In vivo and in vitro reactions of toluene diisocyanate isomers with guinea pig hemoglobin

Guinea pig hemoglobin (Hb) adducts of 2,4- and 2,6-toluene diisocyanate (2,4- and 2,6-TDI) were individually prepared, each at a Hb tetramer to diisocyanate ratio of 1:1, and compared with adducts of Hb from animals exposed to 1 ppm 2,4-TDI vapor. Each Hb sample was subjected to C-4 HPLC chain/heme separation with UV detection. Survey of the LC fractions using an antiserum prepared to a heterologous TDI-protein indicated a difference in chain specificity and product types formed in vitro by the two isomers. Ionspray mass spectrometry (MS) revealed the chemistry of the adduction products. Carbamoylated products, formed from adduction by one TDI molecule (with one isocyanato group hydrolyzed to an amino group), were detected by MS with 2,4-TDI on each chain and with 2,6-TDI on the beta chain. Additionally, a quasi-molecular ion of a bis carbamoylation adduct was noted by MS in the in vitro 2,4-TDI-Hb adduct in the form of TDI-cross-linked alpha and beta chains. Ionspray MS analysis of the Hb isolated from guinea pigs exposed in vivo to 2,4-TDI indicated carbamoylation products with both the alpha and beta chains in which one of the two original isocyanato groups had been hydrolyzed to the amine. We also found evidence of an amine-nitroso adduct on the a chain in the in vivo sample. These results indicate that at least one of the isocyanato moieties (or a masked derivative) of 2,4-TDI survived passage through the lung, into the serum, and through the erythrocyte membrane to form adducts with Hb that were stable to dialysis, gel filtration, and reversed phase HPLC separation under acidic conditions. The presence of an apparent amine-nitroso adduct indicated in vivo formation of 2,4-diaminotoluene, a recognized animal carcinogen.