Rapid, quantitative microassay for the monokine respiration inhibitory factor.

The murine monokine respiration inhibitory factor (RIF) induces lesions at Complex I and Complex II of the mitochondrial electron transport chain (ETC) of tumor cells; these lesions in the ETC appear closely linked with cytostasis of the targets. In this report we describe the use of the sensitive murine mammary adenocarcinoma line EMT-6 in a colorimetric microassay for the effects of RIF on the ETC and target replication. The participation of cytolytic molecules in this assay system was excluded because of the resistance of the target to their effects. The endpoint for the assay was the ETC-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its colored formazan. The two major coupling sites of MTT in the ETC of EMT-6 cells are shown to be proximal to Coenzyme Q, detected either by malate oxidation through Complex I or succinate oxidation through Complex II. The assay was sensitive to both RIF-induced lesions at these dehydrogenases and to the cytostasis-linked reduction in target cell number. The assessment of ETC lesions by this microassay correlated directly with that determined by the less sensitive polarimetric assay based on oxygen consumption. We demonstrate the application of this microassay to parameters for the production of RIF by activated murine macrophages, and to initial molecular characterization of this mediator.