FLOW-CYTOMETRY MEASUREMENT OF MIXED-FUNCTION OXIDASE ACTIVITY AND CELL VIABILITY IN RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) HEPATOCYTES - METHOD DEVELOPMENT

A short-term assay procedure was developed measuring mixed function ox idase (M FO) activity and cell viability in primary hepatocytes cultur es of rainbow trout (Oncorhynchus mykiss), using flow cytometry. Assay s to measure the reaction rate of MFO utilized live hepatocytes (50 x 10(4) cells . mL(-1)) incubated at 22 degrees C in the presence of 5(6 )methoxycarbonyl fluorescein methyl ether methyl ester (MCF) substrate . Results of these experiments enabled optimal enzyme-substrate reacti on conditions to be fixed at 30 mu M MCF for a 20 min period. Hepatocy tes were also subjected to cell volume changes by varying their buffer medium osmolarities. It was shown that the fluorescence intensity of propidium iodide, an indicator of cell viability, was influenced by ce ll volume, but that MFO-related fluorescence intensity was unaffected. Other experiments indicated that simultaneous flow cytometric analysi s of cell viability and MFO activity is possible, as long as interfere nce of MFO activity fluorescence emission on cell viability fluorescen ce emission can be resolved. Exposure of hepatocytes (48 h, 15 degrees C) to beta-naphthoflavone and benzo-[a]-pyrene (known inducers of fis h cytochrome P-450 detoxification systems) resulted in significant exp ression of MFO activity under the experimental conditions described. F urther validation of the end points measured with this procedure is in progress and involves comparisons with similar end points (96 h LC(50 ) and 7-ethoxyresorufin O-deethylase activity) determined with fingerl ing rainbow trout exposed to industrial wastewaters. (C) 1996 by John Wiley & Sons, lnc.