Description: Tissue-specific deletion of the gene for NADPH-cytochrome P450 (P450) reductase (CPR), the essential electron donor to all microsomal P450 enzymes, in either liver or intestine, leads to upregulation of many P450 genes in the tissue with the Cpr deletion. Here, by studying the liver-specific Cpr -null (LCN) mouse, we examined whether an interorgan regulatory pathway exists, such that a loss of hepatic CPR would cause compensatory changes in intestinal P450 expression and capacity for first-pass metabolism of oral drugs. We show for the first time that intestinal expression of CYP2B, 2C, and 3A proteins was increased in LCN mice by 2- to 3-fold compared with wild-type (WT) mice, accompanied by significant increases in small intestinal microsomal lovastatin-hydroxylase activity and systemic clearance of oral lovastatin (at 5 mg/kg). Additional studies showed that the hepatic Cpr deletion, which caused large decreases in bile acid (BA) levels in the liver, intestine, plasma, and intestinal content, led to drastic decreases in the mRNA levels of intestinal fibroblast growth factor 15 (FGF15), a target gene of the BA receptor farnesoid X receptor. Furthermore, treatment of mice with FGF19 (the human counterpart of mouse FGF15) abolished the difference between WT and LCN mice in small intestinal (SI) CYP3A levels at 6 hours after the treatment. Our findings reveal a previously unrecognized direct role of intestinal FGF15/19 in the regulation of SI P450 expression and may have profound implications for the prediction of drug exposure in patients with compromised hepatic P450 function.

Description: The study objectives were 1) to test the hypothesis that the lack of P-glycoprotein (P-gp) and the inhibition of breast cancer resistance protein (Bcrp) at the blood-brain barrier after cassette dosing of potent P-gp and Bcrp inhibitors were due to low plasma concentrations of those inhibitors and 2) to examine the effects of P-gp on the unbound brain ( C u,brain ) and cerebrospinal fluid (CSF) concentrations ( C u,CSF ) of P-gp substrates in rats. In vitro inhibition of 11 compounds (amprenavir, citalopram, digoxin, elacridar, imatinib, Ko143 [(3 S ,6 S ,12a S )-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], loperamide, prazosin, quinidine, sulfasalazine, and verapamil) on P-gp and Bcrp was examined in P-gp– and Bcrp-expressing Madin-Darby canine kidney cells, respectively. An in vivo study was conducted in wild-type and Mdr1a(–/–) rats after subcutaneous cassette dosing of the 11 compounds at 1–3 mg/kg, and the brain, CSF, and plasma concentrations of these compounds were determined. At the maximal unbound concentrations observed in rats at 1–3 mg/kg, P-gp and Bcrp were not inhibited by a cassette of the 11 compounds. For non–P-gp/Bcrp substrates, similar C u,brain , C u,CSF , and unbound plasma concentrations ( C u,plasma ) were observed in wild-type and P-gp knockout rats. For P-gp/Bcrp substrates, C u,brain ≤ C u,CSF ≤ C u,plasma in wild-type rats, but C u,brain and C u,CSF increased in the P-gp knockout rats and were within 3-fold of C u,plasma for six of the seven P-gp substrates. These results indicate that P-gp and Bcrp inhibition at the blood-brain barrier is unlikely in cassette dosing and also suggest that P-gp and Bcrp activity at the blood–CSF barrier is functionally not important in determination of the CSF concentration for their substrates.

Description: Nicotine metabolism is believed to affect not only nicotine’s pharmacological effects but also nicotine addiction. As a key step toward testing this hypothesis, we have studied nicotine metabolism and nicotine’s pharmacological and behavioral effects in a novel knockout mouse model [named Cyp2a(4/5)bgs -null] lacking a number of cytochrome P450 genes known to be or possibly involved in nicotine metabolism, including two Cyp2a and all Cyp2b genes. We found that, compared with wild-type mice, the Cyp2a(4/5)bgs -null mice showed 〉90% decreases in hepatic microsomal nicotine oxidase activity in vitro, and in rates of systemic nicotine clearance in vivo. Further comparisons of nicotine metabolism between Cyp2a(4/5)bgs- null and Cyp2a5- null mice revealed significant roles of both CYP2A5 and CYP2B enzymes in nicotine clearance. Compared with the behavioral responses in wild-type mice, the decreases in nicotine metabolism in the Cyp2a(4/5)bgs -null mice led to prolonged nicotine-induced acute pharmacological effects, in that null mice showed enhanced nicotine hypothermia and antinociception. Furthermore, we found that the Cyp2a(4/5)bgs -null mice developed a preference for nicotine in a conditioned place preference test, a commonly used test of nicotine’s rewarding effects, at a nicotine dose that was 4-fold lower than what was required by wild-type mice. Thus, CYP2A/2B-catalyzed nicotine clearance affects nicotine’s behavioral response as well as its acute pharmacological effects in mice. This result provides direct experimental support of the findings of pharmacogenetic studies that suggest linkage between rates of nicotine metabolism and smoking behavior in humans.

Description: Naphthalene (NA), a ubiquitous environmental pollutant that can cause pulmonary and nasal toxicity in laboratory animals, requires cytochrome P450 (P450)–mediated metabolic activation to cause toxicity. Our recent study using a Cyp2f2 -null mouse showed that CYP2F2 plays an essential role in NA-induced lung toxicity, but not in NA-induced nasal toxicity. The aim of this study was to determine whether mouse CYP2A5, abundantly expressed in nasal olfactory mucosa (OM) and the liver, but less in the lung, plays a major role in the bioactivation and toxicity of NA in the OM. We found, by comparing Cyp2a5 -null and wild-type (WT) mice, that the loss of CYP2A5 expression led to substantial decreases in rates of NA metabolic activation by OM microsomes. The loss of CYP2A5 did not cause changes in systemic clearance of NA (at 200 mg/kg, i.p.). However, the Cyp2a5 -null mice were much more resistant than were WT mice to NA-induced nasal toxicity (although not lung toxicity), when examined at 24 hours after NA dosing (at 200 mg/kg, i.p.), or to NA-induced depletion of total nonprotein sulfhydryl in the OM (although not in the lung), examined at 2 hours after dosing. Thus, mouse CYP2A5 plays an essential role in the bioactivation and toxicity of NA in the OM, but not in the lung. Our findings further illustrate the tissue-specific nature of the role of individual P450 enzymes in xenobiotic toxicity, and provide the basis for a more reliable assessment of the potential risks of NA nasal toxicity in humans.

Description: CYP2A13 is a human cytochrome P450 (P450) enzyme important in the bioactivation of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). CYP2A13 expression levels vary dramatically among lung biopsy samples from patients, presumably owing in part to a suppression of CYP2A13 expression by disease-associated inflammation. Here, we determined whether CYP2A13 expression in the lungs of CYP2A13-humanized mice is suppressed by the presence of lung tumors. Tissues from an NNK lung tumor bioassay were examined. CYP2A13-humanized mice (95–100%) had multiple lung tumors at 16 weeks after NNK (30 or 50 mg/kg) treatment; whereas only ~9% of saline-treated CYP2A13-humanized mice had lung tumor (~1/lung). Mice with lung tumors, from the NNK-treated groups, were used for dissecting adjacent tumor-free lung tissues; whereas mice without visible lung tumors, from the saline-treated group, were used as controls. Compared with the controls, the levels of CYP2A13 protein and mRNA were both reduced significantly (by ≥50%) in the NNK-treated groups. The levels of mouse CYP2B10 and CYP2F2 mRNAs were also significantly lower in the dissected normal lung tissues from tumor-bearing mice than in lungs from the control mice. Pulmonary tissue levels of three proinflammatory cytokines, tumor necrosis factor alpha, interferon gamma, and interleukin-6, were significantly higher in the tumor-bearing mice than in the controls, indicating occurrence of low-grade lung inflammation at the time of necropsy. Taken together, these findings support the hypothesis that CYP2A13 levels in human lungs can be suppressed by disease-associated inflammation in tissue donors, a scenario causing underestimation of CYP2A13 levels in healthy lungs.

Description: The potential involvement of intestinal microsomal cytochrome P450 (P450) enzymes in defending against colon inflammation and injury was studied in mice treated with dextran sulfate sodium (DSS) to induce colitis. Wild-type (WT) mice and mice with intestinal epithelium (IE)–specific deletion of the P450 reductase gene (IE- Cpr -null) were compared. IE- Cpr -null mice have little microsomal P450 activity in IE cells. DSS treatment (2.5% in drinking water for 6 days) caused more severe colon inflammation, as evidenced by the presence of higher levels of myeloperoxidase and proinflammatory cytokines [tumor necrosis factor- α , interleukin (IL)-6, and IL-1 β ], and greater weight loss, colonic tissue damage, and colon shortening, in IE- Cpr -null mice than in WT mice. The IE- Cpr -null mice were deficient in colonic corticosterone (CC) synthesis, as indicated by the inability of ex vivo cultured colonic tissues from DSS-treated IE- Cpr -null mice (in contrast to DSS-treated WT mice) to show increased CC production, compared with vehicle-treated mice, and by the ability of added deoxycorticosterone (DOC), a precursor of CC biosynthesis via mitochondrial CYP11B1, to restore ex vivo CC production by colonic tissues from DSS-treated null mice. Intriguingly, null (but not WT) mice failed to show increased serum CC levels following DSS treatment. Nevertheless, cotreatment of DSS-exposed mice with DOC, which did not restore DSS-induced increase in serum CC, abolished the hypersensitivity of IE- Cpr -null mice to DSS-induced colon injury. Taken together, our results strongly support the notion that microsomal P450 enzymes in the intestine play an important role in protecting colon epithelium from DSS-induced inflammation and injury, possibly through increased local CC synthesis in response to DSS challenge.

Description: CYP2A13 , CYP2B6 , and CYP2F1 are neighboring cytochrome P450 genes on human chromosome 19, and the enzymes that they encode overlap in substrate specificity. A CYP2A13/2B6/2F1-transgenic mouse, in which CYP2A13 and 2F1 are both expressed in the respiratory tract and CYP2B6 is expressed in the liver, was recently generated. We generated a CYP2A13 (only) transgenic mouse so that the specific activity of CYP2A13 can be determined. The CYP2B6 and CYP2F1 genes in the CYP2A13/2B6/2F1 genomic clone were inactivated via genetic manipulations, and CYP2A13 was kept intact. A CYP2A13 (only) transgenic (2A13-TG) mouse was generated using the engineered construct and then characterized to confirm transgene integrity and determine copy numbers. The 2A13-TG mice were normal in gross morphology, development, and fertility. As in the CYP2A13/2B6/2F1-transgenic mouse, CYP2A13 expression in the 2A13-TG mouse was limited to the respiratory tract; in contrast, CYP2B6 and 2F1 proteins were not detected. Additional studies using the CYP2A13-humanized (2A13-TG/ Cyp2abfgs -null) mouse produced by intercrossing between 2A13-TG and Cyp2abfgs -null mice confirmed that the transgenic CYP2A13 is active in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a lung procarcinogen. The 2A13-TG mouse should be valuable for assessing specific roles of human CYP2A13 in xenobiotic toxicity in the respiratory tract.

Description: This study was designed to increase the throughput of rat brain microdialysis studies by administration of compounds as a cassette as opposed to discrete study. Eight compounds (carbamazepine, citalopram, desmethylclozapine, diphenhydramine, gabapentin, metoclopramide, naltrexone, and stavudine) were selected and administered as an intravenous bolus dose at 0.5–3.3 mg/kg each followed by an intravenous infusion at 1 mg/kg per hour for 6 hours in rats in a cassette or discrete dosing. The dialysate, plasma, brain, and cerebrospinal fluid were collected and analyzed using liquid chromatography–tandem mass spectrometry. The microdialysis probe recovery was determined by an in vitro gain method. The recovery between the cassette and discrete dosing was similar, with an average of 1.0 ± 0.10–fold difference. The stavudine interstitial fluid (ISF) concentration, as measured by brain microdialysis, was below the low limit of quantitation and was excluded from the analyses. The ratios of ISF concentration to unbound plasma concentration were within 2-fold for six of the remaining seven compounds, with an average of 0.92 ± 0.51–fold difference between the cassette and discrete methods. The ratios of ISF concentration to unbound brain concentration, as measured by the brain homogenate method, were also similar, with a 1.1 ± 0.7–fold difference. In addition, the ratios of ISF to cerebrospinal fluid concentrations were similar, with a 1.5 ± 0.6–fold difference. The results from this study support the use of a cassette dosing approach to enhance the throughput of rat brain microdialysis studies in drug discovery.

Description: CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13 / 2B6 / 2F1 -transgenic (TG) mouse model (all * 1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (~100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (~0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/ Cyp2f2 -null mice, at levels 10- and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (~0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.