Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Effects of cytochrome P-450 metabolites of arachidonic acid on the epithelial sodium channel (ENaC)

Sodium reabsorption via the epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron plays a central role in the regulation of body fluid volume. Previous studies have indicated that arachidonic acid (AA) and its metabolite 11,12-EET but not other regioisomers of EETs inhibit ENaC activity in the collecting duct. The goal of this study was to investigate the endogenous metabolism of AA in cultured mpkCCD(c14) principal cells and the effects of these metabolites on ENaC activity. Liquid chromatography/mass spectrometry analysis of the mpkCCD(c14) cells indicated that these cells produce prostaglandins, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 12/8-HETE, and 15-HETE, but not 20-HETE. Single-channel patch-clamp experiments revealed that 8,9-EET, 14,15-EET, and 11,12-EET all decrease ENaC activity. Neither 5-, 12-, nor 15-HETE had any effect on ENaC activity. Diclofenac and ibuprofen, inhibitors of cyclooxygenase, decreased transepithelial Na(+) transport in the mpkCCD(c14) cells. Inhibition of cytochrome P-450 (CYP450) with MS-PPOH activated ENaC-mediated sodium transport when cells were pretreated with AA and diclofenac. Coexpression of CYP2C8, but not CYP4A10, with ENaC in Chinese hamster ovary cells significantly decreased ENaC activity in whole-cell experiments, whereas 11,12-EET mimicked this effect. Thus both endogenously formed EETs and their exogenous application decrease ENaC activity. Downregulation of ENaC activity by overexpression of CYP2C8 was PKA dependent and was prevented by myristoylated PKI treatment. Biotinylation experiments and single-channel analysis revealed that long-term treatment with 11,12-EET and overexpression of CYP2C8 decreased the number of channels in the membrane. In contrast, the acute inhibitory effects are mediated by a decrease in the open probability of the ENaC. We conclude that 11,12-EET, 8,9-EET, and 14,15-EET are endogenously formed eicosanoids that modulate ENaC activity in the collecting duct.     

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.     

Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels

Nitric oxide (NO) is an inhibitor of hemoproteins including cytochrome P-450 enzymes. This study tested the hypothesis that NO inhibits cytochrome P-450 epoxygenase-dependent vascular responses in kidneys. In rat renal pressurized microvessels, arachidonic acid (AA, 0.03-1 microM) or bradykinin (BK, 0.1-3 microM) elicited NO- and prostanoid-independent vasodilation. Miconazole (1.5 microM) or 6-(2-propargyloxyphenyl)hexanoic acid (30 microM), both of which are inhibitors of epoxygenase enzymes, or the fixing of epoxide levels with 11,12-epoxyeicosatrienoic acid (11,12-EET; 1 and 3 microM) inhibited these responses. Apamin (1 microM), which is a large-conductance Ca2+-activated K+ (BKCa) channel inhibitor, or 18alpha-glycyrrhetinic acid (30 microM), which is an inhibitor of myoendothelial gap junctional electromechanical coupling, also inhibited these responses. NO donors spermine NONOate (1 and 3 microM) or sodium nitroprusside (0.3 and 3 microM) but not 8-bromo-cGMP (100 microM), which is an analog of cGMP (the second messenger of NO), blunted the dilation produced by AA or BK in a reversible manner without affecting that produced by hydralazine. However, the non-NO donor hydralazine did not affect the dilatory effect of AA or BK. Spermine NONOate did not affect the dilation produced by 11,12-EET, NS-1619 (a BKCa channel opener), or cromakalim (an ATP-sensitive K+ channel opener). AA and BK stimulated EET production, whereas hydralazine had no effect. On the other hand, spermine NONOate (3 microM) attenuated basal (19 +/- 7%; P < 0.05) and AA stimulation (1 microM, 29 +/- 9%; P < 0.05) of renal preglomerular vascular production of all regioisomeric EETs: 5,6-; 8,9-; 11,12-; and 14,15-EET. These results suggest that NO directly and reversibly inhibits epoxygenase-dependent dilation of rat renal microvessels without affecting the actions of epoxides on K+ channels.     

Cyclic AMP-dependent modulation of cardiac L-type Ca2+ and transient outward K+ channel activities by epoxyeicosatrienoic acids

The three major enzyme systems, cyclo-oxygenase, lipoxygenase, and cytochrome P450 (P450/CYP), metabolize arachidonic acid (AA) to biologically active compounds. P450 and its associated monooxygenase activities have been identified in mammalian cardiac tissue, including humans. The four regioisomeric eicosanoids, 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) of AA metabolites derived by P450 epoxygenases have shown to possess potent biological effects in numerous tissues. In the coronary circulation the EETs are leading candidates for endothelial-derived hyperpolarizing factors that hyperpolarize vascular smooth muscle cells by opening Ca2+-activated K+ channels. Recently, the effects of the CYP pathways and their metabolites on cardiac ischemia-reperfusion injury have been evaluated in animal models. Some of these AA metabolites are cardioprotective and some are detrimental. However, EETs appear to be cardioprotective in CYP2J2 transgenic mice and in a canine ischemic model. Multiple effects of EETs on cardiac ion channels have been observed, such as activation of ATP-sensitive K+ channels and L-type Ca2+ channels in cardiomyocytes and inhibition of cardiac Na+ channels and L-type Ca2+ channels reconstructed in planar lipid bilayers. This brief review summarizes EET-induced modulation of cardiac ion channels.     

Stretch of beta 1 integrin activates an outwardly rectifying chloride current via FAK and Src in rabbit ventricular myocytes

Osmotic swelling of cardiac myocytes and other types of cells activates an outwardly rectifying, tamoxifen-sensitive Cl- current, ICl,swell, but it is unclear whether Cl- currents also are activated by direct mechanical stretch. We tested whether specific stretch of beta1-integrin activates a Cl- current in rabbit left ventricular myocytes. Paramagnetic beads (4.5-microm diameter) coated with mAb to beta1-integrin were applied to the surface of myocytes and pulled upward with an electromagnet while recording whole-cell current. In solutions designed to isolate anion currents, beta1-integrin stretch elicited an outwardly rectifying Cl- current with biophysical and pharmacological properties similar to those of ICl,swell. Stretch-activated Cl- current activated slowly (t1/2 = 3.5 +/- 0.1 min), partially inactivated at positive voltages, reversed near ECl, and was blocked by 10 microM tamoxifen. When stretch was terminated, 64 +/- 8% of the stretch-induced current reversed within 10 min. Mechanotransduction involved protein tyrosine kinase. Genistein (100 microM), a protein tyrosine kinase inhibitor previously shown to suppress ICl,swell in myocytes, inhibited stretch-activated Cl- current by 62 +/- 6% during continued stretch. Because focal adhesion kinase and Src are known to be activated by cell swelling, mechanical stretch, and clustering of integrins, we tested whether these tyrosine kinases mediated the response to beta1-integrin stretch. PP2 (10 microM), a selective blocker of focal adhesion kinase and Src, fully inhibited the stretch-activated Cl- current as well as part of the background Cl- current, whereas its inactive analogue PP3 (10 microM) had no significant effect. In addition to activating Cl- current, stretch of beta1-integrin also appeared to activate a nonselective cation current and to suppress IK1. Integrins are the primary mechanical link between the extracellular matrix and cytoskeleton. The present results suggest that integrin stretch may contribute to mechano-electric feedback in heart, modulate electrical activity, and influence the propensity for arrhythmogenesis.     

Activation of rat mesenteric arterial KATP channels by 11,12-epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs), the cytochrome P-450 epoxygenase metabolites of arachidonic acid, are candidates of endothelium-derived hyperpolarizing factors. We have previously reported that EETs are potent activators of cardiac ATP-sensitive K(+) (K(ATP)) channels, but their effects on the vascular K(ATP) channels are unknown. With the use of whole cell patch-clamp techniques with 0.1 mM ATP in the pipette and holding at -60 mV, freshly isolated smooth muscle cells from rat mesenteric arteries had small glibenclamide-sensitive currents at baseline (13.1 +/- 3.9 pA, n = 5) that showed a 7.2-fold activation by 10 microM pinacidil (94.1 +/- 21.9 pA, n = 7, P < 0.05). 11,12-EET dose dependently activated the K(ATP) current with an apparent EC(50) of 87 nM. Activation of the K(ATP) channels by 500 nM 11,12-EET was inhibited by inclusion of the PKA inhibitor peptide (5 microM) but not by the inclusion of the PKC inhibitor peptide (100 microM) in the pipette solution. These results were corroborated by vasoreactivity studies. 11,12-EET produced dose-dependent vasorelaxation in isolated small mesenteric arteries, and this effect was reduced by 50% with glibenclamide (1 microM) preincubation. The 11,12-EET effects on vasorelaxation were also significantly attenuated by preincubation with cell-permeant PKA inhibitor myristoylated PKI(14-22), and, in the presence of PKA inhibitor, glibenclamide had no additional effects. These results suggest that 11,12-EET is a potent activator of the vascular K(ATP) channels, and its effects are dependent on PKA activities.     

Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 microM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose-response curve of the AA effect on ENaC shows that 2 microM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) omega-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase-dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase-dependent metabolite, 11,12-EET.     

细胞色素P450 1A1和1B1靶向抗肿瘤前药研究进展

细胞色素P450（CYP）能催化各种内源性及外源性化合物的代谢,与多种肿瘤发生有关。其中CYP1A1参与多种前致癌物和致突变物的代谢活化,CYP1B1被认为在许多人癌细胞中特异性表达,参与药物的氧化代谢和前药的活化。CYP1A1和181已成为靶向抗肿瘤前药研究的新靶点。相继有大量相关研究报道,本文就近年来文献报道的CYP1A1和1B1靶向抗肿瘤前药研究进展。     

Trans-resveratrol modulates the catalytic activity and mRNA expression of the procarcinogen-activating human cytochrome P450 1B1

The present study was performed to determine if trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) modulates the catalytic activity and gene expression of cytochrome P450 1B1 (CYP1B1). In vitro, trans-resveratrol decreased human recombinant CYP1B1-catalyzed 7-ethoxyresorufin O-dealkylation activity, with an IC50 value of 1.4 +/- 0.2 microM (mean +/- SEM). Enzyme kinetic analysis indicated that trans-resveratrol inhibited CYP1B1 enzyme activity by a mixed-type inhibition and the apparent Ki was 0.75 +/- 0.06 microM. To determine if trans-resveratrol modulates constitutive CYP1B1 gene expression, cultured MCF-7 human breast carcinoma cells were treated with trans-resveratrol. As indicated by RT-PCR analysis, treatment of MCF-7 cells with 10 microM trans-resveratrol decreased relative CYP1B1 mRNA levels after 5 h, but not after 1.5 or 3 h, of exposure. trans-Resveratrol treatment at 5, 7.5, 10, or 20 microM for 5 h produced a concentration-dependent decrease in CYP1B1 mRNA levels. The extent of suppression was approximately 50% at 20 microM concentration. The suppressive effect was not a consequence of a toxic response to the compound as assessed by a cell proliferation assay. Overall, our novel finding that trans-resveratrol inhibits the catalytic activity and suppresses the constitutive gene expression of CYP1B1 leads to the possibility that this nutraceutical confers protection against toxicity and carcinogenicity induced by compounds that undergo CYP1B1-catalyzed bioactivation.     

Reduction of toxic metabolite formation of acetaminophen

Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation.     

Inhibition of heart calcium and chloride currents by sodium iodide. Specific attenuation in cAMP-dependent protein kinase-mediated regulation.

The enzymes cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate the activity of cardiac ion channel proteins. In this study the whole-cell arrangement of the patch clamp technique was used to examine the effect of NaI on PKA-stimulated Cl- and Ca2+ channels in isolated guinea pig ventricular myocytes. Cl- currents (ICl) activated either by the beta-adrenergic agonist isoproterenol or the membrane-soluble cAMP analogue, 8-chlorphenylthio (8-CPT) cAMP, were greatly reduced in amplitude after substitution of an external solution containing 140 mM NaCl with a solution containing 140 mM NaI. This reduction was accompanied by a shift of -7 mV in the reversal potential (Erev) for ICl and could be reversed upon return to the NaCl external solution. Inhibition of ICl by NaI occurred in a concentration-dependent manner and was more pronounced for inward ICl (IC50 = 19 mM at -60 mV) than for outward ICl (IC50 = 60 mM at +60 mV). In contrast to ICl activated by PKA, ICl activated by PKC was slightly augmented in the presence of NaI and the Erev was found to shift by -15 mV. Based on these data, the relative permeability of I- to Cl- (PI/PCl) for this channel was calculated to be 1.79. NaI produced no change in the amplitude of inward calcium currents (ICa) recorded under basal conditions, but strongly inhibited ICa augmented by isoproterenol and 8-CPT cAMP, and during dialysis of cells with the catalytic subunit of PKA (CS). The in vitro incorporation of [gamma-32P]ATP into histone IIA and Kemptide, measured in the presence of PKA and cAMP, was not significantly different in assay mixtures containing salts of Cl- and I-. However, the ability of isoproterenol to augment basal ICa in whole-cell experiments was attenuated when experiments were carried out entirely in NaI external solution. Thus, the reduction in ICl and ICa observed in this study may result from a direct effect of I- on the phosphorylation/dephosphorylation of cardiac ion channel proteins or associated regulatory proteins.     

11,12-epoxyeicosatrienoic acid stimulates heme-oxygenase-1 in endothelial cells

As epoxyeicosatrienoic acids (EETs), particularly 11,12-EET, and the heme oxygenase/carbon monoxide (HO/CO) system share overlapping biological activities, we examined a possible link between 11,12-EET and HO activity in endothelial cells. Confocal microscopy analysis of immunostaining of HO-1 and HO-2 in cultured endothelial cells treated with 11,12-EET (1 microM) showed an increase in florescence of HO-1 protein in the various cellular compartments, but not of HO-2. Incubation of endothelial cells with 11,12-EET (1 microM) for 24 h increased the level of HO-1 protein by about three-fold. Similarly, incubation of endothelial cells with 8,9-EET and sodium nitroprussiate, a known inducer of HO-1, increased HO-1 protein without any effect on HO-2. Upregulation of HO-1 by 11,12-EET, as well as 8,9-EET, was associated with an increase in HO activity, which was inhibited by stannous mesoporphirin (10 microM). Incubation of rat aortas with 11,12-EET (1 microM for 60 min) increased HO activity. These findings identify a novel effect of EETs on endothelial HO-1 and indicate that the signaling pathway of EETs in endothelial cells is possibly via an increase in HO-1 expression and activity.     

Purification and characterization of recombinant rat hepatic CYP4F1.

CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.     

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

Several P450 enzymes localized in the endoplasmic reticulum and thought to be involved primarily in xenobiotic metabolism, including mouse and rat CYP1A1 and mouse CYP1A2, have also been found to translocate to mitochondria. We report here that the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces enzymatically active CYP1A4/1A5, the avian orthologs of mammalian CYP1A1/1A2, in chick embryo liver mitochondria as well as in microsomes. P450 proteins and activity levels (CYP1A4-dependent 7-ethoxyresorufin-O-deethylase and CYP1A5-dependent arachidonic acid epoxygenation) in mitochondria were 23-40% of those in microsomes. DHET formation by mitochondria was twice that of microsomes and was attributable to a mitochondrial soluble epoxide hydrolase as confirmed by Western blotting with antiEPHX2, conversion by mitochondria of pure 11,12 and 14,15-EET to the corresponding DHETs and inhibition of DHET formation by the soluble epoxide hydrolase inhibitor, 12(-3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). TCDD also suppressed formation of mitochondrial and microsomal 20-HETE. The findings newly identify mitochondria as a site of P450-dependent arachidonic acid metabolism and as a potential target for TCDD effects. They also demonstrate that mitochondria contain soluble epoxide hydrolase and underscore a role for CYP1A in endobiotic metabolism.     

Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 microM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 microM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 microM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 microM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking-linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke.