Simultaneous determination of cytosolic glutathione S-transferase and microsomal epoxide hydrolase activity toward benzo[a]pyrene-4,5-oxide by high-performance liquid chromatography.
Cytosolic glutathione S-transferase (GST) and microsomal epoxide hydrolase (EH) are important detoxification enzymes for many epoxide xenobiotics. We have developed a rapid, simple, and convenient HPLC assay which measures both of these enzyme activities toward benzo[a]pyrene-4,5-oxide (BaPO) in tissue homogenates. Tissue fractions were incubated at 37 degrees C in the presence of 5 mM glutathione. Reactions were initiated by addition of BaPO and terminated by the addition of ice-cold acetonitrile containing 2-methoxynaphthalene as an internal standard. Samples were analyzed directly on a 15-cm C18 reverse-phase column at room temperature, with a ternary solvent program which utilized 0.01% ammonium phosphate buffer (pH 3.5), acetonitrile, and water. The uv absorbance (260 nm) was monitored. Baseline resolution of BaPO, BaPO-GSH, and BaPO-diol and the internal standard was accomplished in 10 min. In rat hepatic S9, production of both BaPO-GSH and BaPO-diol was linear with time and protein up to 15 min and 500 micrograms/ml, respectively. Coefficients of variation for replicate analyses were 2.7 and 3.7% for GST and EH activities in S9, respectively. With fluorescence detection (ex, 241; em, 389 nm), this assay was sensitive enough to measure GST and EH activities in mononuclear leukocytes (MNL). GST and EH activities in 109 human MNL samples were 142 +/- 74 (mean +/- SD; range 21-435) pmol/mg/min and 19 +/- 9 (mean +/- SD; range 3-59) pmol/mg/min, respectively. These results demonstrate the simplicity, high sensitivity, and applicability of this assay for a broad range of tissues.
Eaton
D L
DL
Department of Environmental Health, University of Washington, Seattle 98195.
Stapleton
P L
PL
D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D004851:Epoxide Hydrolases / Q000032:analysis*; D005982:Glutathione Transferase / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007962:Leukocytes / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains
D001580:Benzopyrenes; C006707:benzo(a)pyrene 4,5-epoxide; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases