Reversible interaction of a reactive intermediate derived from furazolidone with glutathione and protein

Swine liver microsomes convert the nitrofuran furazolidone into N-(4-cyano-2-oxo-3-butenylidene)-3-amino-2-oxazolidone, a reactive open-chain acrylonitrile derivative. This derivative may be trapped with such thiol-group-containing agents as glutathione and mercaptoethanol. However, this reaction is reversible; e.g., adding an excess of mercaptoethanol to an aqueous solution (pH 7.4) of the glutathione conjugate results in conversion of 43% of this compound into the mercaptoethanol conjugate. In addition, when microsomal protein is added to the glutathione conjugate or the mercaptoethanol conjugate, 36 and 44%, respectively, become covalently bound to the protein. The amount of this covalently bound radioactivity decreases again on prolonged incubation at 37 degrees C (42% disappearance within 24 hr), suggesting that the acrylonitrile derivative also reacts reversibly with thiol groups of microsomal protein. Indeed an excess of mercaptoethanol could remove covalently bound radioactivity from microsomal protein resulting in the formation of the mercaptoethanol conjugate. The reversibility of the reaction is dependent on pH, as is demonstrated for the mercaptoethanol conjugate. Below pH 2 this conjugate is stable; optimal exchange to microsomal protein is found between pH 7 and 10. At very high pH (greater than 11) no binding to protein is found, although the conjugate disappears rapidly. The mercaptoethanol conjugate exhibits mutagenic activity in the Salmonella/microsome test indicating that the acrylonitrile derivative of furazolidone also interacts with DNA.