- Email: [email protected]
The flavin-containing monooxygenase system
Exp Toxic Patho11996; 48: 467-470
Gustav Fischer Verlag lena
Preclinical Division, Pharma Research, F. Hoffmann -La Roche Ltd., Basel, Switzerland
The flavin-containing monooxygenase system RODOLFO GASSER With 2 figures Received: March, 1996
Address for correspondence: RODOLFO GASSER, Preclinical Division, Pharma Research, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland. Key words: Monooxygena se system; Flavin containing monooxygenase system.
The Flavin-Containing Mon ooxygena se (FMO, EC 1.14.13.8) catal yzes the oxygenation of a wide variety of drugs and other xenobiotics that posses a nucleophilic hetero atom (ZIEGLER 1984; ZIEGLER 1988). Examples are: N,N-dimethy1aniline, imipramin e, chlorpromazine, guanethidine , meth ylpyridine, procarb azine (N-oxidation), thiobenzamide, thiourea, sulindac sulfide, cimetidine, parathion (S-oxidation), to name just a few. Of special clinical intere st is the oxidation of trimethylamine in the liver by the FMO, bec ause its deficiency cau ses the "Fish Od or Syndrome" . Many of these compounds, for example, amines, are potential substrates for both the flavin- containing and the cytoc hrome P-450-dependent monooxygenases (P-450). The final oxidation products are usually different, and only rarely is the nitrogen of a specific compound N-oxygen ated by both monooxygenases. The FMO catalyses the direct insertion of oxygen without forming intermediate radicals, as with the P-4S0' s. The me chani sms of the reaction is kno wn in some detail (BEATY et al. 1980 ; BICKEL 1971; ZIEGLER 1980; POULSEN et al. 1995). A flavin hydroperoxide bound to the protein is the oxygenating inte rmediate, and the mechani sm is similar to the one invol ving organic pero xide s (fig. 1). It can be described as a dire ct two electron oxidati on without formation of interm ediate radicals and is only limited by the acce ssibility of the enzy me bound hydrope roxide to the substrate, and specifically by the enz yme structure. As with cytochrome P-4S0, some reacti ons catalyzed by FMO result in the formation of reactive metabolites that are potent ially toxic (ZIEGLER 1988). For exa mple, the nephrotoxicity associated with the glutathio ne co nj ugate of acrol ein
* This work was supported by a grant from the Swiss National Science Foundation .
has been related to renal FMO medi ated metabolism . The FMO form s an S-oxide , subsequently rele asing the cy totoxin acrolein by a base catalyze d elimination react ion (HASHMI et al. 1992; PARK et al. 1992). The function of the FMO with respect to endogenous substrates is not kno wn in detail , but may invol ve maintenan ce of the cellular thiol : disulfide ratio by oxidati on of cysteamine to cys tamine (ZIEGLER et al. 1977). A potent ial role for the FMO in endogenous metabolism has been postulated for the oxidation of meth ionine to its sulfoxide (DUESCHER RJ 1994), and the S-oxidative cleavage of farnesylcysteine and famesylcysteine methyl ester (PARK SB et a1. 1994). The majority of what is known about the molecular characteristics of the FMO co mes from studies of the enzymes purified from pig liver (ZIEGLER et al. 1972). However, expression of distinct isozymes in liver and lung has been described. This possibilit y was first noted with the discovery that FMO activit y in detergent-solubilized preparations from rabbit lung, but not from liver, could be stimulated by Hg2+ (DEVEREUX et al. 1977). More substantial evidenc e was in a subsequent report that imipramine and chlorpromazine, both metabolized by the FMO in liver, do not undergo N-ox idation in rabbit pulmonary microsomal preparations that are active with other FMO substrates (DEVEREUX et al. 1971; OHMIYA et al. 1981). Finally , characterization of the FMO purified from rabbit lung provided direct evidence of major differenc es between the "hepatic" and "pulmonary" forms of the enzyme (WILLIAMS et a1. 1984; TYNES et al. 1985), differing by their lack of immunochemical crossreactivity, the stability of the pulmonary enzyme when subjected to temperature s or concentrations of ionic detergents that denature the hepatic enzyme (TYNES et al. 1985), and the ability of the pulmonary enzyme to metabo lize several primary arylamines that are not substrates for the hepatic enzym e (TYNES et a1. 1986; POULSEN et a1. 1986). Exp Toxic Pathol48 (1996) 5
467
8>-<80 ~-ToH 'A):H'O +
'AD~AD
H + NADPH
FAD
NADP +
Fig. 1. Reaction cycle of the Flavin-containing Monooxygenase. The formation of the flavin peroxiderepresents the rate liming step in the enzyme catalyzedoxidation mechanism.
Isolation and characterization of eDNA's encoding FMO enzymes Human liver FMO activities have been reported (McMANUS et al. 1987; LEMOINE et al. 1990) although the enzymes had never been purified or extensively characterized in regard to substrate specificity, interindividual variations, etc. In the past, little progress in explaining interspecies differences were made because of a lack of information about the structures of these proteins. Although first purified in 1972 (ZIEGLER et al. 1972) from pig liver, the FMO enzyme has proven difficult to sequence. This was apparently due to a blocked N-terminus and/or to the difficulties removing residual detergent from the extremely hydrophobic peptides. Only recently have molecular biology techniques been used to approach this problem. Using antibodies directed against the purified pig liver FMO as well as against the pulmonary rabbit lung FMO form it was possible to identify recombinant cDNA clones expressing the protein in bacteria (GASSER et al. 1990, LAWTON et al. 1990). These cDNA clones formed the basis for identifying the corresponding human counterparts and to identify structural elements important for function and activity. Using molecular biology techniques five distinct FMO isozymes have been cloned from various animal and human tissues and their DNA sequence determined. A nomenclature based on comparisons of amino acid sequences was proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes (LAWTON et al. 1994). A FMO species homolog to the pig and rabbit hepatic FMO forms (GASSER et al. 1990) was found to be expressed mostly in human kidney and not in human liver (DOLPHIN et al. 1991), explaining differences in substrate specificty observed in man (LEMOINE et al. 1990). DNA sequence analysis showed a great degree of identity (87 %) of this FMOl, to both the pig and the rabbit liver FMO protein. The virtual absence
of this form in human liver precludes the involvement of this FMO species in the genetic disease, trimethylaminuria. Northern blot analysis demonstrated that human FMOI mRNA is more abundant in fetal than in adult 468
Exp Toxic Pathol48 (1996) 5
liver, indicating that in man the enzyme is subject to developmental regulation FM02 is expressed predominantly in lung of all mammalian species tested, and in rabbit lung it has been localized in the nonciliated bronchiolar epithelial (Clara) cell predominantly, in addition to ciliated, endothelial, type I, and type II cells and in tracheallinging layer (OVERBY et al. 1992). The human ortholog to the rabbit enzyme is 83 % homologous to rabbit pulmonary FMO (PHILLIPS et al. 1995). Substrates for FM02 include trifluoprerazine, prochlorperazine, N,N-dimethyloctylamine, desmethy1perazine, and N-methyloctylamine, but FM02 does not exhibit detectable substrate activity with primary arylamines while it catalyzes N-oxygenation of alkylamines to oximes (POULSEN et al. 1986). A third FMO species, FM03, was isolated from human liver (LOMRI et al. 1992). It had not been previously characterized and accounts for the majority of FMO expressed in human liver. FMO 3 is the major FMO isoform in methionine sulfoxidation (DUESCHER et al. 1994) and exhibits distinct stereoselectivity for oxygenation (LOMRI et al. 1993) of cimetidine and S-nicotine (CASHMAN et al. 1995). In the case of pargyline, FMOI produced solely the (+)-enantiomer and FM03 predominantly the (-)-enantiomer of the N-oxide (PHILLIPS et al. 1995). These and other studies indicate, that FM03 has a narrower substrate specificity compared to FMOI found in liver of most animal species. FM04 represents a further minor FMO form, present both in rabbit (BURNETT et al. 1994) and human liver (DOLPHIN et al. 1992). Its function and substrate specificity remains unknown. mRNA for FM04 has been detected in human brain (DOLPHIN et al. 1992), and the protein might be involved in the oxidation of antidepressants, such as imipramine, in the brain (BHAMRE et al. 1993). FM05 was observed in rabbit liver (ATTA-AsAFOADJEI et al. 1993) and human liver (PHILLIPS et al. 1995), where it is expressed constitutively to a lesser extent than FM03. Interestingly,it is inactive with methimazole, a general FMO substrate, but highly active with n-octylamine. Figure 2 illustrates the relative tissue distribution of the known human FMOs. From this it seems clear, that FM03 is the predominant human enzyme species. The deduced amino acid sequences of the five human FMOs have 82-87 % identity with their konw orthologues in other mammal but only 51-57 % similarity to each other. From the calculated rate of evolution of FMOs (a 1 % change in sequence per 6 million years) it is possible that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago, prior to speciation. The indivdual FMO genes were all localized to the long arm of human chromosome 1 at lq23-q25 (PHILLIPS et al. 1995). In spite of the marked differences, the FMO enzymes have a number of common features. A common conserved FAD binding site is located at the N-terminus and a NADPH binding site is located at a constant distance from the FAD domain. Both sites are unique signatures
Fig. 2. Distribution of Flavin ContainingMono-oxygenase isozymes in Human Tissues. Individual FMO isozymes were detected by hybridization and/or PCR analysis using isozymespecificoligonucleotides. The letter size correlates roughly to the relative intensities observed. The identity of the products to specific FMO isozymes was confirmed by sequenceanalysis. for this family of enzymes and are requisites for the catalytic activities exhibited by them. The intervening sequences with little primary sequence identity, might modulate the substrate specificities observed, suggesting that the overall structural similarity is probably greater than predicted from the identity of their sequences.
Regulation and genetic variation in FMO expression Variations in activity and specificity in the metabolism of FMO substrates are an important aspect of interindividual differences in response to chemical compounds and tissue specific toxicity. Genetic factors and tissue specificity in expression of FMOs are important contributing factors. FMO is not inducible by the classical cytochrome P450 inducers, such as phenobarbital, polycyclic aromatic hydrocarbons, ethanol or macrolide antibiotics. Evidence does exist from a number of laboratories, however, for developmental and hormonal regulation of FMO. Enhanced FMO activity during mid- and late-gestation in maternal rabbit lung have been demonstrated. This response is due to increased protein and catalytic activity associated with FM02. The time course of expression of FM02 during mid- and late-gestation correlates to plasma peaks of progesterone or cortisol. FM02 also peaks at parturition in maternal kidney, coincident with plasma cortisol levels. FM02 is induced by s.c. administration of either progesterone or dexamethasone in lung, or by dexamethasone in kidney. Correlation of plasma progesterone or cortisol levels during gestation and postpartum support a role for progesterone, but not cortisol in regulation ofFM02 in maternal rabbit lung. The levels of FM01 also appear to be increased during mid- and late-
gestation in liver. FMOI in liver may also be regulated during gestation by progesterone or glucocorticoids as adminstration of these steroids enhanced FMOI mRNA levels 4-fold (LEE et al. 1993; LEE et al. 1995). Also, in mouse, changes elicited by steroid hormones have been described for liver and kidney FMO mediated dimethylamine N-oxidation (DUFFEL et al. 1981). In man the FMO appears to be modulated by steroids too. Recently, a patient treated regularly with a long-acting testosterone ester began to experience strong fish-like odor from his breath, sweath and urine. Subsequently it was established that this patient was heterozygous for the oxidation oftrimethylamine. Steroid treatment was sufficient to manifest the impairment, as discontinuation of treatment resulted in amelioration of his condition (AYESH et al. 1995). Genetically impaired FMO mediated metabolism of trimethylamine to its N-oxide has been described in 1 % of white British subjects (AL-WAIZ et al. 1987). This inborn error in metabolism, associated with a penetrant smell of rotten fish emanating from the afflicted individuals, was attributed to a deficient function of the hepatic form of the FMO. The condition is inherited as a recessive autosomal trait (AYESH et al. 1993). In conclusion, species variation in specific and reactivity of the FMO is associated to differing tissue expression of distinct FMO isozymes, their modulation by sexual hormones and the genetic variability in the FMo expression, specially in man. Because of the differing hepatic composition of individual FMO enzymes in man compared to animal species, human extrapolation from animal data has to be carefully evaluated.
References AL-WEIZ M, AYESH R, MITCHELL SC, et al.: A genetic polymorphism of the N-oxidation of trimethylamine in humans. Clin Pharmacol Ther 1987; 42: 588-594. ATTA-ASAFO-ADJEI E, LAWTON MP, PHILPOT RM: Cloning, sequencing, distribution, and expression in Escherichia coli of flavin-containing monooxygenase 1C 1. Evidence for a third gene subfamily in rabbits. J BioI Chern 1993; 268: 9681-9689. AYESH R, MITCHELL SC, SMITH RL: Dysfunctional N-oxidationof trimethylamine and the influence of testosterone treatment in man. Pharmacogenetics 1995; 5: 244-246. AYESH R, MITCHELL SC, ZHANG AQ, SMITH RL: The fish malodour syndrome: Biochemical, familial and clinical aspects. Br Med J 1993; 307: 655-657. BEATY N, BALLOU DP: Transient kinetic study of liver microsomal FAD containing Monooxygenase. J BioI Chern 1980; 255: 3817-3819. BHAMRE S, BHAGWAT SV, SHANKAR SK, et al.: Cerebralflavin-containing monooxygenase-mediated metabolism of antidepressants in brain: immunochemical propertiesand immunocytochemical localization. J Pharmacol Exp Ther 1993;267: 555-559. BICKEL MH: N-Oxide formation and related reactions in Drug Metabolism. Xenobiotica 1971; 1: 313-319. BURNETT VL, LAWTON MP, PHILBOT RM: Cloning and sequencing of flavin-containing monooxygenases FM03 Exp Toxic Pathol 48 (1996) 5
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and FM04 from rabbit and characterization of FM03. J BioI Chern 1994; 269: 14314-14322. CASHMAN JR, PARK SB, BERKMAN CE, CASHMAN LE: Role of hepatic flavin-containing monooxygenase 3 in drugs and chemical metabolism in adult humans. Chern Biol Interact 1995; 96: 33-46. DEVEREUX TR, FOUTS JR: N-oxidation and demethylation of N,N-dimethylaniline by rabbit liver and lung microsomes: effects of age and metals. Chern-BioI Interact 1971; 8: 91-105. DEVEREUX TR, PHILPOT RM, FOUTS JR: The effect of Hg2+ on rabbit hepatic and pulmonary, partially purified N,Ndiemthylaniline N-oxidases. Chem-Biol Interact 1977, 18: 277-287. DOLPHIN C, SHEPARD EA, POVEY S, et al.: Cloning, primary sequence, and chromosomal mapping of a human flavincontaining monooxygenase (FM01). J BioI Chern 1991; 266: 12379-12385. DOLPHIN CT, SHEPHARD EA, POVEY S, et al.: Cloning, primary sequence and chromosomal localization of human FM02, a new member of the flavin-containing monooxygenase family. Biochem J 1992; 287: 261-267. DUESCHER RJ, LAWTON MP, PHILPOT RM, ELFARRA AA: Flavin-containing monooxygenase (FMO)-dependent metabolism of methionine and evidence for FM03 being the major FMO involved in methionine sulfoxidation in rabbit liver and kidney microsomes. J BioI Chern 1994; 269: 17525-17530. DUFFEL MW, GRAHAM JM, ZIEGLER DM: Changes in dimethylamine N-oxidase acitivity of mouse liver and kidney induced by steroid sex hormones. Mol Pharmacol 1981; 19: 134-139. GASSER R, TYNES RE, LAWTON MP, et al.: The Flavin-Containing Monooxygenase Expressed in Pig Liver: Primary Sequence, Distribution, and Evidence for a Single Gene. Biochemistry 1990,29: 119-124. HASHMI M, VAMVAKAS S, ANDERS MW: Bioactivation mechanism of S-(3-oxopropyl)-N-acetyl-L-cysteine, the mercapturic acid of acrolein. Chern Res Toxicol 1992; 5: 360-365. LAWTON MP, CASHMAN JR, CRESTEIL T, et al.: A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities. Arch Biochem Biophys 1994; 308: 254-257. LAWTON MP, GASSER R, TYNES RE, et al.: The flavin-containing monooxygenase enzymes expressed in rabbit liver and lung are products of related but distinctly different genes. J BioI Chern 1990; 265: 5855-5861. LEE MY, CLARK JE, WILLIAMS DE: Induction of flavin-containing monooxygenase (FMO B) in rabbit lung and kidney by sex steroids and glucocorticoids. Arch Biochem Biophys 1993;302: 332-336. LEE MY, SMILEY S, KADKHODAYAN S, et al.: Developmental regulation of flaving-containing monooxygenase (FMO) isoforms 1 and 2 in pregnant rabbit. Chern Biol Interact 1995; 96: 75-85. LEMONJE A, JOHANN M, CRESTEIL T: Evidence for the presence of distinct flavin-containing monooxygenases in human tissues. Arch-Biochem-Biophys 1990,276: 336-342. LOMRI N, Gu Q, CASHMAN JR: Molecular cloning of the flavin-containing monooxygenase (form II) eDNA from adult human liver. Proc Natl Acad Sci USA 1992; 89: 1685-1689. LOMRI N, YANG Z, CASHMAN JR: Regio- and stereoselective oxygenations by adult human liver flavin-containing mo470
Exp Toxic Pathol48 (1996) 5
nooxygenase. 3. Comparison with forms 1 and 2. Chern Res Toxicol1993; 6: 800-807. McMANUS ME, STUPANS I, BURGESS W, et al.: Flavin-containing Monooxygenase in human liver microsomes. Drug Metab Disp 1987; 15: 256-261. OHMIYA Y, MEHENDALE HM: Pulmonary metabolism of Imipramine in the rat and rabbit. Comparison with hepatic metabolism. Pharmacology 1981; 22: 172-182. OVERBY L, NISHIO SJ, LAWTON MP, et al.: Cellular localization of flavin-containing monooxygenase in rabbit lung. Exp Lung Es 1992; 18: 131-144. PARK SB, HOWALD WN, CASHMAN JR: S-oxidative cleavage of famesylcysteine and famesylcysteine methyl ester by the flavin-containing monooxygenase. Chern Res Toxicol1994; 7: 191-198. PARK SB, OSTERLOH JD, VAMVAKAS S, et al.: Flavin-containing monooxygenase-dependent stereoselective Soxygenation and cytotoxicity of cysteine S-conjugates and mercapturates.Chern Res Toxicol 1992; 5: 193-201. PHILLIPS IR, DOLPHIN CT, CLAIR P, et al.: The molecular biology of the flavin-containing monooxygenases of man. Chern BioI Interact 1995; 96: 17-32. POULSEN LL, ZIEGLER DM: Multisubstrate flavin-containing monooxygenase: applications of mechanism to specificity. Chern BioI Interact 1995; 96: 57-73. POULSEN LL, TAYLOR K, WILLIAMS DE, et al.: Substrate specificity of the rabbit lung flavin-containing monooxygenase for amines: oxidation products of primary alkylamines. Mol Pharmacol 1986; 30: 680-685. TYNES RE, SABOURIN PJ, HODGSON E: Formation of hydrogen peroxide and N-hydroxylated amines catalyzes by pulmonary flavin-containing monooxygenases in the presence of primary alkylamines. Biochem Biophys Res Commun 1985; 126: 1069-1075. TYNES RE, SABOURIN PJ, HODGSON E, PHILPOT RM: Formation of hydrogen peroxide and N-hydroxylated amines catalyzed by pulmonary flavin-containing monooxygenases in the presence of primary alkylamines. Arch Biochern Biophys 1986; 251: 654-664. WILLIAMS DE, ZIEGLER DM, NORDIN DJ, et al.: Rabbit lung flavin-containing Monooxygenase is immunochemically and catalytically distinct from the liver enzyme. Biochem Biophys Res Commun 1984; 125: 116-122. ZIEGLER DM, MITCHELL CH: Microsomal oxidase. IV properties of a mixed function amine oxidase isolated from pig liver microsomes. Arch BiochemBiophys 1972;150: 116-125. ZIEGLER DM, POULSEN LL: Trends in Biochem. Sci 1977;2: 79-83. ZIEGLER DM: Flavin-containing monooxygenases: catalytic mechanism and substrate specificities. Drug Metab Rev 1988; 19: 1-32. ZIEGLER DM: Functional groups activated via flavin-containing monooxygenases. In: Microsomes and Drug Oxidations (MINORS J, BIRKETT DJ, DREW R, McMANUS M, eds.). Taylor and Francis, London 1988; pp 297-304. ZIEGLER DM: Metabolic Oxygenation of Organic Nitrogen and Sulfur Compounds in Drug Metabolism and Drug Toxicity (MITCHEL JR and HORNING MG, eds.). Raven Press, New York 1984, pp 1-31. ZIEGLER DM: Microsomal flavin-containing monooxygenase: oxygenation of nucleophilic nitrogen and sulphur compounds. In: Enzymatic Basis of Detoxication, Vol 1 (edited by W. B. JACOBY). ACADEMIC PRESS, NEW YORK 1980, PP 201-227.
Gustav Fischer Verlag lena
Preclinical Division, Pharma Research, F. Hoffmann -La Roche Ltd., Basel, Switzerland
The flavin-containing monooxygenase system RODOLFO GASSER With 2 figures Received: March, 1996
Address for correspondence: RODOLFO GASSER, Preclinical Division, Pharma Research, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland. Key words: Monooxygena se system; Flavin containing monooxygenase system.
The Flavin-Containing Mon ooxygena se (FMO, EC 1.14.13.8) catal yzes the oxygenation of a wide variety of drugs and other xenobiotics that posses a nucleophilic hetero atom (ZIEGLER 1984; ZIEGLER 1988). Examples are: N,N-dimethy1aniline, imipramin e, chlorpromazine, guanethidine , meth ylpyridine, procarb azine (N-oxidation), thiobenzamide, thiourea, sulindac sulfide, cimetidine, parathion (S-oxidation), to name just a few. Of special clinical intere st is the oxidation of trimethylamine in the liver by the FMO, bec ause its deficiency cau ses the "Fish Od or Syndrome" . Many of these compounds, for example, amines, are potential substrates for both the flavin- containing and the cytoc hrome P-450-dependent monooxygenases (P-450). The final oxidation products are usually different, and only rarely is the nitrogen of a specific compound N-oxygen ated by both monooxygenases. The FMO catalyses the direct insertion of oxygen without forming intermediate radicals, as with the P-4S0' s. The me chani sms of the reaction is kno wn in some detail (BEATY et al. 1980 ; BICKEL 1971; ZIEGLER 1980; POULSEN et al. 1995). A flavin hydroperoxide bound to the protein is the oxygenating inte rmediate, and the mechani sm is similar to the one invol ving organic pero xide s (fig. 1). It can be described as a dire ct two electron oxidati on without formation of interm ediate radicals and is only limited by the acce ssibility of the enzy me bound hydrope roxide to the substrate, and specifically by the enz yme structure. As with cytochrome P-4S0, some reacti ons catalyzed by FMO result in the formation of reactive metabolites that are potent ially toxic (ZIEGLER 1988). For exa mple, the nephrotoxicity associated with the glutathio ne co nj ugate of acrol ein
* This work was supported by a grant from the Swiss National Science Foundation .
has been related to renal FMO medi ated metabolism . The FMO form s an S-oxide , subsequently rele asing the cy totoxin acrolein by a base catalyze d elimination react ion (HASHMI et al. 1992; PARK et al. 1992). The function of the FMO with respect to endogenous substrates is not kno wn in detail , but may invol ve maintenan ce of the cellular thiol : disulfide ratio by oxidati on of cysteamine to cys tamine (ZIEGLER et al. 1977). A potent ial role for the FMO in endogenous metabolism has been postulated for the oxidation of meth ionine to its sulfoxide (DUESCHER RJ 1994), and the S-oxidative cleavage of farnesylcysteine and famesylcysteine methyl ester (PARK SB et a1. 1994). The majority of what is known about the molecular characteristics of the FMO co mes from studies of the enzymes purified from pig liver (ZIEGLER et al. 1972). However, expression of distinct isozymes in liver and lung has been described. This possibilit y was first noted with the discovery that FMO activit y in detergent-solubilized preparations from rabbit lung, but not from liver, could be stimulated by Hg2+ (DEVEREUX et al. 1977). More substantial evidenc e was in a subsequent report that imipramine and chlorpromazine, both metabolized by the FMO in liver, do not undergo N-ox idation in rabbit pulmonary microsomal preparations that are active with other FMO substrates (DEVEREUX et al. 1971; OHMIYA et al. 1981). Finally , characterization of the FMO purified from rabbit lung provided direct evidence of major differenc es between the "hepatic" and "pulmonary" forms of the enzyme (WILLIAMS et a1. 1984; TYNES et al. 1985), differing by their lack of immunochemical crossreactivity, the stability of the pulmonary enzyme when subjected to temperature s or concentrations of ionic detergents that denature the hepatic enzyme (TYNES et al. 1985), and the ability of the pulmonary enzyme to metabo lize several primary arylamines that are not substrates for the hepatic enzym e (TYNES et a1. 1986; POULSEN et a1. 1986). Exp Toxic Pathol48 (1996) 5
467
8>-<80 ~-ToH 'A):H'O +
'AD~AD
H + NADPH
FAD
NADP +
Fig. 1. Reaction cycle of the Flavin-containing Monooxygenase. The formation of the flavin peroxiderepresents the rate liming step in the enzyme catalyzedoxidation mechanism.
Isolation and characterization of eDNA's encoding FMO enzymes Human liver FMO activities have been reported (McMANUS et al. 1987; LEMOINE et al. 1990) although the enzymes had never been purified or extensively characterized in regard to substrate specificity, interindividual variations, etc. In the past, little progress in explaining interspecies differences were made because of a lack of information about the structures of these proteins. Although first purified in 1972 (ZIEGLER et al. 1972) from pig liver, the FMO enzyme has proven difficult to sequence. This was apparently due to a blocked N-terminus and/or to the difficulties removing residual detergent from the extremely hydrophobic peptides. Only recently have molecular biology techniques been used to approach this problem. Using antibodies directed against the purified pig liver FMO as well as against the pulmonary rabbit lung FMO form it was possible to identify recombinant cDNA clones expressing the protein in bacteria (GASSER et al. 1990, LAWTON et al. 1990). These cDNA clones formed the basis for identifying the corresponding human counterparts and to identify structural elements important for function and activity. Using molecular biology techniques five distinct FMO isozymes have been cloned from various animal and human tissues and their DNA sequence determined. A nomenclature based on comparisons of amino acid sequences was proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes (LAWTON et al. 1994). A FMO species homolog to the pig and rabbit hepatic FMO forms (GASSER et al. 1990) was found to be expressed mostly in human kidney and not in human liver (DOLPHIN et al. 1991), explaining differences in substrate specificty observed in man (LEMOINE et al. 1990). DNA sequence analysis showed a great degree of identity (87 %) of this FMOl, to both the pig and the rabbit liver FMO protein. The virtual absence
of this form in human liver precludes the involvement of this FMO species in the genetic disease, trimethylaminuria. Northern blot analysis demonstrated that human FMOI mRNA is more abundant in fetal than in adult 468
Exp Toxic Pathol48 (1996) 5
liver, indicating that in man the enzyme is subject to developmental regulation FM02 is expressed predominantly in lung of all mammalian species tested, and in rabbit lung it has been localized in the nonciliated bronchiolar epithelial (Clara) cell predominantly, in addition to ciliated, endothelial, type I, and type II cells and in tracheallinging layer (OVERBY et al. 1992). The human ortholog to the rabbit enzyme is 83 % homologous to rabbit pulmonary FMO (PHILLIPS et al. 1995). Substrates for FM02 include trifluoprerazine, prochlorperazine, N,N-dimethyloctylamine, desmethy1perazine, and N-methyloctylamine, but FM02 does not exhibit detectable substrate activity with primary arylamines while it catalyzes N-oxygenation of alkylamines to oximes (POULSEN et al. 1986). A third FMO species, FM03, was isolated from human liver (LOMRI et al. 1992). It had not been previously characterized and accounts for the majority of FMO expressed in human liver. FMO 3 is the major FMO isoform in methionine sulfoxidation (DUESCHER et al. 1994) and exhibits distinct stereoselectivity for oxygenation (LOMRI et al. 1993) of cimetidine and S-nicotine (CASHMAN et al. 1995). In the case of pargyline, FMOI produced solely the (+)-enantiomer and FM03 predominantly the (-)-enantiomer of the N-oxide (PHILLIPS et al. 1995). These and other studies indicate, that FM03 has a narrower substrate specificity compared to FMOI found in liver of most animal species. FM04 represents a further minor FMO form, present both in rabbit (BURNETT et al. 1994) and human liver (DOLPHIN et al. 1992). Its function and substrate specificity remains unknown. mRNA for FM04 has been detected in human brain (DOLPHIN et al. 1992), and the protein might be involved in the oxidation of antidepressants, such as imipramine, in the brain (BHAMRE et al. 1993). FM05 was observed in rabbit liver (ATTA-AsAFOADJEI et al. 1993) and human liver (PHILLIPS et al. 1995), where it is expressed constitutively to a lesser extent than FM03. Interestingly,it is inactive with methimazole, a general FMO substrate, but highly active with n-octylamine. Figure 2 illustrates the relative tissue distribution of the known human FMOs. From this it seems clear, that FM03 is the predominant human enzyme species. The deduced amino acid sequences of the five human FMOs have 82-87 % identity with their konw orthologues in other mammal but only 51-57 % similarity to each other. From the calculated rate of evolution of FMOs (a 1 % change in sequence per 6 million years) it is possible that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago, prior to speciation. The indivdual FMO genes were all localized to the long arm of human chromosome 1 at lq23-q25 (PHILLIPS et al. 1995). In spite of the marked differences, the FMO enzymes have a number of common features. A common conserved FAD binding site is located at the N-terminus and a NADPH binding site is located at a constant distance from the FAD domain. Both sites are unique signatures
Fig. 2. Distribution of Flavin ContainingMono-oxygenase isozymes in Human Tissues. Individual FMO isozymes were detected by hybridization and/or PCR analysis using isozymespecificoligonucleotides. The letter size correlates roughly to the relative intensities observed. The identity of the products to specific FMO isozymes was confirmed by sequenceanalysis. for this family of enzymes and are requisites for the catalytic activities exhibited by them. The intervening sequences with little primary sequence identity, might modulate the substrate specificities observed, suggesting that the overall structural similarity is probably greater than predicted from the identity of their sequences.
Regulation and genetic variation in FMO expression Variations in activity and specificity in the metabolism of FMO substrates are an important aspect of interindividual differences in response to chemical compounds and tissue specific toxicity. Genetic factors and tissue specificity in expression of FMOs are important contributing factors. FMO is not inducible by the classical cytochrome P450 inducers, such as phenobarbital, polycyclic aromatic hydrocarbons, ethanol or macrolide antibiotics. Evidence does exist from a number of laboratories, however, for developmental and hormonal regulation of FMO. Enhanced FMO activity during mid- and late-gestation in maternal rabbit lung have been demonstrated. This response is due to increased protein and catalytic activity associated with FM02. The time course of expression of FM02 during mid- and late-gestation correlates to plasma peaks of progesterone or cortisol. FM02 also peaks at parturition in maternal kidney, coincident with plasma cortisol levels. FM02 is induced by s.c. administration of either progesterone or dexamethasone in lung, or by dexamethasone in kidney. Correlation of plasma progesterone or cortisol levels during gestation and postpartum support a role for progesterone, but not cortisol in regulation ofFM02 in maternal rabbit lung. The levels of FM01 also appear to be increased during mid- and late-
gestation in liver. FMOI in liver may also be regulated during gestation by progesterone or glucocorticoids as adminstration of these steroids enhanced FMOI mRNA levels 4-fold (LEE et al. 1993; LEE et al. 1995). Also, in mouse, changes elicited by steroid hormones have been described for liver and kidney FMO mediated dimethylamine N-oxidation (DUFFEL et al. 1981). In man the FMO appears to be modulated by steroids too. Recently, a patient treated regularly with a long-acting testosterone ester began to experience strong fish-like odor from his breath, sweath and urine. Subsequently it was established that this patient was heterozygous for the oxidation oftrimethylamine. Steroid treatment was sufficient to manifest the impairment, as discontinuation of treatment resulted in amelioration of his condition (AYESH et al. 1995). Genetically impaired FMO mediated metabolism of trimethylamine to its N-oxide has been described in 1 % of white British subjects (AL-WAIZ et al. 1987). This inborn error in metabolism, associated with a penetrant smell of rotten fish emanating from the afflicted individuals, was attributed to a deficient function of the hepatic form of the FMO. The condition is inherited as a recessive autosomal trait (AYESH et al. 1993). In conclusion, species variation in specific and reactivity of the FMO is associated to differing tissue expression of distinct FMO isozymes, their modulation by sexual hormones and the genetic variability in the FMo expression, specially in man. Because of the differing hepatic composition of individual FMO enzymes in man compared to animal species, human extrapolation from animal data has to be carefully evaluated.
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