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.

Critical analysis of biomonitoring endpoints for measuring exposure to polymeric diphenyl-methane-4,4'-diisocyanate (MDI) in rats: a comparison of markers of exposure and markers of effect

“…The concomitant decrease of BAL protein and increase in haemoglobin adduction appears to be consistent with upregulated GSH synthesis. This tentative pathway for formation of haemoglobin adducts requires neither the formation of free amines nor an extrapulmonary activation as previously suggested (Sepai et al 1995a;Sabbioni and SchuÈ tze 1998;Sabbioni et al 2000). The haemoglobin binding index (HBI = molecular adducts of compound/gHb : molecular compound/kg body weight), as proposed by Sabbioni and SchuÈ tze (1998), for total MDA was 3 and 0.04 after oral dosing with MDA and MDI, respectively (Sabbioni and SchuÈ tze 1998).…”

“…Accordingly, the yield of adducts or metabolites actually measured is highly dependent on the methodology of hydrolysis. Tentative pathways of adduct formation have been described elsewhere in more detail (Kennedy and Brown 1992;Sabbioni et al 2000).…”

4,4′‐Methylene diphenyl diisocyanate (MDI) [101‐68‐8] and “polymeric” MDI (PMDI) [9016‐87‐9] [MAK Value Documentation, 2008]

“…In Wistar rats exposed to MDI for 3 months in concentrations of up to 2 mg/m 3 for 17 hours a day on 5 days a week, a dose-dependent, specific haemoglobin adduct (MDA-Val-Hyd) was identified after the acid hydrolysis of globin. This was found in concentrations about 12 times higher than the sum of MDA and acetylated MDA after mild alkaline hydrolysis of haemoglobin (Sabbioni et al 2000).…”

Addendum to 4,4'‐Methylene diphenyl diisocyanate (inhalable fraction)[BAT Value Documentation, 2007]

“…A very small amount of MDI also binds to hemoglobin, so that MDI-Hb adducts are detectable in the erythrocytes of workers exposed to MDI (Schütze et al 1995;Sepai et al 1995) as well as in those of rats (Pauluhn 2002;Sabbioni et al 2000).…”