the DNA adduct in blood is still detectable at 14 days after i.v. administration ofsulfur mustard (0.5 LD50), while the adduct to N-terminal valine of hemoglobin can still be analyzed after 56 days. In skin the DNA adduct level is only slowly decreasing
during the first 3 days and is marginal at 17 days after skin exposure ofthe hairless guinea pig. In marmosets the DNA adduct is detectable in blood during 7 days after i.v. administration ofsulfur mustard (4.1 mg/kg). The adduct to N-terminal valine of hemoglobin is still detectable 94 days after exposure. Furthermore, exploratory research has been performed aiming at the development of a fieldable immunochemical assay for sulfur mustard adducts with hemoglobin, albumin and keratin. Upon exposure of human blood to sulfur mustard, the major adducts in albumin are formed with histidine and cysteine-34. Exposure to > 10 nM sulfur mustard was determined by LC-tandem MS analysis of a tripeptide containing this adducted cysteine in a pronase digest of only 3 mg albumin, i.e., the most sensitive marker for exposure to sulfur mustard developed so far. Exposure ofIranian victims ofthe Iran-Iraq conflict was detected by using both this procedure and the modified Edman procedure. Treatment at pH 13 released 80% ofthe bound radioactivity as [
14C]thiodiglycol from keratin isolated from [l4C]sulfur mustard exposed human callus, which suggests that most ofthe adducts formed are esters of glutamic and aspartic acid residues. Exposure of human callus to 10 uM ofsulfur mustard could be detected by GC-MS analysis ofreleased thiodiglycol after derivatization. In a similar way, exposure of human skin to saturated sulfur mustard vapor could be detected. Three partial sequences of hemoglobin, the T5 fragment from albumin, and three partial sequences of keratin were synthesized and used as haptens for raising antibodies, which contain adducted histidine, cysteine, and glutamic- or aspartic acid, respectively. Several clones have been obtained. Some ofthe antibodies directed against keratin adducts showed binding to the horny layer of human skin exposed to sulfur mustard (Ct
1040 mg.min m-3). Such antibodies can directly be applied to human skin, which opens the way for development of an immunochemical kit for field detection ofskin exposure.