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.

Fluorescence-activated cell sorting (FACS) was used to establish clonal populations of a human lymphoblastoid cell strain that contain spontaneously occurring and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in the lambda immunoglobulin gene. Multiple rounds of FACS using a monoclonal antibody specific for the membrane-expressed human lambda immunoglobulin were used to enrich the population fraction of cells lacking a wild-type lambda immunoglobulin on the cell surface. Approximately 20% of the clonal populations established after five rounds of FACS-mediated enrichment did not express the lambda immunoglublin epitope recognized by the monoclonal antibody used for selection. However, evaluation of the FACS-selected mutant clonal populations with a polyclonal antilambda antibody, or a monoclonal antibody directed against a different epitope on the lambda immunoglobulin made by the T5-1 cells, indicated that the mutant clonal populations expressed lambda immunoglobulin on their cell surfaces. Additionally, the presence of lambda mRNA and of both secreted and cytoplasmic lambda protein confirmed the transcription and translation of the lambda immunoglobulin gene. These data suggest that FACS-mediated selection employing epitope-specific monoclonal antibodies provides a powerful technique for isolation of cell populations that express mutations within the coding region of the lambda immunoglobulin gene.

The genomic evolution of a cohort of WB-F344 rat liver epithelial cell lineages undergoing spontaneous neoplastic transformation was followed to define the mechanistic relationship between genomic instability and progression to the neoplastic phenotype. Eighteen independent populations of WB-F344 cells (initiated from a single diploidfounding population) were subjected to 12 cycles of selective growth at confluent cell density, and cellular DNA contents were measured after each selection cycle. Flow cytometry demonstrated significant gains in the amount of G 1 DNA after selection cycles 3, 6, and 7 in 44% (8 of 18), 89% (16 of 18), and 39% (7 of 18) of the cell populations, respectively. All populations subsequently lost DNA and returned to a diploid or pseudo-diploid DNA content within 1 to 2 selection cycles after the appearance of an increased DNA content. Additionally, appearance and subsequent disappearance of aneuploid or tetraploid subpopulations was observed in 11% (2 of 18) and 83% (15 of 18) of the experimental lineages, respectively. Although perturbations of G 1 DNA content were apparent as early as selection cycle 3, at least 8 cycles of selective growth were required for the acquisition of tumorigenicity. While the independent lineages demonstrated significant fluctuations in G 1 DNA content between selection cycles 3 and 8, the majority (11 of 13) of the populations contained a diploid or pseudodiploid DNA content at the time tumorigenicity was expressed. Genomic instability preceded the acquisition of tumorigenic potential in rat liver epithelial cells subjected to selective growth conditions of maintenance at confluence, and may be required for its expression. (HEPATOLOGY 1998;28:78-85.) DNA content analysis by flow cytometry is a useful prognostic indicator of neoplasia. In many solid tumors, a diploid DNA pattern is associated with better patient survival than an aneuploid DNA pattern, which often predicts poor patient survival. Flow cytometric analyses of the relationship between DNA ploidy pattern and patient prognosis in human hepatocellular carcinoma have produced conflicting results. A positive correlation has been reported between a diploid DNA pattern, small tumor size (Ͻ5 cm), and low tumor grade. [1][2][3][4][5] However, the significance of this correlation as it relates to patient prognosis is not clear. Tumor diploidy has been demonstrated to be positively correlated with long-term patient survival in some, [2][3][4]6,7 but not other studies. 1,5,[8][9][10] In fact, Ng et al. 11 found that a diploid DNA pattern was associated with poorer patient survival than was an aneuploid pattern in large hepatocellular carcinomas. A diploid DNA content in a tumor does not preclude the possibility that small chromosomal alterations such as balanced translocations, point mutations, and deletions are present in the tumorigenic cells. Such subtle lesions may not be detected by flow cytometry. Alternatively, the finding of diploidy in a tumor may not preclude that the tumorigenic cells underwent a ...

Four cases of primary non‐Hodgkin's lymphoma (NHL) of the thyroid were studied using flow cytometric (FCM) DNA analysis of propidium iodide‐stained nuclei retrieved from formalin‐fixed, paraffin‐embedded tissue. Two of the four cases were aneuploid and two were euploid. In the two euploid cases, both patients are alive and without evidence of recurrent disease after an average of 4 years follow‐up. Of the two aneuploid cases, one patient is alive and free of recurrent disease after 1 year. In the other aneuploid case, the patient died of disseminated disease 8 months after presentation despite having a low‐grade (follicular, predominantly small cleaved cell type) and low‐stage (tumor confined to thyroid at presentation) lymphoma. These data suggest that the DNA ploidy of primary NHL of the thyroid can be determined using fixed, paraffin‐embedded tissue. Our results also suggest that a large study to assess the prognostic value of this technique is warranted.