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Nrf2

 The effect of Nrf2 knockout on the constitutive expression of drug metabolizing enzymes and transporters in C57Bl/6 mice livers 

Anwar Anwar-Mohamed Steven R. Kleeberger b, a, Ayman Owen O.S. S. El-Kadi Degenhardt a,* 
a, Mohamed A.M. El Gendy a, John M. Seubert a, 
a Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 bLaboratory of Respiratory Biology, National Institutes of Health/NIEHS, Research Triangle Park, NC 27709, USA 
Keywords: Nrf2 Phase I metabolizing enzymes Phase II metabolizing enzymes Cytochrome P450 
a b s t r a c t 

Previous reports have proposed a cross-talk between the nuclear factor erythroid-2 p45-related factor-2 (Nrf2)/antioxidant response element (ARE) and the aryl hydrocarbon receptor (AhR)/xenobiotic response element (XRE) signaling pathways. Therefore, the aim of the current study was to examine the level of phase I, phase II drug metabolizing enzymes (DMEs), and phase III transporters and their related tran- scription factors in the Nrf2 knockout model. Our results showed that phase II DMEs that are under the control of Nrf2 typified by NAD(P)H: quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase (Gst) were significantly lower at the mRNA, protein, and catalytic activity levels in the livers of Nrf2 knockout mice compared to wild type. Furthermore, phase I cytochrome P450s (CYPs), Cyp1, and Cyp2b10 at mRNA, protein, and catalytic activity levels were significantly lower in the livers of Nrf2 knockout mice. Interestingly, our results showed that the transcription factors AhR, constitutive andro- stane receptor (CAR), and pregnane X receptor (PXR) at mRNA, and protein expression levels were signif- icantly lower in the livers of Nrf2 knockout mice compared to wild type. Importantly, phase III drug transporters mRNA levels of the multiple drug resistance associated proteins (Mrp2 and Mrp3), and sol- ute carrier organic anion transporters (Slco1a6 and Slco2b1) were significantly lower in the liver of Nrf2 knockout mice. Co-activators, Ncoa1, Ncoa2, and Ncoa3 mRNA levels were not altered while co-repres- sors, Ncor1 and Ncor2 were significantly lower in the livers of Nrf2 knockout mice. In conclusion, knockout of Nrf2 causes disruption to the coordination of phase I, phase II drug DMEs, and phase III drug transporters through altering the transcription factors controlling them. 

Ó 2011 Elsevier Ltd. All rights reserved. 

1. Introduction 

Humans and higher organisms are simultaneously exposed to different natural and synthetic xenobiotics through the intake of air, water, and food. Therefore, their ability to withstand toxic chemical insults and oxidative stress through a wide array of enzy- matic defense systems has become a necessity for survival (Chanas et al., 2002). The biotransformation and detoxification processes are two sequential reactions that involve phase I and phase II drug metabolizing enzymes, respectively. In phase I reactions, xenobiot- ics are mainly oxidized by cytochrome P450s (CYPs) to become more polar metabolites. Consequently, phase II reactions, facili- tated by key enzymes such as NAD(P)H: quinone oxidoreductase- 1 (Nqo1), glutathione-S-transferases (Gsts), and UDP-glu- curonosyltransferases (UGTs) convert reactive phase I metabolites 

into more hydrophilic secondary metabolites (Nebert and Duffy, 1997). 

It is well documented that different families of CYPs participate in the oxidative metabolism of endo- and xenobiotic substrates (Ramana and Kohli, 1998). Importantly, only the mammalian CYP1, 2, and 3 families are known to be involved in the metabolism of xenobiotics through different signaling pathways (Ramana and Kohli, 1998). In general, transcriptional activation of most CYPs oc- curs through three main nuclear receptor mechanisms: the aryl hydrocarbon receptor (AhR) for CYP1 family, the constitutive androstane receptor (CAR) for the CYP2 family; and the pregnane X receptor (PXR) for the CYP3 family (Ramana and Kohli, 1998). Similarly, the phase II metabolizing enzymes are regulated through one of the most versatile mechanisms that involve the antioxidant response element (ARE). This element was first discovered because of its vital role in regulating the inducible levels of rat GSTA2, and NQO1 gene expressions (Rushmore et al., 1991). Moreover, studies conducted to elucidate the entity of this response element revealed that ARE responds to Michael reaction acceptors, hydroquinones, catechols, isothiocyanates, peroxides, and trivalent arsenicals 

0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.01.014 

Contents lists available at ScienceDirect 

Toxicology in Vitro 

journal homepage: www.elsevier.com/locate/toxinvit 

(Dinkova-Kostova et al., 2001). Further studies on the ARE demon- strated that the basic-region leucine-zipper (bZIP) factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) protein, in combina- tion with other small musculoaponeurotic fibrosarcoma (MAF) proteins, mediate the transcription of genes containing the ARE se- quence in their enhancer region. In unstressed conditions, Nrf2 re- sides in the cytoplasm bound to the actin-associated Kelch-like ECH associating protein 1 (Keap1) (Ma et al., 2004). In response to oxidative stress, the Nrf2-Keap1 interaction becomes less stable causing the former to dissociate and translocate to
phase I biotransforming enzymes might be indirectly under the control of Nrf2 (Shin et al., 2007). Therefore, we hypothesize that Nrf2 knockout mice will possess relatively lower levels of phase I biotransforming enzymes, possibly due to both lowering Nrf2 and the transcription factors controlling them. Therefore, the objectives of the current study were to determine the constitu- tive expression of different CYPs, phase II enzymes, phase III trans- porters, related transcription factors, in addition to different co- activators and co-repressors in both wild type and Nrf2 knockout mice livers.
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