Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: standardization of assay conditions

Rutten, A.A.J.J.L.; Falke, H.E.; Catsburg, J. F.; Wortelboer, Heleen M.; Blaauboer, Bas J.; Doorn, L. G. Van; Leeuwen, F.X.R. van; Theelen, Rob; Rietjens, Ivonne M. C. M.

doi:10.1007/bf02307168

Cited by 70 publications

(28 citation statements)

References 12 publications

(20 reference statements)

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“…The activities of CYP2B1/2 and CYP1A1/2 were assessed by formation of resorufin from benzyloxy-and ethoxy-resorufin as substrates, respectively (Burke et al, 1985;Rutten et al, 1992). The concentrations of resorufin were measured fluorometrically.…”

Section: Methodsmentioning

confidence: 99%

Hepatic Disposition and Effects of Nitric Oxide Donors: Rapid and Concentration-Dependent Reduction in the Cytochrome P450-Mediated Drug Metabolism in Isolated Perfused Rat Livers

Vuppugalla

Mehvar²

2004

J Pharmacol Exp Ther

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Various mechanisms, including high levels of cytokines and nitric oxide (NO), have been proposed as mediators for inflammation-induced cytochrome 450 down-regulation. However, the contribution of each of these mediators to the observed effects is controversial. We used an isolated perfused rat liver (IPRL) model to test the direct effects of NO donors on CYP450 down-regulation in the absence of cytokines or other confounding in vivo factors. Our hypothesis was that NO rapidly and concentration-dependently decreases CYP450 activities in IPRL. Livers were perfused (60 min) with 50 to 500 M sodium nitroprusside (SNP) or 100 to 500 M isosorbide dinitrate (ISDN) as NO donors, and the perfusate and biliary disposition of SNP, ISDN, and generated nitrate/nitrite (NO x ) were determined. Additionally, at the end of perfusion, catalytic activities and protein levels of various cytochrome isoenzymes were measured. Both SNP and ISDN exhibited linear hepatic disposition with extraction ratios of ϳ0.30 and 0.50, respectively. Furthermore, although in small amounts, both NO donors and NO x were found in the bile. Except for CYP2D1, the catalytic activities of all the studied isoenzymes were substantially (up to 85%) decreased by both NO donors. However, the apoprotein levels of isoenzymes remained largely unchanged. Additionally, the inhibitory effects of NO donors were concentration-dependent, with the concentrations of SNP producing one-half of maximum inhibition being in the order of 2C11 Ͼ 2B1/2 Ͼ 2E1 ϭ 3A2 Ͼ 1A1/2. These studies indicate that the effects of NO on the down-regulation of cytochrome 450 catalytic activity are rapid, concentration-dependent, and isoenzyme-selective.

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Section: Methodsmentioning

confidence: 99%

Hepatic Disposition and Effects of Nitric Oxide Donors: Rapid and Concentration-Dependent Reduction in the Cytochrome P450-Mediated Drug Metabolism in Isolated Perfused Rat Livers

Vuppugalla

Mehvar²

2004

J Pharmacol Exp Ther

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“…In both cases the formation of resorufin was monitored fluorometrically (Burke et al, 1985;Rutten et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

Short-Term Inhibitory Effects of Nitric Oxide on Cytochrome P450-Mediated Drug Metabolism: Time Dependency and Reversibility Profiles in Isolated Perfused Rat Livers

Vuppugalla

Mehvar

2004

Drug Metab Dispos

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ABSTRACT:Nitric oxide (NO) is implicated as a mediator in the decreased catalytic activities of cytochrome P450 (P450) enzymes during inflammation or infection. Here, we examined the time course and the reversibility of the NO effect on P450s using isolated perfused rat livers. Livers were perfused at a constant rate with the NO donor sodium nitroprusside (SNP) for 0.5 or 1 h, followed by washout periods of 0 to 2.5 h. At the end of perfusion, microsomes were prepared and analyzed for P450 activities and other metabolic markers. Whereas 0.5 h of NO exposure caused an irreversible decline (ϳ30%) in total P450 content, a greater decline after 1 h of NO (ϳ55%) was mostly (ϳ30%) reversible, a pattern identical to that observed for the microsomal heme content. NO exposure also caused an enzyme-selective and time-dependent decline in P450 activities. Whereas the pattern of decline and reversibility of activities were qualitatively similar for CYP3A2, 2C11, 2E1, and 1A1/2, they differed for 2B1/2 and 2D1 in that the decline in the activity was delayed (1 h) for 2B1/2 and not observed for 2D1. This may be attributed to the accessibility of heme or cysteine thiolate and/or the presence/reactivity of critical cysteinyl amino acid residues in various P450 enzymes. Additionally, for most enzymes, the activity showed a biphasic decline, one within 1 h of SNP perfusion and another after 2 h of washout. This was associated with an identical biphasic decline in the microsomal free thiols, presumably due to the rapid and slow reaction of NO and peroxynitrite, respectively, with critical P450 thiols. The short-term effects of NO on P450 are time-dependent and enzyme-selective, with both reversible and irreversible mechanisms.

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“…and then centrifuged for another 5 min. at 1200 x g. The concentration of resorufin was determined spectrofluorometrically with the excitation and emission settings at 540 nm and 580 nm, respectively (Rutten et al 1992). Standard curves were obtained in the same manner.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the P450 System in Göttingen Minipigs

Skaanild

Friis

1997

Pharmacology & Toxicology

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Absrmcf: It is essential to establish the activity and regulation of the cytochrome P450 system of species selected for toxicological and pharmacological studies. The minipig has become a popular substitute for the traditional non-rodent species although little information is available on its P450 system. The total P450 and the enzyme activity of the most important drug-metabolizing isoenzymes: CYPlA2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were measured in liver microsomes from 4 minipigs and 8 conventional pigs of both sexes. Immunochemical levels were determined for 4 of the isoenzymes. The total P450 activity was slightly higher in minipigs compared to conventional pigs but no sex difference was detected. CYPlA2 activity (7ethoxyresorufin) was 4 times higher in female minipigs than in male minipigs. The activity in male minipigs was almost identical to the activity in conventional pigs. The activity of CYP2E1 (chlorzoxazone) was 4 times higher in female than in male minipigs and 2 times higher in female than in male conventional pigs. No activity of CYP2D6 (debrisoquine) and CYP2C19 (mephenytoin) could be detected.The CYP3A4 activity (testosterone) detected in minipigs was higher than the activity in conventional pigs. A weak sex difference was Seen in both strains. Western blotting using anti-human CYF'2E1 and CYP3A4 confirmed the results obtained in the enzyme activity assays, while only CYPlA2 correlated with the activity in the conventional strain. The total P450 enzyme activity was close to the levels reported for human beings, as were the activities of CYP2El and CYP3A4.The cytochrome P450 enzymes play an important role in the disposition of drugs and other chemicals in the body. Consequently, it is essential to establish the activity and regulation of the cytochrome P450 system of species selected for toxicological and pharmacological studies. During the past the pig and especially the minipig has become a popular alternative to traditional non-rodent species in pharmacological and toxicological testing. However, little information on the P450 system is available for this species. The concentration of total P450 and the activities of CYPlA1/2 and CYP3A4 have been reported in conventional pigs by Monshouwer et al. 1996, Thomsen et al. 1991, Olsen et al. 1997and Witkamp et al. 1993, whereas only total P450 has been described for the minipigs (Fujimori et al. 1986).The objective of the present study was to characterize some of the most important drug-metabolizing P450 enzymes in the Gottingen minipig and the conventional pig and compare these with human data. Materials and MethodsAnimals. Liver microsomes were isolated from 4 Gottingen minipigs (2 males and 2 females, average b.wt. 10 kg) and 8 Danish Landrace x Yorkshire x Durn cross-breed pigs (4 males, average b.wt. 90 kg and 4 females, average b.wt. 30 kg) (Table 1). Pigs were fed commercial chow and provided with water ad libitum.Chemicals. Corticosterone and Hepes were obtained from Sigma, testosterone from Riedl-Hahn and albumin bovine fraction V fro...

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Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: standardization of assay conditions

Cited by 70 publications

References 12 publications

Hepatic Disposition and Effects of Nitric Oxide Donors: Rapid and Concentration-Dependent Reduction in the Cytochrome P450-Mediated Drug Metabolism in Isolated Perfused Rat Livers

Hepatic Disposition and Effects of Nitric Oxide Donors: Rapid and Concentration-Dependent Reduction in the Cytochrome P450-Mediated Drug Metabolism in Isolated Perfused Rat Livers

Short-Term Inhibitory Effects of Nitric Oxide on Cytochrome P450-Mediated Drug Metabolism: Time Dependency and Reversibility Profiles in Isolated Perfused Rat Livers

Characterization of the P450 System in Göttingen Minipigs

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