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 diisocyanat e (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 an imals 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 fra ctions using an antiserum prepared to a heterologous TDI-protein indic ated a difference in chain specificity and product types formed in vit ro by the two isomers. Ionspray mass spectrometry (MS) revealed the ch emistry of the adduction products. Carbamoylated products, formed from adduction by one TDI molecule (with one isocyanato group hydrolyzed t o 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-H b adduct in the form of TDI-cross-linked alpha and beta chains. Ionspr ay 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 hy drolyzed to the amine. We also found evidence of an amine-nitroso addu ct on the a chain in the in vivo sample. These results indicate that a t least one of the isocyanato moieties (or a masked derivative) of 2,4 -TDI survived passage through the lung, into the serum, and through th e erythrocyte membrane to form adducts with Hb that were stable to dia lysis, gel filtration, and reversed phase HPLC separation under acidic conditions. The presence of an apparent amine-nitroso adduct indicate d in vivo formation of 2,4-diaminotoluene, a recognized animal carcino gen.