The metabolism of 7-ethoxycoumarin in primary maintenance cultures of adult rat hepatocytes

Fry, Jeffrey R.; Bridges, James W.

doi:10.1007/bf00500307

Cited by 23 publications

(5 citation statements)

References 20 publications

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“…Although earlier the same conclusion was made by Ullrich and Weber (1972) and by Aitio (1978) for microsomal preparations, the substrate has been added very often in organic solvent. Dimethylformamide has been used for hepatocyte cultures (Fry and Bridges 1980) and for isolated hepatocytes (Paterson et al 1984); methanol has been added to microsomal preparations (Boobis et al 1981, Paterson et al 1984 and to lOOOOg supernatant (Greenlee and Poland 1978).…”

Section: Discussionmentioning

confidence: 99%

Critical evaluation of 7-ethoxycoumarinO-deethylase activity measurement in intact isolated rat hepatocytes

Rogiers¹,

Adriaenssens²,

Vandenberghe³

et al. 1986

Xenobiotica

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In the determination of 7-ethoxycoumarin O-deethylase activity in intact isolated rat hepatocytes various factors influence the assay, including: the decay of 7-ethoxycoumarin fluorescence which is temperature and pH dependent; the measured fluorescence which has to be adjusted for the inner filter effect; glucose addition to the medium which influences the activity; all organic solvents which inhibit the enzymic activity, with dimethylformamide provoking the smallest effect (partial competitive inhibition); the enzymic reaction which is inhibited by the product of reaction; and the presence of bovine serum albumin in the medium which affects the enzymic activity. Biphasic kinetics are observed for the O-deethylation of ethoxycoumarin in intact isolated rat hepatocytes. For the high-affinity component, Km and Vmax values are 5 microM and 43 pmol/min X 10(6) cells and for the low-affinity component are 414 microM and 915 pmol/min X 10(6) cells. Addition of the substrate in dimethylformamide or omitting bovine serum albumin from the medium cause important changes in these kinetic parameters.

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Section: Discussionmentioning

confidence: 99%

Critical evaluation of 7-ethoxycoumarinO-deethylase activity measurement in intact isolated rat hepatocytes

Rogiers¹,

Adriaenssens²,

Vandenberghe³

et al. 1986

Xenobiotica

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“…Metabolism of 7-ethoxycoumarin has been widely studied in rat liver slices [12, 17], lung slices [16], skin strips [18], perfused intestinal loop [19], rat brain [20], and hepatocytes [21, 22]. It was reported that 7-hydroxycoumarin, coumarin-7-O-glucuronide, and coumarin-7-O-sulfate were identified in rat liver slices [12, 16, 17], lung [16], and skin [18].…”

Section: Resultsmentioning

confidence: 99%

“…It was reported that 7-hydroxycoumarin, coumarin-7-O-glucuronide, and coumarin-7-O-sulfate were identified in rat liver slices [12, 16, 17], lung [16], and skin [18]. Among them, coumarin-7-O-sulfate was a major conjugate [12, 16-18, 21], or had a comparable extent with coumarin-7-glucuronide in the intestine [19]. Only 7-hydroxycoumarin was identified in rat brain [20].…”

Section: Resultsmentioning

confidence: 99%

In vitro Drug Metabolism Investigation of 7-Ethoxycoumarin in Human, Monkey, Dog and Rat Hepatocytes by High Resolution LC-MS/MS

Feng

Wen

Stauber

2018

DML

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Most of new metabolites via oxidative ring-opening and glutathionation were identified. Species differences in metabolism of 7-ethoxylcoumarin in hepatocytes were observed. The analysis of metabolites suggests that 7-ethoxylcoumarin may undergo 3,4-epoxidation responsible for formation of glutathione and its derived cysteine conjugates, carboxylic acid and its glucuronides, glucosides and sulfate.

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“…Sulphate and glucuronide 7‐HC conjugates were then de‐conjugated to free 7‐HC by the addition of 25 μl β‐glucuronidase H1 solution (2 mg/ml in 0.1 M acetate buffer, pH 4.5) to each well and incubation of plates at 37 °C for 120 min with gentle shaking. Total 7‐HC production (representing the sum of free 7‐HC and 7‐HC retrieved from de‐conjugation reactions) was then analysed using the principles described by Fry et al (23, 24) and modified by Donato et al (23, 24) for the use of 96‐well plates. Reductions in phase II conjugation activity did therefore not cause a decrease in total 7‐HC production as this reduction caused a corresponding increase in free‐HC and no change in total 7‐HC production from 7‐EC.…”

Section: Methodsmentioning

confidence: 99%

Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°C

Wigg

Phillips

Berry

2003

Liver International

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Isolated hepatocytes in suspension provide a number of advantages for use in bioartificial liver device, however, poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. We therefore investigated the integrity and differentiated function of isolated rat hepatocytes under conditions of mild hypothermia. Isolated hepatocytes were suspended in a bicarbonate buffered saline medium, supplemented with glucose and bovine serum albumin (BSA), and maintained for 48 h at 25 degrees C on a rotary shaker under an atmosphere of 95% O2 and 5% CO2. Under these conditions there was no significant decline in cell viability and good preservation of cellular morphology on transmission electron microscopy for at least 24 h. Isolated hepatocytes in suspension at 25 degrees C were also able to maintain normal Na+ and K+ ion gradients. The cellular energy status ([ATP], ATP/ADP ratio, cytoplasmic and mitochondrial redox potentials), metabolic function (urea synthesis and ammonia removal), albumin synthesis and phase I and phase II drug detoxification activity of these cells were also maintained for at least 24 h post isolation. These observations demonstrate the robust nature of mildly hypothermic isolated hepatocytes in suspension and encourage further studies re-examining the feasibility of using this cell preparation in bioartificial livers.

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The metabolism of 7-ethoxycoumarin in primary maintenance cultures of adult rat hepatocytes

Cited by 23 publications

References 20 publications

Critical evaluation of 7-ethoxycoumarinO-deethylase activity measurement in intact isolated rat hepatocytes

Critical evaluation of 7-ethoxycoumarinO-deethylase activity measurement in intact isolated rat hepatocytes

In vitro Drug Metabolism Investigation of 7-Ethoxycoumarin in Human, Monkey, Dog and Rat Hepatocytes by High Resolution LC-MS/MS

Maintenance of integrity and function of isolated hepatocytes during extended suspension culture at 25°C

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