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Research ArticleArticle

Metabolic Inactivation of Cytochrome P4502B1 by Phencyclidine

Immunochemical and Radiochemical Analyses of the Protective Effects of Glutathione

Uma Sharma, Elizabeth S. Roberts, Ute M. Kent, S. Michael Owens and Paul F. Hollenberg
Drug Metabolism and Disposition February 1997, 25 (2) 243-250;

Immunochemical and Radiochemical Analyses of the Protective Effects of Glutathione

Abstract

Phencyclidine (PCP) inactivates the 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity of P4502B1 in a reconstituted system containing NADPH-cytochrome P450 (P450) reductase (reductase) andl-α-phosphatidylcholine, dilauroyl in a time-, concentration-, and NADPH-dependent manner. Catalytic activity of the enzyme could not be restored upon reconstitution with fresh reductase, indicating that the effect was on the P450 and not on the reductase. Although the kinetics suggested that PCP would be classified as a classical mechanism-based inactivator, protection against inactivation of P450 by PCP by the presence of an exogenous nucleophile, such as glutathione (GSH), indicated otherwise. There was no loss of spectrally detectable P450 associated with inactivation either in the presence or absence of GSH. When radiolabeled PCP was used to inactivate the enzyme and the reaction mixture analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found to be associated with P450, reductase, and catalase that had been added to protect against oxidative damage. When GSH was included in the reaction mixtures, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a marked decrease in the binding to all three proteins. Correspondingly, analysis of the components of the inactivated sample by reversed-phase HPLC demonstrated that radioactivity was associated with P450, reductase, and catalase, and that there was a marked decrease in the labeling of all three proteins in the presence of GSH. The stoichiometry of binding of radiolabeled PCP to the proteins in the incubation mixture in the absence of GSH was 4:1. In the presence of GSH, no significant amount of radioactivity was incorporated into the proteins. An anti-PCP metabolite antibody was used to detect PCP metabolite adducts bound to the inactivated enzyme by Western blot analysis. The antibody recognized adducts bound to P450, reductase, and catalase. In the presence of GSH, there was a decrease in immunoreactivity, although binding of PCP to all three proteins was still detected. Because the added nucleophile protects against inactivation and protein labeling by PCP, these data suggest that the reactive intermediate may escape from the active site and attack other sites on the P450, as well as other proteins in the milieu.

Although PCP1 is not used as an anesthetic because of its serious side effects, it is still a popular drug of abuse with long-term psychotic behavioral effects on some users (1). Therefore, it is of interest to study the kinetics and metabolism of this compound by various P450 isoforms, and this has been the subject of several excellent reviews (2, 3). PCP and a few structurally related PCP analogs are known to be metabolism-dependent inhibitors of P450 enzymes (4-6). PCP inactivates reconstituted P4502B1 in a time-, concentration-, and NADPH-dependent manner (7). Osawa and Coon (5) observed heme loss associated with the inactivation of the major phenobarbital-inducible isoform of rabbit liver microsomes by PCP. However studies in this laboratory (7) have shown that the addition of catalase to the incubation mixture decreases the rate of inactivation and protects completely against heme loss, suggesting that H2O2 contributes to the initial rate of inactivation and loss of heme observed during inactivation. Hoag et al. (6) demonstrated that the presence of cyanide protects against the inactivation of the major phenobarbital-inducible isoform of P450 in rat liver microsomes by PCP. This suggested that the reactive intermediate was released from the active site and was accessible for reaction with the nucleophile. Although the iminium ion of PCP is thought to be the intermediate that reacts with cyanide to give the cyano adduct (8), it has been shown that it is not the ultimate species responsible for inactivation of the phenobarbital-inducible isoforms in rat (7) or rabbit liver (5). Although stoichiometry studies conducted with radiolabeled PCP did not give a 1:1 stoichiometry between PCP and P4502B1 (7), we decided to pursue inactivation studies in the presence of an excess of nucleophile to trap any nonspecific electrophilic species that escaped from the active site and that could then bind to other proteins in the incubation mixture.

The studies herein use several approaches, including for the first time anti-PCP metabolite antibodies (9) to detect binding of PCP metabolites to the components of the incubation mixture. Using radiolabeled PCP and these antibodies, we were able to define the inactivation as metabolism-dependent, but not mechanism-based inactivation.

Materials and Methods

PCP, DLPC, NADPH, and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). PCP [piperidyl-3,4-3H(N)], with a specific activity of 44.7 Ci/mmol, was purchased from Dupont-NEN (Boston, MA). [Phenyl-3-3H(n)]PCP, with a specific activity of 12.68 Ci/mmol, was a generous gift through Dr. J. Woods (University of Michigan, Ann Arbor, MI) from the Basic Neurobiology and Biological Systems Research Branch, National Institute on Drug Abuse. EFC was purchased from Molecular Probes, Inc. (Eugene, OR). HFC was obtained from Enzyme Systems Products (Livermore, CA). The ECL-Western blotting reagent kit and Hyperfilm-ECL were purchased from Amersham Life Sciences (Arlington Heights, IL). Ultima Gold Scintillation cocktail was obtained from Packard Instrument Co. (Meriden, CT). BioBlot-NC nitrocellulose blotting membranes were purchased from Costar Scientific Corp. (Cambridge, MA). All other chemicals used were of reagent grade obtained from commercial sources.

Isolation of Enzymes.

P4502B1 and reductase were purified from liver microsomes of phenobarbital-treated male Long Evans rats (150–175 g; Harlan Sprague-Dawley) according to the methods of Saito and Strobel (10) and Strobel and Dignam (11), respectively. Rats were pretreated with 0.1% phenobarbital in the drinking water for 11 days. Purified reductase primarily consisted of the long active reductase, along with small amounts of the short catalytically inactive enzyme.

Enzyme Assays.

As described previously (12), a discrete time-point assay involving the deethylation of EFC to HFC (13, 14) was used to measure the inactivation of P4502B1 by PCP. The primary reaction mixture contained varying concentrations of the inactivator, 50 pmol P4502B1, 100 pmol reductase, 20 μg/ml DLPC, 210 units catalase, and 50 mM potassium phosphate buffer (pH 7.4) in a final volume of 0.1 ml. After a 3- to 5-min preincubation at 30°C, the reaction was initiated by the addition of NADPH to a final concentration of 0.8 mM. At 0, 5, 7, 10, 15, 20, 25, and 30 min after the addition of NADPH, aliquots of 10 μl were added to a secondary reaction mixture containing 100 μM EFC, 0.2 mM NADPH, 40 μg BSA, and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 1.0 ml. The secondary reaction was allowed to proceed at 30°C for 5 min, at the end of which the reaction was quenched by the addition of 333 μl ice-cold acetonitrile. The amount of product formed was measured at room temperature on an SLM-AMINCO model SPF-500 C spectrofluorometer with the excitation at 410 nm and the emission at 510 nm. The product formed was quantified based on a standard curve constructed using known amounts of HFC.

Effect of Nucleophiles on Inactivation.

The EFC deethylation assay described previously was used with minor modifications to follow the inactivation. In these experiments, the primary reaction mixtures contained either 10 mM KCN (titrated to neutral pH with HCl) or 10 mM GSH. Control incubations, which contained the nucleophile but not PCP, were conducted to study the effect of the nucleophile itself on the deethylation reaction.

Stoichiometry of Binding.

The reaction mixtures contained 0.6 nmol P4502B1, 1.2 nmol reductase, 20 μg/ml DLPC, 105 units catalase, 40 μM PCP, 0.1 μM PCP [piperidyl 3,4-3H(N)], or 0.1 μM [phenyl-3-3H(n)]PCP and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 1.0 ml. One set of incubations included 10 mM GSH in the primary reaction mixtures. The reaction mixtures were preincubated at 30°C for 3–5 min, after which the reactions were initiated by the addition of NADPH to a final concentration of 0.8 mM. At times 0 and 45 min after initiation of the reaction, aliquots (10 μl) were added to secondary reaction mixtures containing 100 μM EFC, 0.2 mM NADPH, 40 μg BSA and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 1.0 ml to measure catalytic activity as described earlier. Aliquots (200 μl) were also added to an ice-cold quench buffer containing 50 mM potassium phosphate buffer (pH 7.7), 40% glycerol, and 0.6% Tergitol NP-10 to give a final volume of 1.0 ml. The difference spectra between 400 and 500 nm were then determined on a DW-2 OLIS spectrophotometer according to the method of Omura and Sato (15). After measuring catalytic activity and spectrally detectable P450, the remaining reaction mixtures were dialyzed against 500 volumes of dialysis buffer containing 50 mM potassium phosphate buffer (pH 7.4), 20% v/v glycerol, and 0.5 mM EDTA in Slide-A-Lyzer dialysis cassettes (Pierce Chemical Co., Rockford, IL) with 10,000 molecular weight cutoff membranes. Experiments were also conducted with 0.4% v/v cholate in the dialysis buffer to reduce noncovalent binding. When the counts in the dialysis buffer were reduced to background levels, the protein samples were analyzed by liquid scintillation counting in a Beckman model LS 5800 liquid scintillation counter to determine the stoichiometry of binding. Because no loss of spectrally detectable P450 was apparent after inactivation, after correcting for volume changes during dialysis, aliquots were added to the quench buffer described earlier to measure spectrally detectable P450 and calculate protein recovery. The stoichiometry of binding was calculated as PCP bound per nanomole of inactivated P450 after subtracting the counts in the samples without NADPH (metabolism-independent binding) and after accounting for recovery.

HPLC Analyses for Specificity of Binding.

After determining the binding stoichiometry, the remaining protein samples were then concentrated using Centricon-30 microconcentrators (Amicon, Inc., Beverly, MA) that had been treated with 2.0 ml of 1% BSA in water to decrease nonspecific binding losses and improve recovery of the proteins (Amicon, Inc., technical notes). An aliquot of each concentrated sample was injected onto a 4.6 × 100 mm POROS R/H perfusive-particle column from Perseptive Biosystems (Framingham, MA). The HPLC system consisted of a Waters 490E programmable multiwavelength detector, Waters 501 HPLC pumps, Waters system interface module, and a Gilson model 201 fraction collector. The system was operated through the Maxima 820 chromatography workstation from Waters Corp. (Milford, MA). The solvent system consisted of buffer A (0.1% TFA) and buffer B (95% acetonitrile/5% water/0.1% TFA). Initial conditions were 20% B with a linear gradient to 75% B in 18 min with the flow rate at 3.0 ml/min and then to 100% B in 2.0 min. Fractions were collected every 0.5 min and monitored by liquid scintillation counting. Elution times for P450, reductase, and catalase were determined by injecting each of the purified proteins separately.

SDS-PAGE Analyses for Specificity of Binding.

Inactivation of P450 by PCP was conducted in a manner similar to that described for the stoichiometry determinations. After inactivation, aliquots containing 0.1 nmol P4502B1 were diluted with sample loading buffer, boiled, and loaded on a 12% polyacrylamide gel with the buffer system described by Laemmli (16). Acid precipitation of the inactivated P4502B1 sample indicated that the radioactivity was still associated with the protein and the adduct was acid-stable. After electrophoresis, the gels were either stained with 0.25% Coomassie blue R-250 or sliced for measuring radioactivity associated with each of the components of the incubation mixture. Sliced gels were finely diced and heated at 50°C in 500 μl of 30% H2O2 for 12–15 hr. Samples were then analyzed by liquid scintillation counting as described previously.

Rabbit Anti-PCP Metabolite Antibodies.

The rabbit anti-PCP metabolite antibodies used in this study were raised against haptens derived from four PCP derivatives. These arylcyclohexylamine haptens were 1-1(-phenylcyclohexyl)-4-hydroxypiperidine,trans-1(1-phenyl-4-hydroxycyclohexyl)piperidine, 4-[(1-piperidinyl)cyclohexyl]benzoic acid, and PCHAP. These hapten-BSA conjugates were designed to generate antibodies capable of recognizing all possible orientations of the PCP molecule (see ref. 9for complete details of the generation and specificity of these antibodies). Inasmuch as antibodies selected by immunization with drug-protein conjugates are directed at parts of the molecule projecting from the protein carrier, it was hoped that the use of several different antiarylcyclohexylamine antibodies could aid in predicting the critical molecular features of the PCP metabolite when it is covalently attached to microsomal proteins.

Western Blot Analyses for Detection of Adducts Using the Anti-PCP Metabolite Antibodies.

The inactivated samples were analyzed by SDS-PAGE as described previously and electroblotted using the method of Towbin et al. (17) with some modifications. A Mini-Trans-Blot Electrophoretic transfer cell (Bio-Rad Labs., Hercules, CA) was used to electroblot the proteins at 4°C onto BioBlot-NC nitrocellulose blotting membranes (0.45 μM) at 22 mV for ∼12–16 hr. The transfer buffer contained 25 mM Tris (pH 8.3), 192 mM glycine, and 20% methanol. The nonspecific binding sites on the nitrocellulose membranes were then blocked by immersing the membranes in TTBS (pH 7.6) and 5% nonfat milk powder for 1.0 hr. Nitrocellulose membranes were subsequently incubated with the different anti-PCP antibodies described earlier for 1 hr. In some cases, the membranes were also exposed to rabbit anti-rat P4502B1. After this incubation, membranes were washed 3 times with the TTBS solution containing 5% nonfat milk and then incubated for 1.0 hr at room temperature with the secondary goat anti-rabbit IgG coupled with horseradish peroxidase (BioRad). Membranes were again washed with the TTBS and 5% milk solution 3 times. After a final wash with Tris-buffered saline (pH 7.6), membranes were incubated with ECL detection reagents for 1.0 min in the dark and then exposed to Hyperfilm-ECL for 1.0–2.0 min.

Specificity of Inactivation.

In these experiments, the primary reaction mixtures contained 1.2 nmol P4502B1, 1.2 nmol reductase, 50 μg/ml DLPC, 210 units catalase, 0.8 mM PCP, and 50 mM potassium phosphate buffer (pH 7.4) in a total volume of 0.5 ml. After a 3- to 5-min preincubation at 30°C, the reactions were initiated by the addition of NADPH to a final concentration of 0.8 mM. At 0 and 40 min after initiation of the reaction, aliquots containing 12 pmol P450 were added to secondary reaction mixtures containing 100 μM EFC, 0.2 mM NADPH, 40 μg BSA, and 50 mM potassium phosphate buffer (pH 7.4) in a final volume of 1.0 ml. This reaction was allowed to proceed at 30°C for 5 min, at the end of which 333 μl ice-cold acetonitrile was added to quench the reaction. The product HFC was quantitated spectrofluorometrically as described previously. At the end of 40 min, 10-μl aliquots of the inactivated samples were reconstituted at 4°C for 30 min with fresh reductase so that the ratio of P450 to reductase was now 1:4. Noninactivated controls were reconstituted in a similar manner. These reaction mixtures were then preincubated at 30°C for 3–5 min, and then secondary reaction mixtures described previously were added to give a final volume of 1.0 ml. After 5 min at 30°C, the reactions were quenched by the addition of 333 μl acetonitrile, and the HFC product formed was measured as described previously.

Results

Inactivation of P4502B1.

Incubation of purified P4502B1 in a reconstituted system containing reductase and lipid with PCP in the presence of NADPH resulted in a time- and concentration-dependent loss of the EFCO-deethylase activity of the enzyme. The requirement for NADPH indicated that PCP had to be metabolized to a reactive species that was responsible for the inactivation. There was no lag time associated with the inactivation. Figure 1 shows the time-dependent loss of EFC O-deethylase activity with increasing concentrations of PCP. Linear regression analysis of the data in fig. 1 was used to determine the rate constants of inactivation. Figure 2 shows a plot of the reciprocals of the initial rate constants of inactivation as a function of the reciprocals of the inactivator concentrations. From this plot, the maximal rate constant at saturating levels of the inactivator (kinactivation) was determined to be 0.023 min−1. The concentration of PCP required for the half-maximal inactivation was 3.7 μM.

Figure 1
Figure 1

Time- and concentration-dependent inactivation of reconstituted P4502B1 by various concentrations of PCP.

Incubation conditions were as described in Materials and Methods. The concentrations of PCP used were (□) 0.0 μM, (▪) 1.0 μM, (○) 2.0 μM, (•) 3.0 μM, (▵) 4.0 μM, (▴) 5.0 μM, and (⊞) 6.0 μM. A 100% activity was 5.5 nmol HFC/min/nmol P450. Values are representative of four experiments that do not differ by >6%.

Figure 2
Figure 2

Double-reciprocal plot of the rate of inactivation of the EFC O-deethylase activity of P4502B1 as a function of the concentration of PCP.

Data are taken from fig. 1.

Effect of Exogenous Nucleophiles.

The requirement for catalytic turnover of PCP for the inactivation suggested that the generation of a reactive species during metabolism was required for the inactivation to occur. This raised the possibility that this reactive species could escape from the P450 active site and bind to nucleophilic sites in the vicinity of the active site resulting in inactivation. To determine if this could be occurring with PCP, the effect of trapping agents such as GSH and KCN on the kinetics of inactivation was studied. When GSH (10 mM) was included in the primary reaction mixture, there was a marked decrease in both the rate and extent of inactivation when compared with the control samples without GSH (fig. 3). This protective effect of GSH was seen even at the increased concentration of inactivator (10 μM). The GSH did not completely prevent the inactivation, and in the stoichiometry experiments, the final inactivation in the incubation mixtures with GSH was ∼40%, compared with >85% inactivation in incubation mixtures without GSH in the presence of PCP. On the other hand, when KCN (10 mM) was included in the inactivation mixture, there was no significant protection against the inactivation (data not shown) at the concentrations used. Neither of the nucleophiles affected the catalytic activity of the enzyme by themselves.

Figure 3
Figure 3

Time- and concentration-dependent inactivation of the EFC O-deethylase activity of P4502B1 by PCP in the absence (A) or presence (B) of 10 mM GSH.

Concentrations of PCP used were (▪) 0.0 μM, (♦) 5.0 μM, and (•) 10.00 μM. A 100% activity was 4.9 nmol HFC/min/nmol P450. Values represent the means ± SD of two sets of duplicate determinations. Standard deviations were smaller than the symbols used.

Stoichiometry of Binding.

Dialysis of the reaction mixture after inactivation of P4502B1 by the radiolabeled inactivator, as described in Materials and Methods, was used to determine the stoichiometry of PCP binding to P4502B1. Reactions were conducted in the presence and absence of GSH to investigate the effect the nucleophile had on the stoichiometry of binding (7). After subtracting the counts in the samples without NADPH (∼17% of the counts in the sample with NADPH) that would account for metabolism-independent binding, we obtained a binding ratio of 4–5 nmol of PCP bound/nmol of P4502B1 inactivated in the absence of GSH. In the presence of GSH, no significant radioactivity was covalently bound to the protein over the metabolism-independent control (binding ratio: 0.03–0.07), indicating that there was no significant covalent binding in the presence of this trapping agent. The addition of cholate to the dialysis buffer did not change the binding in the presence or absence of GSH, and identical results were obtained. These studies were conducted with the PCP radiolabeled on either the piperidyl ring or the phenyl ring. Identical binding ratios were obtained when PCP was labeled at either position, and there was no incorporation of radiolabel from either labeled compound when the reactions were conducted in the presence of GSH.

Specificity of Binding.

To investigate the specificity of the radioactive labeling of the proteins of the incubation mixture, the radiolabeled protein samples from the stoichiometry studies were concentrated after dialysis and analyzed by reversed-phase HPLC. Fractions off the HPLC were collected at 30.0-sec intervals and analyzed by liquid scintillation counting. As shown in fig. 4, radioactivity was primarily associated with the long reductase (eluting at 10.5 min) and with P450 (eluting at 13.5 min) in the samples with NADPH. In the samples where GSH was present during inactivation, radioactivity associated with the reductase or P450 decreased significantly to just over control levels despite ∼40% inactivation. There was no radioactivity associated with the heme that eluted at 5.4 min and that was detected by measuring the absorbance at 405 nm. Some radioactivity was also detected at 21.6 min, which corresponded to the column wash with 100% buffer B.

Figure 4
Figure 4

HPLC analysis of the reconstituted system after incubation with PCP [piperidyl-3,4-3H(N)] in the absence (A) or presence (B) of NADPH, and in the presence of GSH and NADPH (C) with UV detection at 220 and 405 nm and liquid scintillation counting of fractions.

Incubation conditions, sample preparations for HPLC, and chromatographic conditions were as described in Materials and Methods. Retention times were as follows: heme, 5.4 min; catalase, 7.9 min; short reductase, 9.6 min; long reductase, 10.5 min; and P4502B1, 13.4 min.

Specificity for the binding of labeled PCP to the proteins was also analyzed by separating the proteins in the inactivated samples by SDS-PAGE. The radioactivity associated with the gel slices corresponding to the different proteins in the reconstituted system was analyzed by liquid scintillation counting after dissolving the gel slices in H2O2. As shown in fig.5, in the presence of NADPH, radioactivity was found to be primarily associated with P450, reductase, and catalase in descending order. However, when GSH was included in reaction mixtures, radioactivity associated with all three proteins decreased to almost control levels.

Figure 5
Figure 5

Radioactivity associated with the components of the reconstituted system after incubation with PCP [piperidyl-3,4-3H(N)] in the presence or absence of NADPH or with GSH and NADPH after SDS-PAGE analysis, followed by liquid scintillation counting of the gel slices.

Incubation and electrophoretic conditions were as described inMaterials and Methods. Values represent means ± SD from three separate experiments.

Detection of PCP-Metabolite Adduct Using an Anti-PCP Antibody.

An antibody raised against the PCHAP hapten-BSA conjugate detected PCP-metabolite adducts bound to the components of the incubation mixture after SDS-PAGE separation of the components and electroblotting onto nitrocellulose membranes as described in Materials and Methods. As shown in fig. 6, the hapten used to raise this antibody contained the closed phenyl and cyclohexyl rings of PCP, with the piperidyl ring opened to give pentanoic acid. In the absence of NADPH, the antibody did not detect binding to any protein (fig. 7, lane A). The antibody recognized PCP metabolites bound to P450, reductase, and catalase (fig. 7, lane B) in the samples incubated with NADPH. When the incubation mixtures contained GSH and NADPH, there was a significant decrease in the binding of the metabolite adducts to all three proteins in the incubation mixture (fig. 7, lane C), as detected by Western blotting with the anti-PCHAP antibody. The three other antisera did not detect any NADPH-dependent adducts bound to the proteins of the incubation mixture.

Figure 6
Figure 6

Structure of PCP and the hapten-BSA conjugate (PCHAP) used for immunization of rabbits.

Numbers and arrows on the PCP hapten indicate the points of attachment of a protein antigen for use in generating three of the four anti-PCP antibodies used in this study. The antibody specificity would be directed at the parts of the haptens that are distal to the point of antigen conjugation. The antibodies were generated and characterized as described in Materials and Methods and in ref. 9.

Figure 7
Figure 7

Western blot analysis of the incubation mixtures containing PCP without NADPH (lane 1), with NADPH (lane 2), and with NADPH and GSH (lane 3) using the ECL detection method.

Incubation, electrophoretic, and electroblotting conditions were as described in Materials and Methods.

Specificity of Inactivation.

Because reductase was labeled by tritiated PCP, and the PCHAP antibody also recognized PCP-metabolite adducts, it was necessary to determine whether the loss of catalytic activity for the metabolism of EFC was due to inactivation of P450 or inactivation of reductase. As shown in table 1, when protein that had been inactivated by metabolism with PCP was reconstituted with fresh reductase, there was no increase in the catalytic activity measured by the deethylation of EFC to HFC. In comparison, the activity of the noninactivated sample increased by ∼25% when fresh reductase was added to increase the P450 to reductase ratio. Therefore, the loss of activity was due to inactivation of P450.

View this table:
Table 1

Reconstitution of inactivated P450 with fresh reductase to determine if the inactivation was due to inactivation of reductase or the inactivation of P450 1-a

Discussion

The metabolism of PCP leads to inactivation of the catalytic activity of various isoforms of P450. As reported previously (7), rat P4502B1 is inhibited during metabolism of PCP, and the kinetics of inactivation are similar to those seen with typical mechanism-based inactivators (18). These kinetic parameters include time- and concentration-dependent loss of activity, pseudo–first-order kinetics and saturability of inactivation. Several investigators have shown that the presence of cyanide as a trapping agent for the iminium ion inhibits the rate and extent of inactivation (5, 6). In our previous study (7), we did not observe a protective effect of cyanide at concentrations up to 0.1 mM. However, the stoichiometric ratio of radiolabeled PCP bound per nanomole of P450 inactivated was always much greater than the 1:1 ratio expected of classical mechanism-based inactivators (18). Therefore, to determine the basis for the higher level of binding and in an attempt to lower the stoichiometry, we decided to reexamine the effect of trapping agents on the inactivation of P4502B1 by PCP and the stoichiometry of the reaction.

Hoag et al. (6) did not observe any protection against PCP-mediated loss of ketamine N-demethylation by GSH at a concentration of 1.0 mM in microsomes from livers of phenobarbital-treated rabbits. However, at this same concentration of GSH, Ward et al. (19) observed inhibition of the metabolism-dependent binding of 3[H]PCP to rabbit liver microsomal proteins. In our studies, GSH at a concentration of 10 mM decreased both the rate and extent of inactivation of EFCO-deethylase activity of P4502B1. The basal catalytic activity of the enzyme was not altered in the presence of 10 mM GSH. Studies aimed at identifying the specificity of the binding of the reactive metabolite(s) to the components of the incubation mixture revealed that all three proteins in the mixture (P450, reductase, and catalase) were radiolabeled as a result of PCP metabolism. Because all three proteins in the system were radiolabeled, the initial idea behind including high concentrations of GSH in the reaction mixture was to trap the reactive species that escaped from the active site and then bound to reductase and catalase, resulting in the higher stoichiometry of binding. In the presence of GSH, corresponding with the protection against inactivation of P450 catalytic activity during PCP metabolism, we observed a decrease in the binding of the reactive metabolite(s) to the components of the incubation mixture. With a classical mechanism-based inactivator, the reactive species leading to inactivation should bind to the active site, wherein it was generated, and this species would then be inaccessible to GSH. However, if the reactive species were to leave the P450 active site, it would be trapped by GSH, resulting in a decrease in stoichiometry. Although we do not see a complete protection against binding in the presence of GSH, we would like to believe that more than one reactive species is responsible for the inactivation. Another explanation that can be offered is that some of the active metabolite never leaves the active site where it was generated. This metabolite is therefore not accessible by GSH; thus, we see a small amount of inactivation even in the presence of GSH.

KCN did not protect against the inactivation of the EFCO-deethylase activity of P4502B1 by PCP. Although cyanide ions have been reported to trap the iminium ions generated during the metabolism of PCP (19), failure of KCN to protect against inactivation supports the results (5, 7) that the iminium ion is not the ultimate inactivating species of the P4502B1 isozyme. As observed earlier (7), no heme loss was associated with inactivation of the EFCO-deethylase activity of P4502B1 by PCP. This was also true for our experiments, wherein GSH was included in the reaction mixtures (data not shown). This suggests that the inactivation was not the result of heme modification. Osawa and Coon (5) observed loss of spectrally detectable P450 associated with inactivation of the purified major phenobarbital-inducible isoform in rabbit livers by PCP. When catalase was included in the primary incubation mixtures (7), no loss of spectrally detectable P450 was observed. This protection by catalase suggests that P450 inactivation by H2O2 could have been a contributing factor to the overall inactivation.

In our experiments, we used PCP labeled either on the phenyl ring or on the piperidyl ring. Because the metabolism of PCP can follow a variety of pathways (2, 3), using PCP labeled at two different positions ensured detection of binding whether the reactive species was generated through metabolism of the phenyl ring or the piperidyl ring. When GSH was included in the reaction mixtures, we did not detect any binding of the radiolabel to the proteins with PCP labeled either on the phenyl ring or on the piperidyl ring, suggesting that the putative reactive metabolites generated either through the metabolism of the phenyl ring or the piperidyl ring were both available for binding to GSH.

Reductase was also labeled in the binding experiments, giving rise to the possibility that inactivation could be due to the inability of the labeled reductase to transfer electrons from NADPH after initial turnovers to generate the inactivating reactive species. This possibility was investigated by supplementing the inactivated sample with additional reductase. The inability to observe any restoration of the lost catalytic activity upon addition of active reductase ruled out this possibility.

Zorbas and Owens (20) have also studied the irreversible binding of PCP metabolites to proteins using SDS-PAGE and gel slice autoradiography. These studies used liver microsomes prepared from Sprague-Dawley rats after phenobarbital induction. Their data showed a predominant band of radioactivity associated with a PCP metabolite bound to a protein at ∼48–52 kDa (presumably P4502B1) and additional smaller bands at ∼58 and 96 kDa. Their studies demonstrated that irreversible binding occurs even with microsomal fractions and, that, like the present study, the reactive metabolite can escape from the enzyme and target other proteins. In addition, because the metabolite stays associated with the protein after SDS-PAGE analysis (20 and herein) and during Western blot analysis with multiple washing steps and antibody detection (fig. 6), these data suggest the reactive metabolite forms a covalent bond with the proteins.

Reactive metabolites generated during the metabolism of various drugs, environmental pollutants, and endogenous materials are known to cause toxicities in various tissues and organs (21-24). Whereas the radiochemical approach has been the most popular method for identification of protein targets of reactive metabolites, the limitations imposed by this method (25) have increased the use of a complimentary immunochemical approach. For instance, Pumford et al. (26) identified 3-(cystein-S-yl)acetaminophen adducts in proteins of various hepatic subcellular fractions of acetaminophen-treated mice with an affinity-purified antiserum obtained from rabbits immunized with 3-(N-acetyl-l-cystein-S-yl)acetaminophen.

The four anti-PCP antibodies used in these studies were generated against four unique PCP hapten-BSA conjugates (9). Three of the haptens contained all three ring structures of PCP (fig. 6). These haptens were conjugated to the protein antigen at the 4-position on the ring structure of the aromatic ring (position 1; fig. 6), the piperidine ring (position 2), and the cyclohexyl ring (position 3) viaa spacer arm with a carboxylic acid terminus. The PCHAP molecule was coupled directly to the antigen through an existing carboxyl acid terminus (fig. 6). All four haptens were covalently bound to the protein carrier by a peptide bond between the carboxylic acid group and the lysine groups on BSA. By using these four different antiarylcyclohexylamine antibodies in combination with Western blot analysis of the PCP-metabolite adducts, we were able to perform a comprehensive epitope analysis of the PCP metabolite-protein conjugations produced during P450-mediated PCP metabolism.

For Western blot analysis, we used the four different antisera to determine the PCP-metabolite adducts that were bound to the components of the reconstituted P450 system during incubation with PCP. Although the chemical structure of the PCP metabolite-protein adduct is not known, it has been suggested to be a metabolite formed through P4502B1-mediated metabolism of the piperidine ring structure (5, 27). Knowing this, we probed the Western blot gels with two anti-PCP antisera designed to recognize hapten-protein conjugates that were derived from the metabolism of the piperidine ring structure. These antisera contained the antibodies raised against PCHAP and the antibodies generated against the hapten conjugation at position 2 on the piperidine ring structure (fig. 6). Although only the anti-PCHAP antiserum was able to cross-react with the PCP metabolite-protein conjugates on the Western blots, these data suggest that the irreversibly bound PCP metabolite-protein adduct must have an immobilized conformation that is complimentary to the anti-PCHAP antibody binding site. In future studies, this antiserum could also be useful in identifying the PCP metabolite peptide linkage.

The binding studies using the PCHAP antibody also provided further evidence that the binding of PCP to the components of the incubation mixture and the inactivation was metabolism-dependent and not mechanism-based. In the absence of GSH, the antibody recognized adducts bound to all three proteins. When the incubations were conducted in the presence of GSH (10 mM), there was a significant decrease in the binding of PCP metabolites to P450, reductase, and catalase as detected by the antibody (fig. 7). All three proteins were still labeled somewhat, indicating that, even at these high concentrations (10 mM), the nucleophile still does not completely protect against binding.

Owens et al. (9) had previously suggested that the PCHAP antiserum would be useful in detecting covalent adducts formed during the metabolism of PCP. This is the first report of the successful use of this antiserum for the detection of PCP-metabolite covalent binding to liver microsomal proteins. They had also suggested that, if PCP metabolism resulted in covalent binding in human tissues and antibodies were generated against a PCHAP-like hapten, this could lead to potential problems. This is because the antibodies generated against the PCHAP hapten were originally found to have similar binding specificity to the PCP binding site in theN-methyl-d-aspartate receptor complex (9,28-30). If these antibodies or the antiidiotypic antibodies were present in the central nervous system, it could possibly be an explanation for the late-appearing schizophrenic-like condition that has been observed in some PCP abusers (31, 32).

Our goal was to use PCP as a mechanism-based inactivator of P4502B1 in an attempt to obtain covalently modified protein specifically labeled at the active site for studies on the structure of the active site (33,34). Inactivation of P4502B1 was not accompanied with a loss in spectrally detectable P450, suggesting that PCP was a drug of promise for active site studies. However, binding of PCP metabolites to the reductase and catalase presents problems due to escape of the reactive species from the P450 active site. Failure of GSH to decrease selectively formation of the PCP-metabolite adducts to reductase and catalase suggests that, even with P450, the reactive intermediate probably binds outside of the active site, where it is accessible for reaction with GSH before binding to the enzyme.

In conclusion, PCP is metabolized by P4502B1 to a reactive species that inactivates the enzyme and binds to P450 in addition to the other components of the incubation mixture, reductase and catalase. The PCHAP antibody recognizes PCP-metabolite adducts bound to reductase, catalase, and P450. GSH protects against the inactivation and also against binding to the components of the incubation mixture. Studies are currently underway to identify the actual metabolite adduct that binds to proteins and to confirm the structural requirements necessary for detection by the PCHAP antibody.

Acknowledgments

We thank Hsia-lien Lin for the purification of P4502B1 and reductase.

Footnotes

  • Send reprint requests to: Dr. Paul F. Hollenberg, Department of Pharmacology, 2301 Medical Sciences Research Building III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632.

  • This study was supported in part by Grant CA16954 from the National Cancer Institute (U.S. Public Health Service) (to P.F.H.), by Grant DA04136 (National Institute on Drug Abuse), and by a Research Scientist Development Award K02 DA00110 (to S.M.O.).

  • Abbreviations used are::
    PCP
    phencyclidine
    P450
    cytochrome P450
    P4502B1
    the major P450 form from liver microsomes of phenobarbital-treated rats
    DLPC
    l-α-phosphatidylcholine, dilauroyl
    EFC
    7-ethoxy-4-trifluoromethylcoumarin
    HFC
    7-hydroxy-4-trifluoromethylcoumarin
    ECL
    enhanced chemiluminescence
    reductase
    NADPH cytochrome P450 oxidoreductase
    BSA
    bovine serum albumin
    KCN
    potassium cyanide
    GSH
    glutathione
    TFA
    trifluoroacetic acid
    SDS-PAGE
    sodium dodecyl sulfate-polyacrylamide gel electrophoresis
    PCHAP
    5-[N-(1′-phenylcyclohexyl)amino]pentanoic acid
    TTBS
    Tris–Tween-buffered saline
    • Received August 8, 1996.
    • Accepted November 19, 1996.

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Metabolic Inactivation of Cytochrome P4502B1 by Phencyclidine

Uma Sharma, Elizabeth S. Roberts, Ute M. Kent, S. Michael Owens and Paul F. Hollenberg
Drug Metabolism and Disposition February 1, 1997, 25 (2) 243-250;
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