Regulation of bile acid metabolism in mouse models with hydrophobic bile acid composition.

The bile acid (BA) composition in mice is substantially different from that in humans. Chenodeoxycholic acid (CDCA) is an end product in the human liver; however, mouse Cyp2c70 metabolizes CDCA to hydrophilic muricholic acids (MCAs). Moreover, in humans, the gut microbiota converts the primary BAs, cholic acid and CDCA, into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In contrast, the mouse Cyp2a12 reverts this action and converts these secondary BAs to primary BAs. Here, we generated Cyp2a12 KO, Cyp2c70 KO, and Cyp2a12/Cyp2c70 double KO (DKO) mice using the CRISPR-Cas9 system to study the regulation of BA metabolism under hydrophobic BA composition. Cyp2a12 KO mice showed the accumulation of DCAs, whereas Cyp2c70 KO mice lacked MCAs and exhibited markedly increased hepatobiliary proportions of CDCA. In DKO mice, not only DCAs or CDCAs but also DCAs, CDCAs, and LCAs were all elevated. In Cyp2c70 KO and DKO mice, chronic liver inflammation was observed depending on the hepatic unconjugated CDCA concentrations. The BA pool was markedly reduced in Cyp2c70 KO and DKO mice, but the FXR was not activated. It was suggested that the cytokine/c-Jun N-terminal kinase signaling pathway and the pregnane X receptor-mediated pathway are the predominant mechanisms, preferred over the FXR/small heterodimer partner and FXR/fibroblast growth factor 15 pathways, for controlling BA synthesis under hydrophobic BA composition. From our results, we hypothesize that these KO mice can be novel and useful models for investigating the roles of hydrophobic BAs in various human diseases.

Suppressed hepatocyte proliferation via a ROS-HNE-P21 pathway is associated with nicotine- and cotinine-enhanced alcoholic fatty liver in mice.

CYP2A5 is a major enzyme responsible for nicotine and cotinine metabolism in mice. Nicotine and cotinine enhance alcoholic fatty liver in wild type (WT) mice but not in CYP2A5 knockout (KO) mice, and reactive oxygen species (ROS) generated during the CYP2A5-mediated metabolism contributes to the enhancing effect. In combination with ethanol, nicotine and cotinine increased lipid peroxidation end product 4-hydroxynonenal (HNE) in WT mice but not in KO mice. In ethanol-fed KO mice, only 5 and 10 genes were regulated by nicotine and cotinine, respectively. However, in ethanol-fed WT mice, 59 and 104 genes were regulated by nicotine and cotinine, respectively, and 7 genes were up-regulated by both nicotine and cotinine. Plin 2 and Cdkn1a are among the 7 genes. Plin2 encodes adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, which was confirmed to be increased by nicotine and cotinine in WT mice but not in KO mice. Cdkn1a encodes P21 and elevated P21 in nuclei was also confirmed. HNE can increase P21 and P21 inhibit cell proliferation. Consistently, hepatocyte proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 were decreased in WT mice but not in KO mice by nicotine/ethanol and cotinine/ethanol, respectively. These results suggest that inhibition of liver proliferation via a ROS-HNE-P21 pathway is involved in nicotine- and cotinine-enhanced alcoholic fatty liver.

Role of CYP2B in Phenobarbital-Induced Hepatocyte Proliferation in Mice.

Phenobarbital (PB) promotes liver tumorigenesis in rodents, in part through activation of the constitutive androstane receptor (CAR) and the consequent changes in hepatic gene expression and increases in hepatocyte proliferation. A typical effect of CAR activation by PB is a marked induction of Cyp2b10 expression in the liver; the latter has been suspected to be vital for PB-induced hepatocellular proliferation. This hypothesis was tested here by using a Cyp2a(4/5)bgs-null (null) mouse model in which all Cyp2b genes are deleted. Adult male and female wild-type (WT) and null mice were treated intraperitoneally with PB at 50 mg/kg once daily for 5 successive days and tested on day 6. The liver-to-body weight ratio, an indicator of liver hypertrophy, was increased by 47% in male WT mice, but by only 22% in male Cyp2a(4/5)bgs-null mice, by the PB treatment. The fractions of bromodeoxyuridine-positive hepatocyte nuclei, assessed as a measure of the rate of hepatocyte proliferation, were also significantly lower in PB-treated male null mice compared with PB-treated male WT mice. However, whereas few proliferating hepatocytes were detected in saline-treated mice, many proliferating hepatocytes were still detected in PB-treated male null mice. In contrast, female WT mice were much less sensitive than male WT mice to PB-induced hepatocyte proliferation, and PB-treated female WT and PB-treated female null mice did not show significant difference in rates of hepatocyte proliferation. These results indicate that CYP2B induction plays a significant, but partial, role in PB-induced hepatocyte proliferation in male mice.

Absence of cytochrome P450 2A5 enhances alcohol-induced liver injury in mice.

BACKGROUND: Ethanol can induce cytochrome P450 2E1, an active generator of reactive oxygen species, and this cytochrome is considered a risk factor for oxidative liver injury. Recently, we found that in addition to P450 2E1 also cytochrome P450 2A5, another isoform of cytochrome P450, can be induced by ethanol, and that ethanol induction of cytochrome P450 2A5 is P450 2E1-dependent. AIMS: To investigate the role of cytochrome P450 2A5 in alcohol-induced liver injury. METHODS: Cytochrome P450 2A5-knockout mice and wild type mice were fed the Lieber-Decarli ethanol liquid diet to induce liver injury. Controls were fed the Lieber-Decarli control diet. RESULTS: After 4 weeks of feeding with Lieber-Decarli diet, ethanol-induced liver injury was enhanced in the knockout mice compared with wild type mice, as indicated by serum transaminases, hepatic fat accumulation (steatosis), and necroinflammation observed in liver sections with Haematoxylin & Eosin staining. Ethanol-induced oxidative stress was also higher in the knockout mice than the wild types. Ethanol feeding induced cytochrome P450 2A5 in wild type mice but not in the knockout mice, while induction of cytochrome P450 2E1 was comparable in the knockout and wild type mice. CONCLUSION: These results suggest that cytochrome P450 2A5 protects against ethanol-induced oxidative liver injury.

Prediction and validation of apoptosis through cytochrome P450 activation by benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) processed by cytochrome P450 (CYP450) during metabolism is well reported to induce carcinogenesis. The present study has developed a new approach to examine apoptotic activity of a known PAH called benzo[a]pyrene (B[a]P), using protein-ligand and protein-protein interaction through in silico approach, followed by in vitro validation. In silico study showed that the conformational changes and energies involved in the binding of B[a]P to CYP1B1 was crucial with its target proteins. The data showed that activated B[a]P had high affinity to bind with aryl hydrocarbon receptor (AhR) with binding energy of -601.97kcal/mol. Interestingly, B[a]P-CYP1B1 complex showed strong binding affinity for caspase-8, -9, -3 with binding energy of -625.5, -479.3 and -514.2kcal/mol respectively. Moreover, the docking of specific caspase inhibitors in the complex showed weak interaction with low binding energy value as compared to B[a]P-CYP1B1 caspase complexes. To validate our in silico work, we showed B[a]P treated HaCaT cells triggered apoptosis with increase in caspase 8, caspase 9 and caspase 3/7 level. Further, in vitro work confirmed that B[a]P induced apoptosis was significantly suppressed in Ac-DEVD-CMK pre-treated cells. In addition, knockdown of CYP1B1 suppressed B[a]P induced apoptosis in HaCaT cells confirming a pivotal role of CYP1B1 in B[a]P induced apoptosis. Interestingly, through in silico modeling, we screened clotrimazole as a potent CYP1B1 inhibitor which completely inhibited B[a]P mediated activation. This hypothesis was validated by MTT assay, caspase activation measurement and showed remarkable inhibition of B[a]P induced cell death; thereby, highlighting a potent therapeutic role for industrial pollution associated diseases.

Smoke carcinogens cause bone loss through the aryl hydrocarbon receptor and induction of Cyp1 enzymes.

Smoking is a major risk factor for osteoporosis and fracture, but the mechanism through which smoke causes bone loss remains unclear. Here, we show that the smoke toxins benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interact with the aryl hydrocarbon receptor (Ahr) to induce osteoclastic bone resorption through the activation of cytochrome P450 1a/1b (Cyp1) enzymes. BaP and TCDD enhanced osteoclast formation in bone marrow cell cultures and gavage with BaP stimulated bone resorption and osteoclastogenesis in vivo. The osteoclastogenesis triggered by BaP or RANK-L was reduced in Ahr(-/-) cells, consistent with the high bone mass noted in Ahr(-/-) male mice. The receptor activator of NF-κB ligand (RANK-L) also failed to induce the expression of Cyp1 enzymes in Ahr(-/-) cells. Furthermore, the osteoclastogenesis induced by TCDD was lower in Cyp1a1/1a2(-/-) and Cyp1a1/1a2/1b1(-/-) cultures, indicating that Ahr was upstream of the Cyp enzymes. Likewise, the pharmacological inhibition of the Cyp1 enzymes with tetramethylsilane or proadifen reduced osteoclastogenesis. Finally, deletion of the Cyp1a1, Cyp1a2, and Cyp1b1 in triple knockout mice resulted in reduced bone resorption and recapitulated the high bone mass phenotype of Ahr(-/-) mice. Overall, the data identify the Ahr and Cyp1 enzymes not only in the pathophysiology of smoke-induced osteoporosis, but also as potential targets for selective modulation by new therapeutics.

Association of CYP2A6*4 with susceptibility of lung cancer: a meta-analysis.

OBJECTIVES: To assess the association between the variant of Cytochrome P450 2A6 whole gene deletion (CYP2A6*4) polymorphism and risk of lung cancer. METHODS: Two investigators independently searched the PubMed, Elsevier, EMBASE, Web of Science, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI). Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) for CYP2A6*4 and lung cancer were calculated in a fixed-effects model (the Mantel-Haenszel method) and a random-effects model (the DerSimonian and Laird method) when appropriate. RESULTS: This meta-analysis included seven eligible studies, which included 2524 lung cancer cases and 2258 controls (cancer-free). Overall, CYP2A6*4 was associated with the risk of lung cancer (allele*4 vs. allele non-*4, pooled OR  = 0.826, 95% CI  = 0.725-0.941, P-value  = 0.004). When stratifying for population, significant association was observed in Asian (additive model, pooled OR  = 0.794, 95% CI  = 0.694-0.909, P-value  = 0.001; dominant model, pooled OR  = 0.827, 95% CI  = 0.709-0.965, P-value  = 0.016; recessive model (pooled OR  = 0.444, 95% CI  = 0.293-0.675, P-value <0.0001). In the overall analysis, a comparably significant decrease in the frequency of *4/*4 genotype was detected between cases and controls in Asian while no *4/*4 genotype was detected in Caucasian in collected data. CONCLUSION: This meta-analysis suggests that the CYP2A6*4 polymorphism is associated with susceptibility of lung cancer in Asian. The whole gene deletion of CYP2A6 may decrease the risk of lung cancer in Asian samples.

Dexfenfluramine and the oestrogen-metabolizing enzyme CYP1B1 in the development of pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension (PAH) occurs more frequently in women than men. Oestrogen and the oestrogen-metabolising enzyme cytochrome P450 1B1 (CYP1B1) play a role in the development of PAH. Anorectic drugs such as dexfenfluramine (Dfen) have been associated with the development of PAH. Dfen mediates PAH via a serotonergic mechanism and we have shown serotonin to up-regulate expression of CYP1B1 in human pulmonary artery smooth muscle cells (PASMCs). Thus here we assess the role of CYP1B1 in the development of Dfen-induced PAH. METHODS AND RESULTS: Dfen (5 mg kg(-1) day(-1) PO for 28 days) increased right ventricular pressure and pulmonary vascular remodelling in female mice only. Mice dosed with Dfen showed increased whole lung expression of CYP1B1 and Dfen-induced PAH was ablated in CYP1B1(-/-) mice. In line with this, Dfen up-regulated expression of CYP1B1 in PASMCs from PAH patients (PAH-PASMCs) and Dfen-mediated proliferation of PAH-PASMCs was ablated by pharmacological inhibition of CYP1B1. Dfen increased expression of tryptophan hydroxylase 1 (Tph1; the rate-limiting enzyme in the synthesis of serotonin) in PAH-PASMCs and both Dfen-induced proliferation and Dfen-induced up-regulation of CYP1B1 were ablated by inhibition of Tph1. 17ß-Oestradiol increased expression of both Tph1 and CYP1B1 in PAH-PASMCs, and Dfen and 17ß-oestradiol had synergistic effects on proliferation of PAH-PASMCs. Finally, ovariectomy protected against Dfen-induced PAH in female mice. CONCLUSION: CYP1B1 is critical in the development of Dfen-induced PAH in mice in vivo and proliferation of PAH-PASMCs in vitro. CYP1B1 may provide a novel therapeutic target for PAH.

Activity of the estrogen-metabolizing enzyme cytochrome P450 1B1 influences the development of pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a hyperproliferative vascular disorder observed predominantly in women. Estrogen is a potent mitogen in human pulmonary artery smooth muscle cells and contributes to PAH in vivo; however, the mechanisms attributed to this causation remain obscure. Curiously, heightened expression of the estrogen-metabolizing enzyme cytochrome P450 1B1 (CYP1B1) is reported in idiopathic PAH and murine models of PAH. METHODS AND RESULTS: Here, we investigated the putative pathogenic role of CYP1B1 in PAH. Quantitative reverse transcription-polymerase chain reaction, immunoblotting, and in situ analysis revealed that pulmonary CYP1B1 is increased in hypoxic PAH, hypoxic+SU5416 PAH, and human PAH and is highly expressed within the pulmonary vascular wall. PAH was assessed in mice via measurement of right ventricular hypertrophy, pulmonary vascular remodeling, and right ventricular systolic pressure. Hypoxic PAH was attenuated in CYP1B1(-/-) mice, and the potent CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS; 3 mg · kg(-1) · d(-1) IP) significantly attenuated hypoxic PAH and hypoxic+SU5416 PAH in vivo. TMS also abolished estrogen-induced proliferation in human pulmonary artery smooth muscle cells and PAH-pulmonary artery smooth muscle cells. The estrogen metabolite 16α-hydroxyestrone provoked human pulmonary artery smooth muscle cell proliferation, and this mitogenic effect was greatly pronounced in PAH-pulmonary artery smooth muscle cells. ELISA analysis revealed that 16α-hydroxyestrone concentration was elevated in PAH, consistent with CYP1B1 overexpression and activity. Finally, administration of the CYP1B1 metabolite 16α-hydroxyestrone (1.5 mg · kg(-1) · d(-1) IP) caused the development of PAH in mice. CONCLUSIONS: Increased CYP1B1-mediated estrogen metabolism promotes the development of PAH, likely via the formation of mitogens, including 16α-hydroxyestrone. Collectively, this study reveals a possible novel therapeutic target in clinical PAH.

Role of CYP2A5 in the bioactivation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis in mice depends on target-tissue bioactivation by pulmonary cytochrome P450 (P450) enzymes. The present study was designed to test the hypothesis that mouse CYP2A5 plays an essential role in NNK bioactivation in mouse lung. The role of CYP2A5 in NNK bioactivation was studied both in vitro and in vivo, by comparing the kinetic parameters of microsomal NNK metabolism and tissue levels of O(6)-methylguanine (O(6)-mG) (the DNA adduct highly correlated with lung tumorigenesis) between wild-type (WT) and Cyp2a5-null mice. In both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in the apparent K(m) values for the formation of 4-oxo-4-(3-pyridyl)butanone, which represents the reactive intermediate that produces O(6)-mG in vivo. The loss of CYP2A5 did not change circulating levels of NNK or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in mice treated intraperitoneally with NNK at either 20 or 100 mg/kg. However, the levels of lung O(6)-mG were significantly lower in Cyp2a5-null than in WT mice; the extent of the reduction was greater at the 20 mg/kg dose (∼40%) than at the 100 mg/kg dose (∼20%). These results indicate that CYP2A5 is the low-K(m) enzyme for NNK bioactivation in mouse lung. It is noteworthy that the remaining NNK bioactivation activities in the Cyp2a5-null mice could be inhibited by 8-methoxypsoralen, a P450 inhibitor used previously to demonstrate the role of CYP2A5 in NNK-induced lung tumorigenesis. Thus, P450 enzymes other than CYP2A5 probably also contribute to NNK-induced lung tumorigenesis in mice.

Cytochrome P450 1B1 contributes to renal dysfunction and damage caused by angiotensin II in mice.

Cytochrome P450 1B1 contributes to the development of angiotensin II-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the kidney, as well as in salt and water homeostasis, and blood pressure regulation, we determined the contribution of cytochrome P450 1B1 to renal dysfunction and injury associated with angiotensin II-induced hypertension in male Cyp1b1(+/+) and Cyp1b1(-/-) mice. Angiotensin II infusion (700 ng/kg per minute) given by miniosmotic pumps for 13 and 28 days increased systolic blood pressure in Cyp1b1(+/+) mice; this increase was significantly reduced in Cyp1b1(-/-) mice. Angiotensin II increased renal Cyp1b1 activity, vascular resistance, and reactivity to vasoconstrictor agents and caused endothelial dysfunction in Cyp1b1(+/+) but not Cyp1b1(-/-) mice. Angiotensin II increased water consumption and urine output, decreased urine osmolality, increased urinary Na(+) and K(+) excretion, and caused proteinuria and albuminuria in Cyp1b1(+/+) mice that was diminished in Cyp1b1(-/-) mice. Infusion of angiotensin II for 28 but not 13 days caused renal fibrosis, tubular damage, and inflammation in Cyp1b1(+/+) mice, which was minimized in Cyp1b1(-/-) mice. Angiotensin II increased levels of 12- and 20-hydroxyeicosatetraenoic acids; reactive oxygen species; and activity of NADPH oxidase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src in the kidneys of Cyp1b1(+/+) but not Cyp1b1(-/-) mice. These data suggest that increased thirst, renal dysfunction, and injury and inflammation associated with angiotensin II-induced hypertension in mice depend on cytochrome P450 1B1 activity, thus indicating that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension.

Lentiviral-mediated RNAi knockdown yields a novel mouse model for studying Cyp2b function.

There are few in vivo knockout models available to study the function of Cyp2 members involved in the metabolism of endogenous and exogenous chemicals. These models may help provide insight into the cytochrome P450s (CYPs) responsible for the detoxification and activation of drugs, environmental toxicants, and endobiotics. The aim of this work is to produce a potent Cyp2b-knockdown (KD) mouse for subsequent study of Cyp2b function. We made a quintuple Cyp2b-KD mouse using lentiviral-promoted short hairpin RNA (shRNA) homologous to all five murine Cyp2b subfamily members (Cyp2b9, 2b10, 2b13, 2b19, and 2b23). The Cyp2b-KD mice are viable, fertile, and without obvious gross abnormalities except for an increase in liver weight. Expression of the three hepatic Cyp2b members, 2b9, 2b10, and 2b13, is significantly repressed as demonstrated by quantitative real-time PCR and Western blotting. The constitutive androstane receptor activator, 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), was used to determine if shRNA-mediated Cyp2b10 repression could be outcompeted by Cyp2b10 induction. TCPOBOP-treated Cyp2b-KD mice show 80-90% less Cyp2b protein expression than TCPOBOP-treated wild-type (WT) mice, demonstrating that Cyp induction does not outcompete the repressive function of the shRNA. Untreated and TCPOBOP-treated Cyp2b-KD mice are poor metabolizers of parathion compared with WT mice. Furthermore, Cyp2b-KD mice are sensitive to parathion, an organophosphate insecticide primarily metabolized by Cyp2b enzymes, when compared with WT mice. In summary, we designed an shRNA construct that repressed the expression and activity of multiple Cyp2b enzymes. We foresee that this novel Cyp2b-KD mouse model will significantly improve our understanding of the role of Cyp2b enzymes in chemical sensitivity and drug metabolism.

Absence seizures associated with efavirenz initiation.

Efavirenz, used in treating pediatric human immunodeficiency virus infection, has central nervous system side effects. We report on a 5-year-old girl with perinatally acquired human immunodeficiency virus infection, presenting with new onset absence seizures after starting treatment with efavirenz. Plasma efavirenz values were above therapeutic range. The child was homozygous for the CYP2B6-516T/T genotype, which is associated with poor efavirenz clearance. Seizures abated after efavirenz discontinuation.

Proton pump inhibitors in rheumatic diseases: clinical practice, drug interactions, bone fractures and risk of infections.

Platelet activation and aggregation are key elements of the pathogenesis of acute coronary syndromes, of endothelial damage in chronic inflammatory and connective tissue disease (i.e. systemic sclerosis-SSc). Patients affected by chronic inflammatory diseases as well as by connective tissue diseases such as systemic sclerosis, often have the need to take anti-platelet therapy (e.g. ASA or clopidogrel). Current consensus recommendations state that patients prescribed clopidogrel plus aspirin should receive a proton pump inhibitor (PPI) to reduce gastrointestinal bleeding. Although each single PPI has similar efficacy in many cases, differences between them should be considered when choosing a treatment regimen. Many studies show PPI and clopidogrel drug interaction, with clopidogrel non-responsiveness in about 25% of the population. Only pantoprazole, which does not inhibit CYP P450 2C19, doesn't seem to have interaction with clopidogrel or other drugs. Patients affected by systemic sclerosis have high frequency of oesophageal mucosal abnormalities and should take long-term PPI therapy. When addressing long-term therapy safety data are clearly needed. Two recent studies have reported increased hip fracture rates with long-term PPI use, raising concerns about adverse effects of this class of drugs on mineral metabolism. The use of PPIs is also associated with an increase in the risk of development of Clostridium difficile infection (CDI) and the use of PPIs during CDI treatment is associated with an increased risk of recurrence. In order to achieve the desired results and, as with all medications, PPIs should always be used appropriately taking care never to exceed correct dosage and duration. When necessary use of pantoprazole arises as one of the best possible choices.

Direct genotyping of Cytochrome P450 2A6 whole gene deletion from human blood samples by the SmartAmp method.

BACKGROUND: Genetic polymorphisms of the human CYP2A6 gene are considered to be a determinant of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers. We developed a SmartAmp-based genotyping method to detect whole deletion of the CYP2A6 gene directly from blood samples without DNA isolation. METHODS: We validated the new method using CYP2A plasmids, 48 genomic DNA samples and 25 blood samples by utilizing the SmartAmp method, a unique isothermal DNA amplification process. RESULTS: This method could discriminate the CYP2A6 gene from highly homologous CYP2A7 and CYP2A13 genes. CYP2A6*1 (wild-type) and CYP2A6*4 (whole gene deletion) were determined by the new method in perfect accordance with sequence analysis data. CONCLUSIONS: A SmartAmp assay for genotyping the CYP2A6 gene was developed, and the reliability of the method was validated using the conventional PCR method.

CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress.

We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.

Lack of effect of Ginkgo biloba on voriconazole pharmacokinetics in Chinese volunteers identified as CYP2C19 poor and extensive metabolizers.

BACKGROUND: Ginkgo biloba is one of the most popular herbal supplements in the world. The supplement has been shown to induce the enzymatic activity of CYP2C19, the main cytochrome P450 isozyme involved in voriconazole metabolism. Because this enzyme exhibits genetic polymorphism, the inductive effect was expected to be modulated by the CYP2C19 metabolizer status. OBJECTIVE: To examine the possible effects of Ginkgo biloba as an inducer of CYP2C19 on single-dose pharmacokinetics of voriconazole in Chinese volunteers genotyped as either CYP2C19 extensive or poor metabolizers. METHODS: Fourteen healthy, nonsmoking volunteers-7 CYP2C19 extensive metabolizers (2C19(*)1/2C19(*)1) and 7 poor metabolizers (2C19(*)2/2C19(*)2)-were selected to participate in this study. Pharmacokinetics of oral voriconazole 200 mg after administration of Ginkgo biloba 120 mg twice daily for 12 days were determined for up to 24 hours by liquid chromatography-electrospray tandem mass spectrometry in a 2-phase randomized crossover study with 4-week washout between phases. RESULTS: For extensive metabolizers, the median value for voriconazole area under the plasma concentration-time curve from zero to infinity (AUC(0-)(infinity)) was 5.17 microg.h/mL after administration of voriconazole alone and 4.28 microg.h/mL after voriconazole with Ginkgo biloba (p > 0.05). The other pharmacokinetic parameters of voriconazole such as AUC(0-24), time to reach maximum concentration, half-life, and apparent clearance also did not change significantly for extensive metabolizers in the presence of Ginkgo biloba. Pharmacokinetic parameters followed a similar pattern for poor metabolizers. CONCLUSIONS: The results suggest that 12 days of treatment with Ginkgo biloba did not significantly alter the single-dose pharmacokinetics of voriconazole in either CYP2C19 extensive or poor metabolizers. Therefore, the pharmacokinetic interactions between voriconazole and Ginkgo biloba may have limited clinical significance.

Cyp1b1 exerts opposing effects on intestinal tumorigenesis via exogenous and endogenous substrates.

Cytochrome P450 1B1 (Cyp1b1) metabolism contributes to physiologic functions during embryogenesis but also to carcinogenic activation of polycyclic aromatic hydrocarbons (PAH). We generated Cyp1b1-deficient mice carrying the Min allele of the adenomatous polyposis coli gene. These Cyp1b1-deficient Min mice developed twice as many tumors as Min controls, which, however, remained similar in size and histology. Tumors from older (130 days) Cyp1b1-deficient Min mice selectively exhibited focal areas of nuclear atypia associated with less organized epithelia. The metabolism of endogenous substrates by Cyp1b1, therefore, suppresses tumor initiation but also affects progression. Treatment of Min mice with 7,12-dimethylbenzanthracene (DMBA) doubled both tumor multiplicity and size within 20 days but not when mice lacked Cyp1b1. This was paralleled by an abnormal staining of crypts with beta-catenin, phospho-IkappaB kinase, and RelA, which may represent an early stage of tumorigenesis similar to aberrant crypt formation. Cyp1b1 deletion did not affect circulating DMBA and metabolites. Cyp1b1 expression was higher in the tumors compared with normal small intestines. Increased tumorigenesis may, therefore, arise from generation of DMBA metabolites by Cyp1b1 in the developing tumors. Benzo(a)pyrene (BP), which is similarly activated by Cyp1b1 in vitro, did not affect tumorigenesis in Min mice. By contrast, BP and DMBA each suppressed tumor multiplicity in the absence of Cyp1b1. Cyp1b1 metabolism of DMBA and endogenous oxygenation products may each affect a tumor-promoting nuclear factor-kappaB activation, whereas Ah receptor activation by PAH affects suppression. Tumorigenesis may, therefore, depend on activation of PAH by Cyp1b1 and on offsetting suppression by Cyp1b1 of endogenous tumor-enhancing substrates.