Transgenic Mice with a Hypomorphic NADPH-Cytochrome P450 Reductase Gene: Effects on Development, Reproduction, and Microsomal Cytochrome P450

Abstract

A mouse model with a hypomorphic NADPH-cytochrome P450 reductase (Cpr) gene (designated Cpr low allele) was generated and characterized in this study. The Cpr gene in these mice was disrupted by the insertion of a neo gene in intron 15, which led to 74 to 95% decreases in CPR expression in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver, and heart. In the liver, a pattern of pericentral distribution of CPR protein was preserved in the Cpr low/low mice, despite an overall reduction in CPR expression. Genotype distribution in F2 pups indicated limited embryonic lethality associated with the Cpr low allele, a finding that confirms the role of CPR-dependent enzymes in development. Adult male homozygotes had decreased body weight and decreased heart, lung, and kidney weights, whereas homozygous Cpr low females, which had increased serum testosterone and progesterone and decreased copulatory activities, were infertile. Furthermore, adult Cpr low/low mice had decreased plasma cholesterol, and some mice developed mild centrilobular hepatic lipidosis. In addition, despite apparently compensatory increases in total microsomal cytochrome P450 content in the liver and kidney, the decreases in CPR expression were accompanied by reductions in systemic clearance of pentobarbital, as well as in hepatic microsomal metabolism of acetaminophen and testosterone. These phenotypes illustrate the potential impact of a globally decreased CPR activity in human adults, and this novel knock-in mouse model provides a unique opportunity for further explorations of the in vivo roles of CPR and CPR-dependent enzymes.

Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase

“…The decrease in APAP-induced hepatotoxicity in Por +/− mice at ZT12 indicates that POR is an important modulator of APAP hepatotoxicity in vivo. In further support of this model, the trend seen in our data of decreased microsomal APAP bioactivation despite increased total P450 content also is found in POR hypomorphic mice (29). These data led us to conclude that the loss of induction of POR transcription/activity by ablation of the hepatocyte clock is a major contributor to the decreased bioactivation and protection from toxicity seen in Bmal1 fx/fx Cre Alb mice in vivo.…”

“…In keeping with this idea, we observed that enzymatic activity of POR in Bmal1 fx/fx Cre Alb liver microsomes also was decreased compared with WT and correlated with the decrease in APAP bioactivation seen in Bmal1 fx/fx Cre Alb microsomes suggesting that this electron donor may be rate limiting for the P450 activities responsible for NAPQI generation. The activity of POR in vitro has been shown to be limiting with respect to electron donation for several different P450 isoforms and substrates (28,29), and human POR polymorphisms have been reported to contribute to variations in drug metabolism (30). The decrease in APAP-induced hepatotoxicity in Por +/− mice at ZT12 indicates that POR is an important modulator of APAP hepatotoxicity in vivo.…”

“…APAP metabolites were isolated by extraction in methanol as described by Dai et al (36) with some modifications as detailed in SI Experimental Procedures. Microsomal APAP bioactivation was determined by measuring conjugation of APAP to N-acetylcysteine, with modifications detailed in SI Experimental Procedures (29). Microsomal POR-dependent cytochrome c reductase activity was performed as previously described (37).…”

Effects of genetic factors on the pharmacokinetics and pharmacodynamics of amlodipine in primary hypertensive patients

“…The POR gene polymorphism (A503V) reduced the activity of CYP17, but had no effect on the activities of CYP1A2 and CYP2C19 (9). The experiments of transgenic mice have demonstrated that decreased activity of POR decreased the enzymatic activity of CYP (10). One recent study of Caucasian patients demonstrated that POR gene mutation (A503V) significantly increased CYP3A activity in vivo (11).…”

Expression of Constitutive Androstane Receptor, Hepatic Nuclear Factor 4α, and P450 Oxidoreductase Genes Determines Interindividual Variability in Basal Expression and Activity of a Broad Scope of Xenobiotic Metabolism Genes in the Human Liver

“…Additionally, the abundance of common coregulator proteins and RXR may serve as limiting factors to nuclear receptor-mediated transcriptional control. Finally, the rate of regeneration of P450s by P450 oxidoreductase (POR), the obligate P450 regenerative enzyme in mammals (Wu et al, 2005), may dictate phase I metabolism rate. A number of isolated studies have previously identified interindividual variation in expression levels of nuclear receptors such as CAR, SXR, and hepatic nuclear factor 4␣ (HNF4␣) and their correlations with target genes (Pascussi et al, 2001;Chang et al, 2003;Lamba et al, 2003).…”