Inactivation of Rat Hepatic Cytochrome P-450 Isozymes by 3,5

p (CYP3A). Such inactivation involves destruction of P-450 prosthetic heme predominantly by N-ethylation in untreated and PB-pretreated rats, whereas ...
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Chem. Res. Toxicol. 1989,2,400-410

400

Inactivation of Rat Hepatic Cytochrome P-450 Isozymes by 3,5-Dicarbethoxy-2,6-dimethyl-4-ethyl- 1,Cdihydropyridine‘ Katsumi

S u g i y a m a , t K u n q u a n Yao,t

Allan E. Rettie,* a n d M a r i a Almira Correia*it

Department of Pharmacology and Liver Center, University of California, San Francisco, California 94143, and Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195 Received May 30, 1989 We have reported [Correia et al. (1987) Arch. Biochem. Biophys. 258,436-4431 that administration of 3,5-dicarbethoxy-4-ethyl-2,6-dimethyl-l,4-dihydropyridine (DDEP) to untreated, phenobarbital (PB) pretreated, or dexamethasone (DEX) pretreated rats results in relatively selective inactivation of cytochrome P-450 (P-450) isozymes h (CYPBCll), k (CYP2C6), and p (CYP3A). Such inactivation involves destruction of P-450 prosthetic heme predominantly by N-ethylation in untreated and PB-pretreated rats, whereas in DEX-pretreated rats, it also appears to be associated with prosthetic heme alkylation of the apocytochrome presumably a t the active site. T h e cause for this differential course of DDEP-mediated P-450 heme destruction is unclear. Since this process is absolutely dependent on NADPH-mediated DDEP metabolism and can be reproduced in vitro, in search of mechanistic clues, we have examined DDEP metabolism by liver microsomes from the three rat sources as well as by isolated purified rat liver P-450h and P-450k. HPLC analyses of microsomal incubations of DDEP with NADPH, in the presence of an esterase inhibitor, revealed the presence of two major products: deethylated pyridine (DP) and 4-ethylpyridine (4-EDP) with product ratios (DP/4-EDP) of 1.4, 1.4, and 0.7 for reactions catalyzed by liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats, respectively. The corresponding mean product ratios for P-450h- and P-450kcatalyzed reactions were 4.2 and 5.5, respectively. On the other hand, partition ratios ( D P formed/P-450 destroyed) ranged from 12.0, 10.5, and 4.8, respectively, for incubations of liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats to 9.5 and 28.9 for purified P-450h- and P-450k-catalyzed reactions, respectively. However, D P formation in all these microsomal systems was comparable, and although 4-EDP formation was greatly stimulated by DEX pretreatment, it does not appear to be a destructive pathway. In view of this, our findings reported herein suggest that the active site environment of P-450’s h, k, and p apparently determines not only the pattern of DDEP metabolism but also the differential course of prosthetic heme destruction.

Introduction The porphyrinogen 3,5-dicarbethoxy-2,6-dimethyl-4ethyl-1,4-dihydropyridine(DDEP)2is known to destroy several rat hepatic cytochrome P-450 (P-450) isozymes in a mechanism-based “suicidal” process (1-3). The dihydropyridine appears to single out three isozymes [P-450’s h (CYPBCll), k (CYP2C6), and p (CYP3A)I3for inactivation (3). On the basis of in vivo and in vitro studies, we have recently shown that the inactivation processes differ for these three isozymes (3). Thus, DDEP-mediated inactivation of P-450’s h and k largely appears to entail destruction by N-ethylation of the prosthetic heme. On the other hand, that of P-45Op appears to involve destruction of the prosthetic heme to a reactive species that covalently binds to the apocytochrome apparently at the active site (3). Although the precise nature of this process is unclear, it is dependent not only on molecular 02, NADPH, and P-45Op but also on DDEP. To gain some mechanistic insight, we have examined the metabolism of DDEP and/or its structural analogues [4-H (DDHP), 4methyl (DDC),and 4-benzyl (DDBP)] by rat liver micro-

* To whom correspondence should be addressed at the Department of Pharmacology, Box 0450, University of California, San Francisco, San Francisco, CA 94143. ‘University of California. University of Washington. 0893-228x/89/2702-0400$01.50/0

soma1 preparations containing different proportions of P-450’s h, k, and/or p as well as by isolated purified rat liver P-450h and P-450k.4 Our findings, reported herein, This work was reported in ita preliminary form at the 1988FASEB meetings, Las Vegas, NV (FASEB J 2 A1013,1988). Abbreviations: P-450,cytochrome P-450,BNPP, bis(p-nitrophenyl) phosphate, DETAPAC, diethylenetriaminepentaaceticacid; DEX, dexamethasone; DDBP, 3,5-dicarbethoxy-4-benzyl-2,6-dimethyl-1,4-dihydropyridine; DDC, 3,5-dicarbethoxy-4-methyl-2,6-dimethyl-1,4-dihydropyridine; DDEP, 3,5-dicarbethoxy-4-ethyl-2,6-dimethyl-l,4-dihydropyridine; DDHP, 3,5-dicarbethoxy-2,6-dimethyl-1,4-dihydropyridine; DP, 3,5-dicarbethoxy-2,6-dimethylpyridine; 4-EDP, 3,5-dicarbethoxy-2,6-dimethyl-4-ethylpyridine(Cethylpyridine); 4-MDP, 3,5-dicarbethoxy-2,6-dimethyl-4-methylpyridine(4-methylpyridine); PB, sodium phenobarbital; PCN, pregnenolone 16a-carbonitrile; PMSF, phenylmethanesulfonyl fluoride; TAO, troleandomycin; HPLC, high-performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Throughout this report, P-45Op will be used collectively to denote all DEX-inducible and constitutive isoforms, since we have found that DDEP abolishes (>95%) the functional activity (6&testosterone hydroxylase and erythromycin N-demethylase) attributable to such isozymes and thus does not appear to be selective for any P-45Op isoform (3). Furthermore, cDNA-deduced amino acid sequencesof these kmymes reveal a high degree of sequence identity in the regions presumed to be sandwiching the heme. These isozymes have been relegated to the CYP3A P-450 gene subfamily, whereas P-450h and P-450k have been assigned to the CYP2Cll and CYP2C6 gene subfamilies, respectively (43).

Until recently (44), P-45Op isozymes were generally believed to be resistant to functional reconstitution after purification, and therefore, purified P-45Op isozymes were not included in our studies. 0 1989 American Chemical Society

Inactivation of Cytochrome P-450 Isozymes

Chem. Res. Toxicol., Vol. 2, No. 6, 1989 401

ring-CH2Ph),2.17 (6 H, s, ring-CH,), 4.05 (5 H, m, COzCH&H3; 4-H), 5.33 (1 H, br s, NH), 7.15 (5 H, m, benzyl ring-H). Anal. Calcd for C&I%NO4: C, 69.95; H, 7.34; N, 4.08. Found C, 69.96; H, 7.29; N, 4.07. 3,5-Dicarbethoxy-2,6-dimethyl-1,4-dihydropyridine (DDHP). DDHP was prepared by dissolving formaldehyde (0.05mol), Materials and Methods ethyl acetoacetate (0.1 mol), and concentrated NH40H (0.075 mol) in ethanol (50 mL) and then heating under reflux for 3 h. The Animals. Male Sprague-Dawley rats (200-250 g) were fed and mixture was cooled and diluted with an equal volume of HzO,and given water ad libitum. When hepatic microsomes relatively the precipitate was recrystallized from ethanol before purification enriched in P-45Op and/or P-450k content were to be examined, by HPLC. The HPLC-purified compound had the following the animals received either dexamethasone (DEX, 100 mg/kg, properties: mp 186-187 "C [lit. mp 188-189 "C (91;UV (MeOH) ip) in corn oil, daily for 4 days, or phenobarbital sodium (PB, 80 ,A, 231, 252 (shoulder), 373 (e231 = 16; = 7.41 mM-' cm-'); mg/kg, ip), daily for 5 days. Untreated animals served as the EIMS m / z 254 (4), 253 (M, 27), 252 (27), 225 (18), 224 (loo),208 source of hepatic microsomal P-450's h and k. Animals were killed, (52), 206 (22), 196 (57), 180 (19), 152 (18), 106 (18), 67 (16), 45 their livers perfused in situ, and microsomes prepared by con(25), 42 (27), 27 (25); 'H NMR 6 1.28 (6 H, t, C02CHzCH3), 2.18 ventional methods exactly as described previously (3). When (6 H, s, ring-CH,), 3.26 (2 H, s, 4-H), 4.17 (4 H, q, COzCHzCH3), hepatic microsomes containing labeled P-450 heme were used, 5.12 (1H, br s, NH). Anal. Calcd for C13H19N04:C, 61.64; H, heme was labeled by administration of the heme precursor 7.56; N, 5.53. Found: C, 61.60; H, 7.66; N, 5.54. [14C]-b-aminolevulinicacid and quantitated exactly as described 3,5-Dicarbet hoxy-2,6-dimethyl-4-ethylpyridine(4-Ethylpreviously (3). Materials. 3,5-Dicarbethoxy-4-methyl-2,6-dimethyl-1,4-di- pyridine, 4-EDP). 4-EDP was prepared by heating DDEP (4 mmol) in 20% aqueous HNO, at 100 "C for 1 h. After cooling hydropyridine (DDC) was purchased from Eastman Kodak Co., the reaction mixture, solid NaZCO3was added until the pH was Rochester, NY,and purified by HPLC before further use. DEX, basic. The product was extracted with diethyl ether, dried over NADPH, and bis(p-nitrophenyl) phosphate (BNPP) were purNa2S04,and further purified by HPLC. The HPLC-purified chased from Sigma Chemical Co., St. Louis, MO. compound was a liquid with the followingproperties: W (MeOH) Chemical Syntheses of DDEP and Its Analogues and A,, 271 (e271 = 3.3 mM-' cm-'); EIMS m / z 280 (M + 1, 4), 279 Metabolites. These compounds were prepared by slight mod(M, 26), 250 (70), 234 (52), 222 (59), 206 (20), 204 (la), 32 (14), ifications of the previously reported synthetic methods for sub29 (29),28 (100);positive LSIMS m / z 281 (18),280 (M + H, 100), stituted dihydropyridines based on the general Hantzach reaction 234 (38); 'H NMR 6 1.13 (3 H, t, 4-CHZCH,), 1.39 (6 H, t, (4-6). The compounds were purified by repeated HPLC (linear COzCHzCH3),2.51 (6 H, s, ring-CH,), 2.55 (2 H, q, 4-CHzCH3), gradient mode) in a system consisting of two Beckman Model 4.41 (4 H, q, COzCHzCH3).Anal. Calcd for ClSHz1N04:C, 64.49; llOB pumps, a Beckman solvent programmer, and a HewlettH, 7.58; N, 5.01. Found: C, 64.46; H, 7.64; N, 4.99. Packard Model 1040A diode array UV/vis detector. The melting 3,5-Dicarbethoxy-2,6-dimethylpyridine(DP). DP was points reported are uncorrected. The spectral analyses were prepared from DDBP (4 mmol) by the same procedure described carried out with an SLM Amiico 2000 W/vis spectrophotometer. for 4-EDP. The HPLC-purified compound had the following The HPLC-purified compounds were subjected to structural properties: mp 71-72 "C [lit. mp 72-72.5 "C (S)]; UV (EtOH) identification and characterization by 'H NMR and electron ,A, 231, 274, 284 (shoulder) (e274 = 3.9 mM-' cm-'); EIMS m/z impact (EI) and positive LSIMS mass spectrometric analyses, 252 (13),251 (M, 78), 206 (93), 178 (53), 29 (68), 28 (100);positive in order to ascertain their chemical purity and authenticity. NMR LSIMS m / z 252 (M + H, 100); 'H NMR 6 1.41 (6 H, t, spectra were recorded in deuterated chloroform at 80 MHz in a COzCHzCH3),2.84 (6 H, s, ring-CH,), 4.39 (4 H, q, COzCHzCH3), Varian FT-80 spectrometer. Chemical shift values, reported in 8.66 (1H, s, 4 4 . Anal. Calcd for C13H17N0,: C, 62.14; H, 6.82; parts per million relative to tetramethylsilane, were measured N, 5.57. Found: C, 61.74; H, 6.83; N, 5.51. relative to the residual chloroform peak. E1 mass spectra (70 eV) 3,5-Dicarbethoxy-2,6-dimethyl-4-methylpyridine(4were obtained on a Kratos MS-25 mass spectrometer, whereas Methylpyridine, 4-MDP). 4-MDP was prepared by stirring in LSIMS mass spectra were obtained with glycerol as the matrix on a Kratos MS-50 instrument. Elemental analyses of the synsmall portions of sodium nitrite (14.6 mmol) into a solution of DDC (973 "01) in glacial acetic acid (25 mL) at 15-20 "C. After thesized compounds were carried out by the Microanalytical Laboratory, Department of Chemistry, University of California, completing the addition, the mixture was stirred until it was free of the brown fumes. The mixture was poured into 200 mL of Berkeley. ice-watei and then extracted with three 200-mL volumes of ether. 3,5-Dicarbethoxy-4ethyl-%,&dimethyl-l,4-dihydropyridine The combined ether extracts were then extracted with dilute (DDEP). Propionaldehyde (0.1mol), ethyl acetoacetate (0.2 mol), aqueous hydrochloric acid (1:3 v/v). The combined acid extracts and concentrated NHIOH were dissolved in ethanol (50 mL) and were neutralized with sodium bicarbonate, and the product was heated under reflux for 1 h. The mixture was cooled to room reextractedwith ether. The ether-extractedmaterial was subjected temperature, and 100 mL of H20was added and the precipitate to HPLC, and the HPLC-purified compound (a liquid) exhibited collected by vacuum filtration. This filtrate was recrystallized the following properties: UV (MeOH), X 271 (e271 = 3.9 mM-' from ethanol four times and purified by HPLC [Altex Ultrasphere cm-'); EIMS m / z 266 (16), 265 (M, 70), 236 (33), 220 (loo), 219 ODS (5 pm, 1.0 X 25 cm) column; isocratic mobile phase, (32),208 (36),192 (29), 164 (17),77 (30),29 (85),28 (79);'H NMR MeOH/H20, 67% v/v]. The HPLC-purified compound had the following properties: mp 110-111 "C [lit. mp 110-111 "C (5)]; 6 1.37 (6 H, q, COZCHZCH,), 2.24 (3 H, S, 4-CH3), 2.50 (6 H, S, ring-CH,), 4.40 (4 H, q, COzCHzCHJ. Anal. Calcd for C14H1P04: UV (MeOH) &= 235,350 (em = 18.8; em = 8 mM-' cm-'); EIMS C, 63.38; H, 7.22; N, 5.28. Found: C, 62.94; H, 7.23; N, 5.32. m/z 281 (M, 0.4), 252 (loo),251 (14), 236 (17), 224 (34), 196 (35), 32 (16), 29 (19), 28 (61); positive LSIMS m / z 283 (7), 282 (M + Assays. DDEP-mediated liver microsomal P-450 destruction, H, 33), 252 (94), 236 (100); 'H NMR b 0.75 (3 H, t, 4-CH,CHJ, irreversible binding of P-450 prosthetic heme to microsomal protein, and assay of DDEP metabolites were all performed as 1.28 (6 H, t, C02CH2CH3),1.45 (2 H, m, 4-CHzCH3),2.28 (6 H, previously described (3). As discussed below, the inclusion of the s, ring-CH,), 3.91 (1H, t, 4-H), 4.17 (4 H, q, COzCHzCH3),5.70 esterase inhibitor BNPP (0.4 mM) in the incubations limited (1H, br s, NH). Anal. Calcd for Cl5Hz3NO4:C, 64.04; H, 8.24; N, 4.98. Found: C, 63.91; H, 8.41; N, 5.26. DDEP metabolism to two major products, 3,5-dicarbethoxy3,5-Dicarbethoxy-4-benzyl-2,6-dimethyl-l,4-dihydro- 2,6-dimethyl-4-ethylpyridine(4-ethylpyridine,4-EDP),and the pyridine (DDBP). DDBP was synthesized exactly as described deethylated product (3,5-dicarbethoxy-2,6-dimethylpyridine, DP). Catalase (0.5 mg/mL) was also included to minimize any conabove for DDEP, except that phenylacetaldehyde (0.1 mol) replaced propionaldehyde in the reaction mixture. The HPLCcurrent peroxidatic reactions. Accordingly, the complete incubation mixture routinely consisted of DDEP or analogue (0.5 mM), purified compound had the following properties: mp 115-116 "C EDTA (1.5 mM), catalase (0.5 mg/mL), BNPP (0.4 mM), NADPH [lit. mp 115-117 "C (5)];UV (MeOH) ,A, 235, 352 (e235 = 17.0; tm = 6.7 mM-' cm-'); EIMS m / z 298 (17), 252 (loo), 253 (26), (1 mM), and liver microsomes (2 mg of protein/mL) in a final volume of 3 mL of 0.1 M potassium phosphate buffer, pH 7.4. 224 (35), 196 (28);positive LSIMS m / z 344 (M + H, 40), 298 (40), At the end of the incubation, the internal standard nitrendipine 252 (100); 'H NMR 6 1.25 (6 H, t, C02CHzCH3),2.58 (2 H, d,

suggest that the differential mode of inactivation of P-450's h, k, and p by DDEP is governed by the specific active site environment of each isozyme rather than by their differential catalytic processing of the drug.

Sugiyama et al.

402 Chem. Res. Toxicol., Vol. 2, No. 6, 1989

(3.9 pM) and Na2COsbuffer (pH 10.5,0.6 mL) containing NaCl (2 M) were added, and the reaction mixture was extracted three times with CH2C12.The CH2C12extractswere combined and dried over Na+304,and the solvent was evaporated under N2. Residues were dissolved in MeOH and aliquots assayed by HPLC on an Altex Ultrasphere ODS (5 pm, 1 X 25 cm) or Rainin Microsorb (5 pm, 0.46 X 25 cm)column online with either a Hitachi UV/vis detector or a Hewlett-Packard diode array detector, exactly as described previously (3). Isolation and Purification of P-450h. Liver microsomes (3 g of protein) from untreated male Sprague-Dawley rats (2oo-250 g) were diluted to 10mg/mL in 5 mM potassium phoephate buffer, pH 7.4, containing 20% glycerol, 0.1 mM EDTA, 0.2% Emulgen 911, and 0.5% cholate (buffer A) and then solubilized for 2 h at room temperature. The suspension was then centrifuged at 4 "C for 1h at 100000g. The supernatant was applied to a 2.5 X 60 cm DEAE column which had been equilibrated at room temperature with buffer A. After application, the column was washed with 80 mL of buffer A and P-450h was elutzd subsequently with a linear salt gradient from 0 to 100 mM KC1 in a liter of buffer A. The first peak was used for P-450h purification, and the appropriate fractions were pooled on the basis of SDS-PAGE profiles. The pooled fractions containing P-450h were dialyzed overnight against 10 mM Tris acetate buffer, pH 7.4, containing 20% glycerol and 0.1 mM EDTA, and applied to a 1.5 X 16 cm DEAE-Sephacel column equilibrated with 10 mM Tris acetate buffer, pH 7.4, containing 20% glycerol, 0.1 mM EDTA, and 0.2% Emulgen 911 (buffer B). P-450h was only slightly retained on this column and was eluted with the equilibration buffer as a broad peak. The pooled P-450h fractions from the first DEAESephacel column were dialyzed against 10 mM Tris acetate buffer, pH 7.7, containing 20% glycerol and 0.1 mM EDTA, and applied to a second DEAE-Sephacel column which had been previously equilibratedwith the same buffer containing 0.2% Emulgen 911. P-450h was eluted with a linear salt gradient (0-100 mM NaOAc in 200 mL of equilibration buffer), and it was purified to homogeneity as shown by SDS-PAGE. If further purification of the P-450h fraction were required, a hydroxylapatite column was used to remove the residual contaminants. The sample was dialyzed against 10 mM potassium phosphate, pH 7.4, containing 20% glycerol and 0.1 mM EDTA, and applied to a hydroxylapatite column equilibrated with 10 mM potassium phosphate, pH 7.4, containing 20% glycerol, 0.1 mM EDTA, and 0.2% Emulgen 911 (buffer C). After washing the column with buffer C containing 30 mM potassium phosphate, P-450h was eluted with buffer C containing 75 mM potassium phosphate. Detergent was removed from purified P-450h by column chromatography on hydroxylapatite. For this purpose, P-450h was first dialyzed against 10 mM potassium phosphate, pH 7.4, containing 20% glycerol, 0.1 mM D?T, and 0.1 EDTA, and then applied to a hydroxylapatite column equilibrated with the same buffer containing 0.2% Emulgen 911. After sample application, the column was washed with the equilibrationbuffer plus 0.1 % cholateand then washed with the equilibration buffer with 0.1% cholate but no Emulgen 911. P-450h was finally eluted by increasing the concentration of phosphate buffer to 400 mM and dialyzed for 2 days against 2 liters of 100 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol, 0.1 mM EDTA, and 0.1 mM DT". The purified protein so obtained had a specific content of 4 2 nmol/mg of protein and exhibited a single band corresponding to a molecular weight of 51K by SDS-PAGE (Figure 1A). Its functional activity was ascertained after reconstitution (in the presence of dilauroylphosphatidylcholine)with equimolar amounts (nominally, 100 nM) of purified rat liver P-450 reductase and cytochrome b6, with testosterone as the substrate (9). The turnover numbers for different testosterone hydroxylation products were as follows: 2aOH, 9.9; 2/30H, 0.21; 6/30H, 0.41; 7aOH, 0.32; and 16aOH, 14.5. The turnover numbers for P450h-selectivefunctional markers, 2aOH and 16aOH testosterone hydroxylases(ZO), are consistentwith the regie and stemowlective propertiesof the enzyme (11 ) and attest to relatively high P-450h enrichment of the purified preparation. Isolation and Purification of P-450k. The isozyme was purified from liver micm"es from male Long-Evansrats (8Cb120 g) fed sodium phenobarbital (0.15 w/v) in their drinking water for 7 days. A modification of the procedures described by Wolf

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Figure 1. SDS-PAGE analyses of (A) untreated rat liver microsomes and partially and fully purified P-450h and (B) PBpretreated rat liver microsomes and purified rat liver P-450k. SDS-PAGE analyses were carried out as described previously (46). (A) Lanes (left to right): (a) P-450h (20 pmol; specific content 12 nmol/mg), obtained after two sequential DEAE-Sephacel columns; (b) partially purified P-450h (20 pmol, specific content 6-8 nmol/mg), obtained after the first DEAESephacelcolumn; (c) liver microsomes (10 pg, a0.97 nmol/mg of protein) from untreated rats; (d) molecular weight standards (a1pg each; top to bottom, 97.4K, 66.2K, and 42.7K). (B) Lanes (left to right): (a and f) molecular weight standards (-1.5 pg each; top to bottom, 94K, 67K, 43K, 20.1K, and 14.4K); (b) PB-pretreated rat liver microsomes (15pg; specific content 2.5 nmol/mg of protein); (c) partially purified P-450k (0.5% Emulgen 911 eluate from octyl-Sepharose column; 1.5 pg); (d and e) 'purified P-450k" (DEAE-Sepharose CL-6B column eluate; specific content 16.1 nmol/mg of protein, 0.5 and 1.0 pg, respectively). et al. (11) was used,and all steps were carried out at 4 "C unless noted otherwise. Liver microsomes (P-450 content, 2.5 nmol/mg of protein) were diluted to 10 mg of protein/mL in pH 7.4 buffer (buffer D) containing 100 mM potassium phosphate, 20% glycerol, EDTA (1 mM), DTT (1 mM), and PMSF (0.1 mM) and solubilized by the addition of an equal volume of buffer D plus 2% sodium cholate. Following centrifugation to remove insoluble material, the supernatant (total P-450,2400 nmol) was dialyzed against buffer D plus 0.4% cholate and applied to a 2.5 X 40 cm octyl-Sepharose column equilibrated with dialysis buffer. The column was washed with 2 column volumes of dialysis buffer and eluted by the addition of Emulgen 911. The majority of the P-450 (about lo00 nmol) was eluted at an Emulgen concentration of 0.1%. This fraction was highly enriched in P-450b as judged by

Chem. Res. Toxicol., Vol. 2, No. 6, 1989 403

Inactivation of Cytochrome P-450 Isozymes

'' DDEP

DP

+BNPP

-BNPP

4-EDP

Figure 2. Oxidative metabolism of DDEP to DP and 4-EDP. SDS-PAGE analysis (Figure 1B). Raising the Emulgen 911 concentration to 0.5% eluted a second P-450 fraction (about 300 nmol) which contained one major band of lower molecular weight than P-450b. This fraction was concentrated by ultrafiltration and dialyzed extensively against 5 mM potassium phosphate, 20% glycerol, EDTA (0.1 mM), and 0.5% Emulgen 911 (buffer E, pH 7.7) and applied at room temperature to a 1.6 X 20 cm DEAESepharose CL-6B column equilibrated with buffer E. The majority of the P-450 applied was not retained on this column, and the flow-through fraction appeared homogeneous by SDS-PAGE analysis. Detergent was removed from this preparation as described for P-450h. The final preparation had a specific content of 16.1 nmol/mg of protein. It was reconstituted with phosphatidylcholine and an equimolar amount of P-450 reductase and assayed for stereo/regioselective warfarin hydroxylase activity by GC-MS (13). Turnover numbers for (R)-warfarinwere as follows: 4'-OH, 0.06; 6-OH, 0.09; 7-OH, 0.59; and 8-OH, 0.03. The corresponding numbers for @)-warfarinwere as follows: 4'-OH, 0.20; 6-OH, 0.21; 7-OH, 0.89; and 8-OH, 0.15. Thus, the stereo/regioselectivityof warfarin metabolism catalyzed by this preparation is consistent with that reported previously for P-450k (14). P-450 Inactivation and DDEP Metabolism in Reconstituted Enzyme Systems. P-450h (0.36 nmol) was mixed with purified rat liver P-450 reductase [2160 units; 1unit = 16.7 pmol of cytochrome c reduced/(mg of protein-min) at 25 "C, in 0.1 M phosphate buffer, pH 7.41, dilauroylphosphatidylcholine (20 pg), EDTA (1.5 mM), catalase (0.25 mg), and DDEP (0.5 mM) in a final volume of 0.5 mL. After preincubation at 37 "C for 2-3 min, the reaction was initiated with NADPH (1mM) and carried out at 37 "C for 30 min. P-450k (0.16 nmol) was mixed with equimolar amounts of rat liver P-450 reductase ( ~ 2 0 0 units) 5 by preincubation for > 2 min at room temperature. Dilauroylphosphatidylcholine (2 mg/mL, 18.7 pL) was then added and the mixture incubated at room temperature for >2 min. Equimolar amounts of cytochrome b5, catalase (0.5 mg/mL), EDTA (1mM), and DDEP (0.5 mM) were then added, and the reaction mixture (final volume 0.46 mL) was preincubated at 37 "C for 3 min. The reaction was initiated with NADPH (1mM) and carried out for 15 min at 37 "C. Spectrally detectable P-450 loss and DDEP metabolites (following addition of the internal standard nitrendipine)were assayed exactly as described above for the microsomal systems. Chemical Oxidation of DDEP by K3Fe(CN), or NaNOZ. K3Fe(CN), (4 mM) was added to KzCO3 (1mM), KC1 (60 mM), and DDEP (1mM, pH 10.5) in deoxygenated 20% CH3CN/Hz0 solution (5 mL), and the reaction was carried out at 37 "C for 10 min. A large excess of HzO was added to the reaction mixture, and the products were extracted with CHzClz(5 d)The . CHPClz layer was removed, washed with H20,and dried over Na.$04. The residue was dissolved in MeOH and subjected to HPLC as described above. Two-electron oxidation of DDEP by NaNOzwas carried out exactly as described above for the synthesisof 4-MDP from DDC, except that DDEP (0.36 mmol in 5 mL of glacial acetic acid) and NaN02 (23.9 mmol) were mixed at the start, and the products formed were extracted and subjected to HPLC exactly as described above.

Results and Discussion Cytochrome P-450 mediated one-electron oxidation of

DDEP is known to result in extrusion of the 4-ethyl radical (15)and aromatization to the corresponding pyridine (DP) species (6). We (3) and others (16) have reported previously that 4-ethylpyridine (4-EDP) is also a major metabolite of the drug in in vitro hepatic microsomal incubation systems (Figure 2). DP may also be oxidatively

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+NADPH

Time (mln)

Figure 3. HPLC profiles of DDEP metabolites obtained from incubations (A) containing the esterase inhibitor BNPP and (B) in the presence or absence of NADPH. (A) Liver microsomes from PB-pretreated rats were incubated in the presence or absence of BNPP (0.4 mM), exactly as described under Materials and Methods. Nitrendipine (3.9 pM) was added as the internal standard (ST), and the incubates were extracted and assayed by HPLC as described. In the absence of definitive structural identification,we have denoted the putative secondary oxidative metabolites of DDEP as Px and Py. (B) Liver microsomes from DEX-pretreated rats were incubated with DDEP in the presence or absence of NADPH (1 mM) and subjected to HPLC after addition of the intemal standard nitrendipine, exactly as described under Materials and Methods. cleaved by P-450 to yield secondary deesterified products (17,18) and/or hydrolyzed by microsomal esterases at the 3,5-dicarbethoxy side chains. However, we have found that such secondary metabolism is largely limited by inclusion of the microsomal esterase inhibitor BNPP in the incubation systems. Thus, inspection of the HPLC profiles of rat liver microsomal NADPH-dependent DDEP metabolites in the presence or absence of BNPP (Figure 3A) reveals that BNPP not only inhibited the deesterification of DP but did so in a concentration-dependent fashion (not shown), thereby preventing the formation of its secondary metabolites (Px, Py). Indeed, such inhibition of dihydropyridine deesterification by B N P P has been previously reported to account for its potentiation of dihydropyridine-induced porphyrinogenesis (19). Moreover, in the present studies, at the concentrations employed, B N P P did not affect either P-450-dependent oxidative metabo-

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A

1130160; 14012ElDDEP

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I

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20 0 -

,

.

.

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-. .Y ,-. la

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. 15

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DDEP

1

1

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DP

iL CEDP

\

DDEP

DDEP

ihi ;!

,1 i n

ST’

Dp

\I-EDP

-1JL

CEDP

/L

Figure 4. Relative formation of DP and 4-EDP in incubations catalyzed by hepatic microsomes or purified P-450h and P-450k isozymes as well as chemically driven by the one-electron and two-electron oxidants K,Fe(CN), and NaN02,respectively. Panel A illustrates the temporal HPLC profile of DDEP and its metabolites from incubations catalyzed by PB-pretreated rat liver microsomes in its entirety. In panel B, the corresponding relevant “14-20-min window” chromatograms are depicted for DDEP and metabolites from systems catalyzed by liver microsomes from untreated (UT), PB-pretreated (PB), and DEX-pretreated (DEX) rats or by purified P-450h or P-450k isozyme or in chemical systems driven by K,Fe(CN), or NaN02. For experimental details please see Materials and Methods.

lism of DDEP to 4-EDP and D P (not shown) or DDEPmediated destruction of the enzyme (not shown). For these reasons, unless otherwise indicated, BNPP (0.4 mM) was routinely included in all our incubation systems. The HPLC profiles of DDEP metabolites generated by DEX-pretreated rat liver microsomes in these incubation systems in the presence or absence of NADPH indicate that the formation of both major metabolites was NADPH dependent (Figure 3B) and that 4-EDP and D P chromatograph with retention times of 16.5 and 18 min, respectively (Figure 4A). These retention times were found to coincide with those of corresponding chemically synthesized standards that were structurally characterized by mass spectral and NMR analyses and determined to be authentic. Liver microsomes from either untreated or PBpretreated rats when incubated under identical conditions, or systems catalyzed by purified P-450h and P-450k isozymes, gave essentially comparable metabolic profiles, but with quantitatively different yields of the two metabolites (Figure 4B). On the other hand, the reaction chemically

driven by the one-electron oxidant K,Fe(CN), yielded DP as the major product (=95.4%; product ratio 21), whereas that initiated by the two-electron oxidant NaNOz yielded 4-EDP as the predominant product (=98% ; product ratio 0.03) (Figure 4B). The time-dependent formation and the product ratios of the two DDEP metabolites in incubations catalyzed by hepatic microsomes from DEX-pretreated rats are depicted in Figure 5. It is noteworthy that the formation of both metabolites not only exhibited parallel temporal profiles but also reached a maximum by 15 min, which coincided with the complete inactivation of the DDEPsusceptible isozymes in this system (Figure 6). Such findings strongly implicate DDEP-susceptible isozymes in the formation of both products, a possibility further strengthened by the constant product ratios observed after 15 min of incubation (Figure 5B). Furthermore, the changing temporal profile of product ratios (Figure 5B) is characteristic not only of multiple isozyme contributions to DDEP metabolism but also of

Chem. Res. Toxicol., Vol. 2, No. 6, 1989 405

Inactivation of Cytochrome P-450 Isozymes 100

0

]A 4

I

IIP

I

"

I

0

.

I

10

.

I

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.

I

30

.

I

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1

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-

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.-0

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9 0 C

z

E

I /e

-

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3

1

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0

20

10

20

30

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Time (min)

Figure 5. Temporal formation and product ratios of DP and 4-EDP in incubations catalyzed by liver microsomes from DEXpretreated rats. The temporal formation of the two DDEP metabolites [DP (@)and 4-EDP ( O ) ] ,each depicted as nmol/incubation (panel A), and the corresponding product ratios for this system (panel B) are illustrated. Experiments were carried out in triplicate, a prototype of which is depicted. For experimental details please see Materials and Methods.

different metabolic pathways for each isozyme committed to DDEP oxidation. For instance, D P appears to be a major metabolite produced by liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats as well as by purified P-450h and -kisozymes. On the other hand, the other major metabolite, 4-EDP, is formed to the greatest extent in systems catalyzed by liver microsomes from DEX-pretreated rats (Figure 4). Moreover, preliminary findings with liver microsomes from DEX-pretreated rats given the "P-450p-specific inhibitor" troleandomycin (TAO) (20) indicated that such TAO complexation of P-45Op isozymes reduced 4-EDP formation by 56%. A 56% protection from DDEP-susceptible P-450 loss was also observed in parallel, whereas DP formation was inhibited by only 40% (results not shown). Thus, although P-450's h, k, and p equivalently contribute to DP formation, P-45Op isozymes apparently make a relatively greater catalytic contribution to DDEP metabolism to 4-EDP. However, before P-450p-dependent oxidation of DDEP to 4-EDP is implicated in its destruction, it may be worth noting that the corresponding oxidation of DDC, the 4methyl analogue of DDEP, almost exclusively to 4methyl-DP (4-MDP), albeit much more efficient, was not associated with P-450 loss in DEX-pretreated rat liver microsomes (Table I) or PB-pretreated rat liver microsomes (16). Little P-450 loss was also observed (Table I) when rat liver microsomes from DEX-pretreated rats oxidize DDHP (the 4-H analogue) to DP, a reaction that engenders loss of two reducing equivalents. Furthermore, P-450-dependent metabolism of nifedipine [2,6-di-

0

10

0

0 0

10

20

30

40

Time (min)

Figure 6. Time course for DDEP-mediated hepatic P-450 loss and DP formation in incubations catalyzed by rat liver microsomes. The time courses for hepatic microsomal P-450 loss (top panel) and DP formation (bottom panel) in reactions catalyzed by liver microsomes from untreated (UT, O ) ,PB-pretreated (PB, X), or DEX-pretreated (DEX, 0 ) rats are depicted. For experimental details please see Materials and Methods. Experiments were carried out in triplicate, a prototype of which is depicted. Each 3-mL incubation contained liver microsomes (2 mg of protein/mL) from UT or PB- or DEX-pretreatedrats. The P-450 content (nmol/mg of protein) of these preparations was as follows: UT, 0.94; PB, 1.95; and DEX, 2.21, respectively.

Table I. Rat Liver Microsomal Metabolism of 4-Substituted (R)1,4-Dihydropyridinesand Corresponding P-450 Loss" (4W

dihydropyridine DDEP DDC DDHP

P-450 lossb 44.9 f 0.4 0.2 3.8 f 3.2

4-RDPc 58.4 f 3.9 94.6 f 5.6

DPc 40.4 f 3.9 0.7 f 0.5 29.0 9.7

*

" Liver microsomes from DEX-pretreated rats were incubated with DDEP or one of its 4(R)-substitutedanalogues (0.5 mM) at 37 'C for 20 min in reaction mixtures (3.0 mL final volume) containing BNPP, catalase, and NADPH exactly as detailed under Materials and Methods, except that EDTA was replaced with DETAPAC (1.5 mM). *Percent of original microsomal P-450 content (100% = 1.42 f 0.01 nmol/mg of protein). CNanomolesformed per 20-min incubation.

pyridine], or the 4-phenyl analogue of DDEP with loss of H-4 and conversion to the corresponding 4-arylpyridine does not result in P-450 destruction (3,15). Collectively, these precedents strongly support the notion that P-450dependent metabolism of DDEP to 4-EDP is similarly not a destructive pathway. Indeed, the predominant formation of 4-EDP from DDEP by the two-electron oxidant NaNOz and the almost exclusive formation of D P by the oneelectron oxidant K,Fe(CN)6 (Figure 4) argue that P-450dependent 4-EDP formation from DDEP most likely inmethyl-4-(2-nitrophenyl)-3,5-dicarbomethoxy-l,4-di- curs two-electron oxidation, a process characteristic of hydropyridine] , nitrendipine [ 2,6-dimethyl-4-(3-nitronormal, nonsuicidal catalytic turnover of the enzyme. Indeed, these precedents also make it reasonable to assume phenyl)-3-carbethoxy-5-carbomethoxy-1,4-dihydro-

406 Chem. Res. Toxicol., Vol. 2, No. 6, 1989

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Table 11. Product Ratios of DDEP Metabolism by Rat Liver Microsomal P-450 Isozymes and Apparent Partition Ratios for Their DDEP-Mediated Destruction enzvme source DP formedo 4-EDPo P-450 losso DP/4-EDP PRb 27.1 f 5.0 20.1 f 2.9 2.3 f 0.2 1.4 f 0.2 12.0 f 3.5 untreatedc 3.2 f 0.2 1.4 f 0.3 10.5 f 4.0 33.7 f 10.6 24.0 f 3.7 PB treatedc 6.8 f 0.8 0.7 f 0.1 4.8 f 1.8 32.2 f 8.5 50.3 f 16.2 DEX treatedC P-450hd 3.2 f 0.3 0.8 f 0.1 0.3 f 0.0 4.2 f 1.0 9.5 f 1.3 0.1 f 0.0 5.5 f 1.1 28.9 f 3.1 2.6 f 0.2 0.5 f 0.1 P-450ke ‘The values listed for each parameter represent mean f SD (nmol/15-min incubation) obtained from at least 3 individual experiments. For experimental details please see Materials and Methods. *PR = apparent partition ratio [p/E‘,,, DP formed (nmol)/P-450 loss (nmol)] after a 15-min incubation period. ‘Rat liver microsomes (2 mg of protein/mL) 3-mL incubation volume. The P-450 content (nmol/mg of protein) for each microsomal preparation was as follows: untreated, 0.97 f 0.02; DEX pretreated, 1.73 f 0.04; PB pretreated, 1.45 f 0.04. dPurified rat liver P-450h (0.72 wM),0.5-mL incubation volume. “Purified rat liver P-450k (0.35 wM),0.46-mL incubation volume.

that the observed DDEP-mediated P-450 destruction is associated with the one-electron oxidation of DDEP to the cation radical species and subsequent extrusion of its 4ethyl radical to form the aromatized oxidation product, DP (Figure 2). This reasoning is also consistent with the observed inactivation of purified P-450’s h and k, which yield DP as the predominant product of DDEP oxidation (Table 11). With this assumption, the “apparent” partition ratios for DDEP-mediated P-450 inactivation and DP formation, in systems catalyzed by hepatic microsomes from the three sources or by the purified hepatic P-450’s, may be calculated. Thus, if the amount of P-450 inactivated over 15 min is taken to represent the original concentration of DDEP-inactivatable enzyme E b and the concentration of D P formed a t that time is taken as p , then the apparent partition ratios @/E’,,) for DDEP-mediated P-450 isozyme inactivation in DEX- and PB-pretreated and untreated rat liver microsomes are estimated to be ~ 4 . 8 ~, 1 0 . 5 and , r12.0, respectively (Table 11). On the other hand, studies with isolated purified rat liver P-450h and P-450k (reconstituted with P-450 reductase) yield partition ratios of 9.5 f 1.3 and 28.9 f 3.1, respectively, for their DDEPmediated inactivation (Table 11). Thus, these ratios reveal that DEX-inducible P-45Op isozymes are much more susceptible to DDEP-mediated inactivation than the other PB-inducible isozymes or constitutive isozymes. As already stated, P-450h- and P-450k-catalyzed reactions largely yielded DP as the major product, with product ratios (DP/EDP = 4.2 f 1.0 and 5.5 f 1.1,respectively) that are substantially different from those (1.4 f 0.2 and 1.4 f 0.3) of reactions catalyzed by untreated and PBpretreated rat liver microsomes, respectively, and particularly from that (0.7 f 0.1) of reactions catalyzed by liver microsomes from DEX-pretreated rats (Table 11). Furthermore, comparable incubations of liver microsomes from DEX-pretreated rats given TAO (which suppresses 4-EDP formation to a greater extent than that of DP) yielded product ratios of 1.0 f 0.1, which were reverted to 0.7 f 0.1 on TAO decomplexation of P-45Op (results not shown). These findings again support the notion that 4-EDP formation is largely a function of P-45Op isozymes. The substantially different product ratios for DDEP metabolism in systems catalyzed by hepatic microsomes from untreated rats or PB- or DEX-pretreated animals coupled with the fact that P-450h, P-450k, and the oneelectron oxidant K,Fe(CN), generate predominantly, if not exclusively, the pyridine product, whereas DEX-inducible P-45Op isozymes also result in 4-EDP as a major metabolite, are incompatible with the notion that the only step controlled by P-450 enzymes is the one-electron oxidation of the nitrogen (N-1). These findings lead us to suggest that the active site environment of each isozyme, by dictating the fate of the radical cation species and its subsequent collapse to either 4-EDP or DP, must determine

the differential course of DDEP metabolism. This proposal is fully compatible with the notion that each metabolite must be derived from a distinct metabolic intermediate (16). Whether the major DEX-inducible rat liver P-45Op isozyme and its variants3 result in two-electron oxidation of DDEP to produce 4-EDP through either N-1 or C-4 hydroxylation is debatable. The relatively low kinetic isotope effects observed for C-4 hydrogen/proton abstraction tend to exclude C-4 hydroxylation of DDEP by certain P-450 isozymes (16,21), thereby implicating oneelectron oxidation at N-1 as the principal event. Nevertheless, a subtle influence of the active site environment of individual P-450 isozymes on the loss of the C-4 hydrogen/proton cannot completely be ruled out (18,22). Accordingly, the radical cation formed by one-electron oxidation of DDEP may generate 4-EDP by extrusion of the C-4 proton, if the steric environment at the active site of the enzyme were to orient the DDEP molecule in such a way so as to make such proton abstraction by either a strategically placed basic amino acid residue (B:, Figure 7) or the incipient P-450Fe1”=0 species possible. Irrespective of the precise pathway for 4-EDP formation, it appears unlikely that it plays a critical role in P-450 inactivation. Such inactivation appears to be associated predominantly, if not solely, with the one-electron oxidation of DDEP to DP. It is not surprising then that the temporal profiles of DDEP-mediated P-450 loss and DP formation should faithfully mirror each other in each hepatic microsomal system examined (Figure 8). Replacement of DDEP in comparable incubations of liver microsomes from all three sources with its 4-benzyl (DDBP) analogue, which has been previously shown to destroy rat hepatic P-450 without detectable prosthetic heme N-alkylation5 (15, 20, 23), resulted in virtually identical profiles of time-dependent P-450 destruction and DP formation to those observed with DDEP (Figure 8). In parallel, 14C-heme binding to rat liver microsomal proteins was also monitored in all three systems to assess the extent of prosthetic heme alkylation induced by each dihydropyridine analogue. For reasons not readily apparent, the irreversible heme binding observed in these particular experiments was lower than usual (3). Nevertheless, DDBP-induced P-450 destruction and 14C-heme binding to microsomal protein, as in the case of DDEP, were greatly magnified in reactions catalyzed by DEXpretreated rat liver microsomes in comparison with those observed in reactions catalyzed by either untreated or PB-pretreated rat liver microsomes (Figure 9). The roughly equivalent DP formation observed in all three rat hepatic microsomal systems examined (Table 11, It is possible that P-450 heme N-benzylation actually occurs but escapes detection because of the relative instability of the N-benzylporphyrins (45).

Inactivation of Cytochrome P-450 Isozymes

Chem. Res. Toricol., Vol. 2, No. 6, 1989 407

'b'

Table 111. Alignment of "Substrate-BindingnnSegments of Various P-450 Isozymes

FeV=O

DP

FeIV=O

H

H

Et

1

'fl'

R

Et

ti

I

/"

H20

h

380

377

384

F F F F

R R R H

G Y L L N Y L I N Y F I G V Q L

P P P K

K K K K

K D V E I K D I E L K D V E I K D V E I

N D N N

G G G G

P P P P

K K K K

TK D T T C D I T R D I TS D Y

P450pcnl P450pcn2 P450NF P450HLp

K K K K

M K K E

V F M L F I M F I M F I

"chwarze et al. (27). Please note that since no conclusive experimental evidence has been provided in support of such designation, it must be considered entirely speculative. References: P-450b, Fujii-Kuriyama et al., 1982 (38); P-450k, Kimura et al., 1988 (32);P-450h, Yoshioka et al., 1987 (31);P-450pcn1, Gonzalez et al., 1985 (34);P-450pcn2, Gonzalez et al., 1986 (35);P-450HLp, Molowa et al., 1986 (36); P-450NF, Beaune et al., 1986 (37); P450,,, Haniu et al., 1982 (39).

H

\ /

K

372

P450b P450k P450h P450cam

...

'h' 4-EDP

H

Figure 7. Schematic representation of the one-electronoxidation of DDEP to DP and the ethyl radical and plausible routes to 4-EDP formation. The one-electron oxidation of DDEP to DP with concomitant ethyl radical (Et') formation is depicted as proposed by Augusto et al. (15). In this scheme, P-450FeV=0 is converted to (P-450FeN=0),which in its protonated form can be a source of OH', an extremely reactive oxygen species. To rationalize the differential extent of CEDP formation by rat liver microsomes with varying P-450 isozyme complement as well as purified P-450h and P-450k, we propose the presence of a basic amino acid residue (B:)in EDP-forming isozymes capable of abstracting 4-H+ from the cation radical species. Subsequent electron abstraction from the deprotonated species by the ensuing P-450FeNOHand loss of H20 would not only yield 4-EDP but also quench this reactive P-450 species and regenerate the enzyme for a fresh catalytic cycle. Alternatives to this route of 4-EDP formation are also depicted, certain of which may now be excluded (16).

Figure 8), in contrast to the highly variable extent of DDEP-mediated P-450 loss and prosthetic heme alkylation of the apoprotein in these systems (Figure 9) as well as of prosthetic heme N-ethylation ( 3 ) ,suggests that the differential course of prosthetic heme destruction, i.e., heme N-alkylation versus heme alkylation of the apocytochrome, may also be determined by differences in the active site environment of the P-450 isozymes in question. In this regard, it might be instructive to note that the regiospecificity of prosthetic heme N-alkylation by various DDEP analogues varies with different P-450 isozymes (24). Such a finding would suggest that individual apoproteins play a critical role in defining not only their active site perimeters but also the prosthetic heme domains accessible for attack by the alkyl radical. Inaccessibility of such domains for DDEP-mediated heme N-ethylation in the P-45Op isozymes might, as suggested previously ( 3 , 2 5 ) ,lead the extruded radical either to alkylate the heme moiety itself (at the methine bridge to form either an isoporphyrin or a meso adduct, or at the 2- or 4-vinyl side chain) or even to abstract a H' from a readily accessible amino acid residue a t the active site. Alternatively, the prosthetic heme moiety may oxidatively self-destruct, if the incipient P-450FeN=0 species (or its protonated P-450Fer*'OH' counterpart) is not defused by such radical recombination events or by a strategically located oxidizable amino acid residue(s) (26). In this particular context, if indeed the apo-P-450 residues (375-384) not only constitute the "substrate-binding" re-

Table IV. Alignment of Distal Helices of P-450 Isozymes in the Vicinity of the O.-Bindina Siten P450h P450k P450f P450pcnl P450pcn2 P450HLp P450NF P450b P450cam

TDMF TDLF MDLI I I FI VIFI I I F I I I F I LSLF GLLL

GAGTET301 GAGTETjoi GAGTET301 FAGYEP310 FAGYET310 FAGYET310 FAGYET310 FAGTET302 VGGLDT252

TSTTLRY TSTTLLR MSTTLRY TSSTLS F TSSTLSF TSSVLSF TSSVLSF TSSTTLR VVNFLSF

"The distal helical regions of all the P-450 isozymes, except P450,, have been deduced from their cDNAs and assigned on the basis of their sequence similarity to that of P-450,, [derived from its X-ray crystal structure ( 2 8 , 2 9 ) ]and must therefore be considered speculative. References: P-450h, Yoshioka et al., 1987 (31); P-450k, Kimura et al., 1988 (32);P-450f, Gonzalez et al., 1986 (33); P-450pcn1, Gonzalez et al., 1985 (34);P-450pcn2, Gonzalez et al., 1986 (35);P-450HLp, Molowa et al., 1986 (36);P-450NF, Beaune et al., 1986 (37);P-450b, Fujii-Kuriyama et al., 1982 (38);P-450,,, Haniu et al., 1982 (39). gions as purported but also are conserved as proposed ( 2 3 , it is noteworthy that all P-45Op isozymes and their orthologues lack both Phe377 and Tyr380, two potentially oxidizable residues which are conserved in the corresponding regions of P-450's b, h, and k (Table 111). Alternative modes by which the inherent structural composition of the prosthetic heme environment of each DDEP-susceptible P-450 isozyme might intrinsically dictate the differential course of P-450 heme destruction observed in each instance are also conceivable. Indeed, if the active site of P-450, is taken as a model for the mammalian P-450's (28-31), then scrutiny of the reported sequences (32-41) for the key P-450 isozymes in question reveals intriguing differences in another "active site" region which may be critical in determining the course of their P-450 heme destruction, albeit by a different mechanism. Comparative alignment of the distal helices in the vicinity of the 02-bindingsite of these isozymes (Table IV) reveals that those of P-450h and -k are essentially identical, whereas those of P-45Op isozymes, although quite similar, exhibit some striking differences. First, in the immediate vicinity of the usually conserved Thr (Thr252 in P-450,, Thr310 in P-45Op isozymes) a t the 02-binding site is a conspicuous highly conserved tyrosine (Tyr308) residue in all P-45Op isozymes and their orthologues examined to date (34-37, 40, 41). The real functional significance of this residue in normal P-45Op catalytic turnover is unclear. Because it was plausible that, in analogy to prostaglandin

408 Chem. Res. Toxicol., Vol. 2, No. 6,1989

0

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301-7=-

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E 10

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60 ''7

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Tlmr (mln)

i

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1 .o 40 0.8

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0.4 0.2 -

0

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t

1

1

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=

0.0 0

30

Tlmr (mln)

I

50 I

1

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Tlmr (mln)

Figure 8. Temporal profiies of relative DP formation and hepatic microsomal P-450 loss in incubations of DDEP and DDBP. DP formation (closed symbols) and P-450 loss (open symbols) are depicted as nmol/incubationin each instance. UT, PB, and DEX refer to incubation systems catalyzed by liver microsomes from untreated, PB-pretreated,and DEX-pretreatedrats, respectively. For experimental details please see Materials and Methods and the legend of Figure 6. DDEP ( 0 ,0);DDBP (A,A).

H synthetase (42), such a strategically placed tyrosine residue in P-45Op isozymes could be recruited in radicalcatalyzed cooxidation of the prosthetic heme to highly reactive products, we carried out preliminary computer modeling studies of this "putative" 02-binding site (courtesy Dr. Teri Klein, UCSF Computer Graphics Laboratory). Replacement of Leu250 with Tyr308 (the corresponding residue in P-45Op isozymes) indicated no additional disruption of this distorted helical region, when the known coordinates for the P-450, X-ray crystal structure were used. Indeed, the m 3 0 8 hydroxyl was now capable of hydrogen bonding with Met164 in P-450, (Arg211 in P-45Op sequences), which allowed it to nestle in the surrounding structures, conveniently protected from direct assault by the heme iron bound oxo species.

10

20

30

Time (min)

Figure 9. DDEP- and DDBP-mediated hepatic microsomal P-450 loss and I4C-hemeirreversibly bound to microsomal protein in reactions catalyzed by liver microsomes from untreated and PBand DEX-pretreated rats. Cytochrome P-450 loss (nmol/incubation) and 14C-hemeirreversibly bound to microsomal protein (nmol/incubation) were monitored as described previously (3). P-450 heme specific radioactivity was quantitated as detailed previously after prelabeling rats with the heme precursor [14C]-6-aminolevulinic acid (nominally, 10-15 pCi) for 3 h before sacrifice. Each 3-mL incubation contained 2 mg of microsomal protein/mL. The P-450 content of these preparations is given under Figure 6. In each panel, closed symbols refer to values obtained with DDBP, and open symbols to those with DDEP. DEX (m, 0);UT (A,A); PB (0,0).

However, when Thr310 was replaced by Pro310, a natural substituent in the DEX-inducible P-450pcnl sequence (351, we could not escape noticing that this residue could no longer hydrogen bond with the vicinal Ala306 (Gly248 in P-450,), an arrangement thought to convert this distorted helical region of P-450, into a pocket for the heme-bound oxo species (28). The influence of the apparent disruption of such a possibly "protective" pocket in this particular isozyme is unclear. Whether this disruption is responsible for the enhanced oxidative destruction of the prosthetic heme moiety to highly reactive products observed after induction of this particular isozyme by DEX, PB, or PCN remains to be determined. In summary, the diverse product and partition ratios for oxidative metabolism of DDEP by P-450 isozymes h, k, and p and the differential mode for the consequent DDEP-mediated prosthetic heme destruction of these hemoproteins invite the speculation that such differences among isozymes (all bearing identical prosthetic heme moieties) reside in the structure of their active site environment, a proposal to be verified when X-ray crystal structures of these membrane-bound hemoproteins become available. Nevertheless, it is evident that P-45Op isozyme catalyzed one-electron oxidation of certain chemicals (3, 25,26),results in oxidative degradation of its prosthetic heme to highly reactive products. Interception of such products in their trajectory by suitable amino acid residues

Inactivation of Cytochrome P-450 Isozymes

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(17) Guengerich, F. P. (1987) Oxidative Cleavage of Carboxylic Esters by Cytochrome P-450. J. Biol. Chem. 262, 8459-8462. (18) Guengerich, F. P., Peterson, L. A., and Bocker, R. H. (1988) Cytochrome P-450-catalyzedHydroxylation and Carboxylic Acid Ester Cleavage of Hantzsch Pyridine Esters. J. Biol. Chem. 263, 8176-8183. Acknowledgment. We acknowledge the able and de(19) Marks, G. S., Allen, D. T., Johnston, C. T., Sutherland, E. P., voted technical assistance of Ms. Evangeline Soliven. We Nakatsu, K., and Whitney, R. A. (1985) Suicidal Destruction of Cytochrome P-450 and Reduction of Ferrochelatase Activity by also acknowledge the use of the Liver Center Core Facil3,5-Diethoxy-carbony1-1,4-dihydro-2,4,6-trimethylpyridine and Its ities on Spectrophotometry and on Mass Spectrometry (A. Analogues in Chick Embryo Liver Cells. Mol. Pharmacol. 27, L. Burlingame, Director), supported by NIH Grants DK 459-465. 26743 and RR 01614, as well as the UCSF Computer (20) Wrighton, S. 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