Cytochrome P450 Gene Induction in Rats Ex Vivo Assessed by Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (Taqman)

Baldwin, Sandra; Bramhall, Jo L.; Ashby, Charlotte A.; Lin, Yan; Murdock, Paul R.; Hood, Steve R.; Ayrton, Andrew D.; Clarke, Stephen E.

doi:10.1124/dmd.105.008185

Cited by 51 publications

(32 citation statements)

References 36 publications

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“…Indeed, consistent with previous studies in vivo (Heuman et al, 1982;Wrighton et al, 1985;Baldwin et al, 2006) and in vitro (Kocarek et al, 1995;Meredith et al, 2003), a marked increase was …”

Section: Discussionsupporting

confidence: 81%

Liverbeads: A Practical and Relevant In Vitro Model for Gene Induction Investigations

Khansa¹,

Blanck²,

Bars³

2010

Drug Metab Dispos

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ABSTRACT:Cryopreserved rat hepatocytes entrapped within an alginate matrix, commercially available as Liverbeads, were evaluated for their relevance as a screening tool for gene induction in vitro, using quantitative real-time reverse transcriptase-polymerase chain reaction. They were treated with the reference compounds ␤-naphthoflavone (BNF), phenobarbital (PB), pregnenolone 16␣-carbonitrile (PCN), and clofibric acid (CLO) and analyzed for mRNA levels of Cyp1a1, Cyp2b1, Cyp3a1, Cyp4a1, Ugt1a6, and Ugt2b1. In addition, for PB and PCN, the results were compared with those obtained in rat liver in vivo. For each inducer, the gene induction profiles obtained with the Liverbeads in vitro model were time-and dose-dependent. The in vitro gene expression profiles confirmed the corresponding known P450 and UGT induction by each reference compound.

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Section: Discussionsupporting

confidence: 81%

Liverbeads: A Practical and Relevant In Vitro Model for Gene Induction Investigations

Khansa¹,

Blanck²,

Bars³

2010

Drug Metab Dispos

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“…This technique was successfully used to compare basal gene expression levels of CYP1A2 with those induced by 3-MC, using GAPDH as a constitutively expressed control ( Figure 5). As expected, treatment with 3-MC led to induction of CYP1A2 resulting in increased gene expression levels; P ¼ 0.008, two-tailed paired t-test, which was in agreement with Baldwin et al 1 …”

Section: Process Integration On a Microfluidic Devicesupporting

confidence: 79%

“…P450 enzymes, as well as metabolizing drugs, are also involved in drug-drug interactions, in which induction or inhibition of P450 expression can result in a greater production of toxic metabolites, or a decrease in efficacy of a drug due to increased clearance. 1 There are situations in which, instead of assisting in the elimination of drugs, P450s can activate prodrugs or procarcinogens, causing them to become toxic. 3 This is the case with polycyclic aromatic hydrocarbons (PAHs), which are found in compounds such as tar.…”

mentioning

confidence: 99%

“…1 P450s are the major enzymes involved in phase I metabolism of xenobiotics, ie, any foreign chemicals that can be introduced into an organism, including deliberately administered pharmaceutical agents. 2 Phase I metabolism involves the oxidation, hydroxylation, reduction, and hydrolysis of drug molecules making them more water soluble for easy removal from the body.…”

mentioning

confidence: 99%

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Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

Shaw

Hughes

Dyer

et al. 2013

Laboratory Investigation

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This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.Laboratory Investigation (2013) 93, 961-966;

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“…Total RNA was reverse-transcribed using iScript RT-qPCR Supermix (Bio-Rad Laboratories, Hercules, CA) in a 20-l reaction volume. Primer pairs used were as follows: CYP3A2 (forward) 5Ј-GCTCTTGATG-CATGGTTAAAGATTTG-3Ј and (reverse) 5Ј-ATCACAGACCTTGCCAA CTCCTT-3Ј (Baldwin et al, 2006); CYP2C11 (forward) 5Ј-CCCTGAG-GACTTTTGGGATGGGC-3Ј and (reverse) 5Ј-AGGGGCACCTTTGCT CTTCCTC-3Ј; and ␤-actin (forward) 5Ј-ACGAGGCCCAGAGCAAGA-3Ј and (reverse) 5Ј-TTGGTTACAATGCCGTGTTCA-3Ј (Sohi et al, 2011). Real-time PCR was performed using SsoFast Evagreen (Bio-Rad Laboratories), and expression was analyzed using the ⌬⌬C T normalized to ␤-actin.…”

Section: Methodsmentioning

confidence: 99%

Down-Regulation of Hepatic CYP3A and CYP2C Mediated Metabolism in Rats with Moderate Chronic Kidney Disease

Velenosi¹,

Fu²,

Luo³

et al. 2012

Drug Metab Dispos

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ABSTRACT:Expression and activity of drug-metabolizing enzymes are decreased in severe kidney disease; however, only a small percentage of patients with chronic kidney disease (CKD) are at the final stage of the disease. This study aimed to determine the changes in drug-metabolizing enzyme function and expression in rats with varying degrees of kidney disease. Sprague-Dawley rats were subjected to surgical procedures that allowed the generation of three distinct models of kidney function: normal kidney function, moderate kidney function, and severe kidney disease. Forty-two days after surgery, rats were sacrificed and hepatic CYP3A and CYP2C expression was determined. In addition, enzymatic activity was determined in liver microsomes by evaluating midazolam (CYP3A), testosterone (CYP3A and CYP2C), and tolbutamide (CYP2C) enzyme kinetics. Both moderate and severe kidney disease were associated with a reduction in CYP3A2 and CYP2C11 expression (p < 0.05). Likewise, moderate kidney disease resulted in more than a 60% decrease in enzyme activity (V max ) for CYP2C11 and CYP3A, compared with controls (p < 0.05). When the degree of kidney disease was correlated with metabolic activity, an exponential decline in CYP2C-and CYP3A-mediated metabolism was observed. Our results demonstrate that CYP3A and CYP2C expression and activity are decreased in both moderate and severe CKD. Our data suggest that drug metabolism is significantly decreased in the earlier stages of CKD and imply that patients with moderate CKD may be subject to unpredictable pharmacokinetics.

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Cytochrome P450 Gene Induction in Rats Ex Vivo Assessed by Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (Taqman)

Cited by 51 publications

References 36 publications

Liverbeads: A Practical and Relevant In Vitro Model for Gene Induction Investigations

Liverbeads: A Practical and Relevant In Vitro Model for Gene Induction Investigations

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

Down-Regulation of Hepatic CYP3A and CYP2C Mediated Metabolism in Rats with Moderate Chronic Kidney Disease

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