Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocyte suspensions and cultures.
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 alpha-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. Constitutive 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.
Reiners
J J
JJ
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957.
Cantu
A R
AR
Pavone
A
A
Smith
S C
SC
Gardner
C R
CR
Laskin
D L
DL
D015660:7-Alkoxycoumarin O-Dealkylase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005453:Fluorescence; D015603:Keratinocytes / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001571:Benzoflavones; D065607:Cytochrome P-450 Enzyme Inhibitors; D009249:NADP; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1