Microsomal Preparations Research Articles

QSAR of binding of dihydropyridine-type calcium antagonists to their receptor on ileal smooth muscle preparations.

Quantitative structure-activity analysis (Hansch analysis) is applied to elucidate the structural requirement for the binding of dihydropyridine-type calcium antagonists (DHPs) to their receptor in the guinea-pig ileal muscle preparations. It is found that various steric (B1, L), electronic (sigma) and hydrophobic (pi) parameters or their combinations correlate well with the potency of various DHPs to inhibit the binding of [3H]nitrendipine to the microsomal preparations of the guinea-pig ileal muscle. The potency of DHPs increases with the minimum width (B1) of substituent at ortho- or meta-positions, but decreases with the increase in the length of substituent at the meta-position. The potency of DHPs decreases with the increase in both minimum width or length of substituent at the para-position and the optimal values were found to be those for hydrogen. The hydrophobicity (pi) of substituents at different positions in the 4-phenyl ring affects the potency differently, indicating that a different environment exists around each position at the binding site. From the slopes of the pi variable in the regression equations, it is concluded that the receptive environment of the ortho-position of the 4-phenyl ring of DHPs is lipophilic, and for that of the para-position hydrophilic. A good correlation is also observed between the Hammett electronic parameter (sigma) and biological activity of meta-substituted DHPs. It is suggested that in the binding of the substituted 4-phenyl DHPs to their receptor, both electronic and hydrophobic interactions should be considered.

The in vivo metabolism of isomeric methoxyoxindoles

Abstract Isomeric methoxyoxindoles are metabolised by rats, guinea-pigs and rabbits to phenolic metabolites by O-demethylation and hydroxylation. This has been shown by comparison of the physical characteristics of the metabolites with those of standard synthetic hydroxyoxindoles. Negligible O-demethylation of methoxyoxindoles occurs with rat or guinea-pig liver microsome preparations, when compared with those of rabbit, but significant O-demethylation occurs with rat liver slices.

Probing Elongating and Branching β-d-Galactosyltransferase Activities in Leishmania Parasites by Making Use of Synthetic Phosphoglycans

Protozoan parasites of the genus Leishmania synthesize lipophosphoglycans (LPGs), phosphoglycans and proteophosphoglycans that contain phosphosaccharide repeat units of [-6)Gal(β1-4)Man(α1-OPO(3)H-]. The repeat structures are assembled by sequential addition of Manα1-OPO(3)H and β-Gal. In this study, an UDP-Gal-dependent activity was detected in L. donovani and L. major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. Incubation of a microsomal preparation from L. donovani or L. major parasites with synthetic substrates and UDP-[6-(3)H]Gal resulted in incorporation of radiolabel into these exogenous acceptors. The [(3)H]galactose-labeled products were characterized by degradation into radioactive, low molecular mass fragments upon hydrolysis with mild acid and treatment with β-galactosidases. We showed that the activity detected with L. donovani membranes is the elongating β-d-galactosyltransferase associated with LPG phosphosaccharide backbone biosynthesis (eGalT). The eGalT activity showed a requirement for the presence of at least one phosphodiester group in the substrate and it was enhanced dramatically when two or three phosphodiester groups were present. Using the same substrates we detected two types of galactosyltransferase activity in L. major membranes: the elongating β-d-galactosyltransferase and a branching β-d-galactosyltransferase (bGalT). Both L. major enzymes required a minimum of one phosphodiester group present in the substrate, but acceptors with two or three phosphodiester groups were found to be superior.

Postmenopausal hypertension: role of 20-HETE

Blood pressure (BP) increases after menopause. However, the mechanisms responsible have not been elucidated. In this study we tested the hypothesis that 20-hydroxyeicosatetraenoic acids (20-HETE), produced by cytochrome P-450 (CYP450) ω-hydroxylase, contributes to the hypertension in a model of postmenopausal hypertension, aged female spontaneously hypertensive rats (PMR). 1-Aminobenzotriazole, a nonselective inhibitor of arachidonic acid metabolism, for 7 days, reduced BP in PMR but had no effect in young females. Acute intravenous infusion of HET-0016, a specific inhibitor of 20-HETE, over 3 h, also reduced BP in PMR. CYP4A isoform mRNA expression showed no difference in renal CYP4A1 or CYP4A3 but increases in CYP4A2 and decreases in CYP4A8. CYP4A protein expression was decreased in kidney of PMR compared with young females. Endogenous 20-HETE was significantly higher in cerebral vessels of PMR than young females (YF) but was significantly lower in renal vessels of PMR. Omega-hydroxylase activity in cerebral vessels was also higher in PMR but was similar in kidney vessels in both groups. In renal microsomal preparations, endogenous 20-HETE was not different in PMR and young females, but ω-hydroxylase activity was significantly lower in PMR than YF. The data with blockers suggest that 20-HETE contributes to postmenopausal hypertension in SHR. The data also suggest that cerebral production of 20-HETE may be increased and renal tubular production may be decreased in PMR, thus both contributing to their elevated BP.

Analysis of Membrane Topology and Identification of Essential Residues for the Yeast Endoplasmic Reticulum Inositol Acyltransferase Gwt1p

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

Tamoxifen metabolites as active inhibitors of aromatase in the treatment of breast cancer

The mechanism of tamoxifen action in the treatment of breast cancer is believed to be via active metabolites that act as potent estrogen receptor antagonists. Attempts to identify relationships between active metabolite concentrations and clinical outcomes have produced mixed results. Since anti-estrogenic effects may be brought about not only by estrogen antagonism, but also by reduced estrogen synthesis, we tested the ability of tamoxifen and its principal metabolites to inhibit aromatase in vitro. The activity of human aromatase in both recombinant and placental microsomal preparations was measured using the rate of generation of a fluorescent metabolite in the presence and absence of multiple concentrations of tamoxifen, endoxifen, N-desmethyl-tamoxifen, and Z-4-hydroxy-tamoxifen. Aromatase inhibition was further characterized by measuring the inhibition of testosterone metabolism to estradiol. The biochemical mechanisms of inhibition were documented and their inhibitory potency was compared. Using recombinant human aromatase, endoxifen, and N-desmethyl-tamoxifen were able to inhibit aromatase activity with K (i) values of 4.0 and 15.9μM, respectively. Detailed characterization of inhibition by endoxifen and N-desmethyl-tamoxifen indicated non-competitive kinetics for both inhibitors. Similarly, endoxifen-inhibited testosterone metabolism via a non-competitive mechanism. No appreciable inhibition by tamoxifen or Z-4-hydroxy-tamoxifen was observed at similar concentrations. The relative inhibitory potency was: endoxifen>N-desmethyl-tamoxifen>>>Z-4-hydroxy-tamoxifen>tamoxifen. Similar data were obtained in human placental microsomes. Endoxifen and N-desmethyl-tamoxifen were found to be potent inhibitors of aromatase. Inhibition by these tamoxifen metabolites may contribute to the variability in clinical effects of tamoxifen in patients with breast cancer. Relationships between tamoxifen metabolite concentrations and clinical outcomes may be complex, and the biologic mechanisms that underlie these relationships may include aromatase inhibition.

Involvement of Protein Kinase D in Expression and Trafficking of ATP7B (Copper ATPase)

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.

Dicofol degradation to p,p′-dichlorobenzophenone – A potential antiandrogen

In the present investigation, the degradation of the acaricide dicofol (also known as kelthane) was investigated with special emphasis on generation of p,p′-dichlorobenzophenone (DCBP) under alkaline conditions as well as induced by UV-light. Dicofol was also incubated in the presence and absence of microsomal preparations to measure potential metabolic formation of DCBP. The results indicate that the degradation of dicofol to DCBP primarily proceeds as an abiotic process via hydroxide ion catalysed elimination of a trichloromethyl anion. The generated anion picks up a proton from the solvent to generate chloroform. Microsomal metabolism does not appear to play a major role in the degradation of dicofol. DCBP is structurally analogous to the antiandrogen p,p′-dichlorodiphenylethene (DDE). We therefore investigated whether DCBP displays antiandrogenic properties. In an in vitro transactivation system utilising transiently transfected African green monkey kidney (COS-7) cells, DCBP showed potent antiandrogenic efficacy. This finding was confirmed by further studies in T47D human mammary carcinoma cells by measuring mRNA and protein expression of androgen dependent genes i.e. TRMP-2 (testosterone-repressed prostate message-2) mRNA and PSA (prostate-specific antigen) protein.

Proteomic responses in Arabidopsis thaliana seedlings treated with ethylene

Ethylene (ET) is a volatile hormone that modulates fruit ripening, plant growth, development and stress responses. Key components of the ET-signaling pathway identified by genetic dissection in Arabidopsis thaliana include five ET receptors, the negative regulator CTR1 and the positive regulator EIN2, all of which localize to the endoplasmic reticulum. Mechanisms of signaling among these proteins are still unresolved and targets of ET responses are not fully known. So, we used mass spectrometry to identify proteins in microsomal membrane preparations from etiolated A. thaliana seedlings maintained in ambient air or treated with ET for 3 h. We compared 3814 proteins from ET-exposed seedlings and controls and identified 304 proteins with significant accumulation changes. The proteins with increased accumulation were involved in ET biosynthesis, cell morphogenesis, oxidative stress and vesicle secretion while those with decreased accumulation were ribosomal proteins and proteins positively regulated by brassinosteroid, another hormone involved in cell elongation. Several proteins, including EIN2, appeared to be differentially phosphorylated upon ET treatment, which suggests that the activity or stability of these proteins may be controlled by phosphorylation. TUA3, a component of microtubules that contributes to cellular morphological change, exhibited both increased accumulation and differential phosphorylation upon ET treatment. To verify the role of TUA3 in the ET response, tua3 mutants were evaluated. Mutant seedlings had altered ET-associated growth movements. The data indicate that ET perception leads to rapid proteomic change and that these changes are an important part of signaling and development. The data serve as a foundation for exploring ET signaling through systems biology.

Gold nanoparticles uptake and cytotoxicity assessed on rat liver precision cut slices

Nanotechnology research and development have been rapidly growing worldwide for the past decade. Although nanomaterials (NM)s provide great potential of commercial benefits, some of them have been claimed to be toxic in in vivo and in vitro tests and our knowledge on their toxicological properties is far from being comprehensive. With the current technological capabilities toxicological and environmental risk assessment of NPs struggles to keep up with all newly synthesized NMs. For this reason several authors have underlined the need to develop reliable in vitro models for studying their toxic effects. In order to set up a novel tool for the risk assessment of nanoparticles toxicity we evaluated cell uptake and toxicity of 5 nm gold nanoparticles (GNP)s coated with polyvynylpirrolidone (PVP) on an in vitro model system represented by rat liver “precision cut” slices. Rat liver precision cut slices were incubated individually at 37°C in RPMI 1640 medium for up to 24 h in the presence of GNPs at different concentrations. The presence of GNPs inside endocytotic vesicles of hepatocytes was appreciable already after 30 min incubation. From 2h onwards, GNPs cell uptake was substantiated by their presence inside endosome-like vesicles not only in hepatocytes, but also in endothelial and Kuppfer cells. After 6h GNPs could be spotted within lysosomes located close to the biliary pole of the hepatocytes. These results were confirmed in vivo with few experiments carried out upon i.p injection of GNPs. This did not translate into modification of both phase I and phase II of 7-ethoxycoumarin metabolism. Similarly, CYPs activities of slices microsomal preparations towards marker substrates resulted unaffected. Although GNPs were promptly taken up by slices, no signs of cytotoxicity were observed. In fact, LDH release, MTT reduction and GSH levels in GNPs-treated slices were not different from those of control slices. In conclusion, rat liver precision cut slices can be employed for assessing the consequences of the biological impact of nanoparticles.

The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1-5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.

Metabolic Activity and mRNA Levels of Human Cardiac CYP450s Involved in Drug Metabolism

BackgroundTissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.MethodologyCYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68) of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7), samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.Principal FindingsOur results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.ConclusionsThis study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.

Antimalarial Activity of Novel 5-Aryl-8-Aminoquinoline Derivatives

In an attempt to separate the antimalarial activity of tafenoquine (3) from its hemolytic side effects in glucose-6-phosphate dehydrogenase (G6PD) deficiency patients, a series of 5-aryl-8-aminoquinoline derivatives was prepared and assessed for antimalarial activities. The new compounds were found metabolically stable in human and mouse microsomal preparations, with t(1/2) > 60 min, and were equal to or more potent than primaquine (2) and 3 against Plasmodium falciparum cell growth. The new agents were more active against the chloroquine (CQ) resistant clone than to the CQ-sensitive clone. Analogues with electron donating groups showed better activity than those with electron withdrawing substituents. Compounds 4bc, 4bd, and 4be showed comparable therapeutic index (TI) to that of 2 and 3, with TI ranging from 5 to 8 based on IC(50) data. The new compounds showed no significant causal prophylactic activity in mice infected with Plasmodium berghei sporozoites, but are substantially less toxic than 2 and 3 in mouse tests.

Quantitative protein determination for CYP induction via LC‐MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.

Common Variants of Cytochrome P450 4F2 Exhibit Altered Vitamin E-Ω-Hydroxylase Specific Activity

Human cytochrome P450 4F2 (CYP4F2) catalyzes the Ω-hydroxylation of the side chain of tocopherols (TOH) and tocotrienols (T3), the first step in their catabolism to polar metabolites excreted in urine. CYP4F2, in conjunction with α-TOH transfer protein, results in the conserved phenotype of selective retention of α-TOH. The purpose of this work was to determine the functional consequences of 2 common genetic variants in the human CYP4F2 gene on vitamin E-Ω-hydroxylase specific activity using the 6 major dietary TOH and T3 as substrate. CYP4F2-mediated Ω-hydroxylase specific activity was measured in microsomal preparations from insect cells that express wild-type or polymorphic variants of the human CYP4F2 protein. The W12G variant exhibited a greater enzyme specific activity (pmol product ⋅ min−1 ⋅ pmol CYP4F2−1) compared with wild-type enzyme for both TOH and T3, 230–275% of wild-type toward α, γ, and Δ-TOH and 350% of wild-type toward α, γ, and Δ-T3. In contrast, the V433M variant had lower enzyme specific activity toward TOH (42–66% of wild type) but was without a significant effect on the metabolism of T3. Because CYP4F2 is the only enzyme currently shown to metabolize vitamin E in humans, the observed substrate-dependent alterations in enzyme activity associated with these genetic variants may result in alterations in vitamin E status in individuals carrying these mutations and constitute a source of variability in vitamin E status.

Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.

Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

Comparison of cytochrome P450 concentrations and metabolic activities in porcine hepatic microsomes prepared with two different methods

In the present study, porcine liver microsomes prepared by a conventional ultracentrifugation method were compared with microsomes prepared by a calcium aggregation method. Protein concentrations and activities of several cytochrome P450 enzymes were measured. It was concluded that using a calcium aggregation method for microsome preparation resulted in lower activities of porcine 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-pentoxyresorufin O-depentylase (PROD) and p-nitrophenol hydroxylase (PNPH), compared to ultracentrifugation. Protein concentrations of CYP1A2 and CYP2E1, measured by Western blot, were similar in the microsomes prepared by the two methods, whereas CYP2A protein concentrations were significantly lower in the microsomes prepared by the calcium aggregation method. The choice of homogenization buffer (TRIS with addition of either 250 mM sucrose or 2 mM EDTA) did not affect either individual CYP450 protein concentration or the rates of CYP450-mediated reactions. Freeze/thawing of microsomes did not affect the activities of EROD, MROD, COH and PNPH in the microsomes, indicating the stability of the measured isoforms following three cycles of freezing/thawing. A reduction in the activity of PROD was observed after the third freeze/thawing cycles of the microsomes prepared by both methods.

Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids*

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca(2+)-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.

Comparative Use of Isolated Hepatocytes and Hepatic Microsomes for Cytochrome P450 Inhibition Studies: Transporter-Enzyme Interplay

Accurate assignment of the concentration of victim drug/inhibitor available at the enzyme active site, both in vivo and within an in vitro incubation, is an essential requirement in rationalizing and predicting drug-drug interactions. Inhibitor accumulation within the liver, whether as a result of active transport processes or intracellular binding, may best be accounted for using hepatocytes rather than hepatic microsomes to estimate in vitro inhibitory potency. The aims of this study were to compare K(i) values determined in rat liver microsomes and freshly isolated rat hepatocytes of four cytochrome P450 (P450) inhibitors (clarithromycin, enoxacin, nelfinavir, and saquinavir) with known hepatic transporter involvement and a range of uptake (cell/medium concentration ratios 20-3000) and clearance (10-1200 μl/min/10(6) cells) properties. Inhibition studies were performed using two well established P450 probe substrates (theophylline and midazolam). Comparison of unbound K(i) values showed marked differences between the two in vitro systems for inhibition of metabolism. In two cases (clarithromycin and enoxacin, both low-clearance drugs), inhibitory potency in hepatocytes markedly exceeded that in microsomes (10- to 20-fold), and this result was consistent with their high cell/medium concentration ratios. For nelfinavir and saquinavir (high-clearance, extensively metabolized drugs), the opposite trend was seen in the K(i) values: despite very high cell/medium concentration ratios, stronger inhibition was evident within microsomal preparations. Hence, the consequences of hepatic accumulation resulting from uptake transporters vary according to the clearance of the inhibitor. This study demonstrates that transporter-enzyme interplay can result in differences in inhibitory potency between microsomes and hepatocytes and hence drug-drug interaction predictions that are not always intuitive.