Flow cytometric analysis of xenobiotic metabolism activity in isolated rat hepatocytes

Abstract

Flow cytometry offers great potential for the study of xenobiotic metabolism in intact cells. We explored this application by the use of ethoxyfluorescein ethyl ester (EFEE) and isolated rat hepatocytes, a classic system for studying such reactions. EFEE is only weakly fluorescent and it diffuses freely into viable cells, where it is metabolized to fluorescein by a process dependent upon mixed-function oxidase activity. In the current study, viable hepatocytes were first identified by flow cytometric assessment of fluorescein diacetate staining. The viable subpopulation was also identifiable on the basis of forward and right angle light scattering properties alone, and it was in this fraction that EFEE metabolism was measured. Metabolism of EFEE to fluorescein was quantified by flow cytometry. SKF 525A, a-naphthoflavone, and metyrapone, classic inhibitors of mixedfunction oxidation, each inhibited the metabolism of EFEE. These results demonstrate the potential of EFEE for use in flow cytometric studies of drug metabolism, such as in multiparameter mechanistic assays of cellular xenobiotic metabolism and toxicity, and in the isolation by fluorescence-activated cell sorting of subpopulations which differ in this activity. 0 1993 Wiley-Liss, Inc.Key terms: a-naphthoflavone, cell viability, cytochromes P450, esterase activity, ethoxyfluorescein ethyl ester, fluorescence-activated cell sorting, in vitro toxicology, metabolic activation, metyrapone, mixed-function oxidase, SKF 525A Organisms possess several pathways for the metabolism of drugs and other xenobiotics (16). Phase I reactions are oxidations which are catalyzed usually by the family of cytochrome P450 mixed-function oxidases. Phase I1 reactions occur when the products of Phase I or unmodified xenobiotics are conjugated to highly water-soluble small molecules, such as glucuronic acid or sulfate. These 2 groups of enzymatic reactions function to increase the water solubility of xenobiotic substrates, and thus, their ability to be excreted from the body. Conversely, in some instances these reactions generate a toxic species from a previously nonreactive substrate.The use of flow cytometry to measure cellular drug metabolism (6) offers several potential advantages over traditional biochemical methods. With flow cytometry, measurements are made on the individual cells of a population rather than on the population as a whole, as is the case for biochemical assays of cellular extracts. This characteristic of flow cytometry allows the determination of heterogeneity within a population of cells in the capacity to metabolize xenobiotics.

Functional Consequences of Metabolic Zonation in Murine Livers: Insights for an Old Story

“…During the past two decades, successful attempts to isolate pure hepatocyte populations from whole liver by flow cytometry have been made, ( 17‐20 ) overcoming hurdles such as the vast hepatocyte size and intense autofluorescence. However, up to now, flow cytometry has been used predominantly for the isolation and study of hepatocyte populations derived from the whole liver, and early attempts at the acinar zone‐specific isolation of hepatocytes by cell sorting did not prevail.…”

The effect of aging and an oxidative stress on peroxide levels and the mitochondrial membrane potential in isolated rat hepatocytes

“…Fluorochromes were excited at 488 nm with an argon laser at 200 mW. A forward angle light scatter (FALS) and right angle light scatter (90LS) were used to select live or dead hepatocytes [27]. The fluorescence was collected through a 488 nm blocking filter, a 560 nm long pass dichroic mirror and a 520 nm short pass filter (RH‐123, DCHF‐DA) or a 570 nm long pass filter (PI).…”

Mechanisms of action for aryl hydrocarbon receptor ligands in the PLHC-1 cell line

“…Therefore, the aphidicolin double block is presently the method of choice for synchronizing PLHC-l cells, although the potential utility of several other methods has yet to be assessed. (Miller 1983), and the technique has been applied to populations of primary hepatocytes (Black et al 1993).…”