'iiESm LIBRARY Michigan State University This is to certify that the dissertation entitled INFLUENCE OF POLYHALOGENATED AROMATIC HYDROCARBONS ON THE INDUCTION, ACTIVITY, AND STABILIZATION OF CYTOCHROME P450 presented by Richard Voorman has been accepted towards fulfillment of the requirements for Ph .D . degree in Biochemistry W'W Major professor Date/[1:7 : 6am 7 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 IVISSI_J RETURNING MATERIALS: Place in book drop to usumas remove this checkout from “ your record. FINES will be charged if book is returned after the date stamped below. "'/7 ROH + NADP + H2O The activation energy barriers are formidable since the molecule of dioxygen must undergo heterolytic cleavage with 3 incorporation of one atom into the carbon-hydrogen bond, and - reduction of the other atom by two electrons to water. Electrons from NADPH are supplied to the enzyme via the membrane-bound flavoprotein, NADPH-cytochrome P450 reductase. Since the electron transport chain resides in the membrane, there is an essential phospholipid component in the interaction between the P450 enzyme and NADPH-P450-reductase, and electron transport. The term mixed-function oxidase is often applied to cytochrome P450, a reference to the reduction of oxygen to water (oxidase) and oxygenation of the substrate. A substantial problem of nomenclature has developed over the years, such that most names given the enzyme are improper. The enzyme is not a true cytochrome since its primary function involves substrate binding and oxygen activation rather than electron transfer. The wavelength description should include the heme a-band rather than the reduced CO absorption band in the Soret region; even the "450" is incorrect as this can change between 447 and 453 depending on the isozyme. The problem is further compounded in the naming of isozymes, which, according to rules of the Enzyme Commission, should be designated by electrophoretic mobility. Yet isozymes of cytochrome P450 all have Mr values in the range of 48-56 Kdal, and consequently some isozymes are indistinguishable by SDS-PAGE. It has been suggested that isozymes be designated by substrate and position of attack; 4 however most cytochrome P450 isozymes have variable substrate specificity and no known endogenous substrate. In this work the terminology of Ryan g3_§l. (1982) will be used in reference to rat liver microsomal cytochrome P450; in that system isozymes are simply designated by a letter suffix in alphabetical order of first isolation. Cytochrome P450a is a constitutive enzyme of rat liver, cytochrome P450b is induced by phenobarbital, cytochromes P450c and P450d are induced by methylcholanthrene. The number of isozymes by this system now extends to cytochrome P4501 (Ryan g£_§l., 1985). CHEMISTRY any sgaucruas or cvrocnaoug P450 The chemistry and structure of the active site of cytochrome P450 have been the focus of most structural investigations. Cytochrome P450 is a b-type cytochrome; that is, its heme consists of iron protoporphyrin-Ix bound rather loosely in the active site by a combination of hydrophobic and coulombic forces on the porphyrin ring and a coordinate- covalent bond to the penta- or hexacoordinate iron atom. The metal ion is held in place by four nitrogen ligands in the porphyrin ring and a fifth axial bond to a thiolate, probably from a cysteine in the polypeptide. A potential sixth axial coordination site above the plane of the porphyrin ring, the dioxygen binding site, may be either empty or filled with an occupying ligand such as water or a hydroxyl contributed by 5 the protein. Crystallographic data are lacking for most P450's since they are membrane-bound proteins and resist crystallization. However Poulos (1986) has recently carried out a structural analysis of the soluble camphor hydroxylating cytochrome P450 from Pseuggmonas putida to a resolution of 2.20 A. The substrate-free form shows a hexacoordinate iron with sulfur from Cys-357 on one axial ligand and water or hydroxide as the other axial ligand. The substrate pocket and heme appear to be buried deep inside the enzyme, accessible only by a channel, quite unlike the classic model of an enzyme with active site envisioned as a cleft or depression on the surface. In the substrate-free form the pocket is filled with hydrogen bonded solvent molecules. The substrate, camphor, displaces the solvent molecules and the sixth axial iron ligand and although it does not cause a significant conformational change in protein structure it does bring about large decreases in thermal motions of several regions, probably restricting access to the active site. Upon substrate binding, the heme is converted to a high spin state by displacement of the sixth ligand with concomitant redox potential shift from -300 mV to —173 mv, thus allowing for reduction of heme iron. It is likely that mammalian microsomal cytochrome P450's are structurally similar to the bacterial enzyme since the molecular weights are similar and the sequences share enough homology that they were likely derived from the same ancesteral gene. SUBSTRATES AND SUBSTRATE SPECIFICITY The definition of isozyme is used loosely in reference to substrates for cytochrome P450, since isozymes of one enzyme family are confined to one type of reaction and usually on the same substrate; for example the isozymes of lactate dehydrogenase. However the isozymes of cytochrome P450 carry out a variety of reactions on a large number of unrelated substrates. Steroid Hydroxvlases Among cytochrome P450 isozymes which metabolize steroid hormones, the stringency of substrate binding is very high and the reactions very specific. Waxman (1986) has isolated rat hepatic cholesterol 7a-hydroxylase which is highly specific for that reaction and appears the first and rate- limiting step in conversion of cholesterol into bile acids. Cytochrome P450 isozymes involved in steroidogenesis are found in both the mitochondria and endoplasmic reticulum of the adrenal gland. The mitochondrial electron transport system consists of the flavoprotein, NADPH-adrenodoxin reductase. and adrenodoxin, an iron-sulfur protein, both located loosely on the matrix side of the inner mitochondrial membrane, while the cytochrome P450 is membrane bound (Takemori and Kominami, 1984). Mitochondrial cytochrome P450scc catalyses side-chain-cleavage of cholesterol, the rate limiting step in steroidogenesis, to form the steroid 7 nucleus, pregnenolone. This in turn is metabolized in the endoplasmic reticulum by cytochrome P450 17a,lyase. Recently it has been shown that both 17a hydroxylase activity, leading to the formation of cortisol, and 17,20—lyase, which along with the former activity leads to the formation of androstenedione, both reside in the same cytochrome P450 (Zuber gg_§l., 1986). The regulation of these two activities remains to be elucidated. Cytochrome P450-11b, a mitochondrial enzyme, catalyzes the 11b~hydroxylation of either 11-deoxycorticosterone or 11-deoxycortisol leading to the formation of aldosterone and cortisol, respectively. Liver mitochondria contain a cytochrome P450 dependent on electrons from NADPH-ferredoxin reductase and ferridoxin, to catalyze the 26-hydroxylation of a bile acid intermediate in the formation of cholic acid (Atsuta and Okuda, 1977). Fatty Acid Hydroxylases Cytochrome P450 participates in the metabolism of fatty acids by carrying out w, w-1, and w-2 hydroxylation of medium and long chain fatty acids and some prostaglandins. Evidence for physiologic hydroxylation of fatty acids was presented by Das g£_gl. (1968) when they showed that w-hydroxylation of laurate by rat liver microsomes occurred with a Km of 30 uM. This activity also occurs in kidney cortex (Jakobsson §3_§l. 1970) and lung microsomes (Ichihara, 1969). Metabolism of prostaglandins occurs in liver, kidney cortex, adrenals, and 8 lung microsomes, albeit with species specificity in extrahepatic tissues. It is difficult to determine if these reactions are physiologically relevant. Kupfer gt_§l. (1979) showed that inducers of cytochrome P450 affected these reactions. Although the same group has shown that the position of hydroxylation is dependent on the particular .prostaglandin and P450 isozyme, the reaction appears to be relatively fortuitous and non-specific. ProstaglaLLLn 31 is hydroxylated by rabbit cytochrome P450 isozyme 6 (Holm gt al., 1985); however the Km is 140 uM and the Vmax is 2.1/min, not very sensitive for a hormone like compound. Aggggsegggnctions of Cytochrome P450 Although cytochrome P450 appears to function primarily as an oxygenase it can, under the proper conditions, function as a reductase. Certain compounds with highly oxidized substituents such as carbon tetrachloride or halothane can compete with oxygen for electrons from cytochrome P450 and undergo reductive metabolism by cytochrome P450. The facility with which a carbon halogen bond is reduced is dependent on the halogen substituent and the overall structure of the molecule. Carbon tetrachloride can undergo a one electron reduction with loss of chloride to yield the trichloromethyl radical (MacDonald, 1982). This in turn can initiate a radical chain reaction resulting in lipid peroxidation and, given enough substrate, severe liver damage. The anesthetic halothane, is normally metabolized 9 oxidatively by cytochrome P450. However under conditions of' extremely low oxygen partial pressure it can compete with oxygen and be reductively metabolized, resulting in severe membrane damage produced by the halothane-radical initiated chain reaction (Ahr et a1., 1982). Non—Mammalian Cytochrome P450 In plants, cytochrome P450 carries out reactions analogous to those of adrenal steroidogenesis. Murphy and West (1969) showed that the formation of kaurenol in the biosynthesis of gibberllin in cucumber seed endosperm was a cytochrome P450 dependent reaction. Although a detoxification mechanism based on cytochrome P450 might exist in plants, this seems unlikely since plants can simply sequester toxic compounds in vacuoles (Hendry, 1986). Recently, an unusual cytochrome P450 was isolated from Bacillus meggbacterigm (Narhi and Fulco, 1986). It is a soluble, catalytically self-sufficient single polypeptide (119 Kdal) which contains a mol each of FMN, FAD, and heme-- the reductase apparently resides on the same polypeptide as the cytochrome P450 (microsomal P450 is about 52 kdal and the reductase is 79 kdal). This enzyme has an extremely high activity for fatty acid hydroxylation, 4600/min for palmitate and is inducible by xenobiotics. Since it is soluble and catalytically self-sufficient, it will no doubt provide an excellent model for cytochrome P450 chemistry. 10 Two narrow-specificity cytochrome P450 isozymes have been isolated from yeast microsomes (Kappeli, 1986). Study of cytochrome P450 in yeasts could be useful, owing to the similarity with mammalian P450 (being microsomal) and the wealth of information on yeast genetics and biochemistry. Xenobiotic metabolism The most notable activity of cytochrome P450 is the metabolism of xenobiotics. In this capacity it is a protective mechanism, working in analogy to the immune system but on small non-polar molecules which would reside in lipid membranes and continue to exert a pharmacologic effect were it not for their metabolism by this enzyme. As such, cytochrome P450 hydroxylates the xenobiotic thus making it amenable to conjugation with glucuronide or sulfate followed by excretion of the water soluble conjugate. It is striking that, unlike most enzymes which are substrate-specific and operate only on one or a few substrates of structural similarity, cytochrome P450 can act on a number of structurally diverse substrates. Moreover, the enzymatic activity of certain isozymes is enhanced following administration of certain chemicals to the organism. This inducing activity may result in increased excretion of the agent or, in a few cases, enhanced toxicity of the inducer or other agents. The type and magnitude of induction response is dependent on the inducer and dose. 11 Hepatic metabolism of xenobiotics was initially studied from the standpoint of both drug inactivation, as in the metabolism of narcotic analgesics (Brodie, 1956), and activation of procarcinogens, as-in the demethylation of 3- methyl-4-monomethylaminoazobenzene to render it an hepatic carcinogen (Brown g£_§;» 1954). The latter work led to the discovery that treatment of animals with 3-methylcholanthrene resulted in increased demethylation and increased hepatocarcinogenicity. In a similar fashion it was discovered by Remmer (1961) and Conney and Burns (1959) that barbiturates could stimulate drug metabolism and could decrease hexobarbital sleeping time in animals. It is now known that hundreds of compounds are capable of inducing cytochrome P450 and being metabolized by it; however, unlike the immune system, cytochrome P450 copes with this variation by low substrate specificity rather than by highly specific gene prOducts. Early work suggested that there were two major forms of cytochrome P450 induced by the prototypic compounds phenobarbital and 3-methylcholanthrene, the latter compound induces enzyme accompanied by an absorption shift to 448 nm and is frequently referred to as cytochrome P448. We now know that cytochrome P450 is composed of many isozymes: phenobarbital induces primarily cytochromes P450 b and e (Ryan g£_§lq 1982), while 3-methylcholanthrene induces cytochrome P450c,d and perhaps a third form (Seidel and Shires, 1986). Cytochromes P450a,f,g, h, and i are 12 significantly expressed in untreated rats but their levels may rise or fall depending on the inducer (Ryan £1.31» 1985). Cytochrome P450h is specific to male rats while cytochrome P4501 appears to be specific to female rats. The discovery of inducers proceeded largely because they produce significant changes in the total specific content of cytochrome P450. However certain inducers, such as isoniazid which induces cytochrome P450j (Ryan g£_§l., 1985), induce synthesis of one isozyme without significantly altering the total specific content of cytochrome P450. The isozymic composition of cytochrome P450 in a microsomal sample can be differentiated by immunochemical, enzymatic, or spectrophotometric methods. The latter method .is least specific owing to small spectral differences between isozymes but is helpful in regard to certain isozymes (such as the high spin peak and isosafrole complex of cytochrome P450d) and in combination with other methods. Enzymatic charcterization is frequently and successfully used to determine isozyme composition of a microsomal sample. Although there are hundreds of substrates for cytochrome P450, some substrates are restricted to a particular isozyme. In other words, the turnover number for the substrate of one isozyme maybe an order of magnitude or more greater compared to other isozymes. In this regard a few substrates are of note. Benzphetamine, aminopyrine, and pentoxyresorufin are typical diagnostic substrates for dealkylase activity of cytochromes P450 b/e induced by phenobarbital. The enzyme 13 carries out this activity by hydroxylating the substrate's 0' or N linked alkyl group which then leaves as the corresponding aldehyde. The so called arylhydrocarbon hydroxylase activity of cytochrome P450c is very specifically measured with benzo-(a)-pyrene or ethoxyresorufin. Cytochrome P450a is specific to testosterone 7a- hydroxylation. As will be discussed later in this thesis, estradiol 2-hydroxylation is carried out most efficiently by the male specific cytochrome P450h and by the inducible cytochrome P450d. Immunochemical methods are useful in comparing and quantitating isozymes and provide the most direct and potentially most specific probe. The problem is that some isozymes exhibit so much sequence homology that polyclonal antibodies show significant cross reactivity. Cytochromes P450b and P450e cannot be immunochemically quantitated individually because a monospecific polyclonal antibody can not be prepared. EFFECT OF POLYHALOGENATED ARQMAIIC HYQROCARBONS ON CYTOCQRQME 2532 The mechanism of toxicity of the halogenated aromatic hydrocarbons has attracted a great deal of attention over the past 20 or 30 years. These compounds are intriguing because they are chemically rather inert, generally recalcitrant to metabolism, and cause a delayed, although dose-dependent toxicity, the mechanism of which is still not clear. Members 14 of this group include hexachlorobenzene, polychlorinated- and polybrominated biphenyls and chlorinated dibenzodioxins and dibenzofurans. Most notable among the group is 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD; see Figure 2). A minor contaminant of herbicide manufacture, it is the most toxic member of the group having an LD50 in the rat of 100 nmol/kg. Poland and Glover (1973) showed that it was a highly effective inducer of cytochrome P450 arylhydrocarbon hydroxylase and that toxicity was highly correlated with enzyme induction. Based on the correlation of toxicity to steric structure of TCDD isomers and diminished toxicity and enzyme inducibility in certain inbred mouse strains, a cytosolic receptor protein was discovered for which TCDD serves as the tightest fitting ligand known. This protein is frequently referred to as the Ah receptor in reference to its connection to arylhydrocarbon hydroxylase (cytochrome P450c). Thus induction of cytochromes P450c and d is thought to be initiated by interaction of appropriate ligands with the receptor. Enzyme induction has been a hallmark of exposure to toxic polyhalogenated aromatic hydrocarbons; but there appears to be no direct relationship between the activity of induced cytochrome P450's and the ensuing toxicity. The polyhalogenated aromatic hydrocarbons have been divided into 2 or 3 groups based on biological response. One group is relatively non-toxic, does not bind to the Ah receptor and, at high doses, induces cytochrome P450b typically induced by phenobarbital. An example of this group 15 is 2,4,5,2',4',5'-hexachlorobiphenyl (Figure 2). The second ' group, which can be toxic if not metabolized, can serve as ligands for the Ah receptor and induces cytochrome P450c and d. An example of this group is 3,4,5,3',4',5'— hexachlorobiphenyl (Figure 2). A third group is composed of members which show toxicity at high doses, but which induce cytochrome P450 isozymes typically induced by both phenobarbital and methylcholanthrene, so called mixed-type inducers (Dannan g£_§l., 1982); e.g.,2,4,5,3',4',5'- hexachlorobiphenyl (Figure 2). Expression of these characteristics depend on the position and number of halogen substituents, resulting in variable affinity for the Ah receptor and thus appears to depend on the ability of the compound to fit within certain steric restrictions of size and planarity (Poland and Knutson, 1982). LIGANDS AND INHIBITORS OF CYTOCHROM§7P450 Methylenedioxyphenyl (MDP) Compounds This group of compounds is unusual in that they both induce and inhibit cytochrome P450. Piperonyl butoxide is used as an insecticide synergist and functioned in this capacity by inhibiting insectide inactivation by cytochrome P450 (Casida §3_§l., 1966). When used in vitro, piperonyl butoxide can inhibit many microsomal cytochrome P450 reactions including acetaminophen metabolism and aniline hydroxylation (Mitchell et a1., 1973). On the other hand, 16 :‘Z‘ZI 2.3.7.84151RACHL0R00185N20- P-DIOXIN (TCDD) ISOSAFROLE Cl Cl .. o O a Cl Cl 3.4.5.3'.4'.5'-HEXACHL0R0- BIPHENYL (HCB) C] (n .. o o a C c: 2.4.5.3'.4'.5'-HEXACHL0R0- BIPHENYL CH CH .. O O .. CI CI 2.4.5.2'.4'.S-HEXACHLORO- BIPHENYL Figure 2. Representative aromatic hydrocarbons. 17 administration of piperonyl butoxide to mice results in increased metabolism of hexobarbital and increased cytochrome P450 levels (Matthews g3_§l., 1970). Thus, although it can inhibit cytochrome P450, it also has the capacity to induce cytochrome P450 and increase xenobiotic metabolism as well, suggesting that MDP compounds can be displaced by some substrates. The inhibition produced by MDP compounds is of a dual nature consisting of both competitive inhibition, resulting from competition of the MDP compound with the substrate for the enzyme, and non-competitive inhibition, due to formation of a nearly irreversible complex with the enzyme (Philpot and Hodgson, 1972). The latter activity is apparently the result of suicide inhibition, where the enzyme, in an attempt to metabolize the MDPJcompound, activates it to a reactive metabolite which forms a covalent bond to the enzyme forming a metabolite-intermediate complex. Safrole is a constituent of sassafras oil and its isomer, isosafrole (Figure 2), is one of the most intensely studied MDP compounds. The isosafrole metabolite-intermediate complex is manifested optically in the Soret region by absorption peaks at 426 and 455 nm, in the dithionite—reduced difference spectrum, owing to its interaction with heme. The interaction of the metabolite with heme is relatively weak since it can be displaced by a variety of ligands (Dickens g5 a1., 1979). It is thought that metabolism proceeds by hydroxylation of the methylene group yielding an unstable product analogous to an ortho-ester (Anders et a1., 1984) 18 which rearranges and produces either a catechol (loosing carbon monoxide) or a carbene. Carbenes are known to interact with ferrous heme and it has been shown that a carbene derived from 1,3-benzodioxole forms a complex with ferrous-tetraphenylporphyrin and produces a Soret region spectrum very much like that observed for the cytochrome P450 metabolite-intermediate complex (Mansuy g£_§l., 1979). Mechanistically, it appears that cytochrome P450 metabolizes MDP compounds by hydroxylation; the product then rearranges to the carbene, and binds to heme iron, inhibiting or inactivating the enzyme. The stability of the MDP-carbene- P450 complex probably varies with different isozymes of cytochrome P450 since the complex formed in vitro with rabbit cytochrome P450 isozymes tends to degrade over time (Delaforge g£_§l., 1982) whereas in rats it is more stable (Ryan et a1., 1980). This probably results from a property of the active site of cytochrome P450 where the substrate might be held in place by various hydrophobic interactions in addition to ligation to heme iron. Indeed, more carbon monoxide is formed upon incubation of isosafrole with microsomes from phenobarbital treated rats than those from 3- methylcholanthrene treated rats even though isosafrole can form the metabolite intermediate complex with both (Yu 3; gig, 1980). Substitution with one or two methyl groups on the methylene carbon prevents induction of cytochrome P450 in treated rats and also prohibits formation of the metabolite- intermediate complex in vitro (Cook and Hodgson, 1983). 19 Aromatic Hydrocarbons The naturally occurring flavonoids have been extensively studied as inhibitors of cytochrome P450. In particular 5,6- and 7,8-benzoflavone have been widely used as inhibitors of cytochrome P450c (referred to as P448 in the early literature), and were frequently used diagnostically to indicate a P448 activity. Some have recently been shown to be very potent inhibitors not only of cytochrome P450c (Sousa and Marletta, 1985) but also of the substrate-specific estrogen synthase or aromatase (Kellis and Vickery, 1984). Cytochrome P448-1 (equivalent to cytochrome P450d) isolated from S-methylcholanthrene treated rabbits contained up to 0.88 mol a-methylcholanthrene per mol P450 and was apparently tightly bound such that S-methylcholanthrene fluorescence was quenched (Imai gt_§l., 1980). This and related compounds were shown to bind in vitro to P450 in such a way that circular dichroism spectra were significantly changed upon addition of ligand. REGULATION OF CYTOCHROME P450 Catalytic activity of cytochrome P450 in the cell is directly proportional to the amount of polypeptide present, thus changes in the level or activity of cytochrome P450 are thought to be mediated at the level of transcription. Specific chemicals or hormones selectively increase synthesis 20 of one or more forms of cytochrome P450 through presumably specific, though largely unknown, mechanisms. Steroidogenic cytochromes P450 appear to be under the control of cAMP which in turn is under the control of either ACTH or FSH depending on the cell type (reviewed in Whitlock, 1986). Intracellular regulation by cAMP is probably indirect via another receptor protein which then interacts with DNA. Hepatic microsomal cytochrome P450p, while expressed constitutively at significant levels, is induced by pregnenolone-16a-carbonitrile and glucocorticoid-like compounds such as dexamethasone (Gonzalez g£_§l, 1985). The level of this enzyme is increased to very high levels in response to certain macrolide antibiotics. These compounds form a stable metabolic-intermediate complex with the reduced form of the enzyme, similar to isosafrole and cytochrome P450d, and are presumed to increase the level of the enzyme by inhibiting its degradation (Watkins et a1., 1986). The control of isozyme induction by phenobarbital remains a mystery. To date there has been no evidence of a receptor for phenobarbital-induced cytochrome P450, in contrast to the steroid-and aryl hydrocarbon induced enzymes. Indirect induction by accumulation of an unknown endogenous substrate was recently tested by development of a specific suicide substrate for cytochrome P450b. However, although the substrate fully inhibited the enzyme, there was no increase in cytochrome P450b mRNA or protein (Ortiz de Montellano and Costa, 1986). 21 As mentioned earlier, cytochromes P450c and P450d appear to be controlled by the Ah receptor, a cytosolic protein for which a number of aromatic hydrocarbons have an unusually high affinity. Polypeptide and mRNA for cytochrome P450c are virtually undetectable in untreated rats whereas cytochrome P450d is expressed to a level of about 5% of total P450 in microsomes from untreated rats (Ryan et a1., 1982). Both isozymes are induced 20 to 80 fold in rats. Evidence suggests that unmodified substrate binds to the receptor, translocates to the nucleus, interacts with a regulatory segment of DNA and tiggers production of mRNA for P450c and P450d or their species equivalent (Whitlock, 1986). Isosafrole induces primarily cytochrome P450d and some P450c and although increased levels of mRNA were detected in response to isosafrole (Kawajiri g£_§l., 1984) it does not appear to serve as a ligand for the Ah receptor (Cook and Hodgson, 1985). It produces synergistic induction of cytochrome P450d with the Ah receptor ligand, 3- methylcholanthrene (Thomas g£_§l., 1983), and evidence suggests that it might have a stabilizing effect on cytochrome P450d (Steward et a1., 1985). REFERENCES Atsuta, Y., Okuda, Y. (1978). Isolation of rat liver mitochondrial ferredoxin and its reductase active in the 5b- cholestane-Sa,12a-triol 26-hydroxylase. J. Biol. Chem. ggg. 4653-4658. Ahr, H.J., King, L.J., Nastainczyk, W., Ullrich, V. (1982). 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Isolation from adrenal cortex of a non-heme iron protein and a flavoprotein functional as a reductase triphosphopyridine nucleotide cytochrome P450 reductase. Arch. Biochem. Biophys. 111, 660-673. 25 0mura, T., Sato, R. (1962). A new cytochrome in liver microsomes. J. Biol. Chem. 237, 1375-1376. Ortiz de Montellano, P.R., Costa, A.K. (1986). Dissociation of cytochrome P450 inactivation and induction. Arch. Biochem. Philpot, R.M., Hodgson, E. (1972). The effect of piperonyl butoxide concentration on the formation of cytochrome P450 difference spectra in hepatic microsomes from mice. Mol. Pharmacol. 8, 204-214. Poland, A., Glover, E. (1973). Chlorinated dibenzo-p-dioxins: Potent inducers of delta-aminolevulinic acid synthase and aryl hydrocarbon hydroxylase II. A study in structure- activity relationship. Mol. Pharmac. 2, 736-747. Poland, A., Knutson, J.C. (1982). 2,3,7,8-tetrachlorocibenzo- p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Ann. Rev. Pharmacol. Toxicol. 23, 517-554. Poulos, T.L., Finzel, E.C., Howard, A.J. (1986). Crystal structure of substrate-free Pseugpmongs putida cytochrome P450. Biochemistry 25, 5314-5322. Suhara, K. (1978). Cytochrome P450-containing oxygenase systems: bacterial systems In: Sato, R., 0mura, T. Cytochrome P450. Kodansha Academic Press 184-194. Remmer, H. (1961). Drugs as activators of drug enzymes. In: Proceedings fo the First International Pharmacological Meeting: Vol 6, Metabolic factors controlling duration of drug action. Brodie, B.B., Erdos, E.G. (eds) Macmillan, New York Ryan, D.E., Ramanathan, L., Iida, S., Thomas, P., Haniu, M., Shively, J.E., Lieber, C.S., Levin, W. (1985). Characterization of a major form of rat hepatic microsomal cytochrome P450 induced by isoniazid. J. Biol. Chem. 269, 6385—6393. Ryan, D.E., Thomas, P.E., Levin, W. (1980). Hepatic microsomal cytochrome P450 from rats treated with isosafrole: purification and characterization of four enzymic forms. J. Biol. Chem. 255, 7941-7955. Ryan, D.E., Thomas, P.E., Reik, L.M., Levin, W. (1982). Purification, characterization and regulation of five rat hepatic microsomal cytochrome P450 isozymes. Xenobiotica lg, 727-744. 26 Seidel, S.L., Shires, T.K. (1986). Purification and characterization of a previously unreproted form of cytochrome P448 from the liver of S-methylcholanthrene- pretreated rats. Biochem. J. 235, 859-868. Sladek, N.E,, Mannering, G.J. (1966). Evidence for a new P450 hemoprotein in hepatic microsomes from methylcholanthrene treated rats. Biochem. Biophys. Res. Comm. 24, 668-674. Steward, A.R., Wrighton, S.A., Pasco, D.S., Fagan, J.B., Li, D., Guzelian, P.S. (1985). Synthesis and degradation of 3- methylcholanthrene-inducible cytochromes P450 and their mRNAs in primary monolayer cultures of adult hepatocytes. Arch. Biochem. 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Macrolide antibiotics inhibit the degradation of the glucorticoid-responsive cytochrome P450p in rat hepatocytes in vivo and in primary monolayer culture. J. Biol. Chem. 261, 6264-6271. Waxman, D.J. (1986). Rat hepatic cholesterol 7a-hydroxylase: biochemical properties and comparison to constitutive and xenobiotic inducible cytochrome P450 enzymes. Arch. Biochem. Biophys. 241. 335-345. Welton, A.F., Aust, S.D. (1974). Multiplicity of cytochrome P450 hemoproteins in rat liver microsomes. Biochem. Biophys. Res. Comm. 56, 898-906. Whitlock, J.P.,Jr. (1986). The regulation of cytochrome P450 gene expression. Ann. Rev. Pharmacol. Toxicol. 26, 333-369. 27 Yu, L.S., Wilkinson, C.F., Anders, M.W. (1980). Generation of carbon monoxide during the microsomal metabolism of methylenedioxyphenyl compounds. Biochem. Pharmac. gg, 1113- 1122. Zuber, M.X., Simpson, E.R., Waterman, M.R. (1986). Expression of bovine 17a-hydroxylase cytochrome P450 cDNA in nonsteroidogenic (COS 1) cells Science 234, 1258-1261. Zuber, M.X., Maliyaki, J.E., Okamura, T., Simpson, E.R., Waterman, M.R. (1986). Bovine adrenocortical cytochrome P450- 17a: regulation of gene expression by ACTH and elucidation of primary sequence. J. Biol. Chem. 26;, 2475-2482. CHAPTER I SPECIFIC BINDING OF POLYHALOGENATED AROMATIC HYDROCARBON INDUCERS OF CYTOCHROME PUSOd TO THE CYTOCHROME AND INHIBITION OF ITS ESTRADIOL 2-HYDROXYLASE ACTIVITY 28 ABSTRACT Treatment of male Sprague-Dawley rats with 3,A,5,3',A',5'-hexabromobiphenyl (HBB) at 10 umol/kg followed by purification of hepatic microsomal cytochrome P450d revealed that HBB remained specifically bound to PASOd throughout purification. Binding was noncovalent since HBB was removed by extraction with dichloromethane. Although HBB induced both cytochrome'PASOC and PASOd, specific immunoprecipitation of these isozymes from HBB treated rats showed that HBB was associated only with cytochrome PASOd. Quantitation of HBB and cytochrome PASOd in microsomes from HBB treated rats suggested a 0.9:1 ratio of H88 to. cytochrome PASOd. Five other halogenated aromatic hydrocarbon inducers of cytochrome PASOd, bearing steric similarity to BBB (including 2,3,7,8-tetrachlorodibenzo- p-dioxin), were associated with cytochrome PASOd when used to induce cytochrome PASOd in rats. HBB inhibited estradiol 2-hydroxy1ase activity of purified cytochrome P450d in a non-competitive manner with an 150 of 38 nM for 50 nM PASOd whereas its non-coplanar isomer, 2,”,5,2',A',5'-hexa- bromobiphenyl, had an I50 over 700 fold higher. Thus certain polyhalogenated aromatic hydrocarbons with the capacity to induce cytochrome PASOd also bind to the cytochrome when used as inducing agents and inhibit catalytic activity of the cytochrome. 29 INTRODUCTION Cytochrome PASO represents a family of isozymes that catalyze the monooxygenation of numerous substrates, including drugs and some physiologic substrates. The broad substrate specificity of the system and its involvement in toxicity and carcinogenicity has led to the resolution and characterization of the different forms of cytochrome PASO in several species, including humans. The concentrations of some cytochrome PASO isozymes increase dramatically in various tissues in response to treatment of animals with certain chemicals. Cytochromes PHSOc and PASOd (cytochrome Pans isozymes) are induced by many toxic xenobiotics such as 3-methylcholanthrene and certain polyhalogenated aromatic hydrocarbons (PHAH) (Parkinson 33 31., 1983). These isozymes appear to be coordinately induced but differentially regulated, and although they share some sequence homology they have different physical characteristics (Reik gt _l., 1982). Many compounds are able to inhibit specific cytochrome PASO isozymes by binding to active sites in either a covalent or non-covalent manner (Walsh, 198A; Sousa and Marletta, 1985). For example, metyrapone inhibits metabolism catalyzed by the phenobarbital inducible cytochrome PASO isozymes (Hildebrandt gt 31., 1969) while a-naphthoflavone inhibits the MC inducible isozymes (Weibal t 1., 1971). The binding of both inhibitors and substrates is typically and conveniently measured by 30 31 spectral changes in the enzyme brought about by low to high spin state transitions of the heme iron: cytochrome PASOd, however, exists largely in a high spin state (White and Coon, 1982). Isosafrole induces cytochrome PASOd and a metabolite of Isosafrole is bound to the cytochrome giving rise to a characteristic metabolite-inhibitor complex of the reduced cytochrome with a Soret peak at “55 nm (Ryan gt gt., 1980). This is probably a result of metabolite coordination to the 6th axial position on ferrous heme (Mansuy gt gt., 1979). Methylcholanthrene induces rabbit cytochrome PASO LMu (a high spin isozyme analogous to PASOd) and binds tightly to the cytochrome Pu50 LMu such that Methylcholanthrene fluorescence is quenched (Imai gt gt., 1980). We found that 3,A,S,3'M'5'-hexabromobiphenyl (HBB) and five sterically similar compounds were associated with cytochrome PASOd isolated from the livers of rats which had been treated with the chemicals to induce cytochrome PASOd. The binding cannot be measured spectrally, but the compounds inhibit estradiol 2-hydroxylase activity catalyzed by the cytochrome. METHODS AND MATERIALS Materials: 178-[2-3HJEstradiol was purchased from New England Nuclear (Boston, MA). 2',5'-ADP-Sepharose was purchased from Pharmacia (Piscataway, NJ). m-Amino-n-octyl-Sepharose (AO-Sepharose) was prepared by 32 coupling 1,8-diaminooctane (Sigma, St. Louis, M0) to Sepharose AB following cyanogen bromide activation as described (March gt _t., 197”). Cholic acid (Sigma) was decolorized with charcoal, recrystallized from 50% ethanol, converted to the sodium salt and used as a 20% solution (w/v based on cholic acid) for AO-Sepharose chromatography. A 20% solution of sodium cholate (w/v) not recrystallized was used for the remaining chromatography steps. Hydroxylapatite (Hypatite C) was purchased from Clarkson Chemical (Williamsport, PA). Protein A-Sepharose was obtained from Sigma and DEAF-cellulose (DE-52) was obtained from Whatman (Clifton, NJ). Goat anti-rabbit-peroxidase complex was purchased from Cappel Labs (Malvern, PA). Crude 3,4,5,3',A',5'-hexabromobiphenyl was obtained from Ultra Scientific, Inc. (Hope, RI) and purified by alumina column chromatography (Millis, 198A). 2,A,5, 2'A'5'- and 2,A,5,3',A',5'-Hexabromobiphenyl were purified from Firemaster BP-6 (lot 622A-A, Michigan Chemical Corp., St. Louis, MI) as described by Dannan gt _t., 1982. 3,4,5,3',N'-Pentachlorobiphenyl was kindly supplied by Dr. Stephen Safe (Texas A & M University, College Park, TX) and 3,A,S,3',A',5'-hexachlorobiphenyl was purchased from Pathfinder Labs (St. Louis, MO). TCDD was generously donated by Dow Chemical Co. (Midland, MI). Emulgen 911 was obtained from Kao Corporation (Tokyo). The bicinchoninic acid protein assay reagent was purchased from Pierce Chemical (Rockford, IL). 33 Animal Treatment and Microsomal Preparation: Male Sprague-Dawley rats (Charles River, Portage, MI) weighing 150-250 g were treated by oral gavage with the various PHAH dissolved in corn oil and killed 3 days later: or with ISF (150 mg/kg/day) for 3 days and killed 2“ hours following last dose. Rats were starved overnight and killed by decapitation following C02 anesthesia: livers were homogenized in 1.15% KCl, 10 mM EDTA, pH 7.“ (1:“, w/v). Microsomes were prepared by differential centrifugation, (20,000 xg for 30 min, followed by 100,000 xg for 90 min) washed with 100 mM sodium pyrophosphate, pH 7.“, containing 0.1 mM EDTA, pelleted at 100,000 xg for 90 min, and suspended in 20 mM potassium phosphate, pH 7.“, containing 20% glycerol and 0.1 mM EDTA and stored at -20°C. Purification of Cytochromes: Cytochrome b5 was purified according to published methods (Imai, 1976; Spatz and Strittmatter, 1971) to specific content of 32 nmol/mg protein. Epoxide hydrolase was purified to electrophoretic homogeneity as described (Guengerich and Martin, 1980). Cytochromes P“50c and P“50d were purified by a combination of procedures used by others (Guengerich gt gt., 1982; Astrom and DePierre, 1985: Imai gt gt., 1980). Unless otherwise noted, all manipulations were done at “°C. Microsomes were diluted to 1.5 mg protein/ml in 100 mM potassium phosphate, pH 7.“, containing 20% glycerol and 1 34 mM EDTA (100mM PGE). Recrystallized cholate was added slowly to 0.8% (w/v). Approximately 3000 nmol of cytochrome P“50 were loaded on a 2.5 x “0 cm AO-Sepharose column which had been previously equilibrated with three bed volumes of 100mM PGE containing 0.6% cholate. The column was washed with 2 bed volumes of 100 mM PGE 0.“% cholate and eluted with 100 mM PGE containing 0.3% cholate and 0.1% Lubrol PX (Figure 1). Peak fractions of cytochrome P“50 were pooled, dialyzed against 20% glycerol followed by dialysis against 10 mM potassium phosphate, pH 7.6, containing 20% glycerol and 0.1 mM EDTA, (designated 10 mM PCE) with 0.2% sodium cholate and 0.1% Lubrol PX. The cytochrome P“50 was loaded on a DEAF-cellulose column (2.5 x 15 cm) which had been previously equilibrated with 10 mM PGE containing 0.5% cholate and 0.2% Lubrol PX. A small band of mixed hemoproteins was eluted from the column with 2 bed volumes of equilibration buffer and a major band enriched in P“50d migrated halfway down the column. A “00 m1 linear gradient of 0-200 mM NaCl containing the same buffer was applied to elute cytochrome P“50d followed by cytochrome P“50c (Figure 2). The cytochrome P“50-enriched fractions were dialyzed against 30 mM potassium phosphate, pH 7.25, containing 20% glycerol, 0.1 mM EDTA, 0.2% Lubrol PX and separately applied to hydroxylapatite columns (2.5 x 10 cm) equilibrated with the same buffer. After washing with 2 bed volumes of this buffer, cytochrome P“500 was eluted with the same buffer but containing 90 mM phosphate while P“50d eluted with the same 35 ° W} 2.4, L. 15 2.0- ii 53 " .E — E |.6 E \ _ _ a,_O4 O E U7 A l2- ‘2. ((10 t I .5 3 V 0.8.. 33.0.2 0) <1 E! :3 0.4_ °'_ 8 'U 8.3 O o 30 0 Fraction Figure 1. Aminooctyl Sepharose (AD-Sepharose) chromatography of cholate-solubilized rat liver microsomes from isosafrole-treated rats. Microsomes were prepared and loaded onto column as described in "Methods." Following column washing, cytochrome P“50 was eluted with 100 mM potassium phosphate, pH 7.“, containing 20% glycerol, 1 mM EDTA, 0.3% cholate and 0.1% Lubrol PX. 20 ml fractions were collected. Cytochrome P“50 was monitored by Au17; cytochrome P“50 reductase was monitored by reduction of cytochrome C by AA550. Protein was determined by the BCA method (see "Methods"). 36 A4:7 (M). NoCl 20 25 30 35 4O Fraction number Figure 2. DEAE-chromatography of the cytochrome P“50 fraction from an AO-Sepharose column. The dialyzed sample was loaded onto a DEAE-cellulose column, washed and eluted with a gradient of 0-200 mM NaCl in 10 mM PGE containing 0.5% cholate, 0.2% Lubrol PX. 7 ml fractions were collected. 37 buffer containing 180 mM phosphate and 0.2% cholate. Rechromatography of the peak fractions on either hydroxylapatite or DEAF-cellulose resulted in an electrophoretically homogeneous protein solution with specific content of 12-18 nmol/mg protein (Figure 3). Detergent was removed from purified cytochrome P“50 by adsorbing cytochrome P“50 on a small (1.5 x 5 cm) hydroxylapatite column equilibrated with 30 mM potassium phosphate, pH 7.25 and containing 20% glycerol, 0.1 mM EDTA, 0.2% cholate and washing with ten bed volumes of this buffer to remove Lubrol PX (as measured by the method of Garewal, 1973). Cytochrome P“50 was eluted from the column with 300 mM potassium phosphate, pH 7.25, containing 20% glycerol, 0.1 mM EDTA, and 0.6% cholate,dialyzed against 100 mM PCB, and stored at -70°C. Butanol (250 mM) was used to displace the isosafrole metabolite from cytochrome P“50d by a method similar to that described by Fisher gt gt. (1981). White and Coon (1982) showed that oxygen was likely the bonding atom of the axial ligand of heme iron ttggg to thiolate and furthermore showed that oxygen of n-butanol could coordinate to heme of cytochrome P“50LMu, a high spin isozyme in the rabbit, and convert it to low spin. Thus, it seemed likely that such a compound might displace the isosafrole metabolite: the oxygen of butanol displacing the metabolite coordination to heme and the hydrophobic tail of butanol aiding in displacement of the metabolite from the active site. The Figure 3. 38 SDS-polyacrylamide gel electrophoretogram. Slab gel (15 x 20 cm x 0.75 mm) was prepared, run, and stained according to the method of Laemmli (1970) except that arylamide concentration was 7.5%. (A) epoxide hydrase; (B) cytochrome P“50c: (C) cytochrome P“50d: (D) detergent solubilized reductase (upper band), protease solubilized reductase (lower band): (E) partially pure cytochrome P“50 from AO-Sepharose fraction. 39 metabolite was usually displaced before purification of P“50d by incubating n-butanol with the microsomes at 37°C for 30 min prior to addition of cholate and passage through AO-Sepharose. If metabolite-free microsomes were needed, the butanol treated microsomes were passed through a 1.5 x 30 cm Sepharose CL-6B column in 100 mM PGE (Figure “). Assay Methods: Catalytic activity of cytochrome P“50 was measured under conditions in which product formation was proportional to enzyme concentration and incubation time. Metabolic activity was reconstituted using cytochrome P“50, NADPH-cytochrome P“50 reductase, and dilauroylphosphatidylcholine at a 1:2:200 molar ratio. These components were preincubated 3-5 min at high concentration before addition of remaining components. NADPH-cytochrome P“50 reductase was prepared by affinity chromatography on 2',5'-ADP-Sepharose by the methods of Dignam and Strobel (1977) and Yasukochi and Masters (1976). Dilauroyl-phosphatidylcholine was prepared as a 1 mM suspension in 50 mM potassium phosphate, pH 7.“ containing 0.1 mM EDTA and sonicated before use. Bovine gamma globulin (2.5 mg/ml) was included as a carrier. NADPH was used to start the reactions and was generated with a system consisting of (final concentration): 0.5 mM NADP+, 5 mM isocitrate, 5 mM MgClg, 5 uM MnC12 and 0.“ unit isocitrate dehydrogenase. This system was pre-incubated at 37°C for 5 40 455 A 426 426 Figure “. Difference spectrum of dithionite-reduced microsomes from isosafrole treated rats. Microsomes (10 mg protein) were incubated without (A) and with (B) 250 mM n-butanol at 37° for 30 min followed by chromatography on Sepharose CL-6B. An aliquot (1 mg) was divided between two cuvets, the suspension in the sample cuvet was reduced with a few crystals of sodium dithionite and the absorption recorded between “90 and “10 nm. The “55 nm peak is due to the isosafrole metabolite complex with cytochrome P“50; the “26 nm peak is cytochrome b5. 41 min to achieve a steady state level of NADPH before addition as a 100 pl aliquot to the assay mixture. Estradiol 2-hydroxylase was measured by the method of Numazawa gt gt. (1980) as modified by Ryan gt _l. (1982). Protein was determined by the bicinchoninic acid micromethod (Redinbaugh and Turley, 1986) standardized with bovine serum albumin. Cytochrome P“50 concentration was determined based on an extinction coefficient of 91 mM'1 cm"1 for the C0 difference spectrum of dithionite-reduced samples. PHAH were extracted from cytochrome P“50 by preparing a solution of cytochrome P“50 (0.1 nmol P“50 or 50 ug microsomal protein) in 200 pl 0.5 N KOH and extracting 3 times with 800 pl dichloromethane each time. PHAH were transferred to hexane and quantitated by gas chromatography with a 3% 07—1 column and electron capture detector. Immunochemical Methods: Antibodies were raised in New Zealand rabbits by injecting 300 ug antigen (emulsified in Freund's complete adjuvant) intradermally in 15-20 sites along the back. This was followed by monthly subcutaneous injections of 100 ug antigen using incomplete adjuvant. Rabbits were bled 1 week after boosting and the titer was checked by Ouchterlony double diffusion. Serum immunoglobulins (IgG) were prepared by affinity chromatography on protein A-Sepharose (Hjelm gt _t., 1972). An indirect, non-competitive ELISA for cytochrome P“50d was developed based on the method of Paye gt 1. (198“). 42 Antigen was diluted in 50 mM sodium bicarbonate, pH 9.6, 2%- sodium cholate, 0.02% azide and 50 ul/well added to triplicate wells of a microtiter plate. Plates were incubated 3 hr at 37°C or over night at “0C. Antigen solution was replaced with 200 pl 0.1% gelatin in 10 mM potassium phosphate, pH 7.“, 0.15 M NaCl, 0.02% azide and incubated 30 min at 37°C. Plates were then rinsed with tap water with a microtiter plate washer, and IgG was added in 0.1 M potassium phosphate, pH 7.“, containing 0.“ M KCl, 0.“ mM EDTA, 0.5% cholate, 0.1% Emulgen 911, and 0.02% azide (IgG-buffer) containing 1% BSA, at 10 ug/ml and 100 pl per well for 1.5 hr at “°C. Plates were washed and 100 pl goat-anti-rabbit peroxidase conjugate diluted 1:2000 in IgG-buffer containing 1% BSA (without azide) added per well and incubated 30 min at 37°C. After thoroughly washing the plate, 100 pl color reagent (2 mM o-phenylendiamine HCl, 2.5 mM H202 in 0.1 M sodium citrate, pH 5.0) was added. Color development was stopped with 50 ul “N H250“ after 5 min and absorbance read at “90 nm using a Biotech ElA Model 307 plate reader (Burlington, VT). Response to cytochrome P“50d was linear from 0.02 to 0.“ pmol per well. This allowed quantitation of microsomal cytochrome P“50d in and 0.05 to 0.“ ug microsomal protein per well depending on specific content of P“50d (Figure 5). Cross reactivity of anti-P“50d with cytochrome P“500 was minimized by adsorption of the cross reacting antibodies onto cytochrome P“500 coupled to CNBr-activated Sepharose (Reik gt a1., 1982). 1+3 V A H/BB v A ISF l.6 . A 1.2- V D g 0.8_ v A / ); microsom:es from B-naphthoflavone treated rats (El): microsomes from isosafrole treated rats (A1); microsomes from HBB treated rats (V'). 44 For immunOprecipitation, microsomes were solubilized in IgG-buffer at 5 mg/ml. Ten microliters of solubilized microsomes were added to 200 pl of the same buffer containing 1% (w/v) BSA and appropriate amount of specific IgG and incubated overnight at “°C. The immunoprecipitate was washed 3 times with IgC-buffer and assayed for PHAH content. RESULTS Association of Ligands with Cytochrome P“50d When HBB was used to induce cytochrome P“50 in rats, followed by purification of cytochromes b5, P“500 and P“50d, HBB was found to be selectively associated with cytochrome P“50d (Table 1). The association was apparently non-covalent since HBB could be extracted from the cytochrome with dichloromethane and co-chromatographed with an HBB standard. However, HBB was tightly coupled to the cytochrome since it was not fully removed even though the microsomal cytochrome P“50 was solubilized with detergents and had been passed through four different columns. Since it was still possible that HBB simply co-chromatographed with cytochrome P“50d, cytochromes P“500 and P“50d and epoxide hydrase were immunoprecipitated from microsomes of HBB treated rats (Table 2) and the precipitate assayed for HBB. In this case, HBB was significantly associated only with the immunoprecipitate of cytochrome 45 Table 1. Association of HBB with microsomal cytochromes purified from HBB treated rats. Male Sprague-Dawley rats were treated with HBB (10 pmol/kg), microsomal cytochromes were isolated and assayed for HBB as described in "Methods". Cytochrome ' mol HBB/mol cytochrome 05 (0.01 P“50C 0.01 P“50d 0.“9 46 Table 2. Association of HBB with immunoprecipitated microsomal proteins. Detergent solubilized microsomes (50 ug protein) from HBB treated rats were treated with the IgG's shown. The immunoprecipitates were assayed for HBB as described in "Methods". IgG Amt. IgG added (mg) HBB in immuno- precipitate (pmol) pre-immune 1 anti-P“500 0 anti-P“50c 1. anti-P“50d 0 anti-P“50d 1 anti-EH 1 47 P“50d. The amount of immunoreactive cytochrome P“50d was measured in microsomes from HBB treated rats by ELISA and compared to the total microsomal content of HBB (Table 3). The ratio of HBB to cytochrome P“50d was near unity suggesting a 1:1 association of enzyme and ligand. Five other halogenated aromatic hydrocarbons bearing steric similarity to HBB were tested for their ability to form a similar stable complex with cytochrome P“50d. Rats were injected with these compounds, liver microsomes isolated 72 hr later, and cytochrome P“50d was immunoprecipitated. The results, shown in Table “, reveal that all of the compounds tested were associated with the cytochrome P“50d immunoprecipitate. Control experiments with purified cytochrome P“50d with HBB bound at 0.9 mol/mol cytochrome P“50d showed that “2% precipitation occurred using antigen-antibody ratios similar to those used to obtain the data in Table “. Thus it is not surprising that in most cases we could not achieve complete immunoprecipitation of the ligand-P“50 complex. It should be noted the 2,“,5,3',“',5'-hexabromobiphenyl is unlike HBB in that it contains one bromine located ggttg to the biphenyl bridge and is energetically restricted from assuming a cOplanar configuration. In this respect it is only a moderate inducer of cytochrome P“50 (Dannan gt gt., 1983) and moreover, this attribute probably results in the diminished association with cytochrome P“50d. Although 3,“,3'“'-tetrachlorobiphenyl is metabolized by cytochrome 48 Table 3. Correlation of microsomal HBB content with immunologically measured (ELISA) content of microsomal cytochrome P“50d. Rats were treated with HBB (10 pmol/kg), and hepatic microsomes were prepared and assayed for HBB and cytochrome P“50d as described in "Methods". Microsomal P“50d HBB preparation (nmol/mg) (nmol/mg) mol HBB/mol P“50 A 1.“1 1.27 0.901 B 2.11 2.10 0.995 C 1.67 1.“6 0.87“ 49 Table “. Association of five polyhalogenated aromatic hydrocarbons with immunoprecipitated cytochrome P“50d. Animals were treated with the chemicals and hepatic microsomes prepared as described in "Methods". An aliquot of microsomes was extracted and assayed for total ligand content, another aliquot was solubilized and diluted to 5 mg protein/ml: 25 pg microsomal protein was incubated overnight with 1 mg anti-P“50d IgG in 200 ul immunOprecipitation buffer. The resulting precipitate was washed, solubilized with 0.5 N KOH, extracted, and the ligand in the immunoprecipitate was quantitated by gas chromatography. Content of Inducer (pmol/mg protein) Percent DOSE Micro- P“50 Immuno- Inducer Inducer (pmol/kg) somes precipitate In ippt. 3,“,5,3',“',5'- 10 71,4 373 52 hexachlorobiphenyl 2.3.7.8-tetrachloro- 1 195 218 112 dibenzodioxin 3,“,3',“'-tetra- 100 388 102 26 chlorobiphenyl 3,“,5,3',“'-penta- 6 753 "50 60 chlorobiphenyl 2,“,5,3',“'5'-hexa- 10 38“ “3 11 bromobiphenyl 50 P“50c but not P“50d (Voorman gt _t., 198“) it was still associated with cytochrome P“50d although the recovery was low probably due to metabolism by P“50c. Influence of Cytochrome P“50d Ligands on Enzymatic Activity of Cytochrome P“50d Estradiol 2-hydroxylase (E2H) is one of the few well characterized activities associated with cytochrome P“50d (Astrom and DePierre, 1985) and was studied in the following experiments to measure the ability of HBB and other potential cytochrome P“50d ligands to inhibit the catalytic activity of the cytochrome. Cytochrome P“50d was isolated from isosafrole treated rats and the isosafrole metabolite removed from the cytochrome (see Methods). E2H activity in this preparation was compared to activity of a cytochrome P“50d preparation from HBB treated rats which contained 0.8 mol HBB/mol cytochrome P“50d (Figure 6). The cytochrome P“50d-HBB complex exhibited non-competitive inhibition compared to the ligand free cytochrome P“50d. The Km's of the two preparations were nearly equal while the Vmax of the inhibited reaction was 23% of uninhibited cytochrome P“50. Non-competitive inhibition indicates that HBB was either not bound at the active site or had a very slow dissociation rate from the active site. In another experiment HBB was added to ligand free cytochrome P“50d and E2H was measured (Figure 7). Distinct inhibition was observed at 10 nM HBB and increasing HBB concentrations revealed that HBB caused 51 (M l l C) E20H (nmo‘l/min/nmol P450)" “3 l 4 1 . . Figure 6. o 0.1 0.2 0-3 I/[Es’rrodiol], (uM)" Lineweaver-Burk plot of estradiol 2-hydroxylase (E2H) activity in two cytochrome P“50d preparations. Purification and reconstitution of cytochrome P“50d and measurement of E2H were as described in "Methods." Cytochrome P“50d was used at 50 nM in 1 ml incubation; EZH activity measured following 10 minutes incubation. Cytochrome P“50d isolated from isosafrole treated rats where the isosafrole metabolite was displaced from cytochrome P“50d, Km . 19.1 uM, Vmax = 8.7 min"1 ( o ): cytochrome P“50d isolated from HBB treated rats (10 pmol/kg) with 0.8 mol HBB/mol cytochrome P“50d, Km = 17.3 uM, Vmax = 2.0 min-1 ( A ). 52 £5on (nmol/inin/nmol P450)"I C Figure 7. l l L l l 0 0.1 0.2 0.3 0.4 0.5 I/[Es’rrodiol], (uM)"I Lineweaver-Burk plot of effect of H88 on cytochrome P“50d catalyzed estradiol 2-hydroxylase. Ligand-free cytochrome P“50d prepared and reconstituted at 50 nM cytochrome P“50d. HBB was added in 10 pl methanol and the reaction started following 5 min preincubation. The concentrations of HBB were 0 (<3 )3 10 ( D )3 25 ( A ); 100 (:0 ); and 1000 ( V ) nM. Apparent Km values ranged from 9.“ - “1.6 pM and apparent Vmax values ranged from 6.2 - 1.1 min"). 53 both competitive and non-competitive inhibition. These data indicate that HBB interferes with E2H activity and is probably bound at or near the active site of cytochrome P“50d. Cytochrome P“50h, a constitutive P“50, has E2H activity similar to P“50d (Astrom and DePierre, 1985). Since it is possible that HBB somehow interferes with estradiol itself, or perhaps with the reductase binding site on cytochrome P“50, the ability of HBB to inhibit cytochrome P“50h-catalyzed E2H was tested by incubating HBB with microsomes from untreated mature male rats. Although E2H activity in these microsomes was similar to that in microsomes from HBB treated animals no significant change in kinetic constants was observed even when HBB was added at concentrations up to “ pM (Figure 8). Further examination of steric restrictions on the binding site of cytochrome P“50d was done using three isomers of hexabromobiphenyl, differing only in the degree of bromination at carbons gtttg to the phenyl:phenyl bridge. The isomers were tested for their ability to inhibit E2H activity in a reconstituted system using ligand-free cytochrome P“50d. The results, shown in Figure 9, revealed that the ability of the polybrominated biphenyl congener to inhibit cytochrome P“50d decreased with increased ELEEQ bromination of the congener. A fully planar, nonhalogenated hydrocarbon, methylcholanthrene was also found to be a strong inhibitor of P“50d. Methylcholanthrene was shown 54 03 00 I 5on (nmol/mining )" 45 Figure 8. 1/1 ./' l l l l O 0.2 0.4 0.6 0.8 l I /[Eslr0di0l], (uM)-' Lineweaver-Burk plot of effect of H88 on hepatic microsomal estradiol 2-hydroxylase activity in untreated rats. Microsomes were isolated from untreated rats and estradiol 2-hydroxy1ase was measured using 50 pg protein/ml with HBB added at: 0 ( 0.); 0.1 ( D ); 1.0 ( A ) and “ ( V ) uM. 55 :{31005-7 A ' .. B" E BrHBr U - Br Br 33 80.. 2. A ‘2; _ V7 D g 60 .. - 0 CU UJ - E 40_ V7 c: 8. "" Br x A '8 O B V\V' 1.20— . ..,o\ g _ 030 o\8\. .. "I O D\u 0' O 4 L l l I I 109 :0'8 10‘7 106 - 10’5 104 Inhibitor (M) Figure 9. Determination of inhibitory concentrations of selected compounds on E2H activity of cytochrome P“50d. E2H was measured at 50 nM cytochrome P“50d in 1 ml buffer. Inhibitors were added in 10 l methanol. Methylcholanthrene ( U ); HBB ( O ): 2,“,5,3',“',5'-hexabromobiphenyl ( v ); 2,“,5,2',“',5'-hexabromobiphenyl ( A ). 56 earlier to be a ligand for cytochrome P““8, an isozyme isolated from methylcholanthrene-treated rabbits and analogous to cytochrome P“50d (Imai gt 1., 1980). DISCUSSION Our experiments reveal that, when rats were dosed with 10 pmol HBB/kg, HBB was selectively bound to cytochrome P“50d. It was not bound to cytochrome P“500 or cytochrome b5 and appeared to be associated with cytochrome P“50d in a 1:1 ratio. The binding was non-covalent although apparently essentially irreversible since HBB was easily extracted with dichloromethane but not removed by hydrophobic or ion exchange chromatography. Enzymic activity of cytochrome P“50d was clearly inhibited by H88 in what appeared to be a non-competitive or mixed inhibition of cytochrome P“50d at concentrations less than the concentration of enzyme. Two isomers of HBB which are more restricted from co-planarity of the biphenyl rings by the presence of ggttg bromines inhibited catalytic activity to a significantly lesser degree, indicating structural specificity for inhibition. Implicit in these results are that HBB and other polyhalogenated aromatic hydrocarbons that induce cytochrome P“50d are bound non-covalently t0 cytochrome P“50d following induction and that the dissociation rate is so slow that the reaction is essentially irreversible. Isosafrole induces cytochrome P“SOd and an isosafrole metabolite forms a stable complex with cytochrome P“50 heme 57 (Ryan gt gt., 1980). The isosafrole metabolite can be displaced from cytochrome P“50 by n-butanol (see Methods) and other compounds (Dickins gt gt., 1979), as determined by loss of the “55 nm ferrocytochrome P“50 chromophore and recovery of metabolism of selected substrates. When cytochrome P“50d isolated from HBB treated rats was subjected to similar treatment, HBB was not displaced from cytochrome P“50d (data not shown). There was no change in the catalytic capacity of cytochrome P“50d following butanol treatment and chromatography and HBB could still be extracted from cytochrome P“50d. Despite the inability of n-butanol to remove HBB from cytochrome P“50d it seems likely that HBB binds to cytochrome P“50d in a manner like the isosafrole metabolite. This assertion is supported by the fact that both compounds preferentially induce and bind to cytochrome P“50d. Although isosafrole forms the cytochrome P“50-isosafrole metabolite complex tg tttgg with several cytochrome P“50 isozymes (Ryan gt gt., 1980) we found that when cytochrome P“500,d, and h were isolated from isosafrole treated rats, the isosafrole-complex was observed only on cytochrome P“50d (unpublished). Isosafrole inhibits cytochrome P“50d catalyzed estradiol 2-hydroxylase like HBB although it is impossible to determine if the isosafrole alone, and not the metabolite, inhibits the hydroxylase activity since the metabolite forms upon addition of NADPH. It is thought that the cytochrome P“50-isosafrole metabolite complex is formed when the methylene carbon of isosafrole is 58 oxidized to a carbene and this coordinates to the sixth axial position of heme iron (Mansuy _t gt., 1979). This reaction does not occur with HBB since no change in the Soret spectrum was observed with cytochrome P“50-HBB and CO was not inhibited from binding to cytochrome P“50 by HBB as it was by isosafrole (data not shown). It is likely that HBB binds in close proximity to the heme group of cytochrome P“50d. Experiments with rabbit cytochrome P“50 LMu (a high spin cytochrome P“50 analogous to cytochrome P“50d) indicated that methylcholanthrene, pyrene, and similar aromatic hydrocarbons bind to it in such a way that heme circular dichroism absorption bands were shifted (Imai, 1982). Moreover, cytochrome P“50 LMu-bound pyrene was metabolized when activity was reconstituted with reductase and NADPH, and metabolism was inhibited when CO was added. HBB was metabolized little if at all by microsomal cytochrome P“50 (Mills gt gt., 1985), therefore metabolism of H88 would be unlikely even if bound in the active site of cytochrome P“50d. The significance of the phenomenon we have observed is not clear. Certainly, the very tight binding of HBB would affect the metabolism of an endogenous substrate, but a physiologic function of cytochrome P“50d has yet to be identified. Uncoupling of cytochrome P“50 electron transport by certain substrates has been proposed (Kuthan and Ullrich, 1982; Sousa and Marletta, 1985). Presumably a ferrous-oxygen complex is formed following substrate binding 59 and iron reduction. This complex could be: a) released as 02', 0) reduced by one more electron and released as H202, or c) undergo homolytic cleavage to release H20 with subsequent 2 electron reduction of the FeI3-0 complex to release another H20. Although superoxide and peroxide anions would be expected to be deletorious to the cell, damage would occur only if their production overwhelmed the protective activities of superoxide dismutase and catalase. We have not determined the ability of HBB to uncouple cytochrome P“50 electron transport, although we have shown in a preliminary account that cytochrome P“50d has a slightly greater oxidase activity than either cytochromes P“50c or P“50b (Morehouse, 198“). Recently it has been shown that isosafrole (and perhaps some PCB's) stabilized cytochrome P“50 in cultured rat hepatocytes and partially prevented cytochrome P“50d degradation (Steward gt gt. 1985). That is, the bound ligand might directly affect regulation of cytochrome P“50d levels by restricting turnover of the enzyme. Although cytochromes P“50c and P“50d appear to be induced via the Ah receptor, they are differentially regulated depending on the inducer (Fagan gt gt., 1986) and there is evidence for non-Ah receptor mediated induction of P“50d (Cook and Hodgson, 1985: Linko gt gt., 1986). Isosafrole is one of the most effective inducers of cytochrome P“50d, inducing it to considerably higher levels than cytochrome P“50c. Cytochrome P“50d is the major cytochrome P“50 induced by HBB 60 and by 3,“,5,3',“',5'-hexachlorobiphenyl (Parkinson gt a1., 1983). In Tables 1 and 2 we showed that HBB was tightly bound to cytochrome P“50d analogous to isosafrole and that nearly all the cytochrome P“50d could be complexed with HBB. Thus it seems likely that HBB could affect the turnover of cytochrome P“50d in a manner analogous to isosafrole. Such a stabilizing effect is not unprecedented. Cytochrome P“50p appears to be stabilized by a metabolite complex of trolandeomycin, and this cytochrome P“50 is elevated to extremely high levels in rats following treatment with trolandeomycin, probably owing to decreased degradation of the cytochrome P“50p (Watkins gt gt., 1986). REFERENCES Astrom, A., and De Pierre, J.W. (1985). Metabolism of 2-acetyl-amino fluorene by eight different forms of cytochrome P“50 isolated from rat liver. Carcinogenesis 9, 113-120. Cook, J.C., and Hodgson, E. 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Biochem. 92, 57-66. Imai, I., Hoshimoto-Yutsudo, C., Satoke, H., Girardin, A., and Sato, R. (1980). Multiple forms of cytochrome P“50 purified from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rabbits. I. Resolution, purification, and molecular properties. J. Biochem. gt, “89-503. Kuthan, H., and Ullrich, V. (1982). Oxidase and oxygenase function of the microsomal cytochrome P“50 monooxygenase system. Eur. J. Biochem. 126, 583-588. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T“. Nature 227, 680-682. Linko, P., Yeowell, H.N., Gasiewicz, T.A., and Goldstein, J.A. (1986). Induction of cytochrome P“50 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (Ah) - responsive mice. J. Biochem. Toxicol. 1, 95-107. Mansuy, D., Battioni, J.P., Chottard, J.C., and Ullrich, V. (1979) Preparation of a porphyrin-iron-carbene model for the cytochrome P“50 complexes obtained upon metabolic oxidation of the insecticide synergists of the 1,3-benzodioxole series. J. Am. Chem. Soc. 191, 3971-3973. March, S.C., Parikh, I., and Cictrecasas, P. (197“). A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 99, 1“9-152. 63 Millis, C.D. (198“). Studies on the chemical and pharmacotoxicological properties of polybrominated biphenyls. M.S. Thesis, Michgan State University. Mills, R.A., Millis, C.D., Dannan, C.A., Guengerich, F.P., and Aust, S.D. (1985). Studies on the structure - activity relationships for the metabolism of polybrominated biphenyls by rat liver microsomes. Toxicol. Appl. Pharamacol._l§, 88-95. Morehouse, L.A., Bumpus, J.A., Voorman, R., and Aust, S.D. (198“). Superoxide production by reconstituted cytochrome P“50-dependent mixed function oxidases. Fed. Proc. “3, 355. Numazawa, M., Kiyono, Y., and Nambara, T. (1980). A simple radiometric assay for estradiol 2-hydroxylase activity. Anal. Biochem. 10“, 290-295. Parkinson, A., Safe, S.H., Robertson, L.W., Thomas, P.E., Ryan, D.E., Reik, L.M., and Levin, W. (1983). Immunochemical quantitation of Cytochrome P“50 isozymes and epoxide hydrase in liver microsomes from polychlorinated or polybrominated biphenyl-treated rats: A study of structure-activity relationships. J. Biol. Chem. gig, 5967-5976. Paye, M., Beaune, P., Kremers, P., Guengerich, F.P., Letawe-Goujon, F., and Gielen, J. (198“). Quantification of two cytochrome P“50 isoenzymes by an enzyme-linked immunosorbent assay (ELISA). Biochem. Biophys. Res. Commun. lgg, 137-1“2. Redinbaugh, M.G., and Turley, R.B. (1986). Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions. Anal. Biochem. 153. 267-271. Reik, L.M., Levin, W., Ryan, D.E., and Thomas, P.E. (1982). Immunochemical relatedness of rat hepatic microsomal cytochromes P“50c and P“50d. J. Biol. Chem. 257, 3950-3957. Ryan, D.E., Thomas, P.E., and Levin, W. (1980). Microsomal cytochrome P“50 from rats treated with isosafrole: Purification and characterization of four enzymic forms. J. Biol. Chem. £22, 79“1-7955. Ryan, D.E., Thomas, P.E. and Levin, W. (1982). Purification and characterization of a minor form of hepatic microsomal cytochrome P“50 from rats treated with polychlorinated biphenyls. Arch. Biochem. Biphys. gig, 272-288. 64 Sousa, R.L., and Marletta, M.A. (1985). Inhibition of cytochrome P“50 activity in rat liver microsomes by the naturally occurring flavonoid, quercetin. Arch. Biochem. Biophys. gig, 3“5-357. Spatz, L., and Strittmatter, P. (1971). A form of cytochrome 65 that contains an additional hydrophobic sequence of “0 amino acid residues. Proc. Nat. Acad. Sci. gt, 10“2-10“6. Steward, A.R., Wrighton, S.A., Pasco, D.S., Fagan, J.B., Li, D., and Guzelian, P.S. (1985). Synthesis and degradation of 3-methylcholanthrene-inducible cytochromes P“50 and their mRNAs in primary monolayer cultures of adult rat hepatocytes. Arch. Biochem. Biophys. git, “9“-508. Voorman, R., Mills, R.A., Bumpus, J.A., Morehouse, L.A., and Aust, S.D. (198“) Metabolism of PBB congeners by reconstituted monoxygenase systems. The Toxicologist A, 37“. Walsh, C.T. (198“). Suicide substrates, mechanism-based enzyme inactivators: Recent developments. Ann. Rev. Biochem. 53, “93-535. Watkins, P.B., Wrighton, S.A., Schaertz, E.G., Maurel, P., and Guzelian, P. (1986). Macrolide antibiotics inhibit the degradation of the glucocorticoid responsive cytochrome P“50p in rat hepatocytes in vivo and in primary monolayer cultures. J. Biol. Chem. ggl, 626“-6271. White, R.E., and Coon, M.J. (1982). Heme ligand replacement reactions of cytochrome P“50. J. Biol. Chem. 257, 3073-3083. Wiebel, F.J., Leutz, J.C., Diamond, L., and Gelboin, M.V. (1971). Aryl hydrocarbon (benzo(a)pyrene) hydroxylase in microsomes from rat tissues: differential inhibition and stimulation by benzoflavones and organic solvents. Arch. Biochem. Biophys. lit, 78-86. Yasukochi, 7., and Masters, 8.8.8. (1976). Some properties of a detergent solubilized NADPH-cytochrome c (cytochrome P“50) reductase purified by biospecific affinity chromatography. J. Biol. Chem. £21, 5337-533“. CHAPTER II TCDD (2.3.7,8-TETRACHLORODIBENZO-p-DIOXIN) IS A TIGHT BINDING INHIBITOR OF CYTOCHROME P“500 65 ABSTRACT Cytochrome P“50c and P“50d are induced in rat liver endoplasmic reticulum upon treatment of the animal with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or similar compounds thought to exert their effect through the Ah locus. Isosafrole induces cytochromes P“50b, c, and d and forms a displaceble metabolite-intermediate complex with cytochrome P“50d. Based on experiments which indicated that compounds which induce cytochrome P“50d, also bind to the enzyme, we tested the ability of TCDD to inhibit reconstituted cytochrome P“50d-dependent estradiol 2-hydroxylase, using ligand-free cytochrome P“50d from isosafrole treated rats. TCDD was a mixed-type inhibitor of estradiol 2-hydroxylase changing the apparent Km from 9.6 to 2“.5 pM and Vmax from 6.7 to 1.2 min". Since maximum inhibition of estradiol 2-hydroxylase occurred at TCDD concentrations comparable to the concentration of enzyme, a modified form of steady state kinetics was used. Cytochrome P“50d estradiol 2-hydroxylase was inhibited by nearly equimolar concentrations of TCDD. Using I50 = Et/2 + K1 (Et . total enzyme concentration), we showed that TCDD inhibited cytochrome P“50d estradiol 2-hydroxylase with K1 - 8 nM. Association of TCDD with P“50d was rapid; maximum inhibition occurred within two minutes of inhibitor addition. It seems likely that TCDD is a non-covalent, tight binding competitive inhibitor of cytochrome P“50d. 66 INTRODUCTION Cytochrome P“50 catalyses the hydroxylation of many hydr0phobic compounds, facilitating their excretion and thus playing a key role in the metabolism of many xenobiotics and several endogenous compounds. Cytochrome P“50d is one of many isozymes of cytochrome P“50 in the rat liver and is induced coordinately with cytochrome P“500 by chemicals thought to exert their effect through the ah receptor (Whitlock, 1986). Enzymatic activities of cytochrome P“50c have been well characterized using the hydroxylation of benzopyrene or the deethylation of ethoxyresorufin. Recently it has been shown that cytochrome P“50d can catalyze acetanilide “-hydroxylase and estradiol 2-hydroxylase (Tuteja et a1., 1985: Reik gt_gl., 1982; Astrom a1., 1985). Interestingly, cytochrome P“50d is induced by certain methylenedioxyphenyl compounds such as isosafrole which are then metabolized to form a stable inhibitory complex with cytochrome P“50d (Philpot and Hodgson, 1972; Fisher gt_g;., 1981; Ryan gt_g;., 1980). Polycyclic aromatic hydrocarbons are known to form stable complexes with cytochrome P“50LM“, an analogous isozyme in the rabbit (Imai gt_g;, 1980), and a toxic pentachlorodibenzofuran was shown (Kuroki gt_§;., 1986) to reside largely on a cytochrome P“50 equivalent to P“50d. We have recently shown that certain polyhalogenated aromatic hydrocarbons can form a stable complex with 67 68 cytochrome P“50d when the compounds are used as inducers of cytochrome P“50d and that the bound ligand can inhibit the metabolic activity of cytochrome P“50d (Voorman and Aust, 1987). The substrate specificity of cytochrome P“50 is, with the exception of the steroid hydroxylases, relatively broad and non-specific and thus most substrates tend to have low turnover numbers and high Km's. Similarly, many compounds can inhibit cytochrome P“50 and tend to function as classical competitive or non-competitive inhibitors where both inhibitor and substrate exist in vast excess relative to enzyme concentration. Certain enzyme inhibitors function at a concentration similar to the enzyme and can not be evaluated by Michaelis-Menton enzyme kinetic analysis which presupposes steady state conditions with an infinite excess of both substrate and inhibitor (Morrison, 1969). In this report we show that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) functions as a tight binding inhibitor of cytochrome P“50d and have used a modification of steady state kinetics to evaluate the inhibition of cytochrome P“50d dependent estradiol 2-hydroxylase by TCDD. METHODS AND MATERIALS Materials 2[3H]-Estradiol was obtained from New England Nuclear (Boston, MA). TCDD was generously supplied by Dow Chemical (Midland, MI). Isosafrole was purchased from Fluka 69 (Ronkonkoma, NY) and used without further purification. Dilauroylphosphatidylcholine was purchased from Sigma (St. Louis, MO). Animals Male Sprague-Dawley rats (Charles River, Portage, MI) weighing 150-250 g were treated with isosafrole dissolved in corn oil at 150 mg/kg/day for 3 days and killed on the “th day by decapitation following C02 anesthesia. Liver microsomes were isolated and cytochrome P“50d was prepared as described (Voorman and Aust, 1987) using a combination of published methods (Guengerich gt g1. 1982: Astrom and DePierre,1985: Imai)1980) to a specific content of 12 to 18 nmol/mg protein. To prepare ligand-free cytochrome P“50d, the microsomal preparation was incubated with 250 mM n-butanol at 37°C for 30 min, in order to displace the isosafrole metabolite from cytochrome P“50d prior to the first chromatography step (Voorman-Aust, 1987). Enzyme Assays Estradiol 2-hydroxylase activity was measured under conditions where product formation was proportional to enzyme concentration and incubation time. Catalytic activity of cytochrome P“50d was reconstituted by incubating cytochrome P“50d, NADPH-cytochrome P“50 reductase, and dilauroylphosphatidyl-choline at a 1:2:200 molar ratio for several minutes before addition of remaining reagents. 70 NADPH cytochrome P“50 reductase was prepared by affinity chromatography according to the methods of Dignam and Stroebel (1977) and Yasukochi and Masters (1976). Dilauroylphosphatidylcholine was prepared as 1 mM suspenSion in 50 mM potassium phosphate, pH 7.“, containing 0.1 mM EDTA and sonicated before use. Final volume was brought to 1 ml with 50 mM potassium phosphate, pH 7.“ containing 0.1 mM EDTA and included 2.5 mg bovine gamma globulin as a carrier. The reaction was started by addition of either substrate or NADPH generating system (0.5 pmol NADP+, 5 pmol isocitrate, 0.“ unit isocitratate dehydrogenase). Estradiol 2-hydroxylase was measured by the method of Numazawa gt gt. (1980) as modified by Ryan gt gt. (1982). -TCDD was dissolved in methanol and added to the enzyme solution as a 10 ul aliquot. RESULTS In an earlier report we showed that various inducers of cytochrome P“50d formed a complex with the enzyme which was stable throughout purification of the enzyme and, in the case of the halogenated aromatic hydrocarbons, could not be readily removed from the cytochrome (Voorman and Aust, 1987). Isosafrole is also a potent inducer of cytochrome P“50d but the isosafrole metabolite is readily displaced from cytochrome P“50d by n-butanol and can be removed by chromatography. The removal of the isosafrole metabolite 71 from our preparation of cytochrome P“50d was confirmed by both the loss of the “55 nm peak of the reduced difference spectrum (Fig. 1) and by a 210% increase in estradiol 2-hydroxylase activity. The addition of TCDD to the hydroxylase assay resulted in an inconsistent pattern of estradiol 2-hydroxylase inhibition when the kinetic data were displayed in double reciprocal plot (Fig. 2). Significant variation occurred in both slopes and intercepts (calculated by the weighted linear regression method of Cornish-Bowden (1979) using the Wilman3 computer program kindly suppled by C. Suelter) at different inhibitor concentrations making interpretation of the inhibition mechanism difficult. Considerable inhibition occurred at 35 nM TCDD and maximum inhibition at 65 nM when the concentration of cytochrome P“50d was 50 nM. Although an accurate Ki could not be calculated from these data, if non-competitive inhibition was assumed, a Ki of approximately 30 nM was obtained. According to Morrison (1969), since the inhibitor exhibits a Ki comparable to the enzyme concentration it should be considered a tight binding inhibitor and thus steady state conditions do not prevail with respect to the inhibitor and a modified form of steady state kinetics is required. That is, formation of the enzyme-inhibitor complex significantly depletes the pool of free inhibitor, and thus alters the concentration of free inhibitor, a condition not allowed for steady state kinetics. 72 450 nm 450 nm Figure 1. Difference spectra of two dithionite-reduced cytochrome P“50d preparations. A. Cytochrome P“50d purified from isosafrole treated rats. Sample cuvet reduced with dithionite (--"9; both cuvets treated with dithionite and C0 bubbled into sample cuvet ( ). B. Same as A but microsomes were incubated with 250 mM n-butanol prior to the first chromatography step. Each sample was scanned from 500 to “00 nm. 73 T 8 K) VT 55’. , O. a ‘5 4 E F- E 3 .. V . g '1 (O E 22'. ‘$// A 5 /‘ I _ '49: I a I“ . - g3 C) :a“‘” ’ 1 l Lil I I m 0 01 02 03 04 05 l/[Eslrcdiol], mm" Figure 2. Lineweaver-Burk plot of effect of TCDD on cytochrome P“50d estradiol 2-hydroxylase. Enzyme activity was reconstituted as described in "Methods" using 50 nM cytochrome P“50d. TCDD was added to solution in 10 ul methanol followed by estradiol in 10 pl methanol: tubes were incubated 3 min at 37° before starting reaction with NADPH. Activity was determined after a 10 min incubation. Concentrations of TCDD were: 0 ( 0)), 36 ( A ); 65 ( D ); and 135 ( V ) nM. Km ranged from 9.6 to 2“.50 pM and Vmax from 6.7 to 1.2 min". 74 Using the methods set forth by Ackermann and Potter (19“9) and as described by Cha (1975), various concentrations of cytochrome P“50d were titrated with various concentrations of the inhibitor and, after a 10 min pre-incubation, assayed for estradiol 2-hydroxylase activity. The data are displayed in the form of an Ackermann-Potter plot where velocity is plotted against total enzyme concentration (Fig. Be). It can be seen that at sufficient inhibitor concentration virtually all of the enzyme activity was inhibited and only when the enzyme concentration exceeded the concentration of the inhibitor was velocity directly proportional to enzyme concentration. The intercepts of the extended linear portion of each curve should represent the amount of enzyme complexed with TCDD for each titration curve. When the intercepts were plotted against respective concentrations of TCDD (Fig. 30) a linear curve was obtained. The slope of the curve (0.8“) indicated that cytochrome P“50d was inhibited (complexed) by TCDD with nearly 1:1 stoichiometry. By combining the method of Henderson (1972), for depletion of free enzyme and inhibitor due to binding, with those of Cheng and Prusoff (1973) and Chou (197“), who derived detailed analyses for the relation of 150 to inhibition constants, Cha (1975) showed that I50 could be used to estimate the Ki for a tight binding inhibitor using the relationship I50 . Et/2 + Ki where Et = total enzyme concentration. The I50's for TCDD inhibition of E2H were 75 h:‘)\(l"( - .3: + ~\Sm . omH an 2: m an o» omamasodmo mm: coop com fix on» can acouumgucoocoo oomzm osocsoouzo o>fiuooanog umcfimmm coupoHa who: m.omH coumSAunm one any .A um comes osoL2005>o uncfimwm AH>V couuomoc oouqnaccfi on» new Ao>v coduomoc Hocucoo on» we suaooao> on» mcfiuuoad >9 ooumsdumo one: m.omH on .zm.o n oaon .mcoaumcucoocoo nous o>fiuooqnoe uncfimmm couuoan one: Aoa nsoficm> um :oHumLucoocoo oomzm osoccoouao uncfimmm zufiooflo> no soda A5H>Huom ossucm .oooe nag: >5H>Huom onmasxoccsnum Hofiumcuno oomam osocnoouao do :ofiumgufie .m ocsmfim 76 ON. .2: Omvd Om ow .2: 000.- 00_ Om Om 0v ON 0 J 4 2... 000... 00. om om 0..» ON 0 O u _ _ . - O K .\. l \ ON ON m .. O o ov cams... . L u . o m cm 00 W \ .8 10m :25 vowed 0: ON. 8. om .8 .01.. ON 0 q . a \\\w.“\“lfl1“ .‘\“\.-\1. O . .8 .. v.0 .md -md .o._ .2583 (damew! 09w WU Iw/Ulw/IOWU H023 77 estimated by plotting the ratio of uninhibited to inhibited E2H activities against TCDD concentration for a series of fixed enzyme concentrations (Fig. 3c). When Vo/Vi - 2 the reaction was inhibited 50%. By the aformentioned equation, a plot of 150 against enzyme concentration will yield the K1 at the ordinate intercept and -2Ki at the abscissa intercept. The data in Figure 3d revealed an average Ki of 8 nM TCDD. Since TCDD binds very tightly to cytochrome P“50d, evaluation of the association rate is of interest. Preliminary experiments showed that inhibition was maximum following a 10 minute pre-incubation of inhibitor and enzyme. E2H activity was measured under conditions when either the enzyme was pre-incubated with inhibitor and reaction started by substrate addition or by addition of inhibitor immediately following substrate solution (Fig. “). Association of the inhibitor with the enzyme was rapid, apparently occurring within the first two minutes following inhibitor addition. In a similar experiment inhibitor was added several minutes after start of the reaction (Fig. “ inset) which again resulted in an immediate (<2 min) maximum change in reaction velocity. DISCUSSION Based on earlier work (Voorman and Aust, 1987) and the present report, it seems likely that compounds which have a 78 36°- 240. //° $30. D/a/° O A 70 ‘520. /° 13 810, ° 0 60,.” o/ 0 g? 0 5 Ami; b :;// (1. EX)__ "5 E 0 g 40.. / c] D o E 30_ O /‘//A [3 § 20__ /3¢A O “J l0_ [3? O l 1 L J l 0 2 4 6 8 l0 min. Figure “. Effect of TCDD on estradiol 2-hydroxylase activity versus time. Enzyme activity reconstituted as described in "Methods." Using 50 nM cytochrome P“50d (.0 ) NADPH was added and reaction started by addition of substrate, aliquots of mixture were quenched at two minute intervals and product formation measured. ( D ) as above but 18nM TCDD added immediately following substrate addition. ( A.) as above but TCDD incubated with sample 5 min at 37° prior to NADPH and substrate addition. Inset. Reaction started by addition of substrate. ( O ) no TCDD; ( U ) 36 nM TCDD added to mixture at “ min time point. 79 structure approximating that of TCDD are likely to induce and very effectively inhibit cytochrome PHSOd. The present work was undertaken to evaluate the affinity of cytochrome PHSOd for TCDD. Indeed, we have shown that TCDD can act as a titrating inhibitor of cytochrome PHSOd. That is, the inhibitor tends to bind fully to the enzyme when inhibitor concentration is below that of the enzyme. Thus, addition of the enzyme to a comparable solution of inhibitor will significantly deplete the pool of free inhibitor. We used a modified form of steady state kinetics which presupposes non-steady state conditions with respect to the concentration of inhibitor and, by estimation of 150 at infinitely low enzyme concentration, calculated a KI of 8 nM. The significance of this observation is not clear. It is difficult to envision any role it could play in TCDD toxicity: the KI for inhibition is an order of magnitude higher than the KD for TCDD binding to the Ah receptor (Poland et a1., 1976) and the concentration of TCDD required to cause toxicity (10-100 nmol/kg) is below the level which would saturate the binding capacity of cytochrome PHSOd. Nevertheless we have shown that when TCDD is given at 1 nmol/kg it is significantly associated with cytochrome PHSOd (Voorman and Aust, 1987). Others have shown that high concentrations of pentachlorodibenzofuran in the liver following treatment with that compound are associated with cytochrome PMSOd (Kuroki et a1., 1986). 80 In vitro experiments with TCDD and similar hydrophobic compounds pose a problem in that their distribution in a lipid suspension, such as occurs with microsomes or reconstituted cytochrome PhSOd, will result in micro heterogenieity as TCDD would be found only in the lipid phase. In this respect, one could argue that the inhibitor would be directed to the active site of cytochrome PhSOd since the active site is a very hydrophobic domain and cytochrome PHSOd itself would be found in the lipid phase, thus enhancing the binding of TCDD to the cytochrome. Although these factors would contribute to the rapid association of TCDD with cytochrome PHSO, there is considerable structural specificity for inhibition of cytochrome PhSOd that appears to be limited by steric constraints rather than hydrophobicity. We found that isomers of hexabromobiphenyl had inhibition constants that varied over 700 fold depending on chemical structure (Voorman and Aust, 1987). Compounds which exhibit tight binding inhibition are typically substrate or transition state analogues. It is difficult to make any inferences about the structure of TCDD in this regard since so little is known about either the active site of cytochrome PHSOd or the substrate(s) for the enzyme. Johnson et a1. (1986) have developed a mechanism based inhibitor of rabbit cytochrome PHSO 1, an enzyme with high progesterone 21-hydroxylase activity wherein an amino group on the 178 side chain of pregnenolone 81 can coordinate with heme iron. Kellis and Vickery (198R) have shown that certain naturally occurring flavanoids can inhibit human placental aromatase (a cytochrome PHSO isozyme) with a very low 150. Recently the same group (Kellis et a1., 1987) showed that two steroid analogs with oxirane or thiirane side groups bound not only to the substrate binding site, but coordinated to heme iron and showed Ki's of 2-10 nM. It is believed that the halogens of carbon tetrachloride ligate directly to heme iron of cytochrome PHSO, in place of oxygen, and that the chemical is reductively metabolized by cytochrome PHSO (Nastainczyk_et §;,, 1982). The halogens of TCDD could interact specifically with heme iron in a similar manner. TCDD was metabolized in 1112 and in 33333 (Sawahata et a1., 1982; Ramsey et a1., 1982) although metabolic transformation occurred very slowly. Thus the binding and subsequent slow metabolism of TCDD by cytochrome PDSOd remains a possibility. Ligation to heme seems unlikely however, since no spectral perturbations were observed when hexabromobiphenyl was bound to cytochrome PHSOd and CO was not inhibited from binding the reduced cytochrome (unpublished observations). It is most likely that TCDD simply fits the substrate binding site very well but is not susceptible to metabolism. The inhibitor would in this case be competitive, but since binding is tight and the off-rate so slow, the inhibitor 82 inactivates the enzyme and behaves as a non-competitive inhibitor. In effect it acts like a suicide substrate except that a covalent bond is not formed between enzyme and substrate. REFERENCES Ackermann, W.W., and Potter, V.R. (19h9) Enzyme inhibition in relation to chemotherapy. Proc. Soc. Exptl. Biol. Med. 1;. 1-9- Astrom, A., and DePierre, J.W. (1985) Metabolism of 2-acetyl-aminofluorene by eight different forms of cytochrome PHSO isolated from rat liver. Carcinogensis 9, 113-120. Cha, S. (1975) Tight binding inhibitors-I; Kinetic behavior. Biochem. Pharmacol. g3, 2177-2185. Cheng, T.C., and Prusoff, W.H. (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 percent inhibition (150) of an enzymatic reaction. Biochem. Pharmac. 22, 3099-3108. Chou, T.C. (197A) Relationships between inhibition constants and fractional inhibition in enzyme-catalyzed reactions with different numbers of reactants, different reaction mechanisms, and different types and mechanisms of inhibition. Molec. Pharmac. lg, 235-2u7. ' Cornish-Bowden, A. (1979) Fundamentals of enzyme Kinetics. Butterworths, London. Dignam J.B., and Strobel, R.W. (1977) NADPH-cytochrome PHSO reductase from rat liver: Purification by affinity chromatography and characterization. Biochem. 18, 1116-1123. Fisher, G.J., Fukushima, H., and Gaylor, J.L. (1981) Isolation, purification, and properties of a unique form of cytochrome PHSO in microsomes of isosafrole-treated rats. J. Biol. Chem. gig, ”388-R39". Guengerich, F.P., Dannan, C.A., Wright, S.T., Martin, M.V., and Kaminsky, L.S. (1982) Purification and characterization of liver microsomal cytochrome PHSO: Electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or B-Naptho flarone. Biochem. 21, 6019-6030. Henderson, P.J.F. (1972) A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem. J. 121, 321-333. 83 81+ Imai, I., Hashimoto-Yutsudo, C., Satoke, H., Girardin, A., and Sato, R. (1980) Multiple forms of cytochrome PMSO purified from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rabbits. I. Resolution, purification, and molecular properties. J. Biochem. 88, u89-503. Johnson, E.F., Schwab, C.B., Singh, J., and Vickery, L.E. (1986) Active site-directed inhibition of rabbit cytochrome PHSO by amino substituted steroids. J. Biol. Chem. 881, 1ozou-1o2o9. Kellis, J.T., Jr., Childers, W.E., Robinson, C.H., and Vickery, L.E. (1987) Inhibition of aromatase cytochrome PHSO by 10-oxirane and 10-thiirane substituted androgens: Implications for the structure of the active site. J. Biol. Chem. 888, uu21—uu26. Kellis, J.T., and Vickery, L.E. (198R) Inhibition of human estrogen synthetase (aromatase) by flavone. Science 225. 1032-103”. “—— Kuroki, J., Koga, N., Yoshimura, H. (1986) High affinity of 2,3,h,7,8-pentachlorodibenzofuran to cytochrome P450 in the hepatic microsomes of rats. Chemosphere 18, 731-738. Morrison, J.F. (1969) Kinetics of the reversible inhibition of enzyme-catalyzed reactions by tight-binding inhibitors. Biochem. Biophys. Acta 185, 269-286. Morrison, J.F. (1982) The slow-binding and slow, tight-binding inhibition of enzyme-catalysed reactions. Trends Biochem. Sci. 1, 102-106. Nastainczyk, W., Ahr, H.J., and Ullrich, V. (1982) The reductive metabolism of halogenated alkanes by liver microsomal cytochrome PHSO. Biochem. Pharmacol. 81, 391-396. Numazawa, M., Kiyono, Y., and Nambara, T. (1980) A simple radiometric assay for estradiol 2-hydroxylase activity. Anal. Biochem. 10“, 290-295. Philpot, R.M., Hodgson, E. (1972) The effect of piperonyl butoxide concentration on the formation of cytochrome PASO difference spectra in hepatic microsomes from mice. Mol. Pharmac. 8, zou-21u. Poland, A., Glover, E., Kende, A.S. (1976) Stereospecificity high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol: evidence that the binding species is receptor for induction of arlyl hydrocarbon hydroxylase. J. Biol. Chem. 881, u936-u9u6. 85 Ramsey, J.C., Hefner, J.C., Karbowski, R.J., Braun, W.H., and Gehring, P.J. (1982) The 18 vivo biotransformation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat. Toxicol. Appl. Pharmacol. 88, 180-18“. Reik, L.M., Levin, W., Ryan, D.E., and Thomas, P.E. (1982) Immunochemical relatedness of rat hepatic microsomal cytochromes PHSOc and PHSOd. J. Biol. Chem. 257, 3950-3957. Ryan, D.E., Thomas, P.E., and Levin, W. (1980) Hepatic microsomal cytochrome PRSO from rats treated with isosafrole. J. Biol. Chem. 255, 79N1-7955. Ryan, D.E., Thomas, P.E., and Levin, W. (1982) Purification and characterization of a minor form of hepatic microsomal cytochrome PMSO from rats treated with polychlorinated biphenyls. Arch. Biochem. Biophys. 818, 272-288. Sawahata, T., Osoh, J.R., and Neal, R.A. (1982) Identification of metabolites of 2.3.7,8-tetrachlorodibenzo-p-dioxin (TCDD) formed on incubation with isolated rat hepatocytes. Biochem. Biophys. Res. Comm. 122, 3u1-3u6. TuteJa, N., Gonzalez, F.J., Nebert, D.W. (1985) Developmental and tissue-specific differential regulation of the mouse dioxin-inducible P1-450 and P3-u50 genes. Developmental Biology 118, 177-18u. Voorman, R., and Aust, S.D. (1987) Specific binding of polyhalogenated aromatic hydrocarbon inducers of cytochrome PHSOd to the Cytochrome and inhibition of its estradiol 2-hydroxy1ase activity. Toxicol. Appl. Pharmacol., in press. Whitlock, J.P., Jr. (1986) The regulation of cytochrome PHSO gene expression. Ann. Rev. Pharmacol. Toxicol. 88, 333-369. Wroblewski, V.J., and Olson, J.R. (1985) Hepatic metabolism of 2.3.7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat and guinea pig. Toxicol. Appl. Pharmacol. 81, 231-2uo. Yasukochi, Y. (1976) Some properties of a detergent solubilized NADPH cytochrome c (cytochrome PHSO) reductase purified by biospecific affinity chromatography. J. Biol. Chem. 881, 5337-53uu. CHAPTER III INDUCERS OF CYTOCHROME PHSOd: INFLUENCE ON MICROSOMAL I VIVO CATALYTIC ACTIVITY AND DIFFERENTIAL REGULATION BY ENZYME STABILIZATION 86' is :1- f N ABSTRACT The 18 1118 significance of the interaction of isosafrole, 3,4,5,3',A',S'-hexabromo-, and hexachlorobiphenyl on cytochrome P450d was evaluated enzymatically by determination of microsomal estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomes derived from isosafrole treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully removed by incubation with 1 uM HBB. Although isosafrole is capable of forming a complex with many differernt cytochrome PHSO isozymes, it apparently binds significantly only to cytochrome P450d 1Q 1119 as was demonstrated by measuring the enzymatic activity of microsomal cytochrome PhSOd, PHSOc and P450d from isosafrole treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in enzyme activity despite a 20-fold increase in cytochrome PHSOd over the dose range. The ability of cytochrome PMSOd ligands to stabilize the enzyme was investigated in two ways. First, cytochromes PHSOC and Pfl50d were immunochemically measured in microsomes from rats treated with TCDD, at a dose to maximally induce total cytochrome PHSO, followed by a single dose of a potential enzyme stabilizing inducer. The specific content of cytochrome Pu50d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the 87 88 relative turnover of cytochrome PMSOd was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled 1g lilB with 3H-leucine, the second inducer was given and protein was again labeled 3 days later with 1"‘C-leucine. Immunoprecipitation by cytochrome PHSOd from the various treatment groups revealed a higher ratio of 3H/1uC in the ISF- and HCB-treated rats relative to TCDD (control) treated rats suggesting that isosafrole and HCB were able to retard the degradation of cytochrome PASOd, presumably by virtue of being tightly bound to the active site. INTRODUCTION Cytochrome PHSO is a family of monooxygenases which activate dioxygen to hydroxylate a variety of structurally diverse substrates. These may be bound to the enzyme in either a highly specific manner, as in the case of adrenal steroids, or a relatively non-specific manner, as apparently happens with many xenobiotics. Certain cytochrome PHSO isozymes are induced in respone to presumed xenobiotic substrates. Induction of cytochromes PHSOc and PHSOd is thought to be controlled via the Ah receptor, a cytosolic protein for which certain aromatic hydrocarbons have an unusually high affinity (Nebert and Gonzalez, 1985). Recently, it has been shown that certain compounds which induce cytochrome PHSO also bind to the enzyme and inhibit its catalytic activity (Voorman and Aust, 1987). Isosafrole is a potent inducer of cytochrome PHSOd and is metabolized by the enzyme to form a metabolite-intermediate complex which inhibits the catalytic activity of cytochrome PMSOd (Ryan §L_gl., 1980; Murray et_al., 1986). It has been suggested that isosafrole does not induce cytochrome P450d ‘via the Ah receptor but regulates cytochrome PHSOd synthesis Lin another manner (Cook and Hodgson, 1985). Steward g; _1. C 1985) has shown that isosafrole can prolong the half-life 01’ cytochrome PMSOd in primary cultures of rat hepatocytes. HOwever, in rat liver microsomes there was no evidence for a CPIange in the half life of cytochrome PMSOd relative to 89 90 other cytochrome PHSOS induced by 5,6-benzoflavone (Shiraki and Guengerich, 198A). In this report it is shown that isosafrole and 3,“,5,3',H',5'-hexachlorobiphenyl (HCB) induce and inhibit cytochrome PUSOd in the rat liver. Both isosafrole and HCB have an additive inducing effect on cytochrome PNSOd beyond that maximally induced by 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD). The additive effect is probably due to a prolonged half life of the enzyme resulting from stabilization of the enzyme by the bound inducer. METHODS AND MATERIALS Chemicals Isosafrole was obtained from Fluka (Ronkonkoma, NY), 3,H,5,3',N',5'-hexachlorobiphenyl was purchased from Pathfinder Laboratories (St. Louis, MO); 2-[3HJ-estradiol, was obtained from New England Nuclear (Boston, MA). TCDD was generously supplied by Dow Chemical Company (Midland, MI). 3,N,5,3',u',5'-Hexabromobiphenyl was obtained from Ultra Scientific, Inc. (Hope, RI) and purified by alumina column chromatography (Millis, 198A). L-[u,5-3H]Leucine (sp. act. 1A6 Ci/mmol), L-[U-1HCJleucine (sp. act. 3&2 mCi/mmol) and NCS tissue solubilizer were obtained from Amersham (Arlington Heights, IL). Goat anti-rabbit peroxidase complex was purchased from Capped Labs (Malvern, PA). 91 Animal Treatment Male Sprague-Dawley rats (Charles River, Portage, MI) were treated with the inducing chemical dissolved in corn oil by oral gavage as described in figure legends. Rats were starved overnight and killed by decapitation following C02 anesthesia; livers were homogenized in 1.15% r- KCl, 10 mM EDTA, pH 7.u (1 u, w/v). Microsomes were . ’cin":~ .0' prepared by differential centrifugation, washed with 100 mM sodium pyrophosphate, pH 7.“, containing 0.1 mM EDTA and suspended in 20 mM potassium phosphate, pH 7.“, containing Imam. _ "mi -.\- N 20% glycerol, and 0.1 mM EDTA and stored at -20°C. For the pulse labeling studies, animals were treated with a single dose of TCDD followed three days later by an ip dose of 3H-leucine (300 uCi/ml in 0.9% NaCl) at 3 mCi/kg. Three hours after labeling, animals were dosed by oral gavage with the second inducer. Exactly three days after the 3H-leucine dose, animals were treated with 1“C-leucine (50 uCi/ml in 0.9% NaCl) at 500 uCi/kg and killed H hr later. Microsomes were prepared as described above. Assays Ethoxyresorufin-O-dealkylase (EROD) and pentoxyresorufin-O-dealkylase (PROD) were measured by a modified method of Pohl and Fouts (1980). Samples (5-50 ug microsomal protein) were prepared for a final volume of 1 ml in 100 mM potassium phosphate, 0.1 mM EDTA, pH 7.8 buffer. 92 Substrate was added in 10 ul methanol (standardized by EA82 = 22.5 mM‘1 cm'1) and following 3 min incubation at 37°C, the reaction was started by addition of an NADPH generating system. The reaction was terminated after 3 min by addition of 2 ml 50% methanol. Following centrifugation, resorufin fluorescence of the supernatant was measured at 585 nm using excitation at 530 nm and compared to a resorufin standard in buffer (standardized by EH72 =‘73 mM'"1 cm"). Estradiol-Z-hydroxylase was measured by the method of Numazawa (1980) as modified by Ryan (1982). Protein was determined by the bicinchoninic acid (Pierce Chemical, Rockford, IL) micromethod (Redinbaugh, 1986) and stardardized with bovine serum albumin. Cytochrome PHSOC and Pu50d were quantified by ELISA as described (Voorman and Aust, 1987). Immunoprecipitation of cytochrome Ph50d was done by suspending 50-200 ug microsomal protein in 300 pl 0.1 M potassium phosphate, pH 7.u, containing 0.” M KCl, 0." mM EDTA, 0.5% cholate, 0.1% Emulgen 911, 0.02% azide and 1% BSA and adding 100-300 ul anti-serum against cytochrome Pu50d. This mixture was incubated overnight at A°C, and spun in a centrifuge. The immunoprecipitate was washed once with the buffer Just described and once with water. The pellet was dissolved in NCS tissue solubilizer, added to scintillation cocktail and 3H/1”c determined in a Beckman LS 5801 liquid scintillation counter. 93 Total cytochrome PASO content was measured by absorption at -H50 nm of the dithionite-reduced CO difference spectrum using ENSO-A9O - 91 mM’1 cm'1. The isosafrole complex was measured spectrophotometrically by reducing the sample cuvet with dithionite and measuring EH55_q90 -75 mM'1 cm". RESULTS Effect of Isosafrole on Estradiol 2-Hydroxylase Previously it was shown that HCB, when incubated with purified cytochrome PHSOd, could inhibit cytochrome pusoa estradiol 2-hydroxylase by forming a tightly bound complex with cytochrome Pu50d. When microsomes from isosafrole treated rats were incubated with n-butanol and chromatographed on Sepharose Cl-6B to remove the isosafrole metabolite and n-butanol there was a 160% increase in estradiol 2-hydroxylase activity relative to non-butanol treated microsomes (Figure 1). The estradiol 2-hydroxylase activity in the non-butanol treated sample is likely due to other constitutive isozymes of cytchrome Pu50 since another potent inhibitor of cytochrome Pu50d, HBB, was able to titrate out the estradiol 2-hydroxylase due to cytochrome Pu50d. This suggests that HBB is able to inhibit cytochrome PASOd to the same degree as isosafrole even though it has a considerably different structure. 94 A I EZOH nmol/min/mg protein m . it OJ l .5» ”A 1— \Ok . B O 1 1 ' l l .-c O ’ I I0 I00 IOOO. nM HBB Figure 1. Effect of HBB on microsomal estradiol 2-hydroxylase from isosafrole treated rats. Male Sprague-Dawley rats (150-250 gm) were treated with isosafrole (150 mg/kg) by oral gavage once a day for three days and killed on the fourth day following overnight starvation. Hepatic microsomes were isolated, and an aliquot was treated with 250 mM n-butanol at 37° for 30 min followed by chromatography to remove the isosafrole metabolite. Estradiol 2-hydroxylase in 50 pg protein was measured with the isosafrole metabolite intermediate complex still bound (-o-) or following displacement and removal of the metabolite (-D-). —r' t. u l 95 The effect of isosafrole on other microsomal cytochrome P450 isozymes was evaluated using relatively specific substrates for cytochrome Pu50b, c, and d, each isozyme known to be induced by isosafrole (Table 1). In each case there was a significant increase in enzymatic activity following isosafrole displacement by butanol, however the magnitude of increase varied depending on substrate and isozyme contribution. Estradiol 2-hydroxylase, measures both Ph50d and PASOh (Astrom & Depierre, 1985), therefore, the u7z increase in activity is due to cytochrome PASOd. The smaller increase in activity compared to Figure 1 is probably due to a lower specific content of cytochrome Pu50d. Ethoxyresorufin-o-dealkylase is catalyzed almost exclusively by cytochrome Pfl50c (Astrom & DePierre, 1985). Thus, the high rates observed in each treatment group for this activity are in all likelihood due to cytochrome Pu50c, apparently largely uncomplexed with isosafrole prior to treatment with butanol. The 30% increase in. ethoxyresorufin- o-dealkylase following isosafrole displacement is probably due not only to the freeing of some cytochrome Pu50c but also to cytochrome Pu50d which has about 10% the ethoxyresorufin-o-dealkylase activity of cytochrome Ph50c. Pentoxyresorufin-o-dealkylase, specific for cytochrome Pu50b (Burke 35.314. 1985), showed 31% increase in activity following displacement of the isosafrole metabolite. 15W - __. 96 TABLE 1. Catalytic activity of microsomes from isosafrole treated rats. Rats were treated for 3 days with isosafrole (150 mg/kg/day); microsomes were isolated and either used without further treatment or chromatographed with or without preincubation in 250 mM n-butanol. Conditions for enzyme assays were as described in Methods. Numbers in parenthesis are standard deviations (n-3). "in. 4...!!1 Activity (nmol/min/mg protein) Chromatographed Untreated Control +BuOH Estradiol 1.29 1.u8 2.18* 2-hydroxylase (0.06) (0.11) (0.2u) Ethoxyresorufin- 8.29 8.70 11.3* O-deethylase (0.28) (0.“2) (1.1 Pentoxyresorufin- 0.68 0.6H 0.8A* O-depentylase (0.03) (0.07) (0.05) *Significantly different from controls by Duncan's multiple range test at 5% level. 97 Effect of HCB on Microsomal Estradiol 2-Hydroxylase Although an endogenous substrate for cytochrome PHSOd is not known, it was of interest to assess the effect of inducers which bind to Pu50d on the metabolic activity of PhSOd. Thus, estradiol 2-hydroxylase was evaluated over a log-dose range of HCB (Figure 2). It can be seen that, in general, estradiol 2-hydroxylase activity decreased with increasing dose of HCB despite the 20 fold increase in level ‘13. 411‘. H.J‘Vltif. 1 of cytochrome Pu50d and 2.5-fold increase in total cytochrome PHSO. The activity of estradiol 2-hydroxylase at the control dose (no HCB) is probably due to other cytochrome PMSO isozymes including cytochrome Pu50h. The decrease in enzyme activity to below the control level could be due to a repression of cytochrome PHSOh synthesis since it was shown that levels of this isozyme decrease in response to certain polybrominated biphenyls (Dannan 35 31., 1982). We showed earlier that the bromo analogue of HCB did not inhibit cytochrome PHSOh (Voorman & Aust, 1987). Effect of HCB and Isosafrole on Regulation of Cytochrome 115.9 Cytochrome Ph50c and PUSOd are induced in the rat by TCDD in a dose-dependent fashion with maximum induction occurring at 10 nmol TCDD/kg body weight (Fig. 3). Like many inducers of cytochrome PNSO, induction was maximal three days following dosage and did not change significantly over the 12-day period (Figure A). The effect of isosafrole 98 .Amuov caouoga m: om so“: cogsmmme mm: ommaaxogoznum Hofiomzumm .Ao no ucmucoo caufiooam new Aonov acmucoo ouuuomam omzm msogcoouao Hmuou Lou zmmmm new noumaomfi one: Hmeomogofie Lo>fiq .cofium>gmum usmficgo>o mcazoaaom gonna mama moss» uoaafix new czonm momoo one am owm>mm Hugo >9 mu: nag: acumen» one: Asm ms—ev mum; moazmouosmmgqm mam: .mnmamxogoanum Hoqcmgpmo cam ocopcoo oncfiooam omen msotcoosho co ascmnaflnogoanomxmnu.m..z..m.m.=.m Lo somucm .N mgsmfim 99 (ugaimd 6w/|ou.lu) 130de awOJuooiKQ 00. «2 V. N — O o o o’ O l | I I I ' —' (ugaimd Dw/ugwnowu) H023 \ o Ox;;{ 1 1 1 0 OJ L0 10 100 TCDD, mum/kg Figure 3. Effect of TCDD on cytochrome PASO specific content. Male Sprague-Dawley rats (-125 gm) were treated with a single dose of TCDD at the levels shown. Animals, three per treatment group, were killed 12 days later, hepatic microsomes were isolated, and specific content of cytochrome Pu50 was determined. Total cytochrome PNSO (m-u); cytochrome PASOc (A-A); cytochrome PASOd (o-o). ix) .0 .p. Cytochrome P450, nmol/mg protein .0 on Figure A. 101 O 3 6 9 I2 Days post dose Effect of TCDD on cytochrome PNSO specific content over time following single dose of TCDD. Rats were given a single dose of TCDD (10 nmol/kg) as described in Figure 3, and killed 3, 7, and 12 day following dosage. Microsomes were prepared and cytochrome PASO measured as described in "Methods." JS PO Cytochrome P450, nmol/mg protein CH 0 Figure 5. 102 I: TCDD * Oil TCDD * MC [mm TCDD * ISF E TCDD" HCB Total P4500 P450d C ytochrome P450 Effect of TCDD plus 3-methylcholanthrene, isosafrole, and HCB on levels of cytochrome PMSO. Male Sprague-Dawley rats (~150 gm) were treated with TCDD (10 nmol/kg) by oral gavage; three days later the previously treated rats were given a single dose of a second inducer or corn oil control (3 rats per treatment group). Rats were killed 2“ hr later following overnight starvation and liver microsomes were prepared. Total cytochrome PASO represents that determined spectrally by the dithionite-reduced CO difference spectrum and, in the case of isosafrole, also includes the metabolite-intermediate complex. Cytochromes PMSOc and PASOd were quantitated immunochemically as described in "Methods." 103 and HCB on the regulation of cytochrome PASOc and PHSOd was’ examined in rats previously treated with TCDD (Figure 5). Rats given corn oil or 3-methylcholanthrene showed no significant difference in the level of cytochrome Pu50c or Pu50d. However, rats given isosafrole or HCB in addition to the earlier dose of TCDD showed a marked increase in cytochrome PASOd content, but not cytochrome Pu500. Rats were treated with HCB at a concentration to produce maximum induction of cytochrome PASOd (see Figure 2). Quantitation of microsomal HCB content and cytochrome PASOd content in the 100 nmol/kg group in the dose-response experiment (Figure 2) revealed a 0.7:1 association HCB to PhSOd. Microsomes from the isosafrole-treated animals clearly showed the presence of the isosafrole metabolite-intermediate complex when spectrally assayed. The isosafrole-treated rats had significantly increased specific content of total cytochrome Pu50 (including measurement of the metabolite-intermediate complex) but the HCB-treated rats did not. The lower total PASO content in the latter group could be due to the presence of apo-cytochrome PH50. The stabilizing effect of inducing ligands on cytochrome PASOd was investigated by labeling cytochrome PASOd, following its induction by TCDD, using the Schimke method (Arias 38 El-v 1969). It seemed likely that if certain ligands could stabilize cytochrome Pu50d, then labeling of the cytochrome PASOd pool, followed by addition 10]. £00 CI] TCDD + OIL 3 50‘ TCDD + ISF ' m TCDD + HCB E? 1100- } 2.50~ <3 C: 1100— d 1.50— E {,3 E2 y-zg 3: CL ‘LOO- "T g; g; E? 5=E§ E§ 2 =3 its 0-50- g as; :5: 5 ::: it? 5 :5 :3: 4 :92 a. 000 Total P4500 Figure 6. Effect of isosafrole and HCB on cytochrome PN50 specific content following treatment with TCDD. Treatments were as described in Table 2. Cytochrome PMSO was measured as described in Figure 5. 105 of the stabilizing ligand, should result in an increased half life of the labeled cytochrome PN50d. The levels of cytochrome PASOc and PASOd were similar to those of the last experiment, except that now the increased level of cytochrome PA50d in the HCB-treated group is reflected in the total specific content of cytochrome PHSO (Figure 6). Significant differences in the incorporation of radioactivity into cytochrome Pu50d were apparent when the immunoprecipitation was carried out based on either PASOd or protein concentration (Table 2). By virtue of the increased ratio of 3H/1"c in the isosafrole- or HCB treated rats, it seems likely that these compounds increase the half-life of cytochrome pusoa in the rat. DISCUSSION Our results suggest that even though isosafrole can form the metabolite-intermediate complex 12 11812 with several cytochrome PASO isozymes (Ryan 2E 31., 1980), the binding and inhibition were significant only with regard to cytochrome PHSOd. Apparently HBB, a structurally-dissimilar chemical, was able to inhibit estradiol 2-hydroxylase to the same degree as isosafrole. It has been suggested that isosafrole also binds significantly to and inhibits cytochrome PASOb (Murray 22 _1., 1986) as determined by a four-fold increase in androstenedione 16B-hydroxylation following isosafrole displacement. The results in Table I 106 F:......u..r.........1 .. 71.1.. . to... ma.~ mum Fem _ =e.m ea: mmm. ma._ omo.m_ mem.e~ mu: + cove e_.m mom ego oo.m ozm pam— =¢.L Fe..=F mem.em aflogcmmomH + once z_.~ zmm =.e mm.P mam emm mm._ om~.:_ oce.mm file + oooe oqumm oz. mm ofismm 0:. mm oasmm 0:. am scmssmmge .uqaa :H saw .pQQH ca saw :Hmuona ms\sqc oomaa oomza mmaomogofiz .0: new mm Lou coamnnm cam .omnfiafiosaom .omnmmz mm: oumuuaaomea one .c«muogq m: oomuom song counu~q~omgaoczeeH one: oomzm ocm comzm mesonsoouzo .oz_ new mm mcducsoo new Loudafinsaom manna» moz cu :Hmuoga m: ooauoom no :oqumnfiadasaom >2 nogsnmoe mm: :Hmuona Hmsonogofis Hmuou :H >a~>fiuomofiomm =.mco:umz= :« condgommc mm oopmamnq mam: mmeomoLoHs odomqmz .Louma L: z uoaaax ocm ocuosoanoz— venoumficfisom one: mameacm Ham .ocuozoauzm on» Logan unso: mp .Amxxaoe: copy mu: no wa\ms ompv maogummomfi .Hfio :Loo >9 zaoumacossfi omzoaaou .ocaosoaumm no“: Ansogm Lon NV acumen» one: mamsacm .Louma ammo mouse .Amxxaoa: on nook no“: acumen» one: “am oonomv mam; zmazmauosmmzam mam: .omzm meogzoouao no Lo>ocgsu on» no memosocu no uoouum .m magma 107 agree with this in part; however, the magnitude of increase (30%) was not as great as previously observed by Murray 31 31. (1986). The results agree with a previous report indicating that isosafrole forms a stable complex only with cytochrome PASOd (Ryan 31 31., 1980). Also, we found that the isosafrole metabolite was detected only in cytochrome Pu50d but not cytochromes PASOc or Pu50h purified from isosafrole treated rats (data not shown). Hexachlorobiphenyl induces cytochrome PASO in a dose- dependent manner and, in accordance with earlier results, binds to and apparently inhibits cytochrome Ph50d estradiol 2-hydroxylase. A recent report suggests that NADPH-cytochrome PASO-reductase can become limiting in cytochrome Pu50d catalyzed reactions (Graham 31 31., 1987). Although this phenomenon could have occurred in our experiment, there should still have been an increase estradiol 2-hydroxylase as was observed upon displacement of isosafrole from cytochrome PASOd in Figure 1. It has been known for some time that cytochromes PHSOc and PA50d are induced coordinately by numerous xenobiotics. The genetic studies using the mouse (Gonzalez 31 31., 198A) suggest that induction of both proteins is controlled from the Ah receptor at the level of transcription. The receptor has been found in five tested mammalian species and has significant homology across these species (Gasiewicz and Rucci, 198A). In addition, cytochrome PASOc and PHSOd have been identified in variety of species including humans and 108 also share significant homology across species (Thomas 31 31., 198A; Jaiswal 31 31., 1985; Quattrochi 31 31., 1985). Other data, however, suggest that the regulation of the two isozymes is differential, depending on the inducer (Thomas _1 31., 1983; Dannan 31 31., 1983; Parkinson 31 31., 1983). That is, the relative levels of the two isozymes can vary considerably in response to structurally diverse chemicals. Other factors, both 313-acting (on the gene) and 13333- acting (receptor level) have been suggested as controlling gene expression (Jones 31 31., 1985), but demonstration of inducer interaction with these factors resulting in altered gene expression appears limited to the Ah receptor. The induction of cytochrome PA50 elicited by isosafrole is an enigma. This compound has a low degree of structural similarity to the high affinity ligands of the Ah receptor, yet it is a very potent inducer of cytochrome PASOd. Although it has been assumed that it could serve a ligand for the Ah receptor (the methylene dioxyphenyl structure is aromatic in nature), recent data suggest that it is not a competitive ligand (Cook and Hodgson, 1985) and thus, casts doubt on its ability to regulate cytochrome PHSOd synthesis through the Ah receptor. Moreover, other experiments suggest that isosafrole may act in an additive, or even synergistic, manner with the Ah ligand 3-methylcholanthrene (Fennel 31 31., 1979; Thomas 31 31., 1983). In these experiments, animals were treated for one day with isosafrole following three days treatment with . ' 3m.-. 109 3-methylcholanthrene. This resulted in greater levels of cytochrome PASOd than obtained with three days treatment of isosafrole or methylcholanthrene above. Implicit in these results is that isosafrole might have an effect on cytochrome Pu50d beyond that controlled by the Ah receptor, assuming, of course, that methylcholanthrene elicited the maximum response obtainable through the Ah receptor. r Certain polychlorinated and polybrominated biphenyls are 9 also potent inducers of cytochrome Pfl50d, and are unusual in that they induce cytochrome PHSOd to greater levels than cytochrome PASOc (Parkinson 31 31., 1983; Ozawa 31 a1., rn-D.‘3‘:“".I- ‘ x . .1 1979). Several of these inducers can form stable complexes with cytochrome PHSOd (Voorman and Aust 1987; Kuroki 31 31., 1986). It seemed possible that compounds which induce and bind to cytochrome Ph50d might also stabilize the enzyme against degradation and prolong its half life. To test this possibility, TCDD was used to induce cytochrome Ph50d followed by treatment with potential enzyme-stabilizing ligands. The dose of TCDD was sufficient to induce maximum levels of cytochrome Pu50d, and presumably saturate the Ah receptor, yet the dose was low enough that there was approximately a hundred-fold excess of hepatic cytochrome P450d over the total body burden of TCDD. Therefore, even though TCDD can bind to cytochrome PASOd, it could not . stabilize a significant part of the pool of cytochrome PASOd. TCDD treatment will result in prolonged occupation of the Ah receptor and continued induction of cytochrome PHSOd. Thus, 110 an additional ligand for Ah receptor should not have an additive inducing effect with TCDD. Indeed, 3-methylcholanthrene, a ligand for the Ah receptor had no additional inducing effect when given to rats following TCDD treatment. This was in contrast to the additive induction of cytochrome PASOd by both isosafrole and HCB suggesting control of cytochrome PHSOd beyond the level of the Ah F‘ receptor. To test the possibility that the stabilizing ligands might effect the turnover or half life of the protein, the double label technique of Schimke was used to measure 13 II 1113 protein degradation. The double label technique allows measurement of protein degradation in a single animal, eliminating significant inter-animal variation and also measures relative rather than absolute rates of change (Arias 31 31., 1969). There was greater retention of the first label [3H] relative to the second label [1"C] in the isosafrole- and HCB-treated rats. These differences in ratio were apparent in the specific activity of total microsomal protein, and the differences were magnified upon precipitation of cytochrome PMSOd. Primary cultures of rat hepatocytes have the capacity to synthesize cytochrome PASOc but, for unknown reasons, not cytochrome Pu50d (Steward 31 _1., 1985). When hepatocytes were isolated from rats previously treated with agents to induce cytochrome PMSOd, the level of the enzyme decreased to zero over three days. However, if isosafrole was added 111 to the culture medium degradation was significantly retarded, indicating stabilization of cytochrome PASOd by isosafrole. The macrolide antibiotic, tricetyloleandomycin (TAO), greatly increases levels of cytochrome Pu50p, a glucocorticoid responsive enzyme (Watkins 31 _1., 1986). TAO forms a metabolic intermediate complex with PHSOp and when given with dexamethasone, increases levels of cytochrome Pu50p about three fold over that maximally induced by dexamethasome alone. Measurement of cytochrome PASOp degradation by both 13 1113 and 13 11133 pulse labeling, revealed a significant decrease in that rate of cytochrome PASOp degradation in response to TAO treatments. The mechanism by which these compounds could inhibit cytochrome PASO degradation is unknown. Substrate-stabiliza- tion of enzymes has been demonstrated with arginase and tryptophane pyrrolase (Schimke 196A; Schimke 31 31., 1965). It is possible that degradation of cytochrome PHSO is controlled in part by heme oxygenase (Sadler 31 31., 1986). Cytochrome P450 and its heme turn over at the same rate (Parkinson 31 _1., 1983a). Thus, a substrate (or inhibitor) that prevents access to heme or prohibits its removal, could control degradation of the enzyme. 112 REFERENCES Arias, I.M., Doyle, D. and Schimke, R. (1969) Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J. Biol. Chem. 2AA:3303-3315. Astrom, A., and DePierre, J.W. (1985) Metabolism of 2-acetyl-aminofluorene by eight different forms of cytochrome PASO isolated from rat liver. Carcinogenesis 6:113-120. Burke, M.D., Thompson, S., Elcombe, C.R., Halpert, J., Haaparanta, T., and Mayer, R.T. (1985) Ethoxy-, pentoxy- and benzyl-oxyphenazones and homologues: a series of substrates to distinguish between different induced cytochromes PASO. Biochem. Pharmacol. 3fl:3337-33M5. Cook, J.C., and Hodgson, E. (1985). The induction of cytochrome PASO by isosafrole and related methylene dioxyphenyl compounds. Chem.-Biol. Interactions 5h:299-315. Dannan, G.A., Gungerich, F.P., Kaminsky, L.S. and Aust, S.D. (1983) Regulation of cytochrome PASO. Immunochemical quantitation of eight isozymes in liver microsomes of rats treated with polybrominated biphenyl congeners. J. Biol. Chem. 258:1282-1288. ' Fennell, T.R., Dickens, M., and Bridges, J.W. (1979) Interaction of isosafrole 13 vivo with rat hepatic microsomal cytochrome Pu50 following treatment with phenobarbitone or 20-methylcholanthrene. Biochem. Pharmac. 28:1“27'1u29. Gasiewicz, T.A. and Rucci, G. (198A) Cytosolic receptor for 2.3.7,8-tetrachlorodibenzo-p-dioxin. Evidence for a homologous nature among various mammalian species. Mol. Pharmac. 26:90-98. Gonzalez, F.J., Tukey, R.H. and Nebert, D.W. (198“) Structural gene products of the Ah locus. Transcriptional regulation of cytochrome P1-u50 and P3-h50 mRNA levels by 3-methylcholanthrene. Mol. Pharmac. 26:117-121. Graham, M.J., Lucier, G.W., Rickenbacher, U. and Goldstein, J.A. (1987) Induction of rat hepatic microsomal estradiol 2-hydroxylase (E2-0Hase) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Fed. Proc. N6:361. Jaiswal, A.K., Gonzales, F.J. and Nebert, D.W. (1985) Human dioxin-inducible cytochrome P1-u50: complementary DNA and amino acid sequence. Science 228:80-83. 113 Jones, P.B.C., Galeazzi, D.R., Fisher, J.M., and Whitlock, J.P., Jr. (1985) Control of cytochrome P1-u50 gene expression by dioxin. Science 227:1h99-1502. Kuroki, J., Koga, N., Yoshimura, H. (1986) High affinity of 2,3,",7,8-pentachlorodibenzofuran to cytochrome PASO in the hepatic microsomes of rats. Chemosphere 15:731-738. Millis, C.D. (198A) Studies on the chemical and pharmacotoxicological properties of polybrominated biphenyls. M.S. Thesis, Michigan State University. Murray, M., Zaluzny, L., and Farrell, G.C. (1986) Selective reactivation of steroid hydroxylases following dissociation of the isosafrole metabolite complex with rat hepatic cytochrome PMSO. Arch. Biochem. Biophys. 251:"71-u78. Nebert, D.W. and Gonzales, F.J. (1985) Cytochrome Pu50 gene expression and regulation. Trends Pharmacol. Sci. 6:160-16”. Numazawa, M., Kiyono, Y., and Nambara, T. (1980) A simple radiometric assay for estradiol 2-hydroxylase activity. Anal. Biochem. 10H:290-295. Ozawa, N., Yoshihara, 3., Kawano, K., Okada, Y., and Yoshimura, H. (1979) 3,",5,3',A'-pentachlorobiphenyl as a useful inducer for purification of rat liver microsomal cytochrome PAA8. Biochem. Biophys. Res. Commun. 91:11A0-11H7. ~ Parkinson, A., Safe, S.H., Robertson, L.W., Thomas, P.E., Ryan, D.E., Reik, L.M., and Levin, W. (1983) Immunochemical quantitation of cytochrome PABO isozymes and epoxide hydrase in liver microsomes from polychlorinated or polybrominated biphenyl-treated rats. J. Biol. Chem. 288:5967-5976. Parkinson, A., Thomas, P.E., Ryan, D.E. and Levin, W. (1983a) The 13 vivo turnover of rat liver microsomal epoxide hydrase and both the apoprotein and heme moieties of specific cytochrome PASO isozymes. Arch. Biochem. Biophys. 225:216-233. Pohl, R.J. and Fouts, J.R. (1980) A rapid method for assaying the metabolism of 7-ethoxyresorutin by microsomal subcellular fractions. Anal. Biochem. 107:150-155. Quattrochi, L.C., Okino, S.T., Usha, R.P., and Tukey, R.H. (1985) Cloning and isolation of human cytochrome PASO cDNAs homologous to dioxin-inducible rabbit mRNAs encoding PHSO u and PHSO 6. DNA 4:395-AOO. Redinbaugh, M.G. and Turley, R.B. (1986) Adaptation of the bicinchoninic acid protein assay for use with microtiter 114 plates and sucrose gradient fractions. Anal. Biochem. 153:267-271. Rose, J.Q., Ramsey, J.C., Wentzler, T.R., Hummel, R.A., and Gehring, P.J. (1976) The fate of 2.3.7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Toxicol. Appl. Pharmacol. 36:209-226. Ryan, D.E., Thomas, P.E., and Lewin, W. (1980) Microsomal cytochrome Pu50 from rats treated with isosafrole. Purification and characterization of four enzymic forms. J. Biol. Chem. 255:79u1-7955. F“ Ryan, D.E., Thomas, P.E., and Levin, W. (1982) Purification and characterization of a minor form of hepatic microsomal cytochrome PHSO from rats treated with polychlorinated biphenyls. Arch. Biochem. Biophys. 216:272-288. Sadler, E.M., Reddy, V.R., and Piper, W.N. (1986) Increased rat testicular heme oxygenase activity associated with t depressed microsomal heme and cytochrome PASO levels after " repeated administration of human chorionic gonadotrOpin. Arch. Biochem. Biophys. 2u9z382-387. El Schimke, R.T. (196A) The importance of both synthesis and degradation in the control of arginase levels in rat liver. J. Biol. Chem. 239:3808-3817. Schimke, R.T., Sweeney, E.W., and Berlin, C.M. (1965) The roles of synthesis and degradation in the control of rat liver tryptophan pyrrolase. J. Biol. Chem. 2MO:322-331. Shiraki, H. and Guengerich, E.P. (198A) Turnover of membrane proteins: kinetics of induction of rat liver microsomal cytochrome PASO, NADPH-cytochrome PASO reductase, and epoxide hydrolase. Arch. Biochem. Biophys. 235:86-96. Steward, A.R., Wrighton, S.A., Pasco, D.S., Fagan, J.B., Li, D., and Guzelian, P.S. (1985) Synthesis and degradation of 3-methylcholanthrene-inducible cytochromes PHSO and their mRNAs in primary monolayer cultures of adult rat hepatocytes. Arch. Biochem. Biophys. 2A1:A9A-508. Thomas, P.E., Reik, L.M., Ryan, D.E., and Levin, W. (1983) Induction of two immunochemically related rat liver cytochrome PASO isozymes, cytochromes PASOc and P450d by structurally diverse xenobiotics. J. Biol. Chem. 258zu590-A598. Thomas, P.E., Reidy, J., Reik, L.M., Ryan, D.E., Koop, D.R., and Levin, W. (198") Use of monoclonal antibody probes against rat hepatic cytochromes PHSOc and PNSOd to detect 115 immunochemically related isozymes in liver microsomes from different species. Arch. Biochem. Biophys. 235:239-253. Voorman, R., and Aust, S.D. (1987) Specific binding of polyhalogenated aromatic hydrocarbon inducers of cytochrome PASOd to the cytochrome and inhibition of its estradiol 2-hydroxylase activity. Toxicol. Appl. Pharmacol., in press. Watkins, P.B., Wrighton, S.A., Schuetz, E.G., Maurel, P. and Guzelian, P. (1986) Macrolide antibiotics inhibit the degradation of the glucocorticoid responsive cytochrome PHSOp in rat hepatocytes in vivo and in primary monolayer culture. J. Biol. Chem. 261:626fl-6271. CONCLUSION The focus of this thesis has been the interaction of planar polyhalogenated aromatic hydrocarbons with cytochrome P450d. My experiments revealed a specific, non-covalent, yet essentially irreversible association of HBB and sterically similar structures with cytochrome P450d. The ligands apparently bind to the active site of cytochrome P450d since its estradiol 2—hydroxylase activity was inhibited. Indeed, TCDD functioned as a tight-binding inhibitor having a Ki = 8 nM. Furthermore I showed that HCB and isosafrole, both E capable of forming a stable complex with cytochrome P450d, could increase the hepatic content of P450d beyond that maximially induced through the Ah receptor. The increased level of cytochrome P450d was shown to due to reduced turnover of the enzyme, presumably a result of enzyme stabilization by the ligands. It has been known for many years that isosafrole forms a stable complex with cytochrome P450d. My results suggest that HCB and other polyhalogenated aromatic hydrocarbons capable of binding to the Ah receptor can bind to cytochrome P450d in a manner similar to isosafrole. The reason for this binding is not clear. The inhibitors might be functioning as psuedo-substrates or transition—state analogues. Likewise the mechanism of turnover inhibition is not clear. It might be due to blockage of access to P450 heme; removal of heme might be a controlling step in P450 turnover. 116 117 Thus although this work has answered some questions it has also raised others. Much work is being done on the receptor-mediated mechanism of cytochrome P450 induction and much is already known about protein synthesis. However, very little is known about the mechanisms controlling protein degradation; it is in this context that further research on cytochrome P450 regulation could be informative and rewarding.