Knockout Mice in Xenobiotic Metabolism

Henderson, Colin J.; Otto, Diana M. E.; McLaren, Aileen; Carrie, Dianne; Wolf, C. Roland

doi:10.1081/dmr-120026869

Cited by 14 publications

(9 citation statements)

References 17 publications

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“…5B). A similar staining pattern has been observed in the HRN model at early developmental stages and attributed to low levels of Cre expression (Henderson et al, 2003b;Gu et al, 2007). Consistent with previous reports, the loss of POR was accompanied by a marked increase in the expression of cytochromes P450 (Fig.…”

Section: Fig 3 Hepatic Deletion Of Murine Cytochrome P450 Oxidoredusupporting

confidence: 78%

Conditional Deletion of Cytochrome P450 Oxidoreductase in the Liver and Gastrointestinal Tract: A New Model for Studying the Functions of the P450 System

Finn¹,

McLaren²,

Carrié³

et al. 2007

J Pharmacol Exp Ther

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We have previously described a mouse model, where hepatic cytochrome P450 oxidoreductase (POR) expression has been deleted, resulting in almost complete ablation of hepatic P450 function [Hepatic P450 Reductase Null (HRN)]. HRN mice grow normally but develop fatty livers, and they have increased cytochrome P450 levels. Associated with the hepatic lipid accumulation are significant changes in the expression of genes controlling lipid homeostasis. We have characterized this model extensively and demonstrated its value in drug efficiency testing, in toxicokinetics, and in evaluating the role of the hepatic P450 system in drug pharmacokinetics. To extend the deletion of POR, and P450 inactivation, to other tissues, and to develop the utility of this model, we have generated a mouse where POR can be deleted conditionally in the liver and gastrointestinal tract using the rat cytochrome P450 CYP1A1 promoter to drive Cre recombinase expression. Administration of the CYP1A1 inducers tetrachlorodibenzo-p-dioxin or ␤-naphthoflavone resulted in both hepatic and gastrointestinal deletion of POR, whereas administration of 3-methylcholanthrene resulted specifically in loss of hepatic POR expression. In all cases, the resulting hepatic phenotype seemed identical to that of the HRN model, including increased cytochrome P450 expression. Hepatic deletion of POR and the subsequent increase in P450 expression were dependent on inducer dose, with maximal POR deletion occurring at a single dose of 3-methylcholanthrene of 40 mg/kg. This model provides a powerful approach for studying the functions of POR as well as in the evaluation of the role of hepatic and gastrointestinal P450s in drug deposition and chemical toxicity.Mammalian cytochromes P450 (P450s) play a major role in the metabolism and deposition of drugs and environmental chemicals (Rushmore and Kong, 2002). Understanding their functions in drug deposition is now a key factor in drug development and use. In the past, the only means of evaluating the P450 system in vivo in the deposition and toxicity of a drug was through the use of cytochrome P450 inhibitors (Fontana et al., 2005). This approach presents many difficulties for the interpretation of results, particularly as these inhibitors do not inhibit all P450 activity (Emoto et al., 2003), their effects are transient (Mugford et al., 1992), and they will inhibit other pathways, such as the function of drug transporters (Weiss et al., 2006). To circumvent these problems, we have developed a mouse where hepatic P450 activity is reduced by greater than 95% by the conditional deletion of cytochrome P450 oxidoreductase (POR). This was achieved by crossing mice carrying the Por gene flanked with loxP sites with mice expressing Cre recombinase under the control of the rat albumin promoter (Gu et al., 2003;Henderson et al., 2003a). Associated with the loss of hepatic POR, profound changes in the metabolism and deposition of a wide range of drugs and environmental chemicals have been documented (Arlt et al., 2005;Pass et a...

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Section: Fig 3 Hepatic Deletion Of Murine Cytochrome P450 Oxidoredusupporting

confidence: 78%

Conditional Deletion of Cytochrome P450 Oxidoreductase in the Liver and Gastrointestinal Tract: A New Model for Studying the Functions of the P450 System

Finn¹,

McLaren²,

Carrié³

et al. 2007

J Pharmacol Exp Ther

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“…Therefore, we looked at the in vivo situation. Mice carrying a deletion in the hepatic POR gene (30,38), and thus lacking POR and POR-mediated cytochrome P450 activity in the liver, were treated with 3-NBA. No differences in DNA adduct formation by 3-NBA were observed in liver, lung, kidney, bladder, or colon of hepatic POR-null and wild-type mice, emphasizing the major importance of cytosolic nitroreductases and phase II enzymes in the activation of 3-NBA.…”

Section: Discussionmentioning

confidence: 99%

Environmental Pollutant and Potent Mutagen 3-Nitrobenzanthrone Forms DNA Adducts after Reduction by NAD(P)H:Quinone Oxidoreductase and Conjugation by Acetyltransferases and Sulfotransferases in Human Hepatic Cytosols

et al. 2005

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3-Nitrobenzanthrone (3-nitro-7H-benz [de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the 32 P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA-DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,Oacetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA-DNA adduct formation increased 10-to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs. (Cancer Res 2005; 65(7): 2644-52)

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“…In this study, a higher level of P450R was observed in the normal tissue compared to the RCC. Henderson et al (2003) have recently reported an almost five-fold increase in P450 levels in P450R knockout mice, although the mechanism of action is unknown. Little is known about the regulatory mechanisms governing P450 enzymes in tumour tissue, and much remains to be elucidated about both the function and the mechanisms regulating their expression.…”

Section: Discussionmentioning

confidence: 99%

Cytochrome P450 CYP1B1 activity in renal cell carcinoma

2004

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Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n ¼ 11). Measurable levels of active P450R were determined in all normal (n ¼ 11) and tumour samples (n ¼ 26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.

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Knockout Mice in Xenobiotic Metabolism

Cited by 14 publications

References 17 publications

Conditional Deletion of Cytochrome P450 Oxidoreductase in the Liver and Gastrointestinal Tract: A New Model for Studying the Functions of the P450 System

Conditional Deletion of Cytochrome P450 Oxidoreductase in the Liver and Gastrointestinal Tract: A New Model for Studying the Functions of the P450 System

Environmental Pollutant and Potent Mutagen 3-Nitrobenzanthrone Forms DNA Adducts after Reduction by NAD(P)H:Quinone Oxidoreductase and Conjugation by Acetyltransferases and Sulfotransferases in Human Hepatic Cytosols

Cytochrome P450 CYP1B1 activity in renal cell carcinoma

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