Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocyte suspensions and cultures.
Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocyte suspensions and cultures.
复制标题
用于评估角质形成细胞悬浮液和培养物中细胞色素 P-450 依赖性单加氧酶活性的每细胞荧光测定。
DOI：
10.1016/0003-2697(90)90613-e
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发表时间：
1990
影响因子：
2.9
通讯作者：
Laskin,DL
中科院分区：
文献类型：
--
作者：
ReinersJr,JJ;Cantu,AR;Pavone,A;Smith,SC;Gardner,CR;Laskin,DL
一种便于对小鼠表皮角质形成细胞(MEKS)全细胞悬液和培养物中细胞色素P-450依赖的单加氧酶活性进行逐细胞估计的方法具有一定的特点。7-乙氧基香豆素O-脱乙基酶(7-ECD)、7-乙氧基间苯二酚O-脱乙基酶(7-ERD)和7-戊氧基间苯二酚O-脱乙基酶(7-PRD)的活性通过荧光检测进行检测。通过冷冻-解冻循环使水貂具有渗透性，异物代谢在原位发生。用贴在培养皿上的细胞对培养的水貂进行分析。产物的形成与MEK细胞数量和检测时间成正比，并依赖于NADPH生成系统。这三种单加氧酶活性均受到α-萘黄酮和美曲酮的不同程度抑制，且呈剂量依赖关系。从一只老鼠身上获得的MEK数量足以进行多次分析。该方法还用于测定大鼠肝细胞全细胞悬液中的单加氧酶活性。每肝细胞的7-ECD、7-PRD和7-ERD的构象活性分别是背部鱼悬液中测得的活性的357、96和1926倍。
An assay was characterized that facilitated per-cell estimation of cytochrome P-450-dependent monooxygenase activities in whole-cell suspensions and cultures of murine epidermal keratinocytes (MEKs). 7-Ethoxycoumarin O-deethylase (7-ECD), 7-ethoxyresorufin O-deethylase (7-ERD), and 7-pentoxyresorufin O-deethylase (7-PRD) activities were monitored by fluorescent detection of their products. MEKs were made permeable by a freeze-thaw cycle, and xenobiotic metabolism occurred in situ. Analyses of cultured MEKs were made with the cells attached to the culture dishes. Product formation was proportional with MEK cell number and assay time and was dependent upon a NADPH-generating system. The three monooxygenase activities were inhibited to various degrees, in a dose-dependent manner, by the P-450 inhibitors α-naphthoflavone and metyrapone. The number of MEKs obtained from a single mouse was sufficient for multiple analyses. The assay was also used to determine monooxygenase activities in whole-cell suspensions of rat hepatocytes. Consitutive per hepatocyte 7-ECD, 7-PRD, and 7-ERD activities were 357-, 96-, and 1926-fold greater, respectively, than the activities measured in suspensions of dorsal MEKs.