Fluorometric microassay to quantify microsomal epoxide hydrolase in 96-well plates.
The microfluorometric assay, suitable for measuring microsomal epoxide hydrolase activity in cultured cells, is based on the conversion of benzo[a]pyrene-7,8-epoxide to the corresponding trans-benzo[a]pyrene-7,8-dihydrodiol, a compound that fluoresces at 403 nm when excited at 365 nm. Activity is determined by incubating S9 fractions obtained from microcultures with this epoxide and monitoring the fluorescence with a microplate reader. Under the assay conditions selected, the photodecomposition of the reaction product was minimized and the linearity of the reaction was extended. The major advantages of this method are: (1) high sensitivity with a detection limit of 5 pmol/well of trans-benzo[a]pyrene-7,8-dihydrodiol formed, which is comparable to the most sensitive radioactive methods; (2) minimal sample requirement (1-5 micrograms liver microsomes; 10-50 micrograms S9 fraction from cultured cells); (3) reduced consumption of hazardous reagents; and (4) a considerable reduction in assay time and facility for simultaneous determination of enzyme activity in multiple samples.
Herrero
M E
ME
Unidad de Hepatología Experimental, Hospital Universitario La Fe, Valencia, Spain.
Castell
J V
JV
D000818:Animals; D001580:Benzopyrenes / Q000737:chemistry*; D002478:Cells, Cultured; D003592:Cytophotometry / Q000295:instrumentation / Q000379:methods*; D004101:Dihydroxydihydrobenzopyrenes / Q000737:chemistry*; D004790:Enzyme Induction; D004851:Epoxide Hydrolases / Q000032:analysis* / Q000096:biosynthesis; D006801:Humans; D016014:Linear Models; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D015203:Reproducibility of Results
D001580:Benzopyrenes; D004101:Dihydroxydihydrobenzopyrenes; C017228:benzo(a)pyrene 7,8-dihydrodiol; C023393:benzo(a)pyrene 7,8-oxide; D004851:Epoxide Hydrolases