Isocyanate-specific hemoglobin adduct in rats exposed to 4, 4'-methylenediphenyl diisocyanate.

Abstract

4,4'-Methylenediphenyl diisocyanate (MDI) is the most important of the isocyanates used as intermediates in the chemical industry. Among the main types of damage after exposure to low levels of MDI are lung sensitization and asthma. Protein adducts of MDI might be involved in the etiology of sensitization reactions. It is therefore necessary to have sensitive and specific methods for monitoring the isocyanate exposure of workers. To date, urine metabolites or protein adducts have been used as biomarkers in workers exposed to MDI. However, with these methods it is not possible to determine if the biomarkers result from exposure to MDI or to the parent aromatic amine 4,4'-methylenedianiline (MDA). This work presents a procedure for quantitating isocyanate-specific hemoglobin adducts. Blood proteins are used as markers of exposure and possibly as markers of dose size for the modifications of macromolecules in the target organs where the disease develops. For the quantitation of hemoglobin adducts, N(1)-[4-(4-isocyanatobenzyl)phenyl]acetamide (AcMDI) was reacted with the tripeptide valyl-glycyl-glycine and with valine yielding N-[4-(4-acetylaminobenzyl)phenyl]carbamoyl]valyl-glycyl-glycine and N-[4-[4-(acetylaminobenzyl)phenyl]carbamoyl]valine, respectively. N-[4-[4-(Acetylamino-3,5-dideuteriobenzyl)-2, 6-dideuteriophenyl]carbamoyl]valine was synthesized from valine, as was N(1)-[4-(4-isocyanato-3,5-dideuteriobenzyl)-2, 6-dideuteriophenyl]acetamide, for use as an internal standard. These adducts were cleaved in 2 M HCl to yield the corresponding hydantoins, 3-[4-(4-aminobenzyl)phenyl]-5-isopropyl-1, 3-imidazoline-2,4-dione (MDA-Val-Hyd) and 3-[4-(4-amino-3, 5-dideuteriobenzyl)-2,6-dideuteriophenyl]-5-isopropyl-1, 3-imidazoline-2,4-dione, respectively. In globin of rats exposed to MDI, MDA-Val-Hyd could be found in a dose-dependent manner. The adduct was identified by HPLC/MS/MS and quantified by GC/MS after derivatization with heptafluorobutyric anhydride. The amount of MDA-Val-Hyd found after acid hydrolysis of globin at 100 degrees C is about 12 times larger than the sum of N-acetyl-4, 4'-methylenedianiline (AcMDA) and MDA obtained from mild base hydrolysis of hemoglobin. The MDA-Val-Hyd is an isocyanate-specific adduct. MDA and AcMDA released after mild base hydrolyses result most likely from a sulfinamide adduct which is a typical adduct of arylamines. According to these results, higher amounts of isocyanate adducts than arylamine adducts should be expected in workers exposed to isocyanates.