The effect of isoniazid on CYP2E1- and CYP4A-mediated hydroxylation of arachidonic acid in the rat liver and kidney.

Cytochrome P450 (P450) bioactivation of arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) has been reported to be isoform- and tissue-specific. To determine whether altered P450 expression affects the production of these metabolites, the formation of HETEs after isoniazid-mediated CYP2E1 induction was evaluated in the rat liver and kidney. Male Sprague-Dawley rats received isoniazid (200 mg/kg) or saline intraperitoneally once daily for 5 days. Chlorzoxazone, lauric acid, and arachidonic acid hydroxylation was measured in liver and kidney microsomes with and without preincubation with the specific CYP2E1 inhibitor, trans-1,2-dichloroethylene (DCE). P450 isoform content and tissue HETE metabolite concentrations were also determined. Isoniazid increased CYP2E1 protein, and the 6-hydroxychlorzoxazone formation rate was increased by 2.7 +/- 0.3- and 2.2 +/- 0.5-fold in liver and kidney, respectively. Formation of 19-HETE and 11-hydroxylauric acid was induced 2.3 +/- 0.6-fold and 2.2 +/- 0.4-fold in the liver, respectively, with no difference in the kidney. All of the induced activities were attenuated by DCE. An unanticipated decrease in liver CYP4A expression and in vitro 20-HETE formation rate was observed after isoniazid administration. Isoniazid decreased liver and kidney 20-HETE content to 34 +/- 10% and 15.6 +/- 5.3% of control, respectively, without significantly altering tissue 19-HETE concentration. Based on these findings, we conclude that under induced conditions, CYP2E1 is a primary enzyme involved in liver, but not kidney, formation of 19-HETE. In addition, formation of both CYP4A and 20-HETE is reduced in the liver by isoniazid. It was also demonstrated that tissue concentrations parallel in vitro inhibited formation rates for 20-HETE, but not the induced 19-HETE formation in the liver.     

HET0016, a potent and selective inhibitor of 20-HETE synthesizing enzyme

The present study examined the inhibitory effects of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016) on the renal metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes. HET0016 exhibited a high degree of selectivity in inhibiting the formation of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) in rat renal microsomes. The IC(50) value averaged 35+/-4 nM, whereas the IC(50) value for inhibition of the formation of epoxyeicosatrienoic acids by HET0016 averaged 2800+/-300 nM. In human renal microsomes, HET0016 potently inhibited the formation of 20-HETE with an IC(50) value of 8.9+/-2.7 nM. Higher concentrations of HET0016 also inhibited the CYP2C9, CYP2D6 and CYP3A4-catalysed substrates oxidation with IC(50) values of 3300, 83,900 and 71,000 nM. The IC(50) value for HET0016 on cyclo-oxygenase activity was 2300 nM. These results indicate that HET0016 is a potent and selective inhibitor of CYP enzymes responsible for the formation of 20-HETE in man and rat.     

omega-Hydroxylation of 15-hydroxyeicosatetraenoic acid by lung microsomes from pregnant rabbits

15-Hydroxyeicosatetraenoic acid (15-HETE) was converted by lung microsomes from pregnant rabbits to a polar metabolite that was identified by mass spectrometry as the 15,20-dihydroxyeicosatetraenoic acid. The formation of the 20- or omega-hydroxylated product was NADPH dependent, with a specific activity of 1.87 +/- 0.53 nmol/min/mg of microsomal protein. Other hydroxylated derivatives of eicosatetraenoic acid that possessed hydroxy groups at the 5- and 12-carbon atoms were not metabolized by the lung microsomes. This hydroxylation of 15-HETE was observed in lung microsomes of pregnant rabbits and only minor amounts were formed by nonpregnant rabbits. The specific activity for 15-HETE omega-hydroxylation was similar to the value obtained for prostaglandin E1 (1.48 +/- 0.33 nmol/min/mg). It is known that rabbit lungs possess a cytochrome P-450 that is induced during pregnancy and catalyzes the 20-hydroxylation of prostaglandins. The addition of the antibody to cytochrome P-450 prostaglandin omega-hydroxylase or prostaglandin E1, a substrate of this enzyme, resulted in potent inhibition of 15-HETE omega-hydroxylation, providing strong evidence that a common cytochrome P-450 catalyzes the omega-hydroxylation of both prostaglandins and 15-HETE.     

20-Hydroxyeicosatetraenoic acid inhibits the apoptotic responses in pulmonary artery smooth muscle cells

20-Hydroxyeicosatetraenoic acid (20-HETE), a omega-hydroxylation product of arachidonic acid catalyzed by cytochrome P450 4A (CYP4A), plays a role in vascular smooth muscle remodeling. Although its effects on angiogenic responses are known, it remains unclear whether 20-HETE acts on apoptosis of pulmonary arterial smooth muscle cells (PASMC), an important step in PASMC remodeling, and what pathways are involved in the process. Here we show evidence for the missing information. The effect of 20-HETE on PASMC apoptosis and the apoptosis-associated signaling pathways were determined with cell viability assay, Annexin V and propidium idodide binding, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), mitochondrial potentials assay, caspase activity assay and Western blots. We found that exogenous 20-HETE suppressed the serum deprivation-induced loss of bovine PASMCs and prevented Annexin V binding, DNA nick end labeling and chromatin condensation. The effect was worsened by 17-octadecynoic acid (17-ODYA), which inhibited the production of endogenous 20-HETE. Furthermore, 20-HETE induced the expression of bcl-2, maintained the stability of mitochondria membrane, and relieved the activation of caspase-9 and caspase-3. Such effects were reversed in the presence of 17-ODYA. Thus, these findings indicate that 20-HETE protects PASMCs against apoptosis by acting on, at least in part, the intrinsic apoptotic pathway.     

In vitro metabolism of midazolam, triazolam, nifedipine, and testosterone by human liver microsomes and recombinant cytochromes p450: role of cyp3a4 and cyp3a5.

Midazolam, triazolam (TRZ), testosterone, and nifedipine have all been widely used as probes for in vitro metabolism of CYP3A. We used these four substrates to assess the contributions of CYP3A4 and CYP3A5 to in vitro biotransformation in human liver microsomes (HLMs) and in recombinant enzymes. Recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) both produced 1-OH and 4-OH metabolites from midazolam and triazolam, 6 beta-hydroxytestosterone from testosterone, and oxidized nifedipine from nifedipine. Overall, the metabolic activity of CYP3A5 was less than that of CYP3A4. Ketoconazole potently inhibited midazolam, triazolam, testosterone, and nifedipine metabolite formation in HLMs and in rCYP3A4. The inhibitory potency of ketoconazole in rCYP3A5 was about 5- to 19-fold less than rCYP3A4 for all four substrates. In testosterone interaction studies, testosterone inhibited 1-OH-TRZ formation, but significantly activated 4-OH-TRZ formation in HLMs and rCYP3A4 but not in rCYP3A5. Oxidized nifedipine formation was inhibited by testosterone in rCYP3A4. However, in rCYP3A5, testosterone slightly activated oxidized nifedipine formation at lower concentrations, followed by inhibition. Thus, CYP3A4 and CYP3A5 both contribute to midazolam, triazolam, testosterone, and nifedipine biotransformation in HLMs, with CYP3A5 being metabolically less active than CYP3A4 in general. Because the inhibitory potency of ketoconazole in rCYP3A5 is substantially less than in rCYP3A4 and HLMs, CYP3A5 is probably less important than CYP3A4 in drug-drug interactions involving ketoconazole and CYP3A substrates.     

Cytochrome P450 4A isoform inhibitory profile of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016), a selective inhibitor of 20-HETE synthesis

We examined the effect of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (HET0016), an inhibitor of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthesis on the omega-hydroxylation and epoxidation of arachidonic acid (AA) catalyzed by recombinant cytochrome P450 4A1 (CYP4A1), CYP4A2 and CYP4A3, and characterized the enzyme inhibitory profile of HET0016. The IC50 values of HET0016 for recombinant CYP4A1-, CYP4A2- and CYP4A3-catalyzed 20-HETE synthesis averaged 17.7 nM, 12.1 nM and 20.6 nM, respectively. The IC50 value for production of 11,12-epoxy-5,8,14-eicosatrienoic acid (11,12-EET) by CYP4A2 and 4A3 averaged 12.7 nM and 22.0 nM, respectively. The IC50 value for CYP2C11 activity was 611 nM which was much greater than that for CYP4As. The initial velocity study showed the Ki value of HET0016 for CYP4A1 was 19.5 nM and a plot of Vmax versus amount of recombinant CYP4A1 added shows HET0016 is an irreversible non-competitive inhibitor. These results indicate that HET0016 is a selective, non-competitive and irreversible inhibitor of CYP4A.     

Localization of rat cytochrome P450 in various tissues and comparison of arachidonic acid metabolism by rat P450 with that by human P450 orthologs

Metabolites of arachidonic acid produced by P450 are interesting substances with prominent physiological functions. To elucidate the physiological function of P450, it is necessary to identify a specific P450 in a particular tissue or organ and to characterize its catalytic activities. In this study, the expression of CYP2A1, 2B1, 2C23, 2J3, and 4F1 was investigated in liver, lung, kidney, spleen, heart, brain, and testis of rats by RT-PCR. Furthermore, arachidonic acid metabolism was investigated using the rat P450s described above and human CYP2A6, 2B6, 2C9, 2C18, 2C19, 2J2, and 4F2. Among the rat P450s, CYP2B1 and 2C23 efficiently produced EETs and CYP4F1 produced 19/20-HETE in abundace. CYP2B1 was specifically expressed in the lung. CYP2C23 was detected in all tissues used in this study. CYP4F1 was expressed in the kidney as well as in the liver. Among the human P450s, CYP2C9 and 2C19 efficiently produced EETs. CYP4F2 produced 19/20-HETE. The catalytic properties of rat CYP2C23 were similar to those of human CYP2C9 and 2C19. The catalytic properties of CYP4F isoforms were also similar between humans and rats. A systematic analysis of P450 expression in various tissues and of its catalytic property may provide valuable information on the physiological roles of P450s in each tissue.     

Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine.

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.     

细胞色素P450(CYP450)4A11与CYP4F2基因多态性与心血管疾病的相关性

细胞色素P450(CYP)ω-羟化酶主要包括CYP4A11和CYP4F2,代谢花生四烯酸(AA)生成20-羟化二十碳四烯酸(20-HETE),20-HETE具有收缩血管和促进尿钠排泄等作用。CYP4A11和4F2基因具有高度多态性,这种多态性导致酶活性降低,并可能引起某些心血管疾病。     

20-hydroxyeicosatetraeonic acid: a new target for the treatment of hypertension

Arachidonic acid is metabolized by enzymes of the CYP4A and 4F families to 20-hydroxyeicosatetraeonic acid (20-HETE), which plays an important role in the regulation of renal function, vascular tone, and the long-term control of arterial pressure. In the vasculature, 20-HETE is a potent vasoconstrictor, and upregulation of the production of this compound contributes to the elevation in oxidative stress and endothelial dysfunction and the increase in peripheral vascular resistance associated with some forms of hypertension. In kidney, 20-HETE inhibits Na transport in the proximal tubule and thick ascending loop of Henle, and deficiencies in the renal formation of 20-HETE contributes to sodium retention and development of some salt-sensitive forms of hypertension. 20-HETE also has renoprotective actions and opposes the effects of transforming growth factor β to promote proteinuria and renal end organ damage in hypertension. Several new inhibitors of the synthesis of 20-HETE and 20-HETE agonists and antagonists have recently been developed. These compounds along with peroxisome proliferator-activated receptor-α agonists that induce the renal formation of 20-HETE seem to have promise as antihypertensive agents. This review summarizes the rationale for the development of drugs that target the 20-HETE pathway for the treatment of hypertension and associated cardiovascular complications.     

Alteration of cardiac cytochrome P450-mediated arachidonic acid metabolism in response to lipopolysaccharide-induced acute systemic inflammation

Cytochrome P450 (CYP) generated cardioprotective metabolites, epoxyeicosatrienoic acids (EETs), and cardiotoxic metabolites, hydroxyeicosatetraenoic acids (HETEs) levels are determined by many factors, including the induction or repression of the CYP enzymes, responsible for their formation. Therefore, we examined the effect of acute inflammation on the expression of CYP epoxygenases and CYP ω-hydroxylases in the heart, kidney, and liver and the cardiac CYP-mediated arachidonic acid metabolism. For this purpose, male Sprague–Dawley rats were injected intraperitoneally with LPS (1 mg/kg). After 6, 12, or 24 h, the tissues were harvested and the expression of CYP genes and protein levels were determined using real time-PCR, and Western blot analyses, respectively. Arachidonic acid metabolites formations were determined by liquid chromatography–electron spray ionization-mass spectrometry LC-ESI-MS. Our results showed that inflammation significantly decreased the CYP epoxygenases expression in the heart, kidney and liver with a concomitant decrease in the EETs produced by these enzymes. In contrast to CYP expoxygenses, inflammation differentially altered CYP ω-hydroxylases expression with a significant increase in 20-HETE formation. The present study demonstrates for the first time that acute inflammation decreases CYP epoxygenases and their associated cardioprotective metabolites, EETs while on the other hand increases CYP ω-hydroxylases and their associated cardiotoxic metabolites, 20-HETE. These changes may be involved in the development and/or progression of cardiovascular diseases by inflammation.     

Role of cytochrome P450 metabolites of arachidonic acid in hypertension

Considerable evidence has accumulated over the last decade implicating a role of cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA) in the pathogenesis of hypertension. Indeed, 20-hydroxyeicosatetraenoic acid (20-HETE) is produced by vascular smooth muscle (VSM) cells and is a potent vasoconstrictor that depolarizes VSM by blocking large conductance Ca+-activated K2+ channels. In contrast, epoxyeicosatrienoic acids (EETs) are synthesized by the vascular endothelium and have opposite effects on VSM (hyperpolarization and vasodilatation). Inhibition of the synthesis of 20-HETE attenuates myogenic tone and autoregulation of blood flow and modulates vascular responses to vasodilators (NO and CO) and vasoconstrictors (angiotensin II, endothelin). In the kidney, 20-HETE inhibits sodium transport in the proximal tubule by blocking Na+-K+-ATPase activity. In the thick ascending limb of the loop of Henle, 20-HETE inhibits Na+-K+-2Cl- transport, in part, by blocking a 70 pS apical K+ channel. EETs are produced in the proximal tubule where they inhibit Na+-H+ exchange and in the collecting duct where they inhibit sodium and water transport. Numerous studies have established that the formation of EETs and 20-HETE and the expression of CYP enzymes are altered in the kidney in many genetic and experimental animal models of hypertension and in some forms of human hypertension. However, the functional significance of these changes remains to be determined. Given the importance of this pathway in the control of renal function and vascular tone, it is likely that alterations in the renal formation of CYP-dependent metabolites of AA will be shown to participate in the development of hypertension in many of these models.     

16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes

Intact human polymorphonuclear leukocytes (PMNL) incubated with substimulatory amounts of arachidonic acid in the absence of a calcium ionophore formed four metabolites that were isolated by reverse-phase HPLC and characterized structurally by GC/MS. A major metabolite eluting as the most abundant peak of radioactivity lacked UV chromophores above 215 nm, and its formation was sensitive to 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF525A) but not 3-amino-1-[m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), suggesting that it was likely to be a product of cytochrome P450. The GC/MS analysis revealed the presence of two components: 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) in an approximate ratio of 4:1. The minor metabolites were identified as 15-HETE and 5-HETE. Although 20-HETE has been observed previously as a product of arachidonic acid metabolism in PMNL, the occurrence of 16-HETE was a novel finding. The stereochemistry of the hydroxyl group in PMNL-derived 16-HETE was established by analysis of 1-pentafluorobenzyl-16-naphthoyl derivatives on a chiral-phase chromatographic column and comparison with authentic synthetic stereoisomers. The PMNL-derived radioactive metabolite co-eluted with the synthetic 16(R)-HETE stereoisomer. Analysis of the total lipid extracts from intact PMNL followed by mild alkaline hydrolysis resulted in detectable amounts of 16-HETE (108+/-26 pg/10(8) cells) and 20-HETE (341+/-69 pg/10(8) cells), which suggested that these HETEs were formed from endogenous arachidonic acid and esterified within PMNL lipids. Thus, in contrast to calcium ionophore-stimulated neutrophils that generate large amounts of 5-lipoxygenase products, the intact PMNL generate 20-HETE and 16(R)-HETE via a cytochrome P450 omega- and omega-4 oxygenase(s).     

Participation of epoxygenase activation in saikogenin D-induced inhibition of prostaglandin E(2) synthesis

We examined the effect of saikogenin D on arachidonic acid metabolism in C6 rat glioma cells to clarify its anti-inflammatory mechanism. Incubation of C6 cells with saikogenin D for 20 min resulted in the inhibition of prostaglandin E(2) production and the accumulation of an arachidonic acid metabolite that was found to be 11,12-dihydroxyeicosatrienoic acid, a metabolite of 11,12-epoxyeicosatrienoic acid. C6 cells expressed rat epoxygenase mRNAs, CYP1A1, CYP2B1 and CYP2J3, which converted arachidonic acid to epoxyeicosatrienoic acids. 11,12-Epoxyeicosatrienoic acid inhibited A23187-induced prostaglandin E(2) production and SKF-525A, an inhibitor of epoxygenase, attenuated the saikogenin D-induced inhibition of prostaglandin E(2) production in C6 cells. Furthermore, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid, but not saikogenin D, inhibited the activity of cyclooxygenase in a cell-free condition. These data suggest that saikogenin D activates epoxygenases that rapidly convert arachidonic acid to epoxyeicosanoids and dihydroxyeicosatrienoic acids, and then the metabolites secondarily inhibit prostaglandin E(2) production.     

Drug interaction of efavirenz and midazolam: efavirenz activates the CYP3A-mediated midazolam 1'-hydroxylation in vitro

CYP3A4 and CYP3A5 are the most important drug-metabolizing enzymes. For several drugs, heteroactivation of CYP3A-mediated reactions has been demonstrated in vitro. In vivo data suggested a possible acute activation of CYP3A4-catalyzed midazolam metabolism by efavirenz. Therefore, we aimed to investigate the effect of efavirenz on the in vitro metabolism of midazolam. The formation of 1'-hydroxymidazolam was studied in pooled human liver microsomes (HLM) and recombinant human CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) in the presence of efavirenz (0.5, 1, and 5 μM). Product formation rates (V(max)) increased with increasing efavirenz concentrations (～1.5-fold increase at 5 μM efavirenz in HLM and ～1.4-fold in rCYP3A4). The activation in rCYP3A4 was dependent on cytochrome b5, and the activating effect was also observed in rCYP3A5 supplemented with cytochrome b5, where Vmax was ～1.3-fold enhanced. Concomitant inhibition of CYP3A activity with ketoconazole in HLM abolished the increase in the 1'-hydroxymidazolam formation rate, further confirming involvement of CYP3A. The results of this study represent a distinct acute activation of midazolam metabolism and support the in vivo observations. Moreover, only efavirenz, but not its major metabolite 8-hydroxyefavirenz, was responsible for the activation. The increase in 1'-hydroxymidazolam formation may have been caused by binding of efavirenz to a peripheral site of the enzyme, leading to enhanced midazolam turnover due to changes at the active site.     

Effects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by human cytochrome P450 enzymes

1. Effects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by 12 recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes have been investigated. 2. Arachidonic acid (50 microM) significantly inhibited CYP1A1- and 1A2-dependent 7-ethoxycoumarin O-deethylations, CYP2C8-dependent taxol 6alpha-hydroxylation and CYP2C19-dependent R-warfarin 7-hydroxylation. This chemical also inhibited slightly the xenobiotic oxidations catalysed by CYP1B1, 2B6, 2C9, 2D6, 2E1 and 3A4 in recombinant enzyme systems. 3. Retinol, retinoic acid and cholecalciferol were strong inhibitors for xenobiotic oxidations catalysed by recombinant CYP1A1, 2C8 and 2C19. 4. Dixon plots of inhibitions of CYP1A1-, 1A2-, 2C8- and 2C19-dependent xenobiotic oxidations by arachidonic acid, of CYP1A1-, 2B6- and 2C19-dependent activities by retinol, and of CYP1A1- and 2C19-dependent activities by cholecalciferol indicated that these chemicals inhibit P450 activities mainly through a competitive mechanism. 5. In human liver microsomes, arachidonic acid inhibited CYP1A2-dependent theophylline hydroxylation, CYP2C8-dependent taxol 6alpha-hydroxylation and CYP2C19-dependent omeprazole 5-hydroxylation. Taxol 6alpha-hydroxylation was also inhibited by retinol and retinoic acid, and omeprazole 5-hydroxylation was inhibited by retinol in human liver microsomes. 6. These results suggest that xenobiotic oxidations by P450 enzymes are affected by endobiotic chemicals and that the endobiotic-xenobiotic interactions as well as drug-drug interactions may be of great importance when understanding the basis for pharmacological and toxicological actions of a number of xenobiotic chemicals.     

In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (K_i = 4.98 μM). Cryptotanshinone (K_i = 6.87 μM) and tanshinone I (K_i = 0.42 μM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (K_i = 0.09 μM), and noncompetitive inhibition on CYP4F2 (K_i = 4.25 μM) and CYP4F3B (K_i = 3.08 μM). Salvianolic acid A had competitive inhibition on CYP4A11 (K_i = 19.37 μM), and noncompetitive inhibition on CYP4F2 (K_i = 15.28 μM) and CYP4F3B (K_i = 6.45 μM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (K_i = 5.70 μM) and CYP4F3B (K_i = 18.64 μM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 μM) and salvianolic acid A (20 and 50 μM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.     

The role of lithocholic acid in the regulation of bile acid detoxication, synthesis, and transport proteins in rat and human intestine and liver slices

The effects of the secondary bile acid, lithocholic acid (LCA), a VDR, FXR and PXR ligand, on the regulation of bile acid metabolism (CYP3A isozymes), synthesis (CYP7A1), and transporter proteins (MRP3, MRP2, BSEP, NTCP) as well as nuclear receptors (FXR, PXR, LXRα, HNF1α, HNF4α and SHP) were studied in rat and human precision-cut intestine and liver slices at the mRNA level. Changes due to 5 to 10 μM of LCA were compared to those of other prototype ligands for VDR, FXR, PXR and GR. LCA induced rCYP3A1 and rCYP3A9 in the rat jejunum, ileum and colon, rCYP3A2 only in the ileum, rCYP3A9 expression in the liver, and CYP3A4 in the human ileum but not in liver. LCA induced the expression of rMRP2 in the colon but not in the jejunum and ileum but did not affect rMRP3 expression along the length of the rat intestine. In human ileum slices, LCA induced hMRP3 and hMRP2 expression. In rat liver slices, LCA decreased rCYP7A1, rLXRα and rHNF4α expression, induced rSHP expression, but did not affect rBSEP or rNTCP expression; whereas in the human liver, a small but significant decrease was found for hHNF1α expression. These data suggests profound species differences in the effects of LCA on bile acid transport, synthesis and detoxification. An examination of the effects of prototype VDR, PXR, GR and FXR ligands showed that these pathways are all intact in precision cut slices and that LCA exerted VDR, PXR and FXR effects. The LCA-induced altered enzymes and transporter expressions in the intestine and liver would affect the disposition of drugs.     

Effects of arachidonic acid,prostaglandins, retinol,retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by human cytochrome P450 enzymes

1. Effects of arachidonic acid, prostaglandins, retinol, retinoic acid and cholecalciferol on xenobiotic oxidations catalysed by 12 recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes have been investigated. 2. Arachidonic acid (50 μM) significantly inhibited CYP1A1- and 1A2-dependent 7-ethoxycoumarin O-deethylations, CYP2C8-dependent taxol 6α-hydroxylation and CYP2C19-dependent R-warfarin 7-hydroxylation. This chemical also inhibited slightly the xenobiotic oxidations catalysed by CYP1B1, 2B6, 2C9, 2D6, 2E1 and 3A4 in recombinant enzyme systems. 3. Retinol, retinoic acid and cholecalciferol were strong inhibitors for xenobiotic oxidations catalysed by recombinant CYP1A1, 2C8 and 2C19. 4. Dixon plots of inhibitions of CYP1A1-, 1A2-, 2C8- and 2C19-dependent xenobiotic oxidations by arachidonic acid, of CYP1A1-, 2B6- and 2C19-dependent activities by retinol, and of CYP1A1- and 2C19-dependent activities by cholecalciferol indicated that these chemicals inhibit P450 activities mainly through a competitive mechanism. 5. In human liver microsomes, arachidonic acid inhibited CYP1A2-dependent theophylline hydroxylation, CYP2C8-dependent taxol 6α-hydroxylation and CYP2C19- dependent omeprazole 5-hydroxylation. Taxol 6α-hydroxylation was also inhibited by retinol and retinoic acid, and omeprazole 5-hydroxylation was inhibited by retinol in human liver microsomes. 6. These results suggest that xenobiotic oxidations by P450 enzymes are affected by endobiotic chemicals and that the endobiotic-xenobiotic interactions as well as drug-drug interactions may be of great importance when understanding the basis for pharmacological and toxicological actions of a number of xenobiotic chemicals.