Analysis of biomarkers in rats and dogs exposed to polymeric methylenediphenyl diisocyanate (pMDI) and its glutathione adduct

Hemoglobin adducts (Hb-MDX) of monomeric methylenediphenyl diisocyanate (MDI) are often interpreted as indirect evidence of hydrolysis of the diisocyanate moiety to the respective amine (diphenylmethane-4,4'-diamine, 4,4'-MDA) which constitutes the rationale of using this biomarker as an internal dosimeter of exposure to putatively formed MDA. In contrast, more recently published data suggest that following inhalation the high concentration of glutathione (GSH) present in lungs favor an adduct formation with GSH and/or peptides/proteins rather than hydrolysis. The focus of this study was to test this alternate hypothesis, viz. whether Hb-MDX can also be formed by the GSH bis-adduct of monomeric MDI. The synthesized mMDI-GSH bis-adduct was administered to rats by single intratracheal instillation. Additional groups were dosed by gavage and intraperitoneal injection. Biomarkers of exposure were determined in blood (plasma protein and hemoglobin adducts) and urine after harsh alkaline and acid hydrolysis, respectively. Data from previous single inhalation exposure studies with aerosols of MDI and 4,4'-MDA in rats served as reference. As to whether N-acetylation plays any modifying role to yield these mMDI-specific biomarkers was addressed in similarly head-only exposed dogs, a species with no appreciable N-acetylation capacity whereas rats are strong N-acetylators. The results obtained suggest that biomarkers in blood from controlled exposures above current workplace standards of mMDI appear not to be suitable for reliable assessments of past exposures. The biomarkers typically used to assess past exposures to MDI were also identified following exposure to the MDI-GSH bis-adduct. Their yield was low but quite similar for MDI aerosol and the MDI-GSH bis-adduct, whilst that of MDA was distinctively higher. The findings of this study are supportive of a conceptual pathway that the MDI-derived biomarkers of exposure are formed through MDI-GSH adducts rather than MDA. Data from dogs support the findings from rats and show that N-acetylation does not appear to be an essential modifying factor. It is concluded that the yield of MDI-related markers of exposure is relatively low and dependent on the exposure dose (and route). MDA originating from hydrolyzed serum protein or hemoglobin appear to be confounded by false-positive background levels which are surmised to be associated with the method of hydrolysis. The determination of urinary biomarkers might be a useful tool to identify recent exposures (by any route). Due methodological uncertainties associated with the harsh hydrolysis of biological specimens may be reduced substantially when using incremental pre- to post-shift changes rather than relying solely on absolute data.