Identification of novel reaction products of methylene-bis - phenylisocyanate (“MDI”) with oxidized glutathione in aqueous solution and also during incubation of MDI with a murine hepatic S9 fraction

Abstract

Methylene diphenyl diisocyanate (MDI) is an important industrial chemical and asthmagenic respiratory sensitizer, however its metabolism remains unclear. In this study we used LC-MS and LC-MS/MS to identify novel reaction products of MDI with oxidized glutathione (GSSG), including an 837 m/z [M+H]+ ion corresponding to GSSG bound (via one of its N-termini) to partially hydrolyzed MDI, and an 863 m/z [M+H]+ ion corresponding to GSSG cross-linked by MDI (via its two γ-glutamate N-termini). Further studies with heavy isotope labeled and native reduced glutathione (GSH) identified an [M+H]+ ion corresponding to previously described mono(GSH)-MDI, and evidence for “oligomeric” GSH-MDI conjugates. This study also investigated transformational changes in MDI after incubation with an S9 fraction prepared from murine liver. LC-MS analyses of the S9 reaction products revealed the formation of [M+H]+ ions with m/z’s and retention times identical to the newly described GSSG-MDI (837 and 863) conjugates and the previously described mono(GSH)-MDI conjugates. Together the data identify novel biological transformations of MDI, which could have implications for exposure-related health effects, and may help target future in vivo studies of metabolism.

Dilysine-Methylene Diphenyl Diisocyanate (MDI), a Urine Biomarker of MDI Exposure?

“…The reaction mixture was rotated end-over-end for 2 h at 37 °C, after which time the samples were microcentrifuged (10000 g ), 0.2 μm-filtered, and stored on ice until used within 1 h of preparation. The total MDI–GSH reaction mixture and control solutions used in the present study were similar to those previously published ,, and were characterized by LC–UV, MS/MS, and 1 H NMR analyses (Figures S1–S3). The reaction mixture contained primarily bis­(GSH)–MDI ( m / z 865.25 [M + H] + ions) and lower levels of mono­(GSH)–MDI in cyclized form ( m / z 558.17 [M + H] + ions) along with unreacted GSH and limited amounts of oxidized glutathione (GSSG).…”

“…Such molecules may be stabilized by MDI conjugation to the amine terminus(i) of GSH, which may promote chemical excretion (vs potentially pathogenic carbamylation of self-protein) and have been described previously in microsomal studies of MDI. 48 Cyclized MDI−GSH was a minor component of the MDI−GSH reaction products to which mice were exposed in this study and may have been eliminated unchanged. Like dilysine-MDI, the relevance of cyclized MDI−GSH and MDI− GSSG to occupational exposure in human workers will require translational investigation.…”

“…47 The reaction mixture was rotated end-over-end for 2 hours at 37°C, after which time the samples were microcentrifuged (10,000 g), 0.2 μm filtered and stored on ice until used within 1 hour of preparation. The total MDI-GSH reaction mixture and control solutions used in the present study were similar to those previously published 36,42,48 Control solutions were equivalent amounts of GSH treated with a 1:100 dilution of acetone without MDI, or MDI in acetone reacted in phosphate buffer without GSH at 37°C for 2 hours and identically processed.…”

“…LC–MS/MS analysis of urine from mice exposed via the respiratory tract to MDI–GSH contained specific [M + H] + ions not found in mice dermally exposed to MDI in acetone or control mice. One molecule was an m / z 558.17 [M + H] + ion with (a) an isotope distribution expected for a molecule with the chemical formula C 25 H 27 N 5 O 8 S (see Figure S6); (b) a collision-induced dissociation (CID) fragmentation pattern upon MS/MS (Figure S7) expected for mono­(GSH)–MDI in a cyclized formation, with daughter ions of partially hydrolyzed and fragmented MDI ( m / z 225.10 and 106.06), MDI with the thiol group from GSH ( m / z 283.05), Cys-Gly from GSH ( m / z 179.05), or loss of glycine from the parent ion ( m / z 483.14 m / z ); and (c) an LC–MS retention pattern showing multiple retention times (Figure ) identical to that observed with cyclized MDI–GSH synthesized in vitro as previously described. , The MDI–GSH reaction mixture to which the mice were exposed contained low levels (see Figure S1) of cyclized MDI–GSH that we hypothesize may have been excreted unchanged or formed in vivo.…”

“…A second molecule uniquely found in urine from mice exposed via the respiratory tract to MDI−GSH was an m/z 863.23 [M + H] + ion with (a) an isotope distribution pattern (Figure S6) predicted for a molecule with the chemical formula C 35 H 42 N 8 O 14 S 2 ; (b) a CID fragmentation pattern upon MS/ MS (Figure S8) consistent with that expected for MDI−GSSG (daughter ions with m/z 788.21, 564.11, 334.07, 259.04, and 130.05); and (c) an LC−MS retention pattern matching that of MDI−GSSG synthesized in vitro (Figure 8) as previously described. 48…”