Ethoxycoumarin-O-deethylase

An assay for ethoxycoumarin O-deethylase is useful in as a part of a battery of tests for cytochrome P450 activities in a variety of tissues. The deethylase activity results in the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin. A postcolumn pH shift allows the product to be detected by fluorescence. 

The product, 7-hydroxycoumarin, was separated on a Nov-Pak C8 column (3.9 mm X 150 mm). The mobile phase, adjusted to pH 3.5, was a 35 65 mixture of methanol and 1% acetic acid. The flow rate was 1 mL/min. The postcolumn eluate was mixed with 1.0JV NaOH pumped at a flow rate of 0.5 mL/min. Complete mixing occurred via a T-joint followed by 1.5 m of Teflon tubing. The fluorometer was set to use excitation and emission wavelengths of 368 and 456 nm, respectively. 

SURVEY OF ENZYMATIC ACTIVITIES ASSAYED BY THE HPLC METHOD 

The reaction mixture contained in a total volume of 1.0 mL 80 /u.mol of potassium phosphate buffer (pH 7.4), 5 /nmol of magnesium chloride, 1 mg of bovine serum albumin, 0.5 /nmol of NADPH, 0.5 /nmol of NADH, and 0.1 mL of microsomal suspension. The reaction was initiated by the addition of 20 /nL of 7-ethoxycoumarin (0.43 /nmol) dissolved in 50% methanol. After incubation for 10 to 20 minutes at 37°C, the reaction was stopped by the addition of 125 /nL of ice-cold 15% (w/v) trichloroacetic acid. The resulting mixture was extracted with 2 mL of chloroform. An aliquot (0.75 mL) of the organic phase was dried under nitrogen and the residue resuspended in 50 /nL of HPLC-grade methanol. Formation of product was linear with up to 5 fig of protein. 

The source of the enzyme assayed was microsomes prepared from liver and intestinal epithelial cells. 

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Whole liver homogenates had increases of 300-fold in 7-ethoxycoumarin O-deethylase and 75-fold in AHH activities between days 5 and 10 LD50 by time of hatch... 

Brunstrom, B. 1986. Activities in chick embryos of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon (benzo[a]pyrene) hydroxylase and their induction by 3,3, 4,4 -tetrachlorobiphenyl in early embryos. Xenobiotica 16 865-872. 

In this study, P-450-related enzyme activities (benzphetamine N-demethylase, 7-ethoxycoumarin O-deethylase) were also measured in liver homogenates (prepared 24 hours after the last treatment) from rats treated orally with MEK for 1-7 days and compared to the activity obtained with phenobarbital treatment (80 mg/kg intraperitoneally for 3 days) (Robertson et al. 1989). Total cytochrome P-450 was also measured. No consistent change was noted in benzphetamine N-demethylase activity as the result of MEK treatment, while 7-ethoxycoumarin O-deethylase was over 3 times higher than controls and comparable to phenobarbital induction. Total P-450 levels were increased to approximately 150-200% of controls with MEK and to 570% of control by phenobarbital. The authors concluded that the potentiating effects of MEK on the neurotoxicity of -hexane appear to arise, at least in part, from the activating effects of MEK on selected microsomal enzymes responsible for -hexane activation. 

Moon, J.Y., Lee D.W., and Park, K.H., Inhibition of 7-ethoxycoumarin O-deethylase activity in rat liver microsomes by naturally occurring flavonoids structure-activity relationships, Xenobio-tica, 28, 117, 1998. 

Chui et al, [96] have reported that PCDE 74 (2,4,4, 5-tetraCDE) induce 7-ethoxycoumarin O-deethylase activities in trout and PCDE 28 (2,4,4-triCDE) and PCDE 74 (2,4,4, 5-tetraCDE) in rats. The effects of PCDEs were studied by administering PCDEs 100 mg kg 1 day 1 to rats and trout for three days. Chui et al. [96] classified PCDE 28 as a phenobarbital (PB)-type inducer and PCDE 74 a mixed-type inducer. Due to the fact that PCDEs cannot adopt planar configuration, it was suggested that PCDEs cannot act as 3-methyl chloranthrene(MC)-type inducers unlike non-orfho-PCBs. PCBs that are not acutely toxic can still induce toxic and biochemical responses and are PB-type inducers of hepatic drug-metabolizing enzymes [79]. 

AHH aryl hydrocarbon hydroxylase ECOD ethoxycoumarin O-deethylase... 

Ito, T., Y. Aoyama, K. Ishida, M. Kudoh, K. Hori, S. Tsuchiya et al. (1994). Selectivity of isoprenoid-containing imidazole antifungal compounds for sterol 14-demethylase P450 (P450(14)DM) and 7-ethoxycoumarin O-deethylase P450 of rat liver microsomes. Biochem. Pharmacol. 48, 1577-1582. 

P-450 or P-450 reductase, induction responses, spectral and binding studies, and microsomal (endoplasmic reticulum) location and requirement for NADPH, O2 etc. (Burke 1981). The induction of a specific isoenzyme of cytochrome P-450 can be detected in crude microsomal preparations by antibodies or MFO reactions specific to that isoenzyme, e.g. the 3-methylcholanthrene (3MC)-inducible type (P4501A subfamily) is detected by the ethoxyresorufin 0-deethylase (EROD) assay (Phillipson et al. 1984,1985 Stegeman et al. 1986). The presence of more than one isoenzyme of cytochrome P-450 in crude microsomes can result in multiphasic kinetics towards a particular substrate, e.g. 7-ethoxycoumarin O-deethylase activity (ECOD) (Lu and West 1980). 

BPH, benzo[a]pyrene hydroxylase (measured fluorometrically except for James and Little (1984) which was radiometric) EROD, 7-ethoxyresorufin O-deethylase ECOD, 7-ethoxycoumarin O-deethylase. 

Large sex differences have been reported in the cytochrome P450 concentration of mouse kidney microsomes and in the hydroxylation of testosterone by kidney microsomes (Hawke etal. 1983, Hawke and Welch 1985). When assayed at 500 j M, male renal 7-ethoxycoumarin-O-deethylase activity was 3-fold greater than female 7-ethoxycoumarin-O-deethylase activity, although this difference was less than that observed in cytochrome P450 concentration as indicated by an approximately 2-fold greater turnover value for female renal microsomes. 

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