A Practical Approach, Second Edition=Ronald D. Ho.pdf

536 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONSimilarly, tert-butyl hydroperoxide or cumene hydroperoxide are less efficient in supporting peroxidaseactivity of PGS. The peroxidase activity of PGS requires the presence of PGG 2 or PUFAhydroperoxide in the medium. 27,28 Since these hydroperoxides are generated by the dioxygenaseactivity, cooxidation of xenobiotics by PGS and LO is routinely assayed using reaction mediasupplemented with PUFA alone. Peroxide specificity of LO is relatively broad. Not only H 2 O 248and PUFA hydroperoxides 29–31 but either tert-butyl hydroperoxide or cumene hydroperoxide 49 canalso efficiently support the cooxidase activity of LO.7. Epoxide HydraseThe procedure of Oesch et al., 50 which employs styrene oxide, is commonly used to assay epoxidehydrase (EH; E.C. 3.3.2.3) activity of tissue microsomes. The assay can also be performed using[ 3 H]BP-4,5-oxide.a. Reagents1. 7-[ 3 H]styrene oxide, 16 mM, in tetrahydrofuran containing 0.1% triethylamine2. 0.5 M tris-HCl buffer, pH 8.7b. ProcedureAdd substrate solution (20 µl) to the suitable amount of enzyme in buffer (300 µl final volume)and incubate for 5 to 10 min at 37˚C. The reaction is terminated by addition of 5 ml petroleumether (bp 30˚C to 60˚C) to the test tube. The mixture is vortexed and then centrifuged for 5 minat 1000 × g. The tube is then cooled by immersion in an ice-acetone bath. After the aqueous phaseis frozen, the upper organic phase is discarded. This extraction procedure is repeated twice. Finally,2 ml of ethyl acetate is added, and the contents are mixed and centrifuged as before. An aliquot(300 µl) of the ethyl acetate extract containing styrene glycol is transferred to a scintillation vialfor radioactivity counting. The incubation mixture containing boiled enzyme (100˚C for 10 min)or no enzyme is used as the control.c. RemarksEH activity has been observed in rodent liver nuclei, mitochondria, and microsomes (mEH) as wellas cytosol (sEH). Of these, the microsomal and cytosolic forms have received the most attention.According to some reports, selective substrates for mEH include phenanthrene oxide, BP-4,5-oxide,and cis-stilbene oxide while sEH hydrolyzes trans-stilbene oxide and trans-β-benzyl styrene oxidemore efficiently. Most substrates are potentially carcinogenic; therefore, routine precautions forcarcinogen and radioisotope handling should be taken.8. Glutathione TransferaseA number of assay procedures are available for assaying GST activity. This includes estimation ofdepletion of GSH or test substrate from the medium or quantitation of specific product formationfrom the test substrate, either by radiometry, spectrophotometry, or HPLC. The most commonlyused spectrophotometric method, described by Habig et al. 51 and modified by Radulovic andKulkarni 52 for human placental GST (GSTP1-1), is described below.a. Reagents1. 0.2 M 1-chloro-2,4-dinitrobenzene (CDNB) solution in acetone or ethanol2. 0.1 M potassium phosphate buffer, pH 6.53. 25 mM reduced glutathione solution in buffer4. Enzyme: purified or cytosol (100,000 × g supernatant)© 2006 by Taylor & Francis Group, LLC