Flow cytometry measurement of mixed function oxidase activity and cell viability in rainbow trout (Oncorhynchus) hepatocytes: Method development

✍ Scribed by Gagn�, Fran�ois ;Blaise, Christian

• Book ID: 102655708
• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 875 KB
• Volume: 11
• Category: Article
• ISSN: 1053-4725
• DOI: 10.1002/(sici)1098-2256(1996)11:1<53::aid-tox8>3.0.co;2-8

✦ Synopsis

A short-term assay procedure was developed measuring mixed function oxidase (MFO) activity and cell viabilityvn primary hepatocytes cultures of rainbow trout (Oncorhynchus mykiss), using flow cytometry. Assays to measure the reaction rate of MFO utilized live hepatocytes (50 x lo4 cells . mL-') incubated at 22°C in the presence of 5(6)methoxycarbonyl fluorescein methyl ether methyl ester (MCF) substrate. Results of these experiments enabled optimal enzyme-substrate reaction conditions to be fixed at 30 p M MCF for a 20 min period. Hepatocytes 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 cell volume, but that MFOrelated fluorescence intensity was unaffected. Other experiments indicated that simultaneous flow cytometric analysis of cell viability and MFO activity is possible, as long as interference of MFO activity fluorescence emission on cell viability fluorescence emission can be resolved. Exposure of hepatocytes (48 h, 15°C) to p-naphthoflavone and benzo-[a]-pyrene (known inducers of fish cytochrome P-450 detoxification systems) resulted in significant expression of MFO activity under the experimental conditionsdescribed. Further validation of theend points measured with this procedure is in progress and involves comparisons with similar end points (96 h LCS0 and 7-ethoxyresorufin 0-deethylase activity) determined with fingerling rainbow trout exposed to industrial wastewaters. 0 7996 by John Wiiey & Sons, fnc.