Ethanol-Inducible Cytochrome P-450: Assessment of Substrates'' Specific Chemical Probes in Rat Liver Microsomes

The capacity of liver microsomes to oxidize various substrates known to be specific of alcohol-inducible cytochrome P-450 was studied in rats treated with different xenobiotics such as 3-methylcholanthrene, phenobarbital, acetone, and ethanol. Analysis of results showed a significantly marked increase following ethanol and acetone treatments of the p-nitrophenol hydroxylation (283 +/- 19% and 304 +/- 21%), N-nitrosodimethylamine (NDMA) demethylation (280 +/- 105% and 228 +/- 95%), benzene hydroxylation (258 +/- 60% and 236 +/- 61%), butanol oxidation (173 +/- 34% and 154 +/- 32%), aniline hydroxylation (147 +/- 22% and 95 +/- 8%), and ether de-ethylation (95 +/- 17% and 83 +/- 17%) and a not significant increase of N-nitrosodiethylamine (NDEA) de-ethylation (34 +/- 11% and 9 +/- 8%) in rat microsomes, respectively, versus control animals (mean +/- SD, values expressed as nmol/min/nmole P-450). All of these activities significantly decreased after 3-MC treatment, except for the p-nitrophenol hydroxylation. PB treatment markedly enhanced NDEA de-ethylation, p-nitrophenol, and benzene hydroxylations (106 +/- 38%, 109 +/- 14%, and 153 +/- 62%, respectively) versus controls. These results suggest that NDMA and especially 1-butanol are the most specific and useful probes of alcohol-inducible cytochrome P-450 in crude liver microsomes.     

Magnetic Circular Dichroism Studies XXXV. A Comparison of Cytochromes P-450 and P-448

Cytochromes P-450 and P-448 in microsomal suspensions have been shown to be spectrally distinct by magnetic circular dichroism spectroscopy. Furthermore, this technique can be used to measure induction of these two cytochromes by phenobarbital and 3-methyl-cholanthrene. Magnetic circular dichroism spectroscopy is thus at least as useful as difference spectroscopy for the investigation of P-450 and P-448 and more informative because the presence of cytochrome b₅ and hemoglobin can be detected concurrently. We have also shown that the molar magnetic ellipticity for reduced + CO treated cytochrome P-450 of Pseudomonas grown on camphor is of similar value to that of reduced + CO treated microsomal P-450 and P-448.     

Linear Electric Field-Induced Shifts in Electron Paramagnetic Resonance: A New Method for Study of the Ligands of Cytochrome P-450

The linear electric field shift in paramagnetic resonance has, for the first time, been observed in frozen solutions. The magnitude of the shift parameters, measured at g(max) of the electron paramagnetic resonance absorption envelope, has been used to characterize lowspin ferric heme mercaptide complexes where the nonmercaptide ligand was varied. The magnitude of the shift provides a measure of the difference between the crystal field contribution of the mercaptide and nonmercaptide ligands. In low-spin ferric cytochrome P-450 of rat liver, the presence of an electric field shift indicates a deviation from inversion symmetry and proves that the axial ligands to the heme are not the same. From the magnitude of the shift and its dependence upon the angle between applied electric and magnetic fields, it is suggested that the non-axial ligand to the heme has a crystal field contribution greater than imidazole and smaller than guanidine.     

Electron Paramagnetic Resonance Study of the High- and Low-Spin Forms of Cytochrome P-450 in Liver and in Liver Microsomes from a Methylcholanthrene-Treated Rabbit

The high- and low-spin forms of cytochrome P-450 were observed by electron paramagnetic resonance (epr) in a liver slice and in hepatic microsomes from a rabbit injected with methylcholanthrene. Quantitation of the epr absorption of these two chemical species and comparison with a preparation from a control rabbit showed that the single injection of drug increased the concentration of cytochrome P-450 to more than ten times its control value. Analysis of the epr spectrum for the high-spin compound showed that it is the most rhombically distorted ferric heme iron site yet observed (E/D = 0.10). It is suggested that microsomal cytochrome P-450 can exist in two interconvertible forms, in which the heme iron can either be high-spin or low-spin, depending upon the nature of the nonporphyrin ligands of the metal. There seems to be no need to postulate the existence of two different cytochrome P-450 proteins.     

A Role of the Putidaredoxin COOH-terminus in P-450cam (Cytochrome m) Hydroxylations

Methylene hydroxylation by cytochrome P-450(cam) (cytochrome m) can be resolved into four distinct steps: substrate addition, m(o) --> m(os); reduction, m(os) --> m(rs); dioxygen addition, m(rs) --> m(O2) (rs); followed by a second putidaredoxin (Pseudomonas putida ferredoxin)-mediated reduction and product formation. The isolated ferrous oxy-substrate complex exhibits first-order decay kinetics with the relatively slow rate constant of k [unk] 0.01 sec(-1), at 25 degrees , without product release. Putidaredoxin addition accelerates the decomposition with second-order kinetics, k [unk] 51,000 M(-1) sec(-1), and initiation of product formation. Cytochrome m forms a complex with putidaredoxin with dissociation constant of K(D) = 3 muM. In the complete three-protein hydroxylase system, consisting of cytochrome m, putidaredoxin, and the reductase (a DPNH-specific flavo-protein), camphor hydroxylation occurs with a stoichiometry of 1 mole each of DPNH and O(2) used per mole of product formed; the K(M) for putidaredoxin is about 4.2 muM.Putidaredoxin, on treatment with carboxypeptidase A, loses one molecule each of tryptophan and glutamine sequentially from the carboxy terminus to expose a terminal arginine. The tryptophan-free product has been separated from native putidaredoxin and other impurities, and retains the visible and electron paramagnetic resonance spectra and the redox potential of the active center of native putidaredoxin. This modified redoxin binds less tightly to cytochrome m, K(D) [unk] 150 muM, and is 50 times less effective in stimulation of the m(O2) (rs) decay rate. A similar decrease in specific activity is observed in the complete hydroxylase system.     

Cytochrome P-450 gene expression in the functional units of the fetal liver

Hepatocytes of the right and left lobes of the fetal liver are surrounded by different microenvironments. The right and left lobes of the fetal liver are perfused by vascular systems carrying different concentrations of oxygen and constitute distinct functional units. The aim of this study was to assess the expression of the phenobarbital-inducible cytochrome P-450 b,e genes in hepatocytes of the right and left fetal liver lobes in mice. Northern-blot analysis using [32P]cDNAs and quantitative dot-blot hybridization were performed to assess the size and levels of these mRNAs in the right and left fetal liver lobes. In fetal mice, the levels of cytochrome P-450, b,e mRNAs were higher in the left than in the right fetal liver lobe. During the last days of gestation and in the immediate postnatal period, the levels of liver cytochrome P-450 b,e mRNAs increased predominantly in the left liver lobe. In contrast, the levels of albumin and alpha-fetoprotein mRNAs (genes studied to assess the specificity of these findings) were similar in each of functional units of the fetal liver. Phenobarbital induction of cytochromes P-450 b,e mRNAs was not observed in either of the fetal liver lobes. Postnatally, phenobarbital induced these cytochromes similarly in the right and left liver lobes. Therefore, the microenvironment surrounding fetal hepatocytes seems to influence the expression of the cytochrome P-450 b,e genes. This lobar heterogeneity of expression disappears as the pattern of adult liver circulation is attained.     

Changes in Microsomal Phospholipases and Arachidonic Acid in Experimental Alcoholic Liver Injury: Relationship to Cytochrome P-450 2E1 Induction and Conjugated Diene Formation

We evaluated the role of changes in microsomal phospholipases (A and C) and arachidonic acid in the intragastric rat feeding model. The experimental animals (male Wistar rats), divided into 4–5 rats/group, were fed the following diets: corn oil and ethanol and corn oil plus dextrose. One set of groups was killed after 2 weeks of feeding, and the second set was killed after 1 month. For each animal, microsomal analysis of cytochrome P-450 2E1 (CYP 2E1) and fatty acids was done. Fourteen animals had analyses of phospholipase C (PLC) and phospholipase A (PLA), and 10 animals had measurements of conjugated dienes. A significant correlation was obtained between the level of CYP 2E1 and the decrease in arachidonic acid (AA) from baseline levels ( r = 0.69, p < 0.01). The decrease in AA also correlated with the increase in conjugated dienes ( r = 0.70, p < 0.05). PLA and PLC activities were both significantly increased in the corn oil and ethanol groups. The activity of PLC correlated with the decline in AA ( r = 0.69, p < 0.01). The correlations noted between the decrease in microsomal AA and CYP 2E1 induction and conjugated diene formation suggest that these processes may be interlinked especially in regard to generation of lipid peroxides that may play a role in alcoholic liver injury.     

Cytochrome P-450/pseudosubstrate interactions and the role of antioxidants in the adrenal cortex

The adrenal cortex is the site of the synthesis of the steroid hormones such as the glucocorticoid cortisol and the mineralocorticoid aldosterone. The pathway of biosynthesis of these steroids from cholesterol involves a sequence of transformations using cytochrome P-450 enzymes. The hypothesis presented here is that damage to cytochrome P-450 enzymes on interaction with certain steroids, synthesized by the adrenal cortex itself, may be of pathological and perhaps physiological importance. The interaction between cytochrome P-450 enzymes and these steroids, which act as pseudosubstrates, may form part of the pathogenesis of some steroidogenic enzyme deficiencies, with consequent overproduction of precursor steroids, leading to mineralocorticoid or androgen excess. This interaction is dependent on achieving high concentrations of the pseudosubstrate steroids in the adrenal cortex, which probably occurs as a result of the arrangement of the vasculature in the adrenal gland. High concentrations of steroids may be expected to accumulate in steroidogenic cells, both in culture and in vivo, and may have autoregulating effects. The high content of antioxidant compounds in the adrenal cortex, principally ascorbate, may serve to protect cytochrome P-450 enzymes from the damaging effects of oxygen radical species formed as a result of cytochrome P-450/pseudosubstrate interactions.     

Cytochrome P-450scc: enzymology, and the regulation of intramitochondrial cholesterol delivery to the enzyme

The mechanism and properties of the adrenal cortex enzyme system which catalyzes the side chain cleavage of cholesterol to form pregnenolone are summarized. Cytochrome P-450scc, an integral inner mitochondrial membrane protein, interacts with its electron donor adrenodoxin via an aqueous-exposed (matrix side) site, and with its substrate cholesterol via an active site in communication with the hydrophobic phospholipid milieu. In a purified, phospholipid vesicle-reconstituted system, membrane-dissolved cholesterol interacts rapidly with and can be readily metabolized by the membrane-associated cytochrome, and thus represents a readily accessible cholesterol pool. Evidence for a rapidly metabolizable mitochondrial substrate pool (presumably that in the inner mitochondrial membrane) and the regulation by ACTH of cholesterol movement from other site(s) (presumably the outer mitochondrial membrane) into the reactive pool is reviewed; additional evidence is provided which supports the idea that the outer mitochondrial membrane/intermembrane space provides the rate-limiting block to cholesterol utilization. Possible mechanisms by which ACTH might regulate intramitochondrial cholesterol movement are discussed. ACTH has been found to regulate intramitochondrial aqueous volumes (both the matrix and the intermembrane space) in a cycloheximide-inhibitable manner, and it is proposed that these volume changes reflect an altered relationship of outer and inner membranes which may promote movement of cholesterol.     

Induction of Cytochrome P-4502E1 by Ethanol in Rat Kupffer Cells

Ethanol has been shown to affect several Kupffer cell functions, but the mechanisms underlying these changes are unknown. One possible mediator is cytochrome P-4502E1 (CYP2E1), an ethanol-inducible enzyme that has been associated with toxic effects in the liver, as well as in many extrahepatic organs. To assess whether CYP2E1 can be induced by ethanol in Kupffer cells, male rats pair-fed ethanol-containing or control Lieber-DeCarli diets for 3 weeks were studied. Immunoblotting experiments showed that ethanol-treatment caused a 7-fold increase in CYP2E1 content both in Kupffer cells and hepatocytes. When expressed per milligram of S9 protein, the content of CYP2E1 in Kupffer cells was, however, 10 times lower than in hepatocytes. Immunohistochemical studies revealed that CYP2E1 is located in the endoplasmic reticulum of Kupffer cells in vivo and that it is also present in isolated Kupffer cells. In both Kupffer cells and hepatocytes, ethanol feeding increased the hydroxylation of p -nitrophenol, a relatively specific substrate for CYP2E1, demonstrating that the induced CYP2E1 was catalytically active. This reaction was significantly inhibited by anti-CYP2E1 IgG in both types of cells. Although CYP2E1 may not be the predominant pathway for ethanol metabolism in hepatocytes, it is possibly the major one in Kupffer cells. Thus, the induction of CYP2E1 by ethanol in these cells could cause significant changes in intracellular acetaldehyde concentrations which, together with increased lipid peroxidation, may contribute to the development of alcoholic liver injury.     

Cytochrome P-450 mediates tissue-damaging hydroxyl radical formation during reoxygenation of the kidney.

Renal reperfusion injury results from oxygen radical generation. During reoxygenation of hypoxic kidney cells, xanthine oxidase produces superoxide radical, which eventuates in hydroxyl radical formation by the Fenton reaction. This reaction, catalyzed by transition metals such as iron, is particularly important because hydroxyl radical is highly reactive with a wide variety of biomolecules. We tested the hypothesis that this catalytic function is fostered by iron released from the heme moiety of cytochrome P-450. Primary cultures of rat proximal tubule epithelial cells studied in a subconfluent stage were subjected to 60 min of hypoxia and 30 min of reoxygenation. When cells were pretreated with one of three cytochrome P-450 inhibitors (piperonyl butoxide, cimetidine, or ketoconazole), lethal cell injury was attenuated. There was the expected increase in O2-. production during hypoxia/reoxygenation that cytochrome P-450 inhibitors did not prevent; on the other hand, inhibitors did prevent reoxygenation-induced hydroxyl radical formation. Analogously, the increase in catalytic iron (bleomycin-detectable iron) that accompanies hypoxia/reoxygenation did not occur in the presence of cytochrome P-450 inhibitors. In vivo studies confirmed a protective effect of cytochrome P-450 inhibition because glomerular filtration rate was better preserved in rats pretreated with cimetidine and then subjected to renal artery occlusion. In summary, several chemically distinct cytochrome P-450 inhibitors reduced iron release, and thereby, hydroxyl radical formation and reoxygenation-induced lethal cell injury, without inhibiting superoxide radical formation. We conclude that highly labile P-450 may act as an Fe-donating catalyst for Fenton reaction production of HO.-mediated reperfusion injury.     

Immunohistochemical Determination of Hepatic Cytochrome P-4502E1 in Formalin-Fixed, Paraffin-Embedded Sections

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. lmmunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 ± 0.3 vs. 0.8 ± 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 ± 2.1 vs. 7.9 ± 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (r_s= 0.67, p < 0.005) or intensity ± zonal staining scores (r_s= 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embedded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.     

花生四烯酸的CYP代谢途径与心肌缺血再灌注损伤

花生四烯酸（AA）主要由环氧化酶（COX）、脂加氧酶（LOX）及细胞色素P450（CYP）途径代谢。近年来发现，AA的CYP代谢途径与心肌缺血再灌注（MIR）损伤的关系密切。本文就AA的CYP代谢酶在心脏中的表达、CYP代谢途径对MIR的影响及作用机制做一综述。     

P-450 -Dependent Metabolism of Laurie Acid in Alcoholic Liver Disease: Comparison between Rat Liver and Kidney Microsomes

Monooxygenase enzymatic activities were measured in liver and kidney microsomes of control and ethanol-treated rats. Animals were administered alcohol by using a model for alcoholic liver injury. Several in vitro approaches were used to compare the laurate metabolism in liver and kidney microsomes: correlation studies between specific P-450 catalytic activities, immunoblot analysis, and chemical and immunoinhibitions. Ethanol treatment increased the liver and renal hydroxylations of chlorzoxazone and 4-nitrophenol. Moreover, lauric acid (ω-1)-hydroxylation was found to be significantly increased (～6-fold) after ethanol treatment in liver, but not in kidney microsomes. The laurate ω-1/ω ratio increased from 1.52 ± 0.49 to 4.11 ± 1.01 in liver microsomes of control and ethanol-treated rats, and from 0.29 ± 0.06 to 0.44 ± 0.07 in kidney microsomes. Immunoblot analysis using polyclonal anti-cytochrome P-450 (CYP) 2E1 or CYP4A antibodies showed an increase of CYP2E1 and CYP4A contents in both organs, but the increase was higher in liver than in kidney microsomes. Chemical inhibitions using CYP2E1 competitive inhibitors (such as chlorzoxazone and ethanol) led to a nonsignificant inhibition of the renal (ω-1)-hydroxylation of lauric acid. In contrast, 17-octadecynoic acid (a mechanism-based inhibitor of ω -hy-droxylase) was able to inhibit both to- and (ω -1)-hydroxylations of lauric acid in kidney microsomes. Immunoinhibitions specific to CYP2E1 significantly decreased the (ω -1)-hydroxylation of lauric acid in liver, but not in kidney microsomes, whereas the polyclonal anti-CYP4A1 antibody inhibited to- and (ω -1)-hydroxylations of lauric acid in kidney microsomes. All of these results show that lauric acid hydroxylations in liver and kidney respond in different manners to ethanol treatment. Lauric acid (ω -1)-hydroxylation, a highly specific probe for CYP2E1 in rat and human liver microsomes, is mediated by a CYP4A isoform in rat kidney microsomes.     

Evidence that Cytochrome P-4502E1 Contributes to Ethanol Elimination at Low Doses: Effects of Diallyl Sulfide and 4-Methyl Pyrazole on Ethanol Elimination in the Perfused Rat Liver

The roles of cytochrome P-4502E1 and alcohol dehydrogenase (ADH) on ethanol (EtOH) hepatic elimination was examined in the perfused rat liver. EtOH concentration-time curves of outflow after instantaneous administration (0.46 mg) through the portal vein with or without perfusion of diallyl sulfide (DAS), a selective cytochrome P-4502E1 inhibitor, and/or 4-methyl pyrazole (4-MP), a classical ADH inhibitor, were analyzed by the statistical moment analysis and the compartment dispersion model. Recovery ratios obtained by moment analysis significantly changed with perfusion of inhibitors ( p < 0.01). Values of the hepatic volume of distribution and the relative dispersion were significantly higher by the perfusion of DAS and 4-MP ( p < 0.01). In the two-compartment dispersion model, the partition ratio ( K ') and the first-order elimination constant ( K _e) were decreased significantly by DAS ( p < 0.05). By the addition of 4-MP, the blood volume of distribution ( V _B ) and the backward partition rate constant ( k ₂₁) were increased significantly ( p < 0.05). K _e values were decreased significantly to 0 ( p < 0.001). The decrease of elimination rates by DAS and/or 4-MP shows the inhibition of metabolic pathways. The change of V _B and k ₂₁ caused by DAS and 4-MP indicates that EtOH taken into hepatic tissues was not metabolized and flowed out into the perfusates. Inhibition rates calculated from the efficiency number with addition of DAS and DAS + 4-MP were 40.7 and 99.3%. Therefore, cytochrome P-4502E1 and ADH accounted for 40 and 60% of the hepatic EtOH elimination at low doses.     

Cytochrome P-450scc a review of the specificity and properties of the cholesterol binding site

Cytochrome P-450scc is unusual among members of this class of enzymes in showing a high degree of substrate specificity. Features of the cholesterol structure which are particularly important for binding include the 3 beta-hydroxyl, the delta 5-ring configuration, and the side-chain organization in the 20-22 region. Regarding the ring system, binding appears to require planarity and limited size at the 4-5-6 carbons (the A-B ring juncture). In the region of the 3 beta-hydroxyl, a "cleft" in the binding site extends about 4 A beyond the hydroxyl and can accommodate two additional ether-linked carbons. Evidence indicates that an enzyme residue hydrogen-bonds to the oxygen of the 3 beta hydroxyl, providing much of the energy for the initial enzyme-substrate interaction. The cytochrome shows less specificity for the side-chain structure, except in the region of carbons 20-22 where hydroxylation/side-chain cleavage takes place. The binding cleft for the side-chain is limited to approximately the length of the isocaproic group but can accommodate structural variations beyond the 22-position. Evidence indicates that the region near the 20-22 bond is more limited in size, and that an amino acid residue near the heme iron binds strongly and stereospecifically to the 22R-hydroxyl of the cleavage intermediates, 22R-hydroxycholesterol and 20 alpha, 22R-dihydroxycholesterol. The 22R-hydrogen of cholesterol is very close to the heme iron (approximately 3 A), while the 22S-hydrogen is slightly further (about 4 A). The size and bonding properties of the steroid binding/active site suggest a mechanism which accounts for the stereospecificity and sequence of reactions catalyzed by cytochrome P-450scc.     

Cytochrome P-450-dependent vasodilator responses to arachidonic acid in the isolated, perfused kidney of the rat

Pretreatment of phenylephrine (0.5 microM)-preconstricted, isolated perfused kidneys of the male rat with indomethacin (2.8 microM) or BM 13.177 (20 microM) abolished the vasoconstrictor response to arachidonic acid (AA), uncovering a vasodilator response. BW 755C (25 microM), a dual cyclooxygenase/lipoxygenase inhibitor, did not modify the vasodilator effect of AA, whereas 5,8,11,14-eicosatetraynoic acid (10 microM), which blocks all pathways of AA metabolism, abolished AA-induced vasodilation, thus suggesting the involvement of nonlipoxygenase AA metabolites. Clotrimazole (0.7 microM) and 7-ethoxyresorufin (1 microM), both considered to be specific inhibitors of the cytochrome P-450 monooxygenase enzymes, inhibited the vasodilator effect, suggesting that AA-induced renal vasodilation is mediated by one or more cytochrome P-450-derived AA metabolites. None of these interventions affected the vasodilator responses to acetylcholine (100 ng) and nitroprusside (1 microgram). Denudation of the endothelium with CHAPS (10 mg/l) reduced the vasodilator responses to AA, suggesting a requirement of an intact endothelium, whereas inhibition of guanylate cyclase with methylene blue (10(-4) M) was without effect, suggesting that cGMP was not involved in the vasodilator response to AA. The AA-induced renal vasodilation was accompanied by the generation of biologically active material or materials released into the renal effluent, which relaxed endothelium-intact and endothelium-denuded rings of isolated aorta and mesenteric and celiac arteries of the rabbit. These results suggest that in the rat kidney, AA is metabolized by endothelial cytochrome P-450-dependent enzymes to vasodilator metabolites.