A Simple and Sensitive Assay of 7-Ethoxycoumarin in Deethylation

Aitio, Antero

doi:10.1007/978-3-642-66896-8_53

Cited by 71 publications

(80 citation statements)

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“…After 24-96 h in culture, cells were scraped, washed, suspended in 200 l l PBS and sonicated. COH activity was measured as described previously (Aitio 1978) using 100 l M coumarin as substrate. CYP 1 A 12-mediated 7-ethoxyresorufin O-deethylase (EROD) and CYP 2 B 9/10-mediated 7-pentoxyresorufin O-deethylase (PROD) activities were measured as described (Burke et al 1977), using 1 l M ethoxyresorufin and 1 l M penthoxyresorufin as substrates.…”

Section: Methodsmentioning

confidence: 99%

Regulation of CYP2A5 induction by porphyrinogenic agents in mouse primary hepatocytes

Salonpää

Kottari

Pelkonen

et al. 1996

Naunyn-Schmiedeberg's Arch Pharmacol

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All cytochrome P450 (CYP) enzymes contain heme as a prosthetic group. In contrast to other CYP enzymes, murine CYP 2 A 5 is upregulated in vivo by several agents that disturb cellular heme balance. To test the hypothesis that porphyrinogenic agents have the common feature of being able to increase CYP 2 A 5 expression, mouse liver primary hepatocytes were exposed to various porphyrinogenic chemicals and changes in CYP 2 A 5 catalytic activity and levels of mRNA were monitored. Phenobarbital increased hepatic CYP 2 A 5-mediated coumarin 7-hydroxylase (COH) activity (13.2-fold) and the amount of CYP 2 A 5 steady-state mRNA (10.6-fold). Hepatocyte COH activity was increased also by the ferrochelatase inhibitor griseofulvin and the protoporphyrinogen oxidase inhibitor acifluorfen (about 9-fold induction). Of these inducers, only phenobarbital affected CYP 1 A 12 and CYP 2 B 10 expression. In contrast, many other porphyrinogenic agents such as cobalt, 2,2,4-trimethyl-1,2-dihydroquinoline (TMDQ), 1-[4-(3-acetyl-2,4,6-trimethylphenyl)-2,6-cyclohexanedionyl]-O-eth yl propionaldehyde oxime (ATMP), aminotriazole, and thioacetamide either decreased or had no effect on CYP 2 A 5. The increases in COH activity and CYP 2 A 5 mRNA were unaffected by combined treatment with the inducers and heme arginate, suggesting that heme is not a regulator of CYP 2 A 5 induction. Treatment with actinomycin D totally abolished both constitutive CYP 2 A 5 expression and its inducibility, suggesting that a transcriptional component is involved. These data suggest that, in mouse primary hepatocytes, CYP 2 A 5 induction is not a universal response to disturbed cellular heme biosynthesis.

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Section: Methodsmentioning

confidence: 99%

Regulation of CYP2A5 induction by porphyrinogenic agents in mouse primary hepatocytes

Salonpää

Kottari

Pelkonen

et al. 1996

Naunyn-Schmiedeberg's Arch Pharmacol

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“…All CYP2A isoforms appear to catalyze coumarin 7-hydroxylation (a marker substrate) but exhibit regioselectivity of the hydroxylation at all six possible positions [117]. Using coumarin 7-hydroxylase activity as a measure of overall CYP2A function [118] in mouse, rabbit, dog, and monkey liver microsomes, it was found that these species had approximately 18%, 8.7%, 2.9%, and 75%, respectively, of the activity found in HLM preparations. In the same study, coumarin 7-hydroxylase activity in rats was found to be below the limit of quantification [6], and in a similar study, it was found to be detectable only when large amounts of substrate were used [119].…”

Section: Cyp1bmentioning

confidence: 99%

Species Differences of Drug‐Metabolizing Enzymes

DeKeyser

Shou

2012

Encyclopedia of Drug Metabolism and Interactions

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The prediction of human pharmacokinetics, efficacy, and toxicity from preclinical data remains an important goal in the drug discovery and development process to minimize risk to participants during first in human studies. Animal models are commonly used in the preclinical development of new drugs; however, significant differences exist between species. These are due largely to alterations in isoform composition, regulation, expression, and catalytic activities of drug‐metabolizing enzymes (DMEs), particularly cytochromes P450 (CYPs), which play a critical role in numerous oxidative reactions of drugs. Although the reliability of animal testing to predict concentration‐dependent efficacy and toxicity in humans has been questioned; it is generally believed that data from animal studies can be reasonably extrapolated to humans using appropriate drug metabolism and pharmacokinetic principles. In this chapter, the authors describe the similarities and differences of major CYPs and other enzymes in their gene and protein structure, catalytic activity, enzyme kinetics and inhibition, and induction response between the various preclinical species and humans. These comparisons help to better understand underlying mechanisms responsible for the differences and address the question of whether animal data can be extrapolated to humans with regard to drug absorption, distribution, metabolism, and excretion. In conclusion, the DMEs show appreciable interspecies differences from structure to function, and caution must be taken when extrapolating metabolism data from animal models to humans.

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“…EROD was measured in the 9000g supernatants according to Pohl and Fouts [43] with modifications, PROD as described by Lubet et al [44] fluorometrically determining the metabolite resorufin. With ECOD the main metabolite 7-hydroxycoumarin was measured fluorometrically [45]. PNPH was performed according to Chang et al [46] determining the pnitrocatechol metabolite spectrophotometrically.…”

Section: Cytochrome P4s0 Mediated Monooxygenase Functionsmentioning

confidence: 99%

Single and chronic administration of ciprofibrate or of ciprofibrate-glycinate in male Fischer 344 rats: Comparison of the effects on morphological and biochemical parameters in liver and blood

Lupp

Karge

Danz

et al. 2005

European Journal of Drug Metabolism and Pharmacokinetics

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Fibrates lead to a reduction of serum triglycerides and cholesterol in hyperlipidemic patients. Their therapeutic use, however, can be associated with adverse effects like gastrointestinal disorders, myalgia, myositis and hepatotoxicity. Large doses can even cause hepatocellular carcinoma in rodents. Additionally, interactions with the biotransformation of other compounds at the cytochrome P450 (CYP) system have been observed. Thus, the discovery of new derivatives with less of these side effects is of great interest. In the present study a single (10 mg/kg body weight) or a 4-week (1 or 10 mg/kg body weight daily) oral administration of ciprofibrate or of the newly synthesized ciprofibrate-glycinate was investigated in adult male Fischer 344 rats. Serum lipid concentrations were distinctly decreased after single but only slightly after chronic administration of the two fibrates, whereas liver parameters revealed a slight concentration and time dependent hepatotoxicity. Histologically, a hypertrophy, an eosinophilia, a reduced glycogen content and also an apoptosis of the hepatocytes was observed. Effects were more pronounced after chronic treatment and after application of the higher dosage. All CYP enzymes investigated were induced in a time and concentration dependent manner. Resulting CYP mediated monooxygenase and oxidase activities showed a dependency both on enzyme induction and hepatotoxic effects. With no parameter investigated major differences were seen between ciprofibrate and ciprofibrate-glycinate. Thus, the present investigations revealed no noticeable advantages of ciprofibrate-glycinate over its parent compound ciprofibrate.

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A Simple and Sensitive Assay of 7-Ethoxycoumarin in Deethylation

Cited by 71 publications

References 0 publications

Regulation of CYP2A5 induction by porphyrinogenic agents in mouse primary hepatocytes

Regulation of CYP2A5 induction by porphyrinogenic agents in mouse primary hepatocytes

Species Differences of Drug‐Metabolizing Enzymes

Single and chronic administration of ciprofibrate or of ciprofibrate-glycinate in male Fischer 344 rats: Comparison of the effects on morphological and biochemical parameters in liver and blood

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