NADPH DKF’ENDKNT CYTOCHROME P-450 REACTIONS r MODE OF INHIBITION BY THE N—BUTANOL FRACTION OF DESMODIZJM ADSCTEHDENS A THESIS SUBMITTED BY AUGUSTUS APE KU KAMASSAH TO THE DEPARTMENT OF BIOCHEMISTRY, FACULTY OF SCIENCE, UNIVERSITY OF GHANA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF PHILOSOPHY (M.PHIL) DEGREE MARCH, 1996 University of Ghana http://ugspace.ug.edu.gh DECLARATION THE EXPERIMENTAL WORK DESCRIBED IN THIS PROJECT WAS DOSE BY ME, AT THE DEPARTMENT CF BIOCHEMISTRY, UNIVERSITY CF GHANA, LE3CN, UNDER THE SUPERVISION OF PROF. M.E. ADDY. REFERENCES CITED IN THIS WDRK HAVE BEEN FULLY ACKNOWLEDGED. AUGUSTUS APEKU KAMASSAH (CANDIDATE) (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh DEDICATION T O ©AID) A N P M U J M University of Ghana http://ugspace.ug.edu.gh I acknowledge rry deep gratitude to ny supervisor, Prof. M.E. Add/ of the Biochemistry Departirent to vton I owe a great deal, not only for the kind encouragement, but also for the insight and perspective that only the real expert can convey. I also wish to express ny sincere appreciation to Messrs Lambert Faabeloun, Evan Fosu, Eric Chipeni and John Addipa, v\ho took time off their busy schedule on many occasions to help with this work. I would also like to thank all the lecturers at the Department of Biochanistry, especially Dr. Robert Acquaah and Dr. Alex N/arko (Chemical Pathology Unit, NMEMR) for their ccmments and suggestions for irrprovement. My sincere thanks go to the technical staff of the Dept. of Biochemistry, especially Mr. Eososrrpsn for the assistance they gave me. I am grateful to the staff of the Animal, Electron Microscopy and Chemical Pathology Units of the Noguchi Manorial Institute for Medical Research, especially Messrs Boakye and Ayixn and Sister Susie for the technical assistance offered me in their various Units. Tto the following; Edward, William, Ephraim, Abraham and Fred (ny colleagues at the Biochanistry Dept.), John, Robert, Cliff, Bartho, Cee Brown, David, ACKNOWLEDGEMENT i i i University of Ghana http://ugspace.ug.edu.gh Fabio, Dora, Jos^hine, Emily and the Executive members of the Legon Pentecostals Union (1994-1996) I give rty most sincere thanks for their spiritual and moral support. A very special note of appreciation is extended to Mrs Christiana Nette/ for helping with the typing, Mr. Saaliah of the Dept, of Food Science & Nutrition for the graphs and to Mr. Bernard Gatagbui of the University of Bergen (Lab. of Marine Molecular Biology) for providing the bottle of carbon monoxide used in this stud/. Tb a supportive and accomodating family, through vtose motivation I have made it to another stage of the acadsnic ladder, I say I am thankful. Most importantly, I thank the Almighty Lord for sustaining and bringing me this far. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION................................................. DEDICATION......... 11 ACKNOWLEDGEMENT............................................. Table of Contents ....................................................v List of Tables.............................................. vi.i.i List of Figures.......................................... Vlx' List of Abbreviations.................................. Jfi-i Abstract..................................................... xiv CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW.............1 1.1 GENERAL INTRODUCTION.......................... .1 1.2 CYTOCHRCME P-450 MONOOXYGENASES............ .5 1.3 REACTION MECHANISM....................... . .8 1.4 CYTOCHRCME P-450s................................. 14 1.4.1 Isoenzymes..................................15 1.4.2 Conversion to P-420......................... 17 1.4.3 Spectral Characteristics........ 19 1.4.4 Binding to Substrates........................20 1.5 OTHER COMPONENTS OF THE MICROSOMAL MCNOOXYGENASE SYSTEM................... .23 1.5.1 Reductases................................ 23 1.5.2 Lipid Factor................................26 1.5.3 Non-hare Iron Protein....................... 27 v University of Ghana http://ugspace.ug.edu.gh 1.6 INHIBITORS OF CYTOCHROME P-450s MEDIATED REACTIONS.......................................... 28 1.6.1 Formation of Metabolic Intermediate Cctrplexes....................................29 1.7 N-BUTANOL FRACTION (nBF) OF DESMODIUM ADSCENDENS................................... 32 CHAPTER TWO: MATERIALS AND METHODS..................... . .38 2.1 MATERIALS............................................ 38 2.1.1 Chemicals and Reagents................. ........38 2.1.2 Animals.......................................39 2 .2 METHODS.............................................. .3 9 2.2.1 Preparation of Buffers and Solutions.................................... 39 2.2.2 Preparation of n-butanol fraction of D. adscendens..................................39 2.2.3 Fractionation of nBF........................... 40 2.2.4 Induction of CYP Monooxygenase in Mice...................................... 41 2.2.5 Preparation of Microscmes.......................41 2.2.6 Protein Concentration Estimation................................... 42 2.2.7 Ethoxyresorufin O-Deethylase (ERCD) Assay..................................43 2.2.8 Determination of Cytochrons P-450 Content................................. 46 2.2.9 Reduction of CYP...............................46 2.2.10 Reduction of Cytochrcme c................. . A ' 1 vi University of Ghana http://ugspace.ug.edu.gh 2.2.11 Cytochrane (c) P-450 Reductase Assay........................................ 48 2.2.12 Binding studies............................... 48 CHAPTER THREE: RESULTS....................................... 50 3 .1 PROTEIN CONCENTRATION.................................50 3.2 CYP AND P-420 CONTENT............................... 50 3.3 EROD ACTIVITY.........................................55 3.3.1 Effect of Flash Chraratograph/ fractions of nBF.............................. 58 3.3.2 Effect of salsolinol........................... 58 3 . 4 REDUCTION OF CYTOCHRCME C ............................ -62 3.4.1 Effect of nBF............................ .64 3.4.2 Effect of Flash Chrciratography fractions of nBF....................................... 64 3 . 5 REDUCTION OF CYP..................................... 67 3.5.1 Direct Reduction of CYP........................ 67 3.5.2 Indirect effect on the Rate of Reduction of CYP....................... . . . 67 3.6 REDUCTASE ACTIVITY..................... . . . 81 3.7 BINDING STUDIES............................... .82 CHAPTER FOUR: DISCUSSION.................... .91 BIBLIOGRAPHY............................................ ...105 APPENDICES.................................................... 114 vii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES 1 Selected gene families of CYP................................... 8 3.1 Absorbance measurements at 750 ran and protein concentrations of the microsomal preparations from the different laboratory animals...................................................... 50 3.2 Values obtained frcm EROD assay................................ 55 3.3 Effect of nBF on CYP and P-420 levels in mice liver microscmes pre-incubated with nBF........................................ 77 3.4 Effect of nBF on NADPH-cytochrcme c (P—450) reductase activities of microsciral preparations...........................82 vii i University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES FIG. 1.1 A scherre for microscrral electron transport reactions of P-450...................................... 11- FIG. 1.2 Proposed schema for the mechanism of action of CYP in hydroxylation reactions.................................. H FIG. 1.3 Difference spectra of type I, type II and reverse type I corpounds........................................ 21 FIG. 1.4 Pathway of prostaglandin biosynthesis...................... 34 FIG. 2.1 Schsre used in the fractionation of the crude extract from D. adscendens............................... 41 FIG. 2.2 Diagram of printed tracing fran SFM-25 fluoriireter printer............................... 45 FIG. 3.1 Calibration curve for protein determination................ 51 FIG. 3.2 Ttotal liver microscrral protein............................ 52 FIG. 3.3 CYP and P-420 contents of microscrral preparations........... 53 FIG. 3.4 CO-Difference spectra of microscrral preparations using sodium dithionite as reducing agent............... .54 FIG. 3.5 Emission scan of resorufin................................56 FIG. 3.6 Fluorescence (EROD) response of microscrral preparations............................................ 57 FIG. 3.7 Effect of DAF1 and DAF2 on the fluorescence (EROD) response of microsoral preparations................. 59 FIG. 3.8 Percentage inhibition of EROD activity by different concentrations of DAF1 and DAF2..........................60 FIG. 3.9A Effect of salsolinol on the fluorescence (EROD) response of microscrral preparations...................... 61 FIG. 3.9B Percentage inhibition of EROD activity fcy different concentrations of salsolinol..............................62 ix University of Ghana http://ugspace.ug.edu.gh FIG. 3.10 Spectra of oxidized and reduced cytochrane c using saturated sodium dithionite and salsolinol as reducing agents...................................... 63 FIG. 3.11 Spectra of oxidized and reduced cytochrome c using nBF as reducing agent.............................. 65 FIG. 3.12 Spectra of oxidized and reduced cytochrare c using different DAFs as reducing agent..................... 66 FIG. 3.13 CO-Difference spectra of microscrral preparations using salsolinol, GSH and nBF as reducing agent...................63 FIG. 3.14A CO-Difference spectra of microsaral preparations frcm NIEM before and after nBF addition................... 69 FIG. 3.14B CO-Difference spectra of microsaral preparations frcm BIEM before and after nBF addition................... 70 FIG. 3.15A Levels of CYP and P-420 in NIEM liver microsones before and after nBF addition............................ 71 FIG. 3.15B Levels of CYP and P-420 in BIBM liver microscmes before and after nBF addition............................ 72 FIG. 3.16 Fold increases in CYP and P-420 levels in mice liver microsones after nBF addition....................... 74 FIG. 3.17 CO-Difference spectra of microsaral preparations before and after salsolinol addition...................... 75 FIG. 3.18 Effect of salsolinol on the levels of CYP and P-420 in mice liver microsones........................76 FIG. 3.19 Time-dqpendent reduction of liver microsones by different concentrations of nBF.......................... 78 FIG. 3.20 Effect of pre-incubation on CYP levels in NIEM and BIEM liver microscmes................................ 75 FIG. 3.21 Effect of pre-incubation on P-420 levels in NIEM and BIEM liver microscmes............................. . 8C FIG. 3.22 CYP reductase activities of liver microsaral preparations..................................81 x University of Ghana http://ugspace.ug.edu.gh FIG. 3.23 UV-VIS spectra of resorufin and 7-ethoxyresorufin........... 83 FIG. 3.24 Spectra of resorufin formed and nBF addition................84 FIG. 3.25 Spectra of microsomal preparation, 7-ethoxyresorufin and nBF before and after NADPH addition................... 86 FIG. 3.26 Absolute spectra of oxidized microscmes and nBF addition........................................ 87 FIG. 3.27 Absolute spectra of oxidized microscmes with 7-ethoxyresoruf in and nBF addition........................ 88 FIG. 3.28 Absolute spectra of oxidized microscmes with nBF and 7-ethoxyresoruf in addition............................89 x i University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS M arachidcnic acid BIEM - ENF-injected black mice BIVM - BNF-injected white mice ENF S-naphthoflavcne CD carbon monoxide CYP - cytochrome P-450 DAF - Desmodiun adscendens fraction ENA - deoxyribonucleic acid EDffi. ethylenediaminetetraacetic acid EET - epoxyeicosatrienoic acid ER endoplasmic reticulum EROD - ethoxyresorufin 0 -deethylase Gffl - glutathione (reduced) HEJIE - hydroxyeicosatetraenoic acid LTD* - leukotriene D4 3-m: 3-methylcholanthrene mi metabolic intermediate rrRNA - messenger RNA NADH nicotinamide adenine dinucleotide (reduced) NADFH - nicotinamide adenine dinucleotide phosphate nEF - n- butanol fraction University of Ghana http://ugspace.ug.edu.gh NIHy! - Non-induced black mice niwvi Non-induced white mice PAH - polycyclic anoretic hydrocarbon FB phenobarbi tal KB polychlorinated biphenyls ECN - pregnenolone -a - carbonitrile FGE2 - prostaglandin E, PGF2o prostaglandin F2a PG& - prostaglandin Gj PGH2 - prostaglandin H2 F3s - pros taglandins Rt\R - ribonucleic acid SCCW - sterile double-distilled water SER - smcoth endoplasmic reticulum X ternary ccrrplex THIQ - tetrahydroisoquinoline W«R White Wistar rats WWRL - White Wistar rat liver WWRK - White Wistar rat kidney University of Ghana http://ugspace.ug.edu.gh ABSTRACT The n-butanol fraction (nBF) of Desmxhvw adscendens , a plant used for the management of asthma, is' an inhibitor of NADPH-dependent cytochrcire P-450 (CYP) reactions. Its mechanism of action as an inhibitor is however net knewn. In this study, flavoprotein reductase activity, spectral changes associated with binding and spectral properties of reduced cytochrcme c and CYP were used to investigate the mode of inhibition. nBF reduced cytochrcme c but not CYP directly. In the presence of NADPH, the rate of forrration of CYP2* (the reduced form of CYP) was enhanced fcy the addition of nBF. These effects were observed in reactions without substrate, indicating that in the absence of a substrate, nBF does not prevent the NADPH reduction of CYP, but rather enhances it. In the presence of a substrate, as exemplified b/ a spectrophotorretric assay of the ERCD reaction, nBF was found to change the spectrum of oxidized CYP. The results indicate that in the presence of nBF, the binding site of CYP is altered to prevent substrate binding. x iv University of Ghana http://ugspace.ug.edu.gh Therefore, the node of inhibition of nBF relates to substrate binding. nEF could be inhibiting the NADPH-dependent CYP reactions by binding to CYP and preventing substrate binding. nBF could also inhibit the flew of electrons frcm NADPH to substrate-bound CYP fcy interfering with the flavoprotein reductase activity whan it is bound to CYP. On the other hand, nBF could have the same effect cn the rate of reduction of CYP to CYP^’ in the presence of a substrate as it did in the absence of a substrate, leading to the formation of the CYP-Qj-substrate ternary ccrrplex, in which case the effect of nBF would be to prevent the breaking up of the ternary complex to form products, the build-up of viiich causes the substrate to rorem unmetabolized. xv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW 1.1 GENERAL INTRODUCTION Without appropriate means, living organisms would not be able to eliminate frcm their systems the lipophilic corpounds which are either produced hy their cwn metabolism (i.e. steroid hormones, fatty acids, prostaglandins;, or are accidentally or voluntarily absorbed frcm their environments (i.e. food additives, drugs, pesticides, pollutants etc.). Living organisms have thus developed a number of enzymic systems which transform these substances into more polar and therefore hydrophilic metabolites which can be easily excreted via the urine or faeces. Such enzyme systarB must not be interfered with, since they are very vital to the survival of the organism. One of the steps in the metabolic transformation of lipophilic corrpounds ce more polar cnes is usually catalysed by cytochrome P-450 (CYP)-dependent monoo>ygenases. These microscmal multienzyme ccrrplexes oxidize a great variety of endogenous as wsll as exogenous substrates. The reaction involves the formation of a ternary complex between the iron centre of th? cytochrone, the substrate and molecular oxygen. Within the ternary ccrrplex, electrons donated ty reduced nicotinamide adenine dinucleotide phosphate (NADPH), through a flavoprotein reductase, are received hy CYF and passed on to molecular oxygen. CYP, therefore, goes through a f e m ferrous cycle. The reducing pcwer supplied ty the electrons as they pass University of Ghana http://ugspace.ug.edu.gh through CYP is used to split molecular oxygen, cne atcm of winch gets inserted into the substrate and the other atcm is reduced to water. With this reaction occurring in the liver, xenobiotics are rendered more polar and therefore can be excreted through the aqueous bile. The biochanical and biological properties of these enzyme canplexes have been extensively studied in laboratory anirrals (La and West, 1980) . The different types of cytochrcme P-450 are characterized ky their substrate specificity (Haugen et a l . , 1975). Their quantitative and qualitative proportions in a given tissue may vary largely as a function of physiological, pharmacological and pathological parameters (Lu and West, 1980) . This phenomenon is particularly irrportant with respect to drugs because for many drugs, the nature and toxicity of their different metabolites which are often synthesized ky various types of CYP, are not identical. It is also irrportant with respect to environmental pollutants. Different CYPs are induced ky different pollutants and these differences form the basis for the nomenclature of the CYPs. Apart frcm pollutants, drugs, other xenobiotics and endogenous substrates are also transformed by the monooxygenase enzyme complex. Arachidonic acid (AA), usually derived from membrane phospholipids as a result of phospholipase A2 activity, is cne of the endogenous cctrpounds metabolized by the monooxygenase enzyme ccrrplex. It is thus a major substrate for the NADPH-dependent CYP enzyme ccrrplex in a reaction referred to as the third 2 University of Ghana http://ugspace.ug.edu.gh pathway of AA metabolism (Capdevila et a l . , 1982) after the cyclooxygenase pathway giving rise to prostaglandins (FGs), prostacyclins (PGI2) and thromboxane (TX) (Samuelsson et a l . , 1978), and the lipoxygenase reaction giving rise to leukotrienes and lipoxins (Samuelsson et a l . , 1980). The discovery of renal cytochrcme P-450 monooxygenase vvss related to arachidcnic acid (AA) metabolism. The renal system metabolizes AA by three types of reactions: i) allylic oxidation leading to the formation of hydroxyeicosatetraenoic acids (HETEs); ii) olefin epoxidation leading to the formation of four different epoxyeicosatrienoic acids (EETs); and iii) oxidation at co- and co-1 positions to form the 20-HETEs and 19- HETEs respectively (Schwartzman et a l . , 1985a). Sane of the products of this NADPH-dependent CYP pathway, like 5,6 EET and 11,12(R) HETE, are known to affect the Na+/K+ ATPase activity and to cause diseases associated with changes in volumes of bod/ fluids. Cne of such diseases is renal hypertension . In epithelial cells isolated fron the thick ascending lirrb of Henle's locp of the rabbit kidney, AA is specifically metabolized ky a CYP-dependent pathway to products vAiich affect NaVK* ATPase activity and vascular tone (Schwartzrran et a l . , 1985) Ccnpounds which can inhibit the CYP monooxygenase enzyme ccrrplex so as to prevent the formation of the products v\Mch affect the renal NaVK* ATPase 3 University of Ghana http://ugspace.ug.edu.gh activity could be effective agents for the treatment of hypertension. An n-butanol fraction (nBF) of an aqueous extract of Desmodium adscendens was shewn to inhibit AA metabolism ty this monooxygenase pathway 'Add/ and Schwartzrran, 1992) . Salsolinol (Appendix AI:b) was found to inhibit AA metabolism fcy the NADEH-dependent CYP monooxygenase enzyme corplex of the endoplasmic reticulum, just as the plant extract did fAddy and Schwartzrran, 1992) . With AA as substrate, salsolinol inhibited the forrration of the epoxides, EETs and the HETEs. Salsolinol is the 6,7-dihydroxy analogue of salsoline (Appendix AI:a), a tetrahydroisoquinoline (THIQ) carpound in the plant already listed as an anti-hypertensive agent (Stecher, 1968). The nBF was also found to inhibit ethoxyresorufin-o-deethylase activity (Brookrran-Amissah, 1994), an enzyme activity specific for cne of the pollution-induced isozymes of CYP. nBF has been shown to act as a reducing agent (^ ddy and Schwartzrran, 1995) and it is possible that its reducing ability plays a role in the monooxygenase enzyme system. This inhibition of the rronoaxygenase enzyme fcy nBF could therefore be beneficial for its anti-hypertensive role. However, if all the CYP isozymes, including the constitutive ones are inhibited, it will be potentially dangerous because apart frcm AA, which is not free but usually 4 University of Ghana http://ugspace.ug.edu.gh bound to marbrane phospholipids, steroids also serve as endogenous substrates for several different forms of P-450 related enzyme cctrplexes. A unique set of P-450 isozymes is localized in steroidogenic tissues, i.e., adrenal cortex, testis and ovary. Lipids other than steroids also serve as substrates for these P-450 proteins. Rat liver CYP enzymes are able to catalyze the hydroxylation of fatty acids such as lauric acid (dodecanoic) in both co- and co-1 positions (Das et a l . , 1968; Bjorkhem and Danielsson, 1970) . In addition, studies carried out using porcine and rat ki±iey cortex microscmes (Ichihara et a l . , 1971; Jakobsson et a l . , 1970) suggest that cytochrcme P-450 enzymes are involved in the hydroxylation of lauric acid in these tissues as well. The overall aim of the research reported here was to find out the mechanism of this inhibition so as to understand hew the rronooxygenase enzyme activity can be regulated. With this, one can predict the safety of nBF cn other monoaxy'-rpnase reactions that are of relevance to the general metabolism of the cell. 1.2 CYTOCHROME P-450 MONOOXYGENASES The cytochrcme P-450 d^>endent monooxygenase system is found throughout nature, frcm bacteria to man, where it is involved in the oxidation of many organic ccrrpounds. The requiranent of both a reducing agent and molecular oxygen places the reaction within the external mixed function oxidase 5 University of Ghana http://ugspace.ug.edu.gh classification of Mason (1957, 1965) . The terminology iranooxygenase means that the enzyrre catalyzes the consurrption of one molecule of oxygen per molecule of substrate with one atcm of oxygen appearing in the product and the other undergoing two-equivalent reduction. Direct support of this visn/ v\as given ky Posner et a l . (1961), who erplcyed 1802 and H2180 to shew that the oxygen utilized in the hydroxylation of acetanilide was derived frtm rrolecular oxygen rather than frcm water. The essential carrponents of the iranoaxygenases have been identified as cytochrome P-450, NADPH-cytochrome P-450 reductase (probably the sarre as, or very similar to NADPH-cytochrcme c reductase), and phospholipid (Imai, Y. 1976; Levin et a l . , 1974; Yasukochi and Masters, 1976) . Following the discovery of multiple forms of the cytochrome P-450s and in depth investigation of at least 2 of these enzymes in pure form, it became apparent that slight different spectral properties existed for each isoenzyme catalyzing different reactions. Cytochrome P-450 rronooxygenases catalyze a wide variety of oxidations with a vast nurrber of substrate types. Indeed, many authorities suggest that this fact supports the purported multiplicity of P-450 monooxygenase (Haugen et a l . , 1975). It is thought that few and perhaps no liver microsaral P-450 monooxygenases catalyze a single reaction, but certain Cytochrcmes will have greater activity of a certain reaction type than others. 6 University of Ghana http://ugspace.ug.edu.gh Due to the substrate-nonspecificity of the functional monooxygenase enzyme system in the microscmes of liver and other tissues, determination of its activity depends on the choice of substrate (Burke and lyfeyer, 1974) . This determination is usually decided purely by convenience of assay. Because of the original interest in this enzyme carplex as a detoxifying and drug- nretabolizing systan, most carnally used substrates are drugs or environmentally encountered chenicals. It has became increasingly evident that several forms of CYP exist and rray account for the multiple monooxygenase activities in liver microscmes (Haugen et al., 1975; Ryan et al., 1975) . This is based upon the knavledge that different spectral (Werringloer and Estabrook, 1975), and catalytic (Lu et a l . , 1972) forms of this cytochrome can be induced ky chemicals (Haugen et a l . , 1976). A nomenclature based oi gene sequence information of P-450 forms described a gene superfamily and organized all sequences kncwn ky then into P450 gaie families. This was later revised to include chromosomal localization ( Nebert et a l . , 1991). A selection of mammalian gene families is provided in T&ble 1. There are 27 clearly related species of CYP present in the liver endoplasmic reticulum in rats, each with a wide and scmavbat overlapping substrate specificity, that act on a wide variety of drugs, carcinogens ard other xenobiotics in addition to endogenous carpounds such as certain steroids. This number keeps increasing with new findings. 7 University of Ghana http://ugspace.ug.edu.gh Tctble 1. Selected gene families of CYP Gene Family Gene Subfamily Other Name Species CdTrron Inducers CYPI CYPIA P-450c rat PAH, FCB, Pj-450 mouse BNF etc. CYPII CYPIIB P450b rat EB Form 2 rabbit CYPIII CYPIIIA P-450 PCN, rat PCN CYPIV CYPTVA P-452 rat Clofibrat: PAH = polycyclic aromatic hydrocardon; PCB = polychlorinated biphenyls; PB = phenobarbital; PCN =pregnenolone -a - carbonitrile 1.3 REACTION MECHANISM Cytochrcme P-450 rronooxygenases bind substrate and interact with a flavoprotein reductase and molecular oxygen in a two-step (2-electron sequence resulting in the activation of oxygen (Estabrook et a l . , 1971). Ultimately, one oxygen atom is incorporated into the substrate and the second is reduced to water. Fonration of a CYP-substrate-C^ ternary ccnplex helps to explain the generally suggested substrate specificity irrparted ky the different cytochrcme monooxygenases. It also helps to explain the wide variety of oxidative reactions kncwn to be catalyzed ky monooxygenases frcm liver microscmes which include the following: 8 University of Ghana http://ugspace.ug.edu.gh i) oxidative deamination of arrphetamine; ii) 0-, N-, and S- dealkylations of substances like 7-ethoxyresorufin, 7-ethoxycourrarin, amincpyrin, etbylmorphine and thioesters like methylirercaptan ; iii) hydroxylation of alkyl and aryl hydrocarbons like n-propylbenzene, valproic acid, pentobarbital, debrisoquine and acetanilide; iv) epoxidation of substances like benzene and benzo(a) pyrene; v) N-lydroxylat ion ; vi) N- and S- oxidation of aniline, arrphetamine and thioethers; vii) oxidative debalogenation; viii) oxidation of ethanol (Brodie et al., 1958). Hepatic microscmal cytochrare P-450 dependent metabolism is not restricted to oxidative reactions. A wide variety of azo dyes are cleaved reductively to arcrratic amines, and nitro ccrrpounds such as chloramphenicol and nitrobenzene are reduced to primary amines. Reductive dehalogenation also occurs in the liver microscrres (Mannering, 1972). Although the CYP-catalyzed reaction requires two electrons to accorplish its task of hare iron reduction, o>ygen binding and oxygen cleavage, a basic mechanistic problem is the direct and simultaneous transfer of electrons fran NADPH to the CYP. Pyridine nucleotides are two electron donors, but CYP with its single hare prosthetic group, accepts only cne electron at a time. Thus, a protein that serves to transfer electrons from 9 University of Ghana http://ugspace.ug.edu.gh NADPH to the CYP irolecule rrust have the capacity to accept two electrons but serve as a one electron donor. This problem is solved ty the presence of an NADPH-dependent flavcprotein reductase, which accepts two electrons frcm NADFH simultaneously but transfers the electrons individually to an intermediate iron-sulfur protein or directly to CYP. The electron transport systems reside exclusively in either mitochondria or endoplasmic reticulum (Okita and Masters, 1992). The possible coupling of this reductive process with an NADH mediated electron transfer explains the often suggested synergistic role of NACH as well as cytochrome bs and cytochrore b5 reductase (Nilsson and Johnson, 1963) as shewn in Fig. 1.1. A coupling/decoupling role for hydrogen peroxide has been proposed (Thurman et a l . , 1972) . Thus NADFH (and NADFH- generating systems) can be replaced by hydrogen peroxide and a variety of organic peroxides in the oxidation of certain xenobiotics ty microsonal mixtures (Ellin and Orrenius, 1975; Hrycay et a l . , 1976; Rahimtula and O'Brien, 1975; ) and cytochrare P-450 lm2 (Nordblcm et a l ., 1976) in particular. Hcwever, the role of hydrogen peroxide in intact hepatocyte may be of lesser importance (Jones et a l . , 1978). The details of the cytochrcne P-450 monooxygenase reaction involving the cytochrome P-450 is shown in Fig. 1.2. As indicated in the scheme, substrate binding to native ferric P-450 is followed ty reduction to the ferrous state, thereby allowing oxygen binding. A second reduction results 10 University of Ghana http://ugspace.ug.edu.gh XNA'DH FIG. 1.1 FIG. 1. A SCHEME FOR MICROSOMAL ELECTRON TRANSPORT REACTIONS OF CYP.[Fp, NADPH-cytochrome b5 reductase; FXF2, NADPH- cytochrome P-450 reductase; X, hydroxylatable substrate]. The ferrous oxycytochrome can also decompose to yield H202 (not shown). ROH RH 2 PROPOSED SCHEME FOR THE MECHANISM OF ACTION OF CYP IN HYDROXYIiATION REACTIONS. RH = substrate; ROH = hydroxylatable product 11 University of Ghana http://ugspace.ug.edu.gh in splitting of the oxygen-oxygen bond, one atom being lost as water. The other oxygen atom, presumably now an "activated-oxygen" is inserted into a carbon-hydrogen bond of the substrate to produce the corresponding alcohol which is then released with regeneration of the resting ferric state of the enzyme and ccrrpletion of the catalytic cycle (White and Coon, 1980) . The first stq? in Fig. 1.2 is the binding of the substrate to the active site of the enzyme. The binding of the substrate to the cytochrcme systan has been shewn to be hydrcphobic (Griffin and Peterson, 1972), and is facilitated fcy the perturbation of equilibrium between the high and lew spin state of the heme of the ferric ircn to favor the latter form. Binding of the substrate, therefore, converts the hare frcm a low spin state to a high spin state. Stg? 2 of the reaction cycle is an electron reduction of the substrate-bound CYP and this reducing equivalent ultimately cores frcm NADPH. After reduction, the heme is now able to bind an oxygen molecule (step 3) which is the first actual step in oxygen activation. Stqp 4 is mandatory for the reaction stoichiometry and is also required in the position shown in the reaction sequence as indicated previously in Fig. 1.1. A branch exists at this point in the CYP catalytic cycle and it is interesting to mention here that active turnover fcy the liver microsomal CYP involves the production of hydrogen peroxide (H202); as rruch as 55% of consumed oxygen appears as H202 in the presence of substrate and 12 University of Ghana http://ugspace.ug.edu.gh essentially 100% in its absence (Nordblcm and Coon, 1977) . Thus, in addition to the hydroxylase activity, CYP also exhibits a concurrent oxidase activity with the formation of H^ Oj. Hilderbrandt and Estabrook (1971) suggested that cytochrome bs may supply the second electron at this stage, i.e step 4. Further evidence v\as provided ky Werringloer and Kawani (1980) vto measured both the kinetics and extent of reduction of CYP and cytochrome bs in a carbon monoxide (03) atmosphere, and found that these haroproteins participate in reversible electron transfer reactions whm associated with the microsaral membrane. Electron transfer was examined fron cytochrome bs to P-450 in the presence of NADffl; the equilibria ware independent of the reducing agent used. Their observations indicate that the redox properties of cytochrome bs are favourable for the reduction of ferrous oxy-P450 in the membrane. The cleavage of the oxygen-o>ygen bond in the ccrrplex (step 5) can occur in 2 v\ays; i.e either hcmolytically or heterolytically. The nature of the substrate to be oxidized undoubtedly plays an important role at this stq? of the reaction sequence. Heterolysis would lead to the formation of an iron-oxenoid ccrrplex. Qxene addition to a double bond can form areie oxides or epoxides in the case of AA metabolism. The electrqphilic oxygen intermediate may attack an electron-rich carbon atcm resulting in hydrogen abstraction (step 6) and oxygen insertion (step 7) . Dissociation of the product alcohol frcm the cytochrane P-450 active site (step 8) ccnpletes the cycle. 13 University of Ghana http://ugspace.ug.edu.gh Hemolysis of the o:xygen-o>ygen bond is probably the favoured route vhen an organic peroxide, such as cumene hydroperoxide, is used (Estabrook e t a l., 1982) . Studies of benzo (a) pyrene or the radical-mediated oxidation of amino pyrine are two examples of this type of reaction. Thus one can visualize that the cleavage of the oxygen-oxygen bond of the peroxide rray be assisted by the donation frcm the substrate. As a result water is formed as well as a radical species of the substrate. 1.4 CYTOCHROME P-450s Cytochrome P-450 (CYP) , earlier referred to as the CO-binding pigment, was first described by Klingeriberg (1958), and Garfinkle (1958). Qnura and Sato (1962) later characterized it as a hanqprotein. The term cytochrome P-450 refers to a family of heme proteins present in all marrrralian cell types, except mature blood cells and skeletal rruscle cells which catalyze the oxidation of a wide variety of structurally diverse corpounds. CYP also occurs in prokaryotes. The designation of a protein as CYP originated from its spectral properties before its catalytic function was known. This group of proteins has a unique absorbance spectrum that is obtained b/ adding a reducing agent, such as sodium dithionite, to a resuspension of endcplasmic reticulum vesicles, frequently referred to as microsames, followed by the bubbling of CD gas into the solution. The CD is bound to the reduced hane protein which produces an absorbance spectrum with a peak at 450 nm; thus the name P-450 for a pigment with an absorbance at 450 nm. 14 University of Ghana http://ugspace.ug.edu.gh Specific forms of CYP differ in their maximum absorbance wavelength, with a range between 446 and 452 nm. The many forms of CYP are classified, according to their sequence similarities, into various gene subfamilies; this systan of nomenclature is being adopted almost universally (Nebert et a l . , 1991L In mairmalian cells, CYPs serve as electron acceptors in electron transport systans, which are present either in the ER or inner mitochondrial martorane. The CYP protein contains a single iron protoporphyrin IX prosthetic group, and the resulting heme protein contains binding sites for both an oxygen molecule and the substrate. 1.4.1 Isoenzymes Attarpts by Sladek and Mannering (1966) to measure sate of the differences between the two types of microsaral drug metabolizing system synthesized as a result of stimulation ky polycyclic aromatic hydrocarbons (PAHs) and phenobarbital (PB) led to the conclusion that PAHs cause the synthesis of a modified CYP. For lack of a more suitable nomenclature for the microsaral hemoprotein, it was named cytochrome P!-450. Soon after this evidence for the existence of cytochrome Pi-450, Alvares et a l . , (1967) and Hilderbrandt et a l . , (1968) shewed that the of reduced microsomal protein bound to CD obtained after administration of PAHs differed slightly frcm that observed in microsaies from untreated animals. 15 University of Ghana http://ugspace.ug.edu.gh Alvares et a l . , observed a at 448 nm, therefore cytochrome P!-450 is sanetimes called P-448. Hilderbrandt and associates observed a at 446 nm, thus cytochrcrre Pj-450 is referred to as P-446 by sane investigators. The shift from 450 nm to 448 nm and 446 nm is slight, but real. The administration of PAHs causes the biosynthesis of cytochrome P!-450, a molecular species of CYP not normally detected in appreciable amounts in microsomes of untreated or phenobarbital-treated animals. This does not exclude the possibility that small amounts of cytochrcrre Pj-450 may be found in untreated animals. In the broadest sense, the various P-450 isozymes can be classified as: (1) those forms which metabolize physiological or endogenous substrates and (2) those forms which metabolize xencbiotic or exogenous substrates. It is urportant to note a fundamental difference between these two classes of P- 450. Those P-450 isozymes which metabolize exogenous substrates are inducible i n v iv o and exposure to xenobiotics can lead to increased levels of specific forms of P-450 and/or their respective enzymatic activities. As has been shewn in the case of several different xenobiotic metabolizing forms of P-450, changes in their levels result largely fron changes in the amount of rrRNA encoding these proteins (Adesnick e t a l . , 1981). In contrast, v\hile those P-450 isozymes which metabolize endogenous substrates have the capacity to be induced, the constitutive levels of these enzynes are generally optimal for the particular function they serve. There are 16 University of Ghana http://ugspace.ug.edu.gh instances where a specific form of P-450 contains diverse activities; one involved in xenobiotic rretabolism and the other in metabolism of endogenous substrates. An exairple is the products of AA generated ky P-450-mediated reactions via a lipoxygenase-like mechanism (Capdevila et a l . , 1981) . Such products have been shown to exhibit charotactic activity and to be involved in inflammatory responses (Samuelsson, 1980) . The major phenobarbital- inducible form of P-450 in liver has been found to possess this activity. The level of individual isozymes are strikingly dependent cn the animal1 s age, sex and history of exposure to foreign ccnpounds which can serve as monooxygenase inducers. Iirmunochanical and catalytic studies cn the sex specificity of P-450 isozymes expression have established that P-450 2c is male specific and undergoes a developmental induction at puberty (Waxman, 1984). P-450 2d is developnentally induced in female rats although it is also expressed at significant levels in immature rrales at 3-4 weeks of age (Waxman et a l . , 1985) 1.4.2 Conversion to P-420 Martorane-bound P-450 is affected by surface active agents, chelating agents, sulfkydryl reagents, and lipophilic substances. These agents convert P-450 to an inactive form, P-420 (Qnura and Sato, 1964) . The diversity of such effective agents appears to have caused confusion in the interpretation of the state of P-450. 17 University of Ghana http://ugspace.ug.edu.gh P-450 in liver microscmes acts as a site of both o>ygen and substrate activation for drug hydro>o/lation (Imai and Sato 1966) . Since only those drugs possessing high solubility in lipid solvents are b/droxylated ky the microsaral systan, it seems reasonable to assume that the reactive site of P-450, i.e. the vicinity of the heme, is in contact with, or buried in a highly hydrophobic part of the P-450 protein or the lipids of the microsaral mattorane. The unusual spectral properties of CYP are ascribed to a hydrophobic interaction of the hare with nearby carpanents. Since the conversion of P- 450 to P-420 is always accarpanied fcy the inactivation of the hydroxylase systans, and P-420 is incapable of reacting with the kydrcoylatable substrates in the sane way as P-450 (Imai and Sato, 1966), it is likely that the integrity of the unusual state of P-450 is essential for its function. Depending cn the agent enplcyed, the conversion of P-450 to P- 420 would result frcm the disturbance of the hydrophobic environment around the heme either ky primary action of the agent or ky secondary effects caused by conformational changes in the hemoprotein. The conversion of P-450 to P-420 induced ky neutral salts proceeds more rapidly in the reduced form of the hanoprotein than in the oxidized form (Imai and Sato, 1967). Cytochrcme P-420 formed as a result of treatment of microscmes with the detergent, sodium cholate, was converted back to cytochrcme P-450 ky polyols and reduced glutathione (Ichikawa and Yarrano, 18 University of Ghana http://ugspace.ug.edu.gh 1967) . Dialysis also produces partial reversal. 1.4.3 Spectral Characteristics Cytochrcrre P-450 (CYP) is measured ky the difference spectrum seen vten it is reduced, usually with sodium dithionite, and carbon monoxide (CO) is bubbled through the reduced microsomal suspension. NAEH and NADPH reduce CYP only in the absence of molecular oxygen, the pigment being autooxidizable. About 50% inhibition of hydroxylation reactions are obtained when the CO/Oj ratio is about one. Determination of the absolute spectrum of CYP is complicated ky the presence of cytochrcrre bs and cytochrare P-420. The problem has been solved to a large degree ky removing cytochrome b5 from the microscnes and by stabilizing CYP with glycerol. In Mason's laboratory hepatic microsames were treated with the non-ionic detergent, Lubrol WX, to produce subparticles v\hich gave an absolute spectrum for CYP with no apparent interference fron cytochrome b^ and very little interference from cytochrcrre P-420 (Miyake et a l . , 1968) . A rrethod was proposed by Kinoshita and Horie (1967) for determining the absolute spectrum of CYP in microsames v\hich had not been treated to remove cytochrcrre bj. This method is based on the knowledge that cytochrome k^ is the only microsarHl cytochrcrre other than CYP and that the administration of inducing agents such as phenobarbital causes a large increase in the concentration of CYP in microsames without affecting appreciably the cytochrcrre b5. 19 University of Ghana http://ugspace.ug.edu.gh 1.4.4 Binding to Substrates CYP catalyzes hydroxylation reactions of steroids, drugs and other catpounds. It has been found that CYP combines with these ccrrpounds frequently causing spectral effects. Studies ty Imai and Sato (1966), shewed that drugs and other foreign compounds ccntoine with hepatic CYP to produce spectra of two general types, type I and type II. Ccrrpounds giving type I or II difference spectra with hepatic microscmes have cone to be knewn as type I and type II ccrrpounds (or drugs) . Type I ccrrpounds give difference spectra with a ^ in the general range of 385-390 nm and a A„lri in the general range of 418-427 nm; the and X„in given ty type H ccrrpounds are 425-435 nm and 390-405 nm respectively (Schenkman et a l . , 1967) . The characteristic difference spectra observed when type I or type II drugs are added to microscmes can be seen vten the absolute spectra of CYP are recorded before and after the addition of hexobarbital (type I) or aniline (type II) . Besides these two groups of drugs, there is a third class of ccrrpounds which bind CYP to give a spectrum known as reverse type I (Schenkman et a l . , 1967) . This class of corpounds is the mirror image of the type I spectral change (A^ 420nm, X„,in 385nm) (Jefcoate, 1978) (Fig. 1.3). Depending cn the experimental conditions amines interact with CYP with difference spectra that may vary continuously between two extreme spectra, tented type Ila 425nm, 390nm) and type lib (X,„ax 432nm, \„in 410nm) (Jefcoate and Gay lor, 1969) . However there are a group c£ related drugs which do not give arr/ spectral change v\hen bound to CYP (Wilson and Harding, 1970). Imai and Sato (1967a) pointed out that 20 University of Ghana http://ugspace.ug.edu.gh FIG. 1.3 DIFFERENCE SPECTRA OF TYPE I, TYPE II AND REVERSE TYPE I COMPOUNDS 21 University of Ghana http://ugspace.ug.edu.gh barbital and benzene are hydraxylated b/ microsanes although they do not produce binding spectra. It is also to be noted that the ability of corpounds to form binding spectra with CYP does not guarantee their metabolism; irary n-alkylamines ccrrbine avidly with CYP to produce type II spectra, but are not metabolized (Jefcoate et a l . , 1969). With but one exception, type I and type H corpounds produced their characteristic binding spectra consistently regardless of the source of the microsares. The exception is phenoharbital (PB), Wiich gave a type I binding spectrum with microsames fron rats, but a type II binding spectrum with microsanes fran rabbits (Mannering, 1972) . CYP can be oxygenated cnly vfen in the reduced form and rrust therefore be in the reduced state when the activated oxygen reacts with the substrate. Spectrophotometric studies have shown that in the presence of both csygen and NADFH, CYP is mostly in the oxidized state. The reduction of CYP is believed to be the rate-limiting stg? in the overall process of microsomal metabolism. Eoth type I and type II substrates protect CYP against destruction and react with the ferric forms (Schenkman et a l . , 1969) . Type I substrates accelerate while type II substrates decelerate the reduction to the ferrous form (Kupfer and Orrenius, 1970); type I substrates do not affect re-oxidation while type II substrates may accelerate it (Gigon et a l . , 1968). Schenkman (1970) adduced evidence that most type II changes are acccrrpanied by type I changes and became enlarged and symmetrical if 22 University of Ghana http://ugspace.ug.edu.gh corrected for the type I changes. Leibman and co-workers (1969) believed that many of the inconsistencies in the kinetics of binding and metabolism of CYP could be explained by the interconvertibility of type I and type II CYP carpi exes. This conclusion VvQS drawn partly frcm kinetic studies in which either a type I or n corpound (modifier) was added to a suspension of microscmes placed in both cuvettes of a dual beam spectrophotometer. Graded amounts of either a type I or type II corpound (substrate) were added to the sarrple cuvette and the difference spectrum recorded. The sum of the absorbance change at wavelengths of the peaks and troughs were determined. When the modifier was a type II ccnpound and the substrate a type I ccnpound, competitive inhibition vras observed. When the modifier and substrate were type H ccnpounds, inhibition was never corpetitive, and in certain cases kinetics ware analogous to classical non-corpetitive inhibition. Both competitive and non-corpetitive inhibition were seen vten the modifier and substrate wsre type I carpounds. When the modifier was a type I substance, and the substrate a type H corpound, "stimulation" rather than inhibition was seen. 1.5 OTHER COMPONENTS OF THE MICROSOMAL MONOOXYGENASE SYSTEM 1.5.1 Reductases NADPH-cytochrcsme c reductase and NADPH-cytochrome P-450 reductase are 2 reductases purported to be involved in drug metabolism. NADPH-cytochrcme c 23 University of Ghana http://ugspace.ug.edu.gh reductase is thought to reduce CYP directly or indirectly through a nan- herre iron protein or sore other unidentified carrier. Because cytochrome c is not present in microscmes, and there is no other natural substrate for the reductase in these organelles, NADPH-cytochrcme c reductase was considered particularly eligible to play a role in the transfer of electrons frcm NADFH to CYP. The enhancement of NADPH-cytochrcme c reductase activity sesi in microscmes when microscmal drug metabolizing activity is caused to be increased as a result of phenobarbital administration provided another indirect association of the reductase with the microscmal hydroxylating system (Remmer and Marker, 1965; Qrrenius and Emster, 1964) . NADPH-CYP reductase contains both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) as prosthetic groups. It is the only mammalian flavqprotein known to contain both FAD and FMN (lY&sters and Ckita, 1992) . The FAD serves as the entry point of electrons frcm NADFH, and FMN serves as the exit point, transferring electrons individually to CYP. Because the flavin molecule nay exist as cne or two electron reduced forms, and 2 flavin molecules are bound per reductase molecule, the enzyme nay receive electrons fron NADFH and store them between the 2 flavin molecules before transferring them individually to the heme iron for 02 binding (first electron) and cleavage of the oxygen rrolecule (second electron) (Fig 1.2). 24 University of Ghana http://ugspace.ug.edu.gh In certain reactions catalyzed ky the microscrral P-450, the transfer of the 2 electrons m=y not be directly frcm NADPH-CYP reductase, but may occur fron cytochrcrre b5 which is also present in the ER (Fig 1.1) . Cytochrome be, is reduced either ky NADPH-CYP reductase or another microsaxe-bound flavoprotein, NADH-cytochrcrre bs reductase, which is specific for NADH. NADPH-CYP reductase activity measurement is based cn the knowledge that CD forms a ccrrplex with reduced, but not oxidized CYP to give a Soret peak at 450 nm. Fron a variety of experiments that showed the rate of drug metabolism to be more closely related to the rate of cytochrcrre P-450 reductase than the total amount of CYP or the rate of cytochrcrre c reduction, it was concluded that NADHi-CYP reductase activity is rate- limiting in the overall reaction involving microscrral drug metabolism. Qf considerable interest was the observation that type I binding ccnpounds stimulated NADPH-CYP reductase activity whereas type II binding corpounds either inhibited or had no effect cn the reductase (Gigon et a l . , 1968, 1969). NADPH-CYP reductase activities of microscrres from male and female rats were also determined with the idea that an explanation might be found for the well knewn sex difference in drug metabolism (Gigon et a l . , 1968, 1969). Cnly a slight difference in the reductase activity was found and this vas probably due to the snail difference in CYP content of the microsares. Hcwsver, eth/lmorphine (type I carpound) caused a much greater stimulation 25 University of Ghana http://ugspace.ug.edu.gh of NADFH-CYP reductase in microscmes frcm males than it did in microscmes fran females. This suggested to the authors that the CYP in microscmes from female rats is less capable of participating in the oxidation of substrates than that in microscmes from male rats. Nothing is known about NADPH-CYP reductase other than its activity. As the reduction of CYP is relatively difficult to assay directly, a simplified determination of enzyme activity is widely used, utilizing exogenous cytochrcme c (oxidized, ferric form) as an artificial electron acceptor. Accordingly, the reduction of cytochrcme c ky NADPH-cytochrcme c (P-450) reductase mirrors the reduction of CYP. The assumption is that NADffl- cytochrame (c) reductase reflects the activity of NADBi-CYP reductase, car of seme other flavoprotein, as it functions as part of the microsaral multi-enzyme hydroxylase carplex. The principle of this method is that oxidized (ferric) cytochrcme c has a characteristic absorption spectrum as does the reduced (ferrous) form. However, the reduced form has a characteristic absorption band at 550 nm, a band that is absent in the oxidized form. Therefore the enzyme activity can be conveniently assayed ky measuring the increase in absorbance at 550 nm as a function of time. 1.5.2 Lipid Factor Lu and others (Lu and Coon, 1968; Lu et a l . , 1969) separated solubilized hepatic microscmes into three fractions containing CYP, an NADPH reductase and a heat stable lipid factor. The combination of all three fractions v\as 26 University of Ghana http://ugspace.ug.edu.gh required for maximal drug metabolizing activity. Heating the lipid factor for 2 hours at 100°C at neutral pH or for 10 minutes at 50°C in 0.1M HCl or H2S04 did not affect it. It was however destroyed ty ashing (Lu et a l . , 1969a). Atterrpts to replace the lipid factor fraction with other phospholipid preparations including rat liver lecithin car phosphatidyl ethanolamine, egg yolk lecithin, bovine phosphatidyl ethanolamine, proved unsuccessful. The lipid factor could therefore be considered as acting physically in such a way as to provide access of the drug to CYP. 1.5.3 Non-heme Iron Protein Adrenodoxin, a non-hane ircn protein, is a ccrrponent of the electron transport system that functions in the hydroxylation of steroids by adrenal microscmes and mitochondria (Suzuki and Kirnura, 1965, Kimura and Suzuki, 1965) . In this systan, adrenodoxin acts as the electron carrier between flavin enzyme (adrenodoxin reductase) and CYP. Because of the similarities of the microscmal hydroxylase reactions of the adrenal gland and liver, it has bean, assumed that adrenodoxin or scrre other non-heme iron protein is involved in the microscmal drug metabolizing system. In the presence of NADFH, a mixture of adrenodoxin and adrenodoxin reductase caused the reduction of CYP contained in suhrnicroscmal liver particles, but the essentiality of adrenodoxin vas not determined (Miyake et a l . , 1968). Kinura (1968) concluded that tissues capable of producing steroid hormones 27 University of Ghana http://ugspace.ug.edu.gh (adrenals, testis, ovary) contain adrenodoxin, but liver and other tissues do not. This does not however exclude the possibility that a different non-hene protein nay function in the hepatic hydroxylase system. 1.6 INHIBITORS OF CYTOCHROME P-450S MEDIATED REACTIONS Investigations concerning the inhibition of hqpatic mixed-function monoaxygenases are of direct relevance to toxicology and pharmacology in anticipating and predicting the safety of the environment and of drug therapy. Studies in this area is also of relevance to the concentrations of essential endogenous corpounds produced by this enzyme cotplex. For these reasons, it is essential to understand the possible interactions between various inhibitors and the enzyme corplexes at the molecular level. By the use of conventional enzyire kinetic studies and techniques, interactions have been classified as carpetitive or non-ccnpetitive; but rrary instances have arisen where the results do not conform to either of these classifications. In many cases, the lack of conformity has been attributed to the multi-enzyme nature of mixed-function oxygenation reactions or the inability or unsuitability of using conventional concepts for an enzyme located in a marbrane (endoplasmic reticulum) . CYP monoaxygenases are differently inhibited ky a variety of substances including CD, 2-diethylaminoethyl-2-diphenylvalarate (SKF 525-A), metyrapone, ethyl-isocyanate, the Lilly corpound, 2,4-dichloro-6- phenolphenoxy-etkylamine (DPEA) (Imai and Sato, 1967b; Qnura and Sato, 28 University of Ghana http://ugspace.ug.edu.gh 1964) salsolinol and the n-butanol fraction (nBF) of Desrrvdium adscendens (Add/ and Schwartzrren, 1992) . Indeed varying inhibitory effects cn different microsaiBl preparations suggested as underlying support for nultiple forms of P-450 monooxygenases. Hcwever inhibitor studies rrust be interpreted with caution, especially with heterogenous preparations. For example, microsomal hydroxylase activity is widely thought to be insensitive to cyanide. Hcwever, CYP systans have been found to be inhibited by cyanide in a concentration-dependent rranner. 1.6.1 Formation of Metabolic Intermediate Complexes In the last two decades, a facet of mixed-function oxygenases inhibition has beams apparent which yield an interpretation of sore previously unexplained observations and provides an insight into a new concept of mixed-function oxygenase inhibition. This involves ccnpounds which are not substrates for the mixed-function oxygenase reaction, but intermediates in the reaction, or products of the reaction which do not leave the enzyme. Ihis entity (intermediate or product) binds tightly to the enzyms, preventing its further participation in mixed function oxygenation (Franklin, 1977) . These ccrtpounds form carplexes with CYP which can be detected in a ferrous state by an absorbance maximum in the Soret region. No product of a mixed-function oxygenase reaction has been found which, upon addition to ferrous CYP, immediately shews the samne amount of cctrplex which can be obtained during metabolism. Much of the information presently 29 University of Ghana http://ugspace.ug.edu.gh available suggests that an intermediate generated during the oxygenation reaction forms the ccsrplex with CYP (Franklin, 1977), and thus the term "metabolic intermediate (MI) corplex" is used to describe such ccrrpounds. The ability to detect spectrophotometrically (given certain conditions) the existence of the enzyme-intermediate corplex has facilitated the rapid investigation of this aspect of oxidative drug metabolism. The prerequisites for MI corplex are conditions necessary for mixed- function oxygenation reactions to occur. Thus, Q and NADPH are essential, with the exception of organic peroxides v\hich can provide the equivalent of both (Elccmbe et a l . , 1975) . NADH substitutes only very poorly as a source of reducing equivalents ccsrpared with NADPH (Franklin, 1974). The MC ccnplex is probably a corbination of a metabolic intermediate and the hone iron of CYP. The MI corplex exhibits an absorbance maximum in the Soret region between 448 and 456 nm (for compounds examined to date) when the hone iron is in the reduced (ferrous) state (Werringloer and Estabrook, 1975) . Observation of a 450 nm absorbance maximum needs careful scrutiny, however, since generation of 00 during prolonged aerobic incubation of microsones with NADffl can occur. Binding of the CD thus formed with CYP, will beccme apparent under near anaerobic conditions, as produced ty dithionite addition. Ihe MI is not displaced frcm the corplex ty CD and, thus, interferes with the determination of CYP. The MI ccnplex, once formed, inhibits mrxed-function reactions in a non-corpetitive manner. 30 University of Ghana http://ugspace.ug.edu.gh Corpounds capable of forming MI complexes can be divided into 2 main categories; non-nitrogenous and nitrogenous. A fa^ r other ccrrpounds not readily fitting into these categories (e.g. N-2-ethylhexyl-5-norbomene-2, 3-dicarbcocimide [M3K-264] and fluorine) have also been observed to form MI corplexes (Ullrich and Schnabel, 1973). The non-nitrogenous group consists predominantly of meth/lenedioxybenzene derivatives whose ccrrplexes shew an absorbance maximum at 427 nm, in addition to that at 455 nm, viren in the ferrous state (Franklin, 1971). As a groip, nitrogenous MI ccrrplexes differ frcm the non-nitrogenous in several respects. Foremost is their instability in the ferric state. However, in contrast to non-nitrogenous MI ccrrplexes, the nitrogenous MI ccrrplexes stabilize the CYP in the ferrous state and thus, after in v ivo formation, are iirrrediately visible in the microsanes, that is, they do not require dithionite addition to show the presence of an absorbance rroximum at 455 nm. The main classes associated with the nitrogenous MI forming corpounds are the arrphetamine, SKF-525-A, dithionite unstable and nitroso related corpounds (Franklin, 1974). While the substrate, the metabolic route, and the source of microsanes all play a role in determining the kinetics of inhibition, a key factor is pre­ incubation of the inhibitor prior to substrate addition. For the SKF 525-A class of corpounds the effects of anitting preincubation are clear and 31 University of Ghana http://ugspace.ug.edu.gh indicate straight forward ccrrpetitive inhibition (similar \tex, dissimilar Kin) . Preincubation of SKF 525-A and its analogues with hepatic microscmes change the kinetics of inhibition frcm corpetitive inhibition to ncn- ccrrpetitive inhibition. In particular, clear non-ccrrpetitive inhibition is seai with SKF 525-A and its secondary amine derivative (Testa and Jenner, 1981). 1.7 N-BUTANOL FRACTION (nBF) OF DESMODIUM ADSCENDENS Desmodium adscendens is a plant used in herbal medicine for treating asthrra, to aid in child birth, to irrprove lactation and to relieve the pain associated with dysmenorrhea. In studies to elucidate the scientific basis for the therapeutic effects of the plant, it was found that an extract frcm dried sten leaves of D. adscendens, rrade by solvent extraction with n- butanol, activated the cyclooxygenase enzyme and increased prostaglandin (PG) production (/iddy and Schwartzrran, 1995) . lyramine and hordenine, phenolic ccxrpounds of the p-phenethylamine type, which have been isolated frcm the plant, were evaluated alongside the plant extract as modulators of the cyclooxygenase enzyme. Both ccnpounds were also found to activate the enzyme and increase F3 production, especially prostaglandin E2 (PGE2), depending on the enzyme concentration and availability of co-enzymes. The/ were found to activate the enzyme more than the extract of the plant. Increasing the concentration of the nBF of the plant extract did not increase its activation of the cyclooxygenase enzyme. It was inferred frcm these results that the plant material contained compounds other than the (3- 32 University of Ghana http://ugspace.ug.edu.gh phenethylamines which affected the type and quantity of the second messengers produced ty the cyclooxygenase pathway of arachidonic acid (AA) metabolism. An attenpt was therefore irade at evaluating other ccrrpounds kncwn to be present in the plant extract. Salsoline (Appendix AI:a), a 6- hydroxytetrahydro-isoquinoline derivative reported to be an alkaloid present in D. adscendens (Asante-Poku et a l . , 1988), was not available during the experimental period. Therefore salsolinol (l-methyl-6,7- dihy,droxy-l,2,3,4-tetrahydroisoquinoline), a 6,7-dih/droxy analogue was used. Salsolinol decreased the amount of PGE2 formed and increased that of PGH2 and PGF2a in either the presence or absence of GSH. It was also a much more effective activator of the cyclooxygenase enzyme as indicated ty a higher metabolism of AA in its presence catrpared to that of nBF, tyramine or borderline in the presence of GSH. The increased metabolism was in favor of PGH2 and PGF^ production. In the cyclooxygenase pathway of AA metabolism (Fig. 1.4), the AA is converted to an endoperoxide 15-hydroperoxide prostaglandin G2 (PGG2) . PGG2 is then converted to the hydroxyl derivative PGH2 ty a GSH-dependent peroxidase. This is an indication that formation of PGH2 frcm P3G-, requires a reducing agent. This product of the cyclooxygenase pathway is converted to prostaglandin D, E and F as well as thromboxane (TX) or prostacyclin (PGI2) ty different specific enzymes (Fig. 1.4), whose 33 University of Ghana http://ugspace.ug.edu.gh .COOH arachidonic acid Jo2 |°2 (oxygenase) COOH pgg2 OOH 2 GSH (glutathione peroxidase) 1 COOH /(p ro s ta g lan d in r R-»D isomerase) OH PGHj (prostaglandin \ 2H (reductase' ) R-* E isomerase) \ OH CC Cc a prostaglandin OH prostaglandin E2 OH prostaglandin FIG. 1.4 PATHWAY OF PROSTAGLANDIN BIOSYNTHESIS 34 University of Ghana http://ugspace.ug.edu.gh presence varies depending upon the cell type and tissue (Grew, 1992) . The conversion of PGH2 to PGF2a utilizes a reductase iirplying that an additional reductant is required as a co-enzyme by the cyclooxygenase enzyme system to convert AA to PGF2„. Co-enzymes known to be utilized ky the cyclooxygenase enzyme for AA metabolism to produce various PGs include phenolic compounds, catecholamines, indole compounds and other reducing agents (Lands et a l . , 1971) . The effect of the nBF and other corpounds on PG synthesis indicates that the fraction contains the type of reductant required for the reduction of hydroperoxide v\hen PGG2 goes to PGH2 and that for the reduction of the carbonyl v\hen PGH2 is converted to PGF2a. These results suggest that salsoline, with the same basic structure as salsolinol is likely to be the ccrrpound in the plant vsfoich acts as a reducing co-enzyme in the redox enzyme systans in this pathway of AA metabolism. The nBF has also been shewn to inhibit AA metabolism ky the CYP ironooxygenase pathway (Addy and Schwartzrran, 1992) . Salsolinol, the 6, 7- dihydroxy analogue of salsoline, the tetrahydroisoquinoline corpound in the plant, was found to inhibit AA metabolism ky the NADPH-dependent CYP ironooxygenase enzyme corplex of the ER, just as the plant extract did. With AA as substrate, salsolinol inhibited the formation of the epoxides, EETs and HETEs in this NADPH-dependent CYP monooxygenase reaction. The CYP monoaxygenase-catalysed reaction starts with substrate binding to native ferric P-450 (Fig. 1.2) and this is followed ky reduction to the 35 University of Ghana http://ugspace.ug.edu.gh ferrous state, thereby allowing oxygen binding. If the CYP must be in the ferric (Fe3+) state to bind the substrate, a reducing agent capable of reducing the Fe3* of the cytochrcme will prevent substrate binding and inhibit the reaction. With the reducing property indicated in the cyclooxygenase reactions, the nBF of D. adscendens could contain such an inhibitor. The extract could also contain a type of inhibitor which interferes with the flow of electrons from NADPH to CYP, the rate determining step that activates molecular oxygen for this type of oxygenation reaction. The extract may also react with either the substrate, risking it unavailable, car act as a co-substrate, replacing the NADPH as the electron donor/co- reductant in the mixed-function oxygenase reaction. In a previous stud/ in which an atterrpt was made to find out the mode of action of salsolinol in this inhibition of all the products of the NADffl- dependent AA oxygenation (Addy and Schwartzrtan, 1992), the tetrahydroisoquinoline (THIQ) ccitpound was found to reduce cytochrcme c directly, with a reducing potential similar to that of reduced glutathione (GSH) (Kamassah, 1992) . It was inferred from the results that the mode of action of salsolinol and hence salsoline in the plant, was to reduce CYP and keep it in the reduced state, thereky interfering with the oxidation/reduction cycle required for its function. 36 University of Ghana http://ugspace.ug.edu.gh The hepatic CYPIA1 isoenzyne which is inducible ky p-naphthoflavone v\ss also shewn to be ncn-ccnpetitively inhibited ky the nBF of an aqueous extract of D. adscendens when 7-ethoxyresorufin was used as an artificial substrate in an EROD assay (Brookrran-Amissah, 1994) . In this reaction, 7- ethoxyresorufin is deethylated to resorufin, as shown in Appendix All. The CYP isoenzyme responsible for AA oxygenation is different frcm CYPIAl. Therefore, inhibition of both ky nBF signifies a node of inhibition nore related to the general reaction mechanism such as the reduction frcm Fe^ to Fe2*. The study reported here was designed to test the reducing properties of nBF and its interactions with CYP and substrates using spectral changes as the method of investigation. 37 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 Chemicals and Reagents Butanol, sodium potassium tartrate, potassium chloride, sodium chloride, sodium carbonate, sodium hydroxide, dithiothreitol (COT), glycerol (87%), silica gel 60, methanol and potassium dihydrogen phosphate were purchased frcm Fluka Chemie PG, Switzerland. Sodium dithionite, sodium dih/drogen orthcphosphate and chloroform were obtained frcm British Drug House Chenical Ltd., England. Ethylenediaminetetra-acetic acid (EDTA) and Folin- Ciocalteau's phenol reagent were purchased frcm Hopkins and Williams, England. Ccpper sulphate (anhydrous) was obtained frcm Merck Darmstadt, FRG. Bovine serum albumin, E-naphthoflavone, glutathione (reduced), nicotinamide adenine dinucleotide phosphate (NADPH), salsolinol, resorufin, cytochrcme c and ethoxyresorufin were obtained frcm Sigma Chemical Co., U.S.A. Tris- (hydroxymethyl) amino methane was obtained frcm Eastman Kodak Co., U.S.A. Carbon monoxide gas was purchased frcm Union Carbide, Belgium. Scyabean oil was obtained frcm the local market. Dry stem leaves of Desmodium adscendens were obtained under cocoa trees at the Cocoa Research Institute of Ghana (CRIG) . All chemicals were of the highest ccrrniercial grade available. 38 University of Ghana http://ugspace.ug.edu.gh 2.1.2 Animals White Wistar rats, black C57BL/ks J dkm and white DDY mice were bred at the Animal E^ qperimentation Unit of the Noguchi Memorial Institute for Medical Research (NMIMR) at Legon. All anirrals were fed an pelleted feed frcm the NMIMR. The ccrrposition of the feed was 40% vfaeat bran, 40% maize, 2% fish meal, 17% cod liver oil, and 1% sodium chloride. 2.2 METHODS 2.2.1 Preparation of Buffers and Solutions Buffers and solutions were prepared as described in Appendix B. 2.2.2 Preparation of n-butanol fraction of D . adscendens Crude aqueous extract of D. adscendens was prepared ty boiling the pulverized dry leaves for approximately 4 hours in water and allowing to cool. The mixture v®s centrifuged to remove solid particles. The supernatant was collected and the pellets discarded. The supernatant was concentrated to approximately 20.0ml by a Rotary Evaporator. The crude extract was shaken three times with 3 volurres of water-saturated n-butanol. The organic layer was pooled after 3 extractions, the solvent ramoved (by evaporation) and the residue taken up into 10.0ml of water. It was thai freeze-dried cn an Eyela Freeze Dryer FD-1. The freeze-dried n- fcutanol fraction of D. adscendens (nBF) was stored in a refrigerator whai not in use. 39 University of Ghana http://ugspace.ug.edu.gh 2.2.3 Fractionation of nBF Approximately 0. 4g of the freeze-dried nBF was dissolved in 5. Qml of water and coated cn 1.6g of silica gel 60 (40-60nm, 230-400 mesh). The silica gel was dried in a rotary evaporator and added to the top of a column (30 x 500mn) containing 33g of silica gel (same mesh) packed in n-butanol. 150 ml of five different n-butanol based solvents of graded polarity were flashed through the colutm sequentially. Each eluent v\as collected in bulk, evaporated to get rid of the solvent, and taken up in equal volumes (approx. 5ml) of double distilled water. The solvents used in the flash chroratography v«ere as follows: (1) n- butanol; (2) water-saturated n-butanol; (3) 5% MeCH in water-saturated n- butanol; (4) 10% MsOH in water-saturated n-butanol; (5) 20% MeCH in water- saturated n-butanol. The scheme used in the fractionation of the crude aqueous extract fron Desmodium adscendens is shown below in Fig. 2.1. Values in parentheses are yields expressed as percentage of weight of material taken for the step in the fractionation scheme. Fractions with reported bioactivity (Addy, 1989) are marked with an asterisk. The method used in this separation is that according to Still et a l . , (1978). 40 University of Ghana http://ugspace.ug.edu.gh PLANT MATERIAL water centrifugation CRUDE EXTMCT n-butanol Liquid / liquid extraction ORGANIC PHASE AQUEOUS nBu (20%) Flash Chrcmatography PHASE *F1 (13%) *F2 (29%) *F3 (16%) F4 (12%) *F5 (8 % FIG. 2.1 SCHEME USED IN THE FRACTIONATION OF THE CRUDE EXTRACT FROM D. ADSCENDENS 2.2.4 Induction of CYP Monooxygenase in Mice Eight mice were injected intraperitoneally once daily for 3 days with 6 - naphthoflavone (ENF) (80 irg/kg bod/ weight), prepared ty sonication in soyabean oil. The mice were sacrificed on the fourth day. The control animals ware injected with the same volume of scyabean oil minus 6 - naphthoflavone. 2.2.5 Preparation of Microsomes A modified method of Cmura and Sato, 1964 was used in this preparation. The experimental animals were killed ty strangling. Their livers and kidneys 41 University of Ghana http://ugspace.ug.edu.gh were ranoved and weighed. The tissues were excised into small pieces with a pair of scissors, rinsed in 0.9% NaCl and blotted dry. 2 times volume of homogenizing buffer (Appendix BI) was added in a centrifuge tube and the excised tissues were homogenized with a polytron kinerratic harogenizer (6 strokes). The hanogenate was centrifuged at 10,000g for lOmin. at 4°C using a Hitachi 20ER-52D Centrifuge. The pellet was discarded and the supernatant centrifuged at 16,800g for lOmin. at 4°C. The supernatant obtained, often referred to as the S-9 fraction, was centrifuged at 40,000rpm (105,000g) for 1 hour in a Hitachi 80P-7 autcrratic preparative Ultracentrifuge (Rotor RP65T-453) at 4°C. When rats were used as the experimental animals, the pellets were washed in hatDgenizing buffer to remove any hemoglobin not reroved earlier and to clean microsanes of cytochrare b5 contamination which often interferes with CYP spectral studies. The washed pellets were then re-hcncgenized in 1 volume of resuspension buffer (Appendix BII) and distributed into cryotubes and stored at -84°C until they were used. Preparation of microscmes was carried out cn ice to prevent enzyme degradation. 2.2.6 Protein Concentration Estimation Protein was determined as described ty Lcwry et a l . , (1951) in which absorbances of coloured corplexes resulting frcm a reaction between alkaline cqpper-phenol reagent and tyrosine and tryptophan are measured at 42 University of Ghana http://ugspace.ug.edu.gh 750 nm. Protein reaction was carried out as follows: 0. 4ml microscmal preparation (diluted 1:20 with 0.5M NaCH) was made up to a final volume of 1.0ml with 0.5M KfeCH. 5.0ml of alkaline ccpper reagent (Appendix Bill) ware added, mixed thoroughly ky vortexing, and allowed to stand for 10 minutes. 0.5ml of a 1:1 diluted solution of Folin-Ciocalteau phenol reagent (IN) was added and mixed immediately and corpletely using a Vortex Mixer. After 30 minutes of incubation, the absorbance was read at 750 rm cn a Shimadzu UV- 190 double beam Spectrophotometer, after zeroing with a blank containing all the reagents except the microsames. This determination was done in duplicate. The microsanal protein concentration was directly intrapolated fron a standard curve constructed with bovine serum albumin (BSA) . 2.2.7 Ethoxyresorufin O-Deethylase (EROD) Assay The EROD assay was performed in order to ascertain the induction of CTPIA1. The ERCD measurements were performed according to the method of Mayer and Burke (1974) . The instrument was standardized with resorufin and an emission scan for resorufin was run to determine the wavelength of maxirrum fluorescence. The ERCD reaction was carried out in a fluoranster cuvette as follows: 1.975ml of EROD buffer, 10^ 1 7-ethoxyresorufin (0.41irM) and 5jj.1 of the liver microsanal preparation frcm the non-injected white mice (NIWyR were mixed thoroughly in the cuvette. 10^ .1 of NADPH (lOnM) were added to the mixture, and the amount of resorufin formed with time was recorded for 43 University of Ghana http://ugspace.ug.edu.gh a time interval close to 2 minutes. The reaction was spiked with 10^1 of the working solution of resorufin (17.9pM), and the change in fluorescence recorded for a time interval close to 1 more minute. This served as the control experiment. The reaction vas repeated using 5jj.1 of the microsaral preparation from the BNF-injected white mice (BIWvi) . The volume of the buffer was adjusted accordingly to give a final reaction mixture of 2.0ml. EROD activities of microsaral preparations frcm the non-induced black mice (NISI) and HSIF-injected black mice (BIEM) - both of the C57 BL/ksJ dhm strain were also determined. TWo different volumes of micnoscrral preparations 4.6 and 4.750nl were used for the BIBM and NIEM respectively. The velocity of the reaction using induced CYP monooxygenase was calculated as follows: Velocity = S x c t x R A diagram of a printed tracing frcm the fluorimeter is shown in Fig. 2.2. In order to evaluate the inhibitory effect of certain reducing agents cn this system, the ERCD assay was repeated using the same volumes and concentrations of reagents as in the control assay. After running for about 1 minute, 50|il of 50irM salsolinol were added to the reaction cuvette. The reaction was run for a further 1 minute before spiking with the working solution of resorufin. The assay was repeated, varying the volumes (concentrations) of salsolinol. Different volumes of the DAF1-5 of the nEF 41 University of Ghana http://ugspace.ug.edu.gh obtained after Flash Chromatography were also used. Microscmal preparations frcm the two different strains of mice were used for the inhibition studies. TIME (MIN) FIG. 2.2 DIAGRAM OF PRINTED TRACING FROM SFM-25 FLUORIMETER PRINTER S = fluorescence change due to reaction, measured in mm. R = fluorescence change due to internal spike, measured in mm. c = moles of resorufin in internal spike (179pmol) t = time in min. 45 University of Ghana http://ugspace.ug.edu.gh 2-2.8 Determination of Cytochrome P-450 Content Microsanes ware diluted in the resuspension buffer (Appendix BII) to a final protein concentration of approximately 2mg/ml. A 5ml sanple of the suspension was equally divided into 2 cuvettes with a 1-cm light path. Carbon monoxide was gently bubbled through the sarrple and reference cuvettes for approxirrately 4 minutes at a flow rate of approximately one bubble per second. After establishing a baseline ky scanning between 400 nm and 500 rm at a speed of 0.2nm/sec, a few grains of solid sodium dithionite (Na2S204) were added to the sarrple cuvette. The spectrum was then re-scanned fron 400 nm to 500 nm. The absorbances at 420, 450, and 490 nm were noted and these were used in calculating the CYP and P-420 concentrations as described by Ctnura and Sato (1964), using values of 91 and 110 rriYT1 cm-1 for molar extinction coefficient between 450 and 490 nm, and between 420 and 490 rm respectively. 2.2.9 Reduction of CYP An atteript vas made to ascertain whether the substrates used in this research, could reduce CYP directly. The experiments were performed as described above, but in place of sodium dithionite as the reducing agent, L-salsolinol, GSH and nBF were used. 46 University of Ghana http://ugspace.ug.edu.gh In order to determine the rate of reduction of CYP, experiments were carried out as described in section 2.2.8 with 0.4uM NADPH solution as the reducing agent. After scanning and determining the CYP and P-420 contents, varying concentrations of L-salsolinol and nBF were added iirmediately. After 25 minutes, the CYP and P-420 contents were again determined after re-scanmng the spectrum fron 400 nm to 500 nm. In another set of experiments, varying concentrations of nBF were pre-incubated with the microsomal fraction before NADPH addition, and the rate of reduction of CYP determined for 25 minutes at 5 minutes interval. An equal volume of distilled water was used in place of the reducing agents as controls. In these experiments, after determining the rate of reduction of CYP, the spectrum was re-scanned fron 400 nm to 500 rm and the CYP and P-420 contents determined. 2.2.10 Reduction of Cytochrome c lb 1.5ml of TRIS-HC1 buffer, pH 7.4 and 1.9ml of cytochrome c solution (11.5nM), 0.1ml of saturated Na2S204 was added. The absorption spectrum (between 330-700 nm) of the mixture was obtained using a Shiiradzu UV-190 double beam spectrophotometer against a blank of 3.4ml TRIS-HC1 buffer and an equal amount of saturated Na2S204 (i.e 0.1ml). This absorption spectrum was recorded over one obtained without the addition of Na2S204. The second scanning was done 15 minutes after the addition of the reducing agent. Different concentrations of nBF, salsolinol and DAF1-5 were used in place of sodium dithionite as reducing agents. 47 University of Ghana http://ugspace.ug.edu.gh 2.2.11 Cytochrome (c) P-450 Reductase Assay The method used in this experiment is a rrodification of that according to Masters et al . , (1967). Microscmal preparation (approximately lOmg/ml) was used in this assay. 2.15ml of 0.1M phosphate buffer, pH 7.4 (Appendix BI), 250^1 of oxidized cytochrare c (5mg/ml), and 0.1ml of the microsaral preparation were mixed in a spectrcphotcmeter cuvette. The reaction was initiated ky addition of 25^ 1 (2% NADFH), and the absorbance change at 550 nm measured for 3 minutes at 30 seconds intervals on a Shimadzu double-beam Spectrophotometer UV-190, against a blank containing the phosphate buffer instead of NADPH. The effect of nBF an the reductase activity was studied ky pre-incubating varying concentrations of the nBF for 3 minutes before the addition of NADFH. The reductive role of nBF was also investigated ky initiating the reaction with nBF instead of NADPH. 2.2.12 Binding studies The oxidized spectrum of the microsaral preparation was obtained and its interaction with the substrate 7-ethoxyresoruf in (7-ER) and plant extract investigated. In these binding studies, microsaral preparations frcm mice pre-treated with BNF were used because CYPIA1, which is induced ky E3NF is specific for EROD activity. The metabolism of 7-ER to resorufin was used as a model for the binding studies. The spectrophotcmetric method of Klotz et a l . , 1984 was used. For this method, a mixture consisting of 14.5^1 (0.41nM) of 7-ER, 7.5(jl microsaral preparation (lOmg/ml) and 15|jl NADFH 48 University of Ghana http://ugspace.ug.edu.gh (100.5nM) in 3ml of 0.1M ERCD buffer, pH 7.4 was matched against a control containing all the above except NADFH. The spectrum of the enzymatic product, resorufin v®s obtained b/ scanning the solution between 400-700 nm. Varying concentrations of nBF were added after product formation to determine its effect on resorufin after it has been formed. In another set of experiments, varying concentrations of nBF ware pre­ incubated with the microscmes and substrate for 3-5 minutes before NADPH addition. nBF was added to both cuvettes to offset absorbances due to the plant extract. The spectrum of the microsanes and 7-ER pre-incubated with nBF was obtained before and after NADPH addition. The spectrum of resorufin and 7-ER was obtained by scanning between 300-700 nm. Resorufin and 7-ER were dissolved in 0.1M Tris-HCl pH 8.0 containing 0.1M NaCl to give a final concentration of 1.6(jM resorufin and 8.4|jM 7-ER. 49 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE RESULTS 3.1 PROTEIN CONCENTRATION The protein concentrations of the liver microsomal preparations were calculated from the calibration curve shown in Fig. 3.1 using a dilution factor of 1:20. The X750 absorbances and the resultant protein concentrations are shown in Tkble 3.1. T&ble 3.1 Absorbance measursnents at 750 nm and protein concentrations of the microsaral preparations frcm the liver and kidney of the different laboratory animals Sairple Absorbance Protein concentration (mg/ml) NIEML 0.196 17.0 BIBML 0.235 20.5 NIWML 0.187 16.14 B]>ML 0.217 18.9 WWRL 0.177 15.51 WWRK 0.121 10.2 NIfflL = non-induced black mice liver; BIHYIL = ENF-injected black mice liver; NIVML = non-induced white mice liver; BIMC, = ENF-injected white mice liver; VMRL, = v^ rlte wistar rat liver microsares; V'WRK = white wistar rat kidney microscmes. The amount of protein per gm. wet weight of liver for the microsaral preparations frcm the mice are presented in Fig. 3.2. 3.2 CYP AND P-420 CONTENT Cytochrcme P-448, a molecular species of CYP is induced ty 3- methylcholanthrene and other polycyclic hydrocarbons (e.g. ENF). Its 50 University of Ghana http://ugspace.ug.edu.gh reduced cytochrome-CO cctiplex absorbs maxijnally at 448 nm. CYP of the HXF- injected mice (BIBM and BIWM) refers to P-448. FIG. 3.1 CALIBRATION CURVE FOR PROTEIN DETERMINATION The CYP contents of the microscmal preparations were calculated using an extinction coefficient of 91 irW1 cm-1 for the wavelength couple 450 (or 448) - 490 nm with dithionite as reducing agent. The results are shewn in Fig. 3.3. As shown in Fig. 3.3, administration of HSF to the mice resulted in a greater than 2 fold elevation of the CYP content in both strains of 51 University of Ghana http://ugspace.ug.edu.gh mice. Hie non-induced CYP values in both strains of mice were comparable. There appeared to be a higher induction of CYP in the black mice. FIG. 3.2 TOTAL LIVER MICROSOMAL PROTEIN B IW MN IBM B IB M N IW M NIBM - Non-induced black mice BIBM - BNF-injected black mice NIWM - Non-induced white mice BIWM - BNF-injected white mice 52 University of Ghana http://ugspace.ug.edu.gh FIG. 3.3 CYP AND P-420 CONTENTS OF MICROSOMAL PREPARATIONS NIBM - Non-induced black mice BIBM - BNF - injected black m ice NIWM - Non-induced white mice BIWM - BNF-injected white mice WWRL - White wistar rat (liver) WWRK - White wistar rat (kidney) The amount of cytochrome P-420 present in the microscrral preparations was also determined using an extinction coefficient of 110 irtyr1 cm"1 for the wavelength couple 420 - 490 nm. (See Appendix C for sairple calculation) . The difference spectra of the dithionite reduced CYP-00 ccnplex of the different microscrral preparations are shown in Fig. 3.4. There was a lower 53 University of Ghana http://ugspace.ug.edu.gh FIG. 3.4 CO-DIFFERENCE SPECTRA OF MICROSOMAL PREPARATIONS USING SODIUM DITHIONITE AS REDUCING AGENT. A :NIBML; B :BIBML; C :NIWML; D :BIWML; E:WWRL; F:WWRK 54 University of Ghana http://ugspace.ug.edu.gh concentrations of CYP in the kidney as carpared to that in the liver of the white Wistar rats. 3 . 3 EROD ACTIVITY The excitation and enission maxima for the resorufin used were found to be 560 nm and 583 nm respectively. An enission scan is shewn in Fig. 3.5. There was no response (S=0, TSble 3.2) in the ERCD assay when microsomal preparations frcm the non-induced mice, both black C57BL/ks J dbm and v^ hite EDY strains were used. Characteristic tracings are shewn in Fig. 3.6 (Panels A&B) . The tracings of the ERCD reaction using the microsaral preparations from the ENF-injected mice are shown in Fig. 3.6 (Panels C&D) . These shewed an obvious increase in the amount of resorufin formad in the cuvettes, suggestive of a good enzyme activity. Tkble 3.2 Values obtained frcm EROD assay using microsomal preparation from (A) black C57BL/ks J dbm mice, and (B) white DDY mice Microsaral preparation t (min) S (nm) R (nm) Specific activity (pmol/min/mg. protein) A: NIEM 1.1 0 27 0 BIEM 1.1 15 27..5 1441.3 B: NIVM 2.05 0 37 0 BBM 1.95 23 40 1117.5 S = change in fluorescence due to the reaction; R = change in fluorescence due to the working standard; t = duration (time) of reaction. 55 University of Ghana http://ugspace.ug.edu.gh 5 0 0 FIG. 3.5 5 5 0 £ 0 0 WAVELENGTH EMISSION SCAN OF & 5 0 n m RESORUFIN 56 University of Ghana http://ugspace.ug.edu.gh RE L. FL UO RE SC EN CE IN TE NS IT Y FIG. 3.6 FLUORESCENCE (EROD) RESPONSE OF A:NIBM; C :BIBM; D-.BIWM MICROSOMAL PREPARATIONS B :NIWM; 57 University of Ghana http://ugspace.ug.edu.gh 3.3.1 Effect of Flash Chromatography fractions of nBF Characteristic tracings of scire flash chrcrratography fractions of nBF on the ERCD activity of the microscmal preparations are shewn in Fig. 3.7. Fig. 3.8 shows the percentage inhibition of ERCD activity by the flash chromatograph/ fractions of nBF. As can be seen in the figure, the inhibition by both DAFl and DAF2 were bipbasic for both BIWM and BIBM. The overall % inhibition was greater for DAF1 as ccnpared to DAF2 even though concentrations of DAF2 were higher. Also the effect of DAFl and DAF2 cn the inhibition of ERCD activity in BIWM was greater than in BIBM. For DAF3, the % inhibition increased with increasing concentration for both BIM and BIWyl even though the concentrations were lewer compared to those of DAFl and DAF2. These inhibitions were greater than those of DAFl and DAF2. 3.3.2 Effect of salsolinol Characteristic tracings of the effect of salsolinol on the EROD activity of the microscmal preparations are shown in Fig. 3.9A. Fig. 3.9B shews the percentage inhibition of EECD activity b/ the different concentrations of salsolinol. As shown in the figure, inhibition increased with increasing concentration of salsolinol. At the lower concentrations inhibition was greater in BIW4 but as the concentration increased, that of BIEM became greater. 58 University of Ghana http://ugspace.ug.edu.gh RE L. FL UO RE SC EN CE IH TE NS IT Y FIG. 3.7 EFFECT OF DAFl (0.073%) AND DAF2 (0.11%) ON THE FLUORESCENCE (EROD) RESPONSE OF A:BIBM (DAFl); B (DAFl); C :BIBM (DAF2); D:BIWM (DAF2) MICROSOMAL PREPARATIONS : BIWM 59 University of Ghana http://ugspace.ug.edu.gh FIG. 3.8 PERCENTAGE INHIBITION OF EROD ACTIVITY BY DIFFERENT CONCENTRATIONS OF (A) DAFI, (B) DAF2 AND (C) DAF3 HBIBM □ BIWM 0.025 0.05 0.073 DAFI CONCENTRATION (%) □ BIBM E3 BIWM 0.0S7 0.11 0.162 DAF2 CONCENTRATION (%) □ BIBM ■ BIWM 0.019 0.02a 0.031 0AF3 CONCENTRATION (%) BIBM - BNF - Injected black mice BIWM - BNF - Injected white mice 60 University of Ghana http://ugspace.ug.edu.gh RE L. FL UO RE SC EN CE IN TE NS IT Y FIG. 3.9A EFFECT OF SALSOLINOL (2.38mM) ON THE FLUORESCENCE (EROD) RESPONSE OF A:BIBM; B:BIWM 61 University of Ghana http://ugspace.ug.edu.gh FIG. 3.9B PERCENTAGE INHIBITION OF EROD ACTIVITY BY DIFFERENT CONCENTRATIONS OF SALSOLINOL BBIBM BBIWM 1.22 2.38 3 .49 4.55 SALSOLINOL CONCENTRATION (mM) BIBM - BNF-injected black mice BIWM - BNF-injected white mice 3.4 REDUCTION OF CYTOCHROME C Ibe addition of a reducing agent to an oxidized solution of cytochrcme c leads to reduction of the cytochrcme with a characteristic peak at 550 nm. Fig. 3.10 (F&nel A) shews the spectra of the oxidized and reduced forms c£ cytochrcme c, with characteristic bands at 408 and 530 nm for the oxidized 62 University of Ghana http://ugspace.ug.edu.gh FIG. 3.10 SPECTRA OF OXIDIZED ( ---- ) AND REDUCED (---- ) CYTOCHROME C USING A:SATURATED SODIUM DITHIONITE (100^1) AND B:1.43mM SALSOLINOL AS REDUCING AGENTS 63 University of Ghana http://ugspace.ug.edu.gh (A) and 415, 520, and 550 nm for the reduced (B) form. The reducing agent used was saturated sodium dithionite (100|il). Fig. 3.10 (Panel B) shews the spectra of the oxidized and the reduced forms of cytochrare c, using 1.43irM salsolinol as the reducing agent. The a-band at 550 nm, is characteristic of reduced cytochrome c. 3.4.1 Effect of nBF At loxer nBF concentrations (0.006% and 0.012%) the oxidized solutions of cytochrare c did not show any reduction. However, at higher concentrations of 0.02 and 0.17%, there was a reduction. Fig. 3.11 shows the spectra of the reductions. At the lewer concentrations, the prominent 550 nm peak peculiar to the reduced form of cytochrare c is clearly missing. 3.4.2 Effect of Flash Chromatography fractions of nBF With the exception of Fig. 3.12 (Panels A&B) vdien 0.015% and 0.022% of DAFI were used, all the other fractions did not show any reduction when added to the oxidized cytochrare c. A characteristic spectrum of cytochrare c before and after addition of DAF3 (0.018%) ( one of the fractions which did not give any reduction), is shewn in Fig. 3.12 (Panel C) . 64 University of Ghana http://ugspace.ug.edu.gh lua o i £ Q d)w u u Q ^ W 01 2 ® — 0\° CN rH O h: INCREASING CONCENTRATION OF REDUCTANT 68 University of Ghana http://ugspace.ug.edu.gh FIG. 3.14A CO-DIFFERENCE SPECTRA OF MICROSOMAL PREPARATIONS FROM NIBM (a) AFTER AND (b) BEFORE A:0.002%; B:0.004%; C:0.011% nBF ADDITION 69 University of Ghana http://ugspace.ug.edu.gh FIG. 3.14B CO-DIFFERENCE SPECTRA OF MICROSOMAL PREPARATIONS FROM BIBM (a) AFTER AND (b) BEFORE A:0.011%; B:0.04% nBF ADDITION 70 University of Ghana http://ugspace.ug.edu.gh FIG. 3.15A LEVELS OF (A) CYP AND (B) P-420 IN NIBM LIVER MICROSOMES BEFORE AND AFTER nBF ADDITION ^ before Matter 0.25 in O zo o CL >-o 0.002 0.004 0.011 nBF CONCENTRATION (%) H before Rafter 0.002 0.004 0.011 nBF CONCENTRATION (%) University of Ghana http://ugspace.ug.edu.gh FIG. 3.15B LEVELS OF (A) CYP AND (B) P-420 IN BIBM LIVER MICROSOMES BEFORE AND AFTER nBF ADDITION IS before [El after 0.011 0.04 0.11 nBF CONCENTRATION (%) 72 University of Ghana http://ugspace.ug.edu.gh The fold increases of CYP and P-420 contents of the liver microsaral preparations after nBF addition are shewn in Fig. 3.16. In both the NEEM and RTFM, the levels of CYP increased at Iomst concentrations of nBF mare than P-420. But at a higher concentration, the P-420 level v*as greater than CYP in BIBM, whereas in NIBM they were almost the same. Fig. 3.17 shows the difference spectra of the microsaral preparations before and after the addition of 0.57irM salsolinol. The levels of CYP and P-420 before and after addition of salsolinol to the microsaral preparations are shown in Fig. 3.18. CYP levels increased approximately ty 2-fold and 4.5-fold, whereas P-420 levels decreased ty 0.2-fold and 0.4- fold in NIEM and BIQ4 respectively. Unlike the nBF effect, the P-420 levels decreased with the addition of salsolinol. In another set of experiments, the nBF was pre-incubated with the microsanes before NADPH addition. Immediately after the addition of NADFH, the rate of reduction of CYP was followed ty recording the change in absorbance of the CYP-CO corplex at 450 nm for 25 minutes at 5 minutes interval. The CYP and P-420 contents of the microsaral preparations were determined only once after this period. TSble 3.3 shows the CYP and P-420 levels determined for these pre-incubation experiments. 73 University of Ghana http://ugspace.ug.edu.gh FIG. 3.16 FOLD INCREASES IN CYP AND P-420 LEVELS IN (A) NIBM AND (B) BIBM LIVER MICROSOMES AFTER nBF ADDITION LU O_l O l 0.002 0.004 0.011 nBF CONCENTRATION (%) ■ CYP S P-420 SCYP HP-420 0.011 0.04 nBF CONCENTRATION (%) 74 University of Ghana http://ugspace.ug.edu.gh FIG. 3 17 CO-DIFFERENCE SPECTRA OF MICROSOMAL PREPARATIONS ' FROM A : NIBM AND B : BIBM (a) after AND (b) BEFORE 0.57mM SALSOLINOL ADDITION 75 University of Ghana http://ugspace.ug.edu.gh FIG. 3.18 EFFECT OF SALSOLINOL (0.57mM) ON THE LEVELS OF CYP AND P-420 IN (A) NIBM AND (B) BIBM LIVER MICROSOMES B before E after P-420 0.35 0 before Blatter P-420 76 University of Ghana http://ugspace.ug.edu.gh Table 3.3 Effect of nBF on CYP and P-420 levels in mice liver microscmes preincubated with nBF Sairple Concentration of nBF CYP (nmol mg'1 protein) P-420 (nrrol mg'1 protein) A:NIBM 0.0% 0.242 0.191 0.004% 0.247 0.300 0.008% 0.275 0.295 0.011% 0.341 0.327 0.02% 0.450 0.500 B:BIEM 0.0% 0.929 0.295 0.008% 0.970 0.459 0.011% 0.857 0.486 0.20% 1.044 0.573 0.0% nBF irrplies the addition of only NADPH Fig. 3.19 shows the tine-dependent reduction of CYP in NIEM and BTB4 at differ ait concentrations of the nBF. For NIHtf, the rate of reduction increased with increasing concentration of nBF using NADEH as the reference level. In the case of BIHyI, a similar pattern was observed but these increases were lower than the NADPH (reference level) except at 0.20% which reduced CYP to almost the same level as NADPH. Fig. 3.20 & 3.21 show that pre-incubation increased the levels of CYP and ’ P-420 in both NIHYI and BIEM. Pre-incubation increased the P-420 levels clearly only in microsomes from BIEM. 77 University of Ghana http://ugspace.ug.edu.gh FIG. 3.19 TIME-DEPENDENT REDUCTION OF (A) NIBM AND (B) BIBM LIVER MICROSOMES BY DIFFERENT CONCENTRATIONS OF nBF (%) AND NADPH 0 5 10 15 20 25 TIME (SECS) 0.2 0.18 | 0.16 ^ 0.14 < 0.12 ill jg 0.1 < 0.08 o 0.06 co ffl 0.04 < 0.02 0 w c o a > O i - C M « ^ - w T - T - i - C M C M C M C g C M C M TIME (SECS) University of Ghana http://ugspace.ug.edu.gh FIG. 3.20 EFFECT OF PRE-INCUBATION WITH nBF ON CYP LEVELS IN (A) NIBM AND (B) BIBM LIVER MICROSOMES 0.4 0.35 0. 0.25 0 0.1 0.1 0.05 0 0.004 0.011 nBF CONCENTRATION (%) 0.011 nBF CONCENTRATION (%) np - no pre-incubation p - pre-incubation 79 University of Ghana http://ugspace.ug.edu.gh FIG. 3.21 EFFECT OF PRE-INCUBATION WITH nBF ON P-420 LEVELS IN (A) NIBM AND (B) BIBM LIVER MICROSOMES 0 . 5 0 . 4 5 ­ 0 . 4 » 0 . 3 5 0 . 2 5 0 . 0 5 0 . 0 0 4 0 . 0 1 1 nBF CONCENTRATION (%) 0.6 0 . 5 - o> E 0 . 4 0 . 3 - oM ? 0.2 0.1 0 np - no pre-incubation p - pre-incubation 0.011 nBF CONCENTRATION (%) 80 University of Ghana http://ugspace.ug.edu.gh 3.6 REDUCTASE ACTIVITY The cytochrcme c (P-450) reductase activity of microscmal preparations were calculated using the absorbance change at 550 rm per minute (over a 3 minutes period). Fig. 3.22 shows the change in absorbance with time for the reductase activity of both NlWtf (non-induced white mice) and BlWtf (ENF- injected white mice) . FIG. 3.22 CYP REDUCTASE ACTIVITIES OF LIVER MICROSOMAL PREPARATIONS 0.7 0.6 E S 0.5 LO H ** 0.4 LU o < 0.3 m ° 0.2 m < 0.1 0 0 30 60 90 120 150 TIME (SECS) NIWM - Non-induced white mice BIWM - BNF-injected white mice 81 University of Ghana http://ugspace.ug.edu.gh Tables 3.4 shows the specific reductase activities and the effect of nBF cn the reductase activities. The closeness of the reductase activities obtained fa r both NIKM and EEWM as indicated in Fig. 3.22 was expected since ENF and other polycyclic h y d r o c a r b o n s increase P-448 but not levels of P-450, NADFH cytochrare c reductase, or the rate of P-450 reduction (Smith and Davies, 1980). Table 3.4 Effect of nBF on NADPH-cytochrcme c (P-450) reductase activities of microsaral preparations Sanple Concentration Reductase activity % Inhibition of nBF (nmol/min/mg protein) A:NIV'M 0.00% 26.32 _ 0.004% 25.13 4.50 0.04% 23.52 10.63 0.40% 17.61 33.09 BiBBM 0.00% 27.92 - 0.004% 27.18 2.70 0.04% 25.68 8.02 0.40% 17.45 37.50 3.7 BINDING STUDIES The UV-VIS spectrum of 7-ER and resorufin are shown in Fig. 3.23. Note the absorption maximum at 482 and 572 nm respectively. Fig. 3.24 shows the spectra of resorufin formed and the effect of varying concentrations of nBF cn the resorufin formed. At higher concentration of 0.083%, nBF slightly distorted the resorufin spectrum at wavelengths lower and higher around 572 82 University of Ghana http://ugspace.ug.edu.gh FIG. 3.23 UV-VIS SPECTRA OF RESORUFIN (---- ) AND 7- ETHOXYRESORUFIN (---- ) S3 University of Ghana http://ugspace.ug.edu.gh AB SO RB AN CE j FIG. 3.24 SPECTRA OF (a) RESORUFIN FORMED AND (b) nBF ADDITION TO THE RESORUFIN FORMED [A:0.017%; B:0.083 nBF] 84 University of Ghana http://ugspace.ug.edu.gh nm, though the 572 nm peak itself was unaffected. At a lower concentration of 0.017% the spectrum was about the same as that without nBF. In both cases, there was an increase in absorbance. Based cn these results, nBF was pre-incubated with 7-ER for 3-5 minutes before NADFH addition. In these experiments the prominent 572 nm peak characteristic of resorufin and indicative of product formation, diminished with increasing concentration of nBF (Fig. 3.25) . The spectrum of oxidized CYP solution gave a peak at 409-412 nm. This is not the sane as 414-418 nm reported in the literature (Levin et a l . , 1974). The reported values in the literature are hcwever for partially purified CYP. Increasing nBF concentrations altered the spectrum of the oxidized CYP (Fig. 3.26). The altered spectrum of oxidized CYP in the absence of substrate indicates an interaction between CYP and the plant extract. The addition of substrate (7-ER) altered the spectrum of the oxidized microscmes only slightly (Fig. 3.27a and b) . Hcwever addition of nBF to the CYP-substrate ccrrplex changed the spectrum greatly only at the higher concentration of nBF (Fig. 3.27) . The effect of nBF when added before addition of substrate (7-ER) is shown in Fig. 3.28. Addition of nBF ccnpletely changed the spectrum of the microsaral pr^aratian as observed before (Fig. 3.26). At a lower concentration of nBF (0.017%), there was only a slight modification in the spectra vten nBF was added before and after substrate addition (Fig. 3.27 [Panel B] & 3.28 [Panel A]). 85 University of Ghana http://ugspace.ug.edu.gh ra 9 « £^ W r- 1/1 £ o 2O H Eh a- (fl * [ l 2 n o fl E SSI Ow o OSu cs b o0 w 2 i 1 o H SB pu Eh CO W m c n C5 H Ck § a £ <,* r~W tH (X ' O o E u ••« a 86 University of Ghana http://ugspace.ug.edu.gh f e p l : i I! I1 | ! i i / jTTiif :'1 • \ p 7i li ! • ' ; ? ' 1 ! :i !/ i • 1 |hi • liiil i |; i: • l r n UNI 5 i 1 I I I Mil . itiiiilLU M ||! i I ■ r Jill : t l ' I i rHf !i l i i'll!ilil - LU 3 D H ? a a o s a v i - ! 11 ■ I I I I , ' !11 ijj ! • ! • - 1 W : ! ill.. lA u ill ! I i ! I I ' i l l •! : i ! ! ' I ' I i liiil. a o H v g H o s e v I i • i ’l i i ! & tttttt LUi fir nj ill i ' i l i l l l i i i l l a o H v a a o s a v "ir , £- O Z w J w > <3 -Qj. ju»| J i i : x i h O z w -3 W > < I ' « TTT| x H O z w W t> 4 < r—I 0\° 6 LT) \ CN tJ) rH LO ° 2 n CO • ' W cN a ^ o ’H M o s ? : H < E P X H H tSl H s * H CO o g o — CO (0 o , ° 2 fc H O o 2 H Q H H Q$^ N U H W Q » l H f c w o i w Eh — 0\° D XI ci J m O ■ § ^ ° vo CN o H [H 87 University of Ghana http://ugspace.ug.edu.gh 83 FI G. 3. 27 AB SO LU TE SP EC TR A OF (a ) OX ID IZ ED MI CR OS OM ES (2 5n g/ ml ) AN D (b ) OX ID IZ ED MI CR OS OM ES AN D ET HO XY RE SO RU FI N (2 uM ), (c ) OX ID IZ ED MI CR OS OM ES , ET HO XY RE SO RU FI N, AN D WA TE R OR nB F (A :W AT ER , B : 0. 01 7% ; C: 0. 17 %) AD DI T I O N University of Ghana http://ugspace.ug.edu.gh FTfi 3 28 ABSOLUTE SPECTRA OF (a) OXIDIZED MICROSOMES (25|xg/ml) ' (b) OXIDIZED MICROSOMES (25^g/ml) WITH nBF AND 7-ETHOXYRESORUFIN (2|iM) ADDITION [A: 0.017%; B: 0.0 8 3% nBF] © University of Ghana http://ugspace.ug.edu.gh However at higher concentrations of nBF (Fig. 3.27 [Panel C] & 3.28 [PanelB]) the final spectra were the same, indicating that the effect of nBF is concentration-dependent. 90 University of Ghana http://ugspace.ug.edu.gh CHAPTER POOR DISCUSSION The design of the experiments was to obtain both the inducible and constitutive forms of CYP and to investigate the mode of action of nBF in inhibiting the NADPH-dependent cytochrome P-450 reactions. This was to determine vhether a particular form of CYP was responsible for the inhibitions. The protein concentrations and spectral studies show that the two forms of CYP used were different. S-naphthoflavone (BNF) administration increased the total liver protein content. Within 4 days after administration of BNF, there was a 17.8% and 21% increase in liver protein content for the EDY and C57 EL/ksJ respectively. There was also an increase in the amount of protein per gramme of vet weight liver of both strains of mice (Fig. 3.2). The greater than 2 fold elevation of CYP ccntent (Fig. 3.3) in both strains is indicative of the induction of the CYP monooxygenase system hy S-naphthoflavone. Carbon monoxide interacted with reduced microscmal hsnoprotein obtained from BNF as expected, but the maximum absorption of this catplex was found at 448 nm instead of at 450 nm, which is the maximum for the herrcprotein frcm microscmes of normal mice (Alvares et a l . , 1967, Hilderbrandt et a l . , 1968) . When reduced in the presence of CO, the microsanal preparations had 91 University of Ghana http://ugspace.ug.edu.gh a maximum at 450 rm (448 rm for the BNF-injected mice), with a small peak around 423 nm (Fig. 3.4 [E&nels A&B]) . Indeed, a peak at 423 rm has been observed in all preparations of partially purified microscmal CYP described to date (Levin e t a l . , 1972; Lu and Coen, 1968; Miyake et a l . , 1968), and has been the subject of considerable controversy. The shoulder near 423 rm in the CO-reduced spectra is due to a small amount of cytochrome P-420. Cytochrane P-420 (P-420), the solubilized form of CYP was present in all the microscmal preparations, with a proportionate increase in the CYP contents (Fig. 3.3) of the 2 strains of mice (after S-naphthoflavone injection). .Determination of the P-420 concentration in the final microsaral fraction, ty the method of Qrura and Sato (1964) using the GO-difference spectrum (Fig. 3.4) reveals that 21-29% of the CYP concentration is present as P- 420. The 03 difference spectrum of P-420 is very similar to that c£ hemoglobin (Qmura and Sato, 1964) . Sane of the absorbance at 420 nm nay be attributed to contamination ty blood. Moreover the high P-420 contents could be due to the non-purified nature of the CYP. In the case of the WtfRK, the P-420 content was even greater than the CYP content. This can be seen in the OO-difference spectrum of the dithionite reduced CYP-CO ccnplex in Fig. 3.4 [E&nel F]. Unlike the other 00- difference spectra in Fig. 3.4, the 423 nm shoulder is higher than the 450 nm peak. In microscmal fraction fron rat kidney cortex, the concentration University of Ghana http://ugspace.ug.edu.gh of 00-binding herroprotein estimated cn the basis of absorbance of the CD ccrrplex; is only 10% to 30% (Kato, 1966) of that found in liver microscmes.. As shewn in Fig. 3.3, the CYP content of WBK is approximately a third of that found in VMRL. The ERCD assays were performed to confirm the inhibition of the NADEH- dependait cytochrcme P-450 reactions. iS-naphthoflavone (ENF) is known to induce the CYPIA1 isozyme specific for the arcmatic ring hydroxylation, and hence the ERCD assay. Liver microscmes fron mice injected with HSF, deethylated ethoxyresoruf in to resorufin. The non-responsiveness of microscmes fron the non-induced mice to increase in the amount of resorufin formed observed with the HSF-injected mice (Fig. 3.6) strongly suggests that different isozymes of CYP are induced under different conditions. Extract of nEF of D. adscendens has been shewn to inhibit deethylase activity in EROD assays (Brookman-Amissah, 1994) . The flash chromatography fractions (DAFl, 2 and 3) of D. adscendens were found to inhibit deethylase activity in the ERCD assays using microsanes frcm the HSIF-injected black and white mice. An interesting pattern of inhibition was observed for the ERCD activities vhen DAFl and DAF2 were added to the reaction mixtures (Fig. 3.8). The inhibition was biphasic, with this type of inhibition being pronounced in the black mice. DAF3 inhibited the ERCD activities c£ SB University of Ghana http://ugspace.ug.edu.gh both the black and white mice with increasing concentration. Ihe % inhibition was greater in El'IF—injected white mice than the black mice. The different effects of the flash chromatography fractions cn the black and white mice could be due to genetic differences. The polarity of the solvents used to flash out the nBF of D. adscendens increased frcm DAFI to DAF5. Thus the varying effects of these fractions cn the ERCD activities of the black and white mice could also be due to different ratios of the ccnponents of the plant extract in a particular fraction. This was the reason vihy the effects of DAFI, 2 and 3 were studied alongside the crude nBF. DAF4 and 5 were not used because of the lew activity r^iorted for them in a work done ty AdcV in 1989, cn the 1 effect of the fractions of D. adscendens on the inhibition of smooth muscle contraction. The investigation of the effect of nBF and its flash chrcrratograply fractions cn cytochrome c was carried out based cn a previous stud/ in v\hidh cytochrare c was found to be directly reduced ty salsolinol (Kamassah, 1992) . Addition of varying concentrations of DAF2 - 5 to an oxidized solution of cytochrome c did not show any reduction. DAFI however showed a reduction (Fig. 3.12 [Panels A&B]) . Fran the polarity of the solvents used, DAFI was the least polar fraction and is likely to contain salsoline and therefore EftFl could possibly contain the reductant purported to be responsible for the reducing ability of the plant extract. 94 University of Ghana http://ugspace.ug.edu.gh The nEF did not show arc/ reduction at lcwer concentrations, tut did so at higher concentrations (Fig. 3.11). The inability of the lcv»er concentrations of nBF of the crude extract of D. adscendens to reduce cytochrome c directly could be due to a srraller amount of DAFl present. Ihe interaction of various ccrrpounds with the P-450 emymes has been shewn to result in increased P-450 functions including P-450 reduction, and iranoaxygenase and oxidase activities (Vfeng et a l . , 1993). The nBF of the crude extract of D. adscendens under investigation has been known as an inhibitor of P-450 NADFH-dependent AA metabolism (Adc^ and Schwartzman, 1992). The purpose of the present stud/ was to investigate this, that is, the mechanism(s) underlying the inhibition of the NADPH-dependent CYP reactions. In an earlier stud/, cytochrcnne c was reduced ty salsolinol and GSH (Kartassah, 1992). In that study, salsolinol reduced cytochrome c with cptimal concentration in the millimolar range. The reduction ty GSH and salsolinol were about the same, indicating that, the/ both have the sane redox potentials. It was inferred from these results that the mode of action of salsolinol, and hence salsoline in the plant, was to reduce CYP and keep it in the reduced state, interfering with the first step (Fig. 1.2) where the ferric ion binds the substrate and is reduced to ferrous icn ty the electron flew of NADPH. 95 University of Ghana http://ugspace.ug.edu.gh The present stud/ in which direct redaction of CYP b/ nBF, salsolinol and GSH was investigated revealed that nBF did not reduce CYP directly. Salsolinol and GSH which reduced cytochrcrre c, also did not reduce CYP (Fig. 3.13). This is evidenced b/ the absence of the 450 rm band or peak observed when 00 was bubbled through a suspension of microscmes containing these ccrrpounds (GSH, salsolinol and nBF). This does not however preclude the presence of a reductant in the nBF of D. adscendens since salsolinol and GSH (both kncwn reductants) did not also reduce CYP directly. In these experiments for the direct reduction of CYP, when nBF was added to microsanal preparation and CD was bubbled through, there was a maximum absorbance at 420 nm (Fig. 3.13 [Panel C]), irrplying that the cytochrane is being degraded. Whai GSH and salsolinol were used, the same effect was observed. Considering the fact that GSH naturally occurs in the cell and takes part in axidatian-reduction reactions, the presence of nBF which acted like GSH in this case could also be in the cell and not be harmful to the organism. nBF could ccntain a type of reductant which interferes with the flow cf electrons fron NADEH to CYP. Because of the inability of nBF and salsolinol to reduce CYP directly, the reduction was followed ty an indirect method where CYP was reduced in the presence of NADPH (the natural intracellular reducing agent which donates electrons to CYP) . 96 University of Ghana http://ugspace.ug.edu.gh The rationale behind these investigations was to find out hew nBF vraold interact with CYP in the presence of NADEH. The levels of CYP and P-420 were determined after NADPH was added, prior to the addition of nBF. This v b s to allow for the estimation of the amount of CYP reduced ty NADPH. After the 25 minutes period during which the reduction was allowed to proceed, the concentrations of the two cytochromes were determined. With the exception of CYP from the non-induced mice, increasing concentration of nBF increased both CYP and P-420 levels of the 2 strains of mice (Fig. 3.15A&B) . The increased P-420 levels could be due to the modification of the hemqprotein v\hen in the reduced state, leading to the formation of P-420. The conversion of P-450 to P-420 induced ly neutral • salts has been shewn to proceed more rapidly in the reduced form of the hsncprotein than in the oxidized form (Imai and Sato, 1967) . The incrsrent ves more pronounced in BNF-induced black mice (Fig. 3.15B(B)), suggesting that the CYPIA1 isozyme is more susceptible to nBF reduction in the presence of NADPH. However, the denaturation to P-420 was also greater in BNF-induced black mice. The lesser effect of nBF cn the constitutive CYP is understandable, considering the fact that it would be dangerous to have a constitutive enzyme being affected ty substances taken into the bod/. The absorption spectrum of r®1 showed an increase in absorbance frcm a longer to a shorter wavelength (i.e. 450 nm to 420 nm) (results not shewn) . Thus, the high P-420 contents observed when higher concentrations of riEF 97 University of Ghana http://ugspace.ug.edu.gh were added could be as a result of absorbances due to the nBF itself at 420 nm. The fold increases in CYP and P-420 levels show that CYP levels increased more than those of P-420 in both non-induced black mice and BNF-mduced black mice except at high concentrations of nEF (Fig. 3.16). As stated earlier, the high concentrations of ri®1 could be having deleterious effect ai the CYP systan, taking into account the fact that reduced CYP is more susceptible to degradation. For ccnparison, salsolinol (0.57itM) was used in place of nBF. In both NUM and BTFM there was an increase in CYP with a corresponding decrease in P- 420 levels (Fig. 3.18). Like the nBF, the proportion of increment in CYP was greater for the ENF-induced black mice than the non-induced black mice at the same salsolinol concentration. The action of salsolinol on the fold changes in CYP and P-420 contents suggests a reductive role for this catpound with increasing CYP levels. The results, (decreasing P-420 levels) also inplies that the CYP is not being reduced to P-420, suggesting , that the high P-420 levels with high concentration of nBF is due to the high absorbances of nBF at the lower wavelengths. In one set of experiments, the NADPH was added before nBF addition. It was therefore possible that NADPH could have reduced most of the CYP before nEF was added, thus rendering the effect of the nBF cn the reduction of CYP 96 University of Ghana http://ugspace.ug.edu.gh negligible. An attanpt was therefore made to pre-incubate the microscmal preparations with nBF for 2 minutes before NADEH addition. Fig. 3.19 shews a tune-dependent reduction of CYP after NADEH was added, following the 2 minutes incubation with nBF. For both the non-induced black mice and BNF- induced black mice, the rate of reduction was proportionate with increasing concentration of the plant extract (nBF). A higher concentration of nBF vas needed to reduce the CYP in the BNF-induced mice more than NADEH alone did. Thus it is possible that, high extract concentrations are needed (during pre-incubaticn) to reduce the induced CYP of the BNF-induced mice effectively. Pre-incubation with nBF did, hcwever, have a greater effect on reduction c£ CYP fcy NADEH. At the same concentrations of nBF, pre-incubation as opposed to no pre-incubation increased the levels of CYP (Fig. 3.20). The levels of P-420 also increased (except at a higher concentration of 0.011% for non-induced black mice, Fig. 3.21A) but that of CYP was more significant. Thus pre-incubaticn of microsanal preparations with nH1, before NADEH addition enhances positive effect on reduction of CYP by NADEH. Since nBF did not reduce CYP directly, and also the synergistic effect of nBF and NADPH on the reduction of CYP was not pronounced, the proposal that the plant extract kagps CYP in the reduced state, and thereby interfering with the first step (Fig. 1.2), where the ferric icn binds the substrates, and thus prevents the binding of the substrate to CYP, is not tenable. 99 University of Ghana http://ugspace.ug.edu.gh Hie ironooxygenase enzyrre system catprises CYP, NADPH-CYP reductase and a phospholipid. The NADFH-CYP reductase, which is a flavoprotein transfer the necessary reducing equivalents firm NADFH to CYP. In the reaction cycles shown in Figs 1.1 and 1.2 (step 2), the electrons frcm NADFH through the NADPH-CYP reductase reduce the CYP-substrate cctrplex to allow O2 binding. With the reducing property of nBF, it was ejected that the nBF would enhance CYP reductase activity in the presence of NADFH but this was not so. It rather inhibited the reductase activities (T&ble 3.4). The inhibition of NADFH-CYP reductase activity could be the possible node of action of nBF on the NADFH-dependent monooxygenase reactions. The extract could be inhibiting by interfering with the flow of electrons from NADPH to .CYP, the rate determining step. This would keep CYP in the oxidized state and therefore O2 cannot bind to the CYP-substrate ccnplex to form the ternary CYP-substrate-Q corplex. In coe set of experiments, the reductase reaction was initiated ty the addition of nBF in place of NADEH. Cytochrome c (P-450) was reduced but the rate was very minimal. This was expected since the extract has shewn reducing properties. Cytochrare c, the electron acceptor in this assay for the reductase, is an artificial one, not membrane-bound and therefore differs frcm CYP, the real acceptor. Its direct reduction ty nBF was investigated. As indicated ty the results, there vas a direct reduction of cytochrare c ty nEF (Fig. 100 University of Ghana http://ugspace.ug.edu.gh 3.11). Unis further shows that cytochrome c is different frcm CYP, which was not reduced directly ty nBF. CYP is different frcm cytochrarre c in that CYP is a b type cytochrcme with a protdhane or a related hsne (without fornyl group) as prosthetic group, not covalently bound to the protein; whereas cytochrcme c has covalent linkages between the hone side-chain and the protein. In the assay for the cytochrcme c (P-450) reductase activity, cytochrcme c was used as an artificial electron acceptor in the presence of CYP. In the presence of cytochrcme c it could be that nBF increases electron flew to . CYP (as already indicated ky irore CYP2* formation when nBF is pres ait), therefore reducing electron flew to cytochrcme c. The inhibition of reductase activity therefore is apparent but not real. With this inhibition, the flow of electrons to CYP is not affected. In the spectrqphotametric assay for the ERCD activity, the inhibition c£ the 572 nm peak formation characteristic of resorufin when nBF was pre­ incubated with the microsomal preparation and 7-ER, before co-factor (NADFH) addition confirms the inhibition of the ERCD assay ky nEF and suggests that the extract binds to CYP. However, once 7-ER is deethylated to resorufin, addition of the plant extract (nBF) did not have ary interaction which affected the formed product. This is indicated ky the presence of the 572 nm peak even after addition of higher concentrations of 101 University of Ghana http://ugspace.ug.edu.gh nBF (Fig. 3.24). Addition of nBF ccnpletely changed the spectrum of the microscmal preparation and subsequent addition of substrate (7-ER), affected the resultant spectrum only slightly. Catparing this to the spectrum obtained whai 7-ER was added first before nBF addition (tracing c, Fig. 3.27 and 3.28), the results show that cnce nBF binds CYP, the substrate binds to only a small portion of CYP, hence the slight change of spectrum. In another experiment vhen nBF and NADEH were added to the microsanal preparation before substrate addition, there was no spectrum of the microscmal preparations, nBF and substrate when scanned between 350 and 700 nm. The absorbance remained negative (results not shewn). This supports the proposal that nBF could contain the type of inhibitor acting to prevent donation of electrons frcm NADEH to CYP. The plant extract could also form a metabolic-intermediate "MI" corplex with CYP in the microsanal preparation. The "MI" ccrrplex cnce formed is stable, preventing further participation in the mixed function oxygenation or dealkylation as in the case of the ERCD reactions. The nBF could also bind to the substrate, thus making it unavailable to participate in the reaction. 7-ER did not shew a binding spectrum with 102 University of Ghana http://ugspace.ug.edu.gh oxidized CYP (Fig. 3.27). This does not however iirply that 7-ER does not bind CYP since scire substrates have beai shewn not to have binding spectra but are h/droxylatable (Imai and Sato, 1967a) . In the presence of nBF, the spectrum obtained for the oxidized microsames and the substrate together with nBF changed, indicating an interference with the subsequent steps far product fornmtian. In the absence of a substrate, nBF enhanced CYP2* formation vten NADffl uas present. The CYP2* ccnponent of CYP was further enhanced if the nBF mis pre-incubated with the microsomal preparation before NADPH addition. Thus nEF does not prevent reduction of CYP ty NADEH in the absence of a substrate. In the presence of nBF, the substrate could still bind CYP and form the CYP2+-substrate ccnplex. CYP2*, once formed can be determined ty observing a peak at 450 nm with CD binding. This could however, not be determined due to the unavailability of more CO at this stage of the work. If this CYP2+-substrate is formed and O2 binds to form the CYP2+-substrate-Q2 ternary ccnplex (TC), then nEF could be inhibiting ty preventing the breakdown of the TC to form products. Accurrulation of this ccnplex leads to its breakdown into substrate, Oj and CYP3* and the substrate therefore remains unmetabolized. 103 University of Ghana http://ugspace.ug.edu.gh In the related work ty Add/ and Schwartzrran (1992), during vtoch the formation of products in the monooxygenase pathway was inhibited ty nBF, the substrate (AA)^ which was added to the microsaral preparation containing CYP remained unmetabolized. In conclusion, the stucV indicated that CYP was not reduced directly ty nBF, though nBF reduced cytochrcme c directly. The NADPH-dependent reduction of CYP; in the absence of a substrate^was however enhanced by nBF. nBF is inhibiting the NADFH-dependent CYP reactions ty binding to CYP and preventing either substrate binding or the breakdown of the ternary complex to form products. 104 University of Ghana http://ugspace.ug.edu.gh BIBLIOGRAPHY Addy, M.E. (1989). Several chrcrnatographically distinct fractions of Desmodium adcendens inhibit smooth nuscle contractions. Int. J. Crude Drug Res. 27: (2) 81-91. Addy, M.E. and Schwartzman, M.L. (1992). An extract of Desmodium adscendens inhibits NADPH-dependent oxygenation of arachidonic acid ky kidney cortical microscmes. Phyto. Res. 6: 245-250. Add/, M.E. and Schwartzman M.L. (1995) . An extract of Desmodium adscendens activates cyclooxygenase and increase prostaglandins synthesis ky ram seminal vesicle microscmes. Phyto. Res. 9: 287-293. Adesnick, M., Bar-Nun, S., Maschio, F., Zunich, M., Lippnan, A. and Brad, E. (1981) . 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The involvement of cytochrcme P-450 in hepatic microscmal steroid tydrcoylation reactions supported ty sodium periodate, sodium chloride, and organic hydroperoxides. Eur. J. Biochan 61: 43-52. Ichihara, K., Kusunose, E. and Kusunose, M. (1971). A fatty acid