The mechanisms of toxicity of 1,2--dihaloethanes and bisulfite will be investigated by Drs. Mosbaugh and Reed with the main objective being the elucidation of the structure and function alterations of macromolecules including loss of enzyme fidelity. The specific properties of both protein thiols and the alkylating agents proposed for these studies, should make it possible to identify specific protein thiol alterations that result from alkylation including loss of biological function and the alteration in protein thiol homeostasis. Based on the relative rates of alkylation by S--(2-chloroethyl)glutathione (CEG) of functional groups associated with nucleic acids and proteins, the alkylation if protein molecules is proposed to be an important feature of the mechanism of toxicity of ethyl dichloride. The alkylation occurs via the formation of a reactive episulfonium ion from CEG nonenzymatic decomposition. It is proposed that protein alkylation by CEG is selective both for protein thiols over other functional groups and even selective amongst proteins containing thiols. Further, it is proposed that a major portion of the acute toxicity of 1,2--dihaloethanes is associated with the loss of specific protein functions due to these alkylation events. Bisulfite and sulfite interaction with specific proteins may provide the basis for the cocarcinogenicity shown with polyhaloaromatics or N-- methyl--N'--nitro--N--nitrosoguanidine. Under physiological conditions, bisulfite acts as a comutagen requiring both DNA damage and DNA repair to induce mutations. We propose to investigate the extent to which DNA polymerase fidelity is altered by the formation of a sulfonate adduct(s). Model proteins, carbonic anhydrase III, interleukin 3, thioredoxin, and DNA polymerases will be reacted with CEG or bisulfite, and the structure of the covalent adducts will be identified by electrophoresis, mass spectrometry, and digestion combined with peptide characterization and quantitation. Similar studies will be conducted with cells and in vivo to elucidate the major protein alterations by the 1,2--dihaloethanes and bisulfite.
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