Massaro E J, Elstein K H, Zucker R M, Bair K W
Developmental Toxicology Division, U.S. Environment Protection Agency, Research Triangle Park, North Carolina.
Mol Toxicol. 1989 Oct-Dec;2(4):271-84.
In vitro assessment of the efficacy/capacity of toxicants (e.g., cancer chemotherapeutic agents, environmental pollutants, etc.) to damage/kill cells and/or inhibit growth (cell duplication) requires accurate measurement of target cell viability as a function of exposure. Rapid measurement of viability, such as can be achieved employing fluorescent probes of metabolic function in combination with instrumental analysis, is highly desirable. However, we observe that exposure to chemicals (of unrelated type) complicates the interpretation of viability data and, in the case of perturbed cells, questions the validity of viability growth assays based on intrinsic enzyme activity. Viability commonly is determined flow cytometrically (FCM) by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. Nonfluorescent CFDA is taken up by diffusion and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF), a negatively charged fluorescent molecule that is retained (incompletely) by the cell. As such, if CF fluorescence intensity is a relative measure of enzyme activity, it also can be considered an index of cellular vigor (metabolic rate). It is generally accepted that the viable cell excludes both basic dyes, such as PI, and acidic dyes, such as trypan blue, and uptake is indicative of irreversible cellular injury presaging cell death. We observe that, following incubation for 4 h with 0.5-1.0 microM tributyltin (TBT), a potent environmental toxicant, murine erythroleukemic cells (MELC) exhibit enhanced (supranormal) CF fluorescence compared to control cells. Apparent cell volume (ACV) is unaltered, and because such cells exclude PI, they are considered viable in terms of the CFDA/PI assay. However, rate of growth (increase in cell number over 48 h) is depressed, suggesting that supranormal CF fluorescence, even in the absence of PI uptake, is indicative of cellular perturbation. In effect, although CF fluorescence is the product of an enzyme-catalyzed reaction and, therefore, an indicator of vital function (enzyme activity), it apparently is not a reliable index of cellular vigor. At higher TBT concentrations (greater than 1.0, but less than 50.0 microM), the cells exhibit both increased CF fluorescence and PI fluorescence and are growth inhibited. MELC exposed to the cancer chemotherapeutic agents adriamycin, m-AMSA, or crisnatol (Burroughs Wellcome 770U82) also exhibit increased cellular CF fluorescence. However, rate of growth is decreased and ACV increased. The latter, measured either as a function of electrical resistance (Coulter volume) or by the FCM parameter axial light loss could account for the increase in mean CF fluorescence.(ABSTRACT TRUNCATED AT 400 WORDS)
对毒物（如癌症化疗药物、环境污染物等）损伤/杀死细胞和/或抑制生长（细胞复制）的功效/能力进行体外评估，需要准确测量作为暴露函数的靶细胞活力。快速测量活力，例如使用代谢功能荧光探针结合仪器分析来实现，是非常可取的。然而，我们观察到，暴露于（不相关类型的）化学物质会使活力数据的解释变得复杂，并且在细胞受到干扰的情况下，会质疑基于内在酶活性的活力生长测定的有效性。活力通常通过羧基荧光素二乙酸酯（CFDA）/碘化丙啶（PI）测定法进行流式细胞术（FCM）测定。非荧光的CFDA通过扩散被细胞摄取，并通过细胞质酯酶催化的水解转化为羧基荧光素（CF），一种带负电荷的荧光分子，细胞（不完全）保留该分子。因此，如果CF荧光强度是酶活性的相对度量，它也可以被视为细胞活力（代谢率）的指标。一般认为，活细胞会排斥碱性染料（如PI）和酸性染料（如台盼蓝），摄取这些染料表明细胞受到不可逆损伤，预示着细胞死亡。我们观察到，用0.5 - 1.0微摩尔三丁基锡（TBT，一种强效环境毒物）孵育4小时后，与对照细胞相比，小鼠红白血病细胞（MELC）表现出增强的（超常的）CF荧光。表观细胞体积（ACV）未改变，并且由于这些细胞排斥PI，根据CFDA/PI测定法它们被认为是活的。然而，生长速率（48小时内细胞数量的增加）受到抑制，这表明即使在没有PI摄取的情况下，超常的CF荧光也表明细胞受到了干扰。实际上，尽管CF荧光是酶催化反应的产物，因此是生命功能（酶活性）的指标，但它显然不是细胞活力的可靠指标。在更高的TBT浓度（大于1.0但小于50.0微摩尔）下，细胞同时表现出CF荧光增加和PI荧光增加，并且生长受到抑制。暴露于癌症化疗药物阿霉素、间苯二酚吖啶或克立那托（Burroughs Wellcome 770U82）的MELC也表现出细胞CF荧光增加。然而，生长速率降低，ACV增加。后者，无论是作为电阻（库尔特体积）的函数测量还是通过FCM参数轴向光损失测量，都可以解释平均CF荧光的增加。（摘要截断于400字）
