Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives

Rauschenbach, Reimund; Gieschen, Hille; Husemann, Manfred; Salomon, Birgit; Hildebrand, Michael S.

doi:10.1016/0926-6917(95)00016-x

Cited by 16 publications

(1 citation statement)

References 23 publications

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“…These cells express POR and cytochrome b5 (necessary for CYP3A activity) endogenously but have low CYP levels. Therefore, V79 cells have often been used to study human and rat CYP enzymes 47–50 , a bovine CYP3A4-like isoform 51 and equine isoforms CYP3A89, CYP3A94 and CYP3A95 52 . Despite endogenous cytochrome P450 oxidoreductase (POR), we chose to co-express each CYP3A isoform with POR to guarantee maximal CYP3A activity, for which TST and NIF were selected as marker substrates.…”

Section: Discussionmentioning

confidence: 99%

Functional impact of cytochrome P450 3A (CYP3A) missense variants in cattle

Giantin

Rahnasto-Rilla

Tolosi

et al. 2019

Sci Rep

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Cytochrome P450 3A is the most important CYP subfamily in humans, and CYP3A4/CYP3A5 genetic variants contribute to inter-individual variability in drug metabolism. However, no information is available for bovine CYP3A (bCYP3A). Here we described bCYP3A missense single nucleotide variants (SNVs) and evaluated their functional effects. CYP3A28, CYP3A38 and CYP3A48 missense SNVs were identified in 300 bulls of Piedmontese breed through targeted sequencing. Wild-type and mutant bCYP3A cDNAs were cloned and expressed in V79 cells. CYP3A-dependent oxidative metabolism of testosterone (TST) and nifedipine (NIF) was assessed by LC-MS/MS. Finally, SNVs functional impact on TST hydroxylation was measured ex vivo in liver microsomes from individually genotyped animals. Thirteen missense SNVs were identified and validated. Five variants showed differences in CYP3A catalytic activity: three CYP3A28 SNVs reduced TST 6β-hydroxylation; one CYP3A38 variant increased TST 16β-hydroxylation, while a CYP3A48 SNV showed enhanced NIF oxidation. Individuals homozygous for rs384467435 SNV showed a reduced TST 6β-hydroxylation. Molecular modelling showed that most of SNVs were distal to CYP3A active site, suggesting indirect effects on the catalytic activity. Collectively, these findings demonstrate the importance of pharmacogenetics studies in veterinary species and suggest bCYP3A genotype variation might affect the fate of xenobiotics in food-producing species such as cattle.

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Section: Discussionmentioning

confidence: 99%

Functional impact of cytochrome P450 3A (CYP3A) missense variants in cattle

Giantin

Rahnasto-Rilla

Tolosi

et al. 2019

Sci Rep

View full text Add to dashboard Cite

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CytochromeP450 Enzymes

Hrycay

Bandiera

2010

Pharmaceutical Sciences Encyclopedia

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The cytochrome P450 (CYP) enzymes, also known as the microsomal mixed function oxidase system, are the predominant biotransformation pathway in the body for lipid‐soluble endogenous and xenobiotic compounds. CYP enzymes play a key role in the biotransformation of pharmaceutical agents and thus are major determinants of duration of action and clearance of drugs. This article focuses on the characterization, function, and regulation of CYP enzymes that are relevant to our understanding of how CYP enzymes can metabolize a large variety of drugs.

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Cluster Screening: An Effective Approach for Probing the Substrate Space of Uncharacterized Cytochrome P450s

2013

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Cytochrome P450 monooxygenases (P450s) are versatile enzymes with high potential for biocatalysis. The number of newly annotated P450 genes has been increasing constantly, and these thus represent a rich resource for new biocatalysts. However, the substrate scopes of newly identified P450s are often not known, and thus their exploitation is difficult. Herein we describe an approach, named "cluster screening", and its application for the primary characterization of two P450s: CYP154E1 and CYP154A8. A library comprising 51 compounds was designed and organized into nine groups according to their chemical properties. The activities of both P450s in vitro were maintained with suitable nonphysiological redox partners, and the cluster library was screened with these enzymes for product formation. From this library, 30 compounds tested positive for CYP154E1 and 23 were positive for CYP154A8. Cluster screening distinguishes subtle differences in activity and selectivity of enzymes as closely related as those of the same P450 family. For example, the alkaloid pergolide mesylate was converted by CYP154E1 (4 %) but not by CYP154A8. A building block of vitamin D3 , Grundmann's ketone, was converted by both enzymes, although conversion was higher with CYP154E1 (100 vs 53%).

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Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives

Cited by 16 publications

References 23 publications

Functional impact of cytochrome P450 3A (CYP3A) missense variants in cattle

Functional impact of cytochrome P450 3A (CYP3A) missense variants in cattle

CytochromeP450 Enzymes

Cluster Screening: An Effective Approach for Probing the Substrate Space of Uncharacterized Cytochrome P450s

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