title: Studies on the methyl isocyanate adducts with globin.
abstract: Isocyanates such as methylisocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, or diisocyanates, used in the production of plastics, are highly reactive toxic compounds that spontaneously bind to biological macromolecules. In vivo formation of stable adducts with blood protein globin offers possibilities for biomonitoring of internal exposure to various reactive species. Thus, biomonitoring of the isocyanates through determination of their specific adducts with globin is a challenge. In this study, we characterized the adducts formed in human globin upon treatment with 100-fold molar excess of MIC. The globin was subject to enzymatic hydrolysis with pronase, and the hydrolysate was analysed by high performance liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometric detection (HPLC/APCI-MS). The two major MIC adducts were those with N-terminal Val and side-chain of Lys, as confirmed by comparison with the synthetic standards. About 20 other adducts were observed, and several of them were tentatively identified using their MS and MS/MS spectra. Whereas detection of the adducts with Tyr and His was expected, the adducts with Trp and Phe, and a Lys adduct containing two MIC moieties, were probably analytical artifacts resulting from the transcarbamoylation during globin hydrolysis rather than products of direct carbamoylation. The other detected products were MIC-Val-His, derived from the N-terminal dipeptide of globin beta-chain, and dipeptides consisting of MIC-Lys attached to Gly, Val, Leu, Thr, and Glu. Failure to detect the corresponding non-modified dipeptides suggests that the pronase action may be hampered by the amino acid modification. MIC is known as a metabolic intermediate of the industrial solvents N,N-dimethylformamide (DMF) and N-methylformamide (MF) in humans and rats. The HPLC/APCI-MS analysis of globin from rats injected with DMF or MF, 1000 mg/kg, revealed the presence of the MIC adducts with both Val and Lys. The level of the Val adduct in globin from the DMF-dosed rats, determined using Edman degradation and GC/MS, was ca. 40 nmol/g, which is a level common in workers occupationally exposed to DMF. This suggests that also the Lys adduct in such human globin samples can be feasible to analysis and is therefore considered for further studies as a potential biomarker of exposure to DMF.
authors.affiliation: Centre of Industrial Hygiene and Occupational Diseases, National Institute of Public Health, Srobárova 48, 10042 Prague 10, Czech Republic. jmraz@szu.cz
authors.forename: J
authors.initials: J
authors.lastname: Mráz
authors.forename: P
authors.initials: P
authors.lastname: Simek
authors.forename: D
authors.initials: D
authors.lastname: Chvalová
authors.forename: H
authors.initials: H
authors.lastname: Nohová
authors.forename: P
authors.initials: P
authors.lastname: Smigolová
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004126:Dimethylformamide / Q000493:pharmacokinetics; D004784:Environmental Monitoring / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D005559:Formamides / Q000493:pharmacokinetics; D005914:Globins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D017953:Isocyanates / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D011402:Pronase / Q000737:chemistry; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D005559:Formamides; D017953:Isocyanates; D004126:Dimethylformamide; D005914:Globins; C008461:methyl isocyanate; D011402:Pronase; D014633:Valine; D008239:Lysine; C002950:methylformamide