Chem. Res. Toxicol. 1990,3, 372-376
372
Substrate Specificities of Rabbit Lung and Porcine Liver Flavin-Containing Monooxygenases: Differences Due to Substrate Size Toshiyuki Nagata,t**David E. Williams,#and D. M. Ziegler*p+ Clayton Foundation Biochemical Institute and Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712,and Department of Food Science and Technology and Toxicology Program, Oregon State University, Corvallis, Oregon 97331 Received August 31,1989
Phenothiazine, 2-(trifluoromethyl)phenothiazine,and a series of 10-(N,N-dimethylaminoalkyl)-2-(trifluoromethyl)phenothiazineswith alkyl side chains varying in length from C2to C, were tested for substrate activity with purified rabbit lung and porcine liver flavin-containing monooxygenases (FMO). While all were substrates for the hepatic FMO, only phenothiazines bearing CBand C7alkyl side chains were oxidized at significant rates by the pulmonary FMO. Kinetic constants calculated from reaction velocities for the oxidation of thiourea, phenylthiourea, and naphthylthiourea indicate that a nucleophilic heteroatom on the end of a molecule not much larger than a six-membered ring in cross section is oxidized by both enzymes, but the addition of bulky lipophilic substituents increases the K, of N-substituted thioureas for rabbit lung FMO and 1,3-diphenylthiourea (thiocarbanilide) is excluded entirely. From the dimensions of compounds excluded and from those oxidized, it would appear that the hydroperoxyflavin in rabbit lung FMO lies about 6-8 A below the surface in a channel no more than 8 A in diameter in its longest axis. The channel leading to this oxidant in hepatic FMO appears more open and readily admits compounds bearing a tricyclic ring. Differences in dimensions of the substrate channel appear responsible for some of the differences in substrate specificities between liver and lung FMO.
Introduction Flavin-containing monooxygenases (FMO) purified to homogeneity from porcine liver (1)and rabbit lung (2,3) microsomes catalyze oxygenation of a wide variety of xenobiotic soft nucleophiles. Studies on mechanism (cf. ref 4 for recent review) suggest that the major steps in the catalytic cycle of both enzymes are similar in that oxygenatable substrate is not required for flavin reduction by NADPH or reoxidation of the flavin by molecular oxygen. These enzymes are present in tissues in the 4a-hydroperoxyflavin form and apparently discriminate between physiologically essential and useless nucleophiles by excluding the former rather than by selectively binding the latter. Yet, despite the similarities in catalytic mechanism, some striking differences in substrate specificities between the pulmonary and hepatic FMO's have been described (2,3,5). The studies of Ohmiya and Mehendale (6-9)were the first indication that N-oxidase activities of rabbit lung and liver microsomes were catalyzed by different enzymes. Subsequent studies with FMO purified from rabbit lung showed that the pulmonary enzyme catalyzes Noxygenation of N,N-dimethylaniline and trifluoperazine but not of chlorpromazine, imipramine, thioridazine, or mesoridazine (2,5). The ability of pulmonary FMO to discriminate between trifluoperazine and other tricyclic drugs was unexpected since all are excellent substrates for ~~
* Author to whom correspondence should be addressed.
'The University of Texas at Austin. Present address: Tokyo Institute of Technology, Research Laboratory of Resources Utilization,4259 Nagatsuta, Midori-ku,Yokohama 227, Japan. f Oregon State University. 0893-228x/90/2703-0372$02.50/0
hepatic FMO ( 4 ) . Unlike hepatic FMO, the pulmonary enzyme is apparently capable of excluding tricyclics bearing a propylamine side chain but not those with a piperazine ring attached to the propyl side chain. While the piperazine-substituted phenothiazines are somewhat more basic, the slight difference in charge alone does not appear responsible since rabbit lung FMO also does not catalyze the oxidation of chlorcyclizine (5) which contains a 4-methylpiperazine component. On the other hand, chlorcyclizine, chlorpomazine, and imipramine also differ from trifluoperazine in the distance of the terminal heteroatom on the side chain from substituents larger than a six-membered ring. The structure-activity studies described in this report were carried out to define more precisely the constraints of substrate size that limit access of nucleophiles into the catalytic site of the pulmonary FMO.
Experlmentai Procedures Materials. Commercial reagents of the highest purity available were purchased from the following firms: glucose 6-phosphate, Leuconostoc mesenteroides glucose-&phosphate dehydrogenase, NADP', and triflupromazine were from Sigma; 2-(trifluoromethyl)phenothiazine, phenothiazine, thiourea, 1-phenylthiourea, thiocarbanilide, S-methylthiourea (2-methyl-2-thiopseudourea sulfate), solvents, and all alkyl halides required for the synthesis of (NJ-dimethylaminoalky1)phenothiazines were from Aldrich. 1-Naphthylthioureapurchased from Eastman Kodak was crystallized twice from 95% ethanol and dried under vacuum. Dimethyl sulfoxide was dried over calcium hydride and distilled under reduced pressure, and diethyl ether was distilled over sodium before use. l-Bromo-4-chlorobute, 1-bromod-chloropentane, and 7-bromoheptanenitrile were also distilled under reduced pressure just prior to use. All other reagents were used as supplied. 0 1990 American Chemical Society
Specificities of Liver and Lung FA40 The rabbit lung and porcine liver flavin-containing monooxygenases were isolated by the methods described previously (2, 10). Organic Syntheeis. Except for the C3 analogue (triflupromazine) which is available commercially, all other lO-(N,N-dimethylaminoalkyl)-2-(trifluoromethyl)phenothiazineswith alkyl side chains from C2to C,were synthesized by literature methods or by adaptations of routine synthetic methods. The C2 (11)and C4 and Cs (12) analogues, prepared exactly by the procedures described in the references cited, were converted to hydrochloride salts, dissolved in a minimum of anhydrous ethanol, and precipitated by adding twenty volumes of anhydrous ether. The white, crystalline salts recrystallized twice by the same procedure were collected and dried under vacuum. The salts of the C2, C4, and Cs analogues migrated as a single spot on alumina TLC plates developed in chloroform, and their physical properties appeared to be identical with values reported in the references cited above. Because of facile cyclization of alkyl halide phenothiazinea with more than 5 carbons in the side chains, the C6 and Cl analogues could not be synthesized by adapting methods used for synthesis of the C 2 4 , derivatives. They were, however, readily prepared by reduction and methylation of the corresponding alkyl nitrile phenothiazines. Since the synthesis of neither the C6 nor Cl analogues haa been described previously, the major steps in their synthesis are summarized below. lo-( N,N-Dimethylaminohexyl)-2-( trifluoromet hy1)phenothiazine. 10-(5-Chloropentyl)-2-(trifluoromethyl)phenothiazine (2.5 g) prepared as described in ref 12 was dissolved in 25 mL of dimethyl sulfoxide containing 0.7 g of sodium cyanide. The mixture was heated to 80 OC for 3 h, cooled to room temperature, and extracted with 100 mL of benzene after adding 100 mL of water. The benzene extract, washed with water three timea, was dried over sodium sulfate and concentrated under vacuum. The concentrated material was applied to an alumina column and 10-(nitrilohexyl)-2-(trifluoromethyl)phenothiazineeluted with benzene-hexane (1:l). The nitrile (yield 2.3 g) was dried under vacuum and used in the next step without further purification. The nitrile dissolved in 15 mL of diethyl ether was added dropwise to LiA1H4,suspended in 50 mL of dry diethyl ether at 3-5 OC, and then stirred at room temperature overnight. After excess L u was destroyed by dropwise addition of 6 M KOH, the ether layer was decanted and the residue extracted twice with 15-20 mL of ether. The combined ether extracts were dried under reduced pressure. The waxy solid (yield 2 g) migrated as a single iodine- and ninhydrin-positive spot on the alumina plates developed in methanol and on cellulose plates developed with benzene. The amine was converted to the Nfl-dimethyl derivative by the formaldehydecyanoborohydride procedure described by Borch and Hassid (13). Solid sodium borohydride was slowly added to the amine dissolved in 25 mL of acetonitrile containing 2.5 molar exwas formaldehyde. The reaction mixture was chilled on ice as necessary, and the reducing agent was added until the reaction was no longer detectably exothermic. The mixture was warmed to room temperature, stirred for 4 h, neutralized by dropwise addition of acetic acid, and then concentrated under reduced pressure. The residue, resuspended in 20 mL of 0.01 M KOH, was extracted three times with benzene. The combined extracts were washed with water (three times) and dried over sodium sulfate, and benzene was then removed under vacuum. The waxy solid, redissolved in a minimum volume of benzene, was transferred to a column of alumina and eluted with benzene-chloroform (1:l).The fractions, free from ninhydrin-positive material, with the highest 300-nm absorption (vs the eluting solvent),were combined and concentrated under reduced pressure. The residue was redissolved in a minimum volume of dry ethanol, purged with HC1, and precipitated by adding about 20 volumes of diethyl ether. The residue, collected by centrifugation, was reprecipitated two more times from ethanol-ther and then dried under vacuum overnight. (Yield from the primary amine was 0.9 g.) The Cs analogue could not be crystallized, but it appeared free from detectable impurities by chromatography on alumina TLC plates developed in benzene-hexane (12) on celluloee plates developed in benzene. 'H NMR spectra of the product in CDC13, recorded with a General Electric QE300 spectrometer, expressed in ppm downfield from tetramethylsilane were as follows: S 7.08 (m, 7 H, aromatic H), 3.88 (t, 2 H, CH2on phenothiazine N), 2.90
Chem. Res. Toxicol., Vol. 3, No. 4, 1990 373 (m, 2 H, NCHd, 2.75 (s,6 H, two CH3N), 1.59 (m, 8 H, four CHJ. Elemental composition was determined on a VG analytical ZAB 2-E mass spectrometer: mass 394.17 (Cz1H%N2F3S). lo-(N,N-Dimethylaminoheptyl)-2-(trifluoromethyl)phenothiazine. 2-(Trifluoromethyl)phenothiazine (2.67 g) in 60 mL of dimethyl sulfoxide containing 0.27 g of sodium hydride was coupled to 7-bromoheptanenitrile (2.1 g) by heating to 60 OC for 3 h. The reaction mixture was cooled, diluted with an equal volume of cold water, and extracted three times with benzene. The combined benzene extracts were dried over sodium sulfate, and the solvent along with excess 7-bromoheptanenitrile was removed under vacuum. The residue was redissolved in a small volume of benzene, applied to a column of silica gel, and eluted with benzene. Fractions containing nitrile were combined (solvent was removed under reduced pressure) and used without further purification. Yield was 3.35 g. The lO-(nitriloheptyl)-2-(trifluoromethy1)phenothiazinewas reduced, methylated, and purified by the same procedures described above for the synthesis of the C6 analogue. Like the latter, the Cl analogue failed to crystallize, but it appeared homogeneous by chromatography on alumina and silica gel TLC plates developed in benzene and in chloroformethanol-NH40H (lOl:O.l), respectively. 'H NMR (CDClJ S 7.08 (m, 7 H, aromatic H), 3.88 (t, 2 H, CHz on aromatic nitrogen), 2.90 (m, 2 H, CH2 adjacent to side-chain N), 2.75 (s,6 H, two CH3N), 1.59 (mg, 10 H, five CH2). Elemental composition was determined from analytical mass spectra: mass 408.19 (CnH21NzF3S). Methods. Enzyme Assays. Monooxygenase activities were calculated from substrate-dependent oxygen uptake at 37 "C in media containing 0.2 mM NADP+, 1.5 mM glucose 6-phosphate, 1.0 IU of glucose-6phosphate dehydrogenase, and 0.1 M potassium phosphate, pH 7.5, in a final volume of 2.0 mL. Oxygen uptake was determined in a 2-mL thermostated oxygraph veasel (Gibson Medical Electronics)fitted with a Clark-type electrode. The signal from the electrode was recorded with a Heath Schlumbergerlinear recorder fitted with a EU-200-02 DC offset module. After 3-4 min of temperature equilibration,the monooxygenases were added through the capillary access port and substrate-independent oxygen uptake was recorded for at least 2 min. The substrate dissolved in water (or dioxane) was added in 10 pL or less through the access port and oxygen uptake recorded for an additional 2-4 min. Kinetic constants were calculated from initial reaction velocities at substrate concentrations above and below K,. Activities for the (NJ-dimethylaminoalky1)phenothiazines with rabbit lung FMO were measured independently in both Austin and Corvallis. The kinetic constants for porcine liver FMO were calculated from measurements carried out only in Austin. The hydrochloride salts of the compounds with basic side chaina were dissolved in water just prior to use. Spectra of each solution were recorded to check for possible sulfoxidation and to verify concentration. The millimolar absorptivity of the hydrochloride salts of the C347 derivativesin water appeared relatively constant at 2.4/cm and 21.6/cm at 307 and 258 nm, respectively. Phenothiazine, 2-(trifluoromethyl)phenothiazine,phenylthiourea, naphthylthiourea, and thiocarbanilide were dissolved in dioxane. Up to 10 pL/mL of dioxane had no detectable effect on activities of rabbit lung or porcine liver FMO.
Results All derivatives of lO-(N,N-dimethylaminoalkyl)-2-(trifluoromethy1)phenothiazinewith less than 5 methylenes in the side chain showed no detectable activity with rabbit pulmonary FMO (Table I). At concentrations approaching saturation in aqueous solutions at pH 7.5, activity with the C5 analogue was detectable, but the apparent K, was so high that t h e velocity is limited by the solubility of t h e substrate. The C6and C, derivatives, on the other hand, were excellent substrates for the pulmonary FMO, and the turnover was essentially t h e same at saturating concentrations of both these analogues, although the K , of the C6derivative is about 6 times higher than t h a t of the C, derivative. I n contrast t o rabbit lung FMO, the Cz-C7 analogues were readily oxygenated by porcine liver F M O (Table I).
374 Chem. Res. Toxicol., Vol. 3, No. 4, 1990
Nagata et al.
Table I. Effect of Alkyl Side Chain Length of 10-(N,"-Dimethylaminoalkyl)-2-(trifluoromethy1)phenothiazines on Substrate Activities of Rabbit Lung and Porcine Liver FMOO
Table 11. Effect of Substrate Size on Activities of Sulfur ComDounds with Rabbit Luna and Porcine Liver FMO K,, rM,"for comDound rabbit lune Dorcine liver thiourea 23 23 S-methylthiourea NAb NAb 1-phenylthiourea 220 4 1-naphthylthiourea 112 4 thiocarbanilide SAb I phenothiazine SA 12 2-(trifluoromethy1)phenothiazine NA >5 w ~
/
CH3
n
Km, PM
N
\
CH3
?,k
Rabbit Lune FMO NAc NA NA >>lo00 SA' 130 42 22 44
mol of O2 consumed/mol of substrate 'addedb
I
2
3 4 5 6 7 2 3 4 5
6 7
Porcine Liver FMO 55 56 11 59 11 57 11 60 14 67 15 68
0.98 0.99
1.0 0.96 0.96 0.98 0.98
In units of nmol of substrate oxidized/(minmmol of FAD) at 37
"C,pH 7.5, with saturating NADPH and amine substrate measured as described under Experimental Procedures. Reaction stoichiometry calculated with limiting substrate by the procedure described previously (19). 'NA = no detectable activity. SA = slight activity at concentrations of substrate approaching saturation in aqueous solutions.
Except for the Czanalogue, the concentrationsof the C& derivatives required to half-saturate the liver FMO are (within experimental error) essentially the same as and similar to values reported for other phenothiazines with Nfl-dimethylaminoalkyl side chains (14). The molar ratios of oxygen uptake to substrate added were close to 1:l for all derivatives with measurable activities for both the pulmonary and hepatic FMOs (Table I). This suggests that these monooxygenases catalyze only a single oxygenation of these substrates. While the nature of the products formed was not determined, previous studies (14) with the porcine liver FMO demonstrated that only the side-chain nitrogen of NJV-dimethylalkyl- and of piperazine-substituted phenothiazines is oxidized and the oxygen atom is always added to the nitrogen furthest re* moved from the phenothiazine ring. This interpretation is also consistent with the observation that 2-(trifluoromethy1)phenothiazine does not exhibit any detectable activity with the rabbit lung FMO and only slight activity with the porcine hepatic FMO (Table 11). The effect of substrate size on activity of these enzymes is also evident from kinetic constants for organic sulfur compounds listed in Table 11. The concentration of thiourea required to half-saturate both enzymes appears identical. Addition of more bulky substituents (as in phenylthiourea, naphthylthiourea) increases the apparent K , for the lung FMO and, perhaps due to increased lipophilicity, decreases the K , for the porcine liver enzyme. On the other hand, thiocarbanilide which is close to naphthylthiourea in molecular weight and physical properties shows, at best, only slight activity with rabbit lung FMO. The effect of large groups near the nucleophilic center is also evident with phenothiazine. This compound is an excellent substrate for liver FMO whereas activity with the
~
a K,'s calculated from double-reciprocal plots of initial velocities vs substrate concentration. Velocities based on substrate-dependent oxygen uptake at pH 7.5, 37 "C, with saturating NADPH (generating system) and oxygen. Thiourea and S-methylthiourea were dissolved in water. All other compounds listed were dissolved in dioxane at 0.25 M and diluted ass necessary so that no more than 5 pL/mL of dioxane was present in the assay medium. Activity of neither the lung nor liver FMO is affected by up to 10 pL/mL of dioxane. bNA = no detectable activity at concentrations at or near solubility of the compound in the assay medium. SA = slight activity-less than 10% of rate with thiourea. eActivity detectable but K , far above solubility of compound in the assay medium.
pulmonary FMO is barely detectable. However, size is not the only factor controlling access of a potential nucleophile to the catalytic site. Neither enzyme catalyzes detectable oxygenation of S-methylthiourea (Table 11). This compound, which is at least as nucleophilic as NJV-dimethylalkylamines,shows no detectable activity with either enzyme, whereas the tertiary amines are generally excellent substrates for both these forms of FMO ( 4 , 5).
Discussion Previous studies have shown that FMO's purified from rabbit lung and porcine liver catalyze the oxidation of xenobiotic soft nucleophiles by essentially the same mechanism, but they differ considerably in substrate specificity. Since the enzyme-bound oxygenating intermediate (the 4a-hydroperoxyflavin) formed independently of substrate is identical in both enzymes, it is evident that differences in substrate specificities must be due to differences in structural elements that exclude nucleophiles from each enzyme. The structureactivity measurements described in this report suggest that the substrate channel leading to the hydroperoxyflavin is far more restricted in the pulmonary than in the hepatic FMO. Both enzymes catalyze the oxidation of a nucleophilic heteroatom extending only 2-3 A from a structure not much larger in cross section than a six-membered ring (e.g., phenylthiourea, naphthylthiourea; Chart I, Table 11). On the other hand, nucleophilic centers projecting no more than 3-5 A from the center of a molecule.more than 12 A in its longest dimension (e.g., phenothiazine, thiocarbanilide triflupromazine; Tables I and 11) are excluded by the pulmonary but not the hepatic enzyme. However, a nucleophilic heteroatom on an alkyl side chain 6 or moie carbons removed from the tricyclic ring is readily oxidized by both enzymes (Table I). Thus it would appear that differences in the dimensions of the substrate channels leading to the hydroperoxyflavin in these two forms of FMO is responsible for some of their differences in substrate specificity. From the dimensions (Chart I) of the compounds examined, it would appear that, unlike the more open site of hepatic FMO, the hydroperoxyflavin in the pulmonary enzyme is about 6-8 A below the surface in a channel no more than 8 A across in its longest axis. Although only approximate, these dimensions do provide a structural
Specificities of Liver and Lung FMO
Chem. Res. Toxicol., Vol. 3, No. 4, 1990 375
On the other hand, the data in Table I1 clearly show that rabbit lung FMO does not catalyze the oxidation of thiocarbanilide whereas thiourea, phenylthiourea, and naphthylthiourea are substrates. While the products were not identified, the nature of the oxygenating intermediate suggests that pulmonary FMO, like the hepatic enzyme, catalyzes the oxidation of thiocarbamides to formamidine sulfinates through intermediate formamidine sulfenates (19). Both oxidation products are more reactive than the parent thiourea and may contribute to toxicity. Formamidine sulfinates can react with amine nucleophiles, generating guanidines (20), and potential cell damage due to covalent modification of macromolecules by this mechanism has been discussed previously (21). On the A B C other hand, formamidine sulfenic acids readily oxidize GSH to GSSG, and increased biliary efflux of GSSG in perfused rat liver and conscious animals treated with thioureas has been demonstrated (22). Increased efflux of disulfide was observed only with substrates oxidized to sulfenates via FMO. Inhibitors selective for P-450-de- \v/ 'SH A SH SH pendent monooxygenases had no detectable effect. A potential role of the latter enzymes in the bioactivation of thioureas is based largely on their decreased toxicity in D E F rats treated with SKF-52SA (23). While the biochemical a (A) Promazine; (B) IO-(N,N-dimethylaminohepty1)phenobasis is not known, SKF-52SA like most other lipophilic thiazine; (C)10-[3-(4-methylpiperazin-l-yl)propyl]phenothiazine; tertiary amines is a substrate for liver FMO (4) and is, (D) phenylthiourea; (E) naphthylthiourea; (F)thiocarbanilide. therefore, potentially capable of inhibiting the oxidation of alternate substrates. A recent report by Miller et al. basis for the inability of rabbit lung FMO to catalyze ox(24) suggests that differences in toxicities of N-substituted idation of imipramine, chloropromazine, and other 10thioureas may be due to differences in their chemical re(aminopropyl)phenothiazines,whereas it readily catalyzes activity. Although these differences may contribute, the the oxidation of trifluoperazine and prochlorperazine (5). biochemical mechanisms for the oxidation of thioureas The terminal nitrogen of the piperazine-substituted phemust play a key role. A t present, all available evidence nothiazines is at least 7 A from the tricyclic ring (Chart indicates that the S-oxygenation of thiocarbamides is I), and the dimensions of the side-chain piperazine are well catalyzed largely if not exclusively by FMO's (19,22). The within the limits of other six-membered rings readily acstudies described in this report suggest that the overall size commodated by rabbit lung FMO. of substituents near the nucleophilic heteroatom limits Lack of substrate activity of phenothiazines bearing side substrate access to the active site much more in the rabbit chains less than 7 A in length is probably not due to slight lung than in the pig liver enzyme. Thus, it is possible that differences in charge. Provided the compounds are prethe size of compounds bearing a nucleophilic heteroatom dominately present as neutral species or monocations, the may have predictive value for toxicity of thiocarbamides degree of protonation has little or no effect on kinetic in animals expressing a pulmonary FMO similar to the constants. For example, the pK,'s of NJV-dimethylaniline rabbit lung enzyme. and n-octylamine differ by more than five pH units, but, Acknowledgment. Portions of this work were suptheir Km'sfor the rabbit lung enzyme are similar (330 and ported by grants from the Foundation for Research (to 290 pM,respectively; see ref 5 ) . The pKts of the comD.M.Z.) and from the National Institutes of Health (HLpounds listed in Table I were not measured, but it is likely 38650 to D.E.W.). The assistance of personnel at the J & that they would all be similar to triflupromazineand would B Sausage Co., Waelder, TX, in securing fresh hog liver exist predominantly as monocations at pH 7.5. for enzyme preparation is gratefully acknowledged. The more restricted access to the 4a-hydroperoxyflavin in pulmonary FMO may also be responsible for the difReferences ference in pulmonary toxicity of phenylthiourea and (1) Ziegler, D. M., and Mitchell, C. H. (1972) Microsomal Oxidase thiocarbanilide in some animals (15-17). Thiourea and IV. Properties of a Mixed-Function Amine Oxidase Isolated from many of its N-monosubstituted derivatives produce pulPig Liver Microsomes. Arch. Biochem. Biophys. 150,116-125. monary edema and pleural effusion in susceptible animals (2) Williams, D.E.,Hale, S. E., Muerhoff, A. S., and Masters, B. S. (18) whereas symmetrical diarylthioureas are not pulmoS. (1984) Rabbit Lung Flavin-Containing Monooxygenase. Purification, Characterization and Induction During Pregnancy. nary toxins for rabbits (16)and are considerably less toxic Mol. Pharmacol. 28,381-390. for rats (15). Analysis of metabolic end products of phe(3) Sabourin, P. J., Smyser, B. P., and Hodgson, E. (1984) An Imnylthiourea and thiocarbanilide excreted by rabbits did proved Method for the Purification of the Flavin-Containing not reveal any major quantitative differences although loss Monooxveenase from Mouse and Pie" Liver Microsomes. Znt. J. of sulfur was greater with phenylthiourea than with thioBiochek-16, 713-720. carbanilide (16,17). However, metabolic end products of (4) . . Zieeler. D. M. (1988) Flavin-Containine Monooxveenase: Catacompounds administered orally probably reflects metablytic-Mechanism and Substrate Specifikies. Drug Metab. Rev. 19, 1-32. olism in the liver rather than the lung. In liver, substituted (5) Poulsen, L. L, Taylor, K., Williams, D. E., Masters, B. S. S., and thiocarbamides, including thiocarbanilide, are oxidized to Ziegler, D. M. (1986) Substrate Specificity of the Rabbit Lung formamidine sulfinates by microsomal FMO (19). While Flavin-Containing Monooxygenase for Amines: Oxidation Prodnot impossible, it is unlikely that produds produced in the ucts of Primary Alkylamines. Mol. Pharmacol. 30, 680-685. liver could account for differences in pulmonary toxicity (6) Ohmiya, Y., and Mehendale, H. M. (1982) Metabolism of of different thioureas. Chlorpromazine by Pulmonary Microsomal Enzymes in the Rat Chart I. Dimensions of Phenothiazine Derivatives and of Thiocarbamides Estimated from CPK Molecular Models"
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376 Chem. Res. Tonicol., Vol. 3, No. 4, 1990 and Rabbit. Biochem. Pharmacol. 31,157-162. (7) Ohmiya, Y.,and Mehendale, H. M. (1983) N-Oxidation of N,NDimethylaniline in the Rabbit and Rat Lung. Biochem. Pharmacol. 32, 1281-1285. (8) Ohmiya, Y.,and Mehendale, H. M. (1984) Effect of Mercury on Accumulation and Metabolism of Chlorpromazine and Impramine in Rat Lungs. Drug Metab. Dispos. 12, 376-378. (9) Ohmiya, Y.,and Mehendale, H. M. (1984) Species Differences in the N-Oxidation of Chlorpromazine and Imipramine. Pharmacology 28,289-295. (10) Ziegler, D. M., and Poulsen, L. L. (1978) Hepatic Microsomal Mixed-Function Amine Oxidase. Methods Enzymol. 52,142-151. (11) Kaiser, C., Tedeschi, D. H., Fowler, P. J., Pavloff, A. M., Lester, B. M., and Zirkle, C. L. (1971) Analogs of Phenothiazine. Synthesis and Potential Antidepressant Activity of some Phenothiazine Derivatives and Related Compounds Containing a Carbocyclic Basic Side Chain. J . Med. Chem. 14, 179-186. (12) Yale, H. L., Sowinski, F., and Bernstein, J. (1957) 10-(3-Dimethylaminopropyl)-2-(Trifluoromethyl)-Phenothi~ine Hydrochloride (VESPRIN) and Related Compounds. J. Am. Chem. SOC. 79,4375-4379. (13) Borch, R. F., and Hassid (1972) A New Method for Methylation of Amines. J. Org. Chem. 37, 1673-1674. (14) Sofer, S. S., and Ziegler, D. M. (1978) Microsomal MixedFunction-Amine Oxidase: Oxidation Products of PiperazineSubstituted Phenothiazine Drugs. Drug Metab. Dispos. 6, 232-239. (15) Dieke, S.H., Allen, G. S., and Richter, C. P. (1947) The Acute Toxicity of Thioureas and Related Compounds in Wild and Domestic Norway Rats. J. Pharmacol. Exp. Ther. 90, 260-270. (16) Smith, R. L., and Williams, R. T. (1961) The Metabolism of
Nagata et al. Arylthioureas-I. The Metabolism of 1,3-Diphenyl-2-Thiourea (Thiocarbanilide) and Its Derivatives. J. Med. Pharm. Chem. 4, 97-107. (17) Scheline, R. R., Smith, R. L., and Williams, R. T. (1961) The Metabolism of Arylthioureas-11. The Metabolism of 14C-and "S-Labelled 1-Phenyl-2-Thiourea and Its Derivatives. J. Med. Pharm. Chem. 4, 109-135. (18) Dieke, S. H., and Richter, C. P. (1945) Acute Toxicity of Thiourea to Rats in Relation to Age, Diet, Strain and Species Variation. J. Pharm. Exp. Ther. 85, 195-202. (19) Poulsen, L. L., Hyslop, R. M., and Ziegler, D. M. (1979) SOxygenation of N-Substituted Thioureas Catalyzed by Pig Liver Microsomal FAD-Containing Monooxygenase. Arch. Biochem. Biophys. 198,78-88. (20) Walter, W. (1955) Guanidierende wirkungder formamidinsulfinsaure. Angew. Chem. 67, 275-276. (21) Ziegler, D. M. (1982) Functional Groups Bearing Sulfur. In Metabolic Basis of Detoxication (Jakoby, W. B., Bend, J. R., and Caldwell, J., Eds.) Chapter 9, pp 171-184, Academic Press, New York. (22) Kreiter, P. A., Ziegler, D. M., Hill, K. E., and Burk, R. F. Increased Biliary GSSG Fflux from Rat Livers Perfused with Thiocarbamide Substrates for the Flavin-Containing Monooxygenases. Mol. Pharmacol. 26, 122-127. (23) Van den Brenk, H. A. S., Kelley, H., and Stone, M. G. (1970) Innate and Drug-Induced Resistance to Acute Lung Damage Caused in Rats by Alpha-Naphthyl Thiourea (ANTU) and Related Compounds. Br. J. Exp. Pathol. 57, 621-624. (24) Miller, A. E., Bischoff, J. J., and Pae, K. (1988) Chemistry of Aminomethanesulfnic and -sulfonic Acids Related to the Toxicity of Thioureas. Chem. Res. Toxicol. 1, 169-174.
