Introduction

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The cytochrome P450 (CYP450²) enzymes are heme-containing proteins located in the endoplasmic reticulum and mitochondria of cells (Nedelcheva and Gut, 1994; Guengerich, 1996; Glue and Clement, 1999). Within the past 5 or 6 years, our understanding of apoptotic cell death has been expanded to include the CYP450 enzymes as contributors to the formation of reactive oxygen species (ROS). The findings that 1) ROS can act as one of the myriad triggers inducing apoptosis, 2) CYP450 enzymes can generate ROS, 3) CYP450 enzymes are located in the mitochondria, and 4) the mitochondria are central to the initiation of apoptosis have put the CYP450 enzymes into the web of events leading to apoptosis (Anandatheerathavarada et al., 1997; Dalton et al., 1999; Bhagwat et al., 2000; Nebert et al., 2000).

Further support for the notion that CYP450 enzymes are involved in apoptosis comes from the demonstration that CYP450 inhibitors attenuate oxidative stress-induced apoptotic death of cultured hepatocytes (Shiba and Shimamoto, 1999). Recently, R-deprenyl was found to be an inhibitor or inactivator of CYP2B1 (Sharma et al., 1996). R-Deprenyl and the aliphatic analogs, (R)-N-2-heptyl-N-methyl-propargylamine (R-2HMP) and (R)-N-2-heptylpropargylamine (R-2HPA), reduce apoptotic death in cerebellar granule cells, PC12 cells, and in several in vivo models of apoptosis (Ansari et al., 1993; Tatton et al., 1996; Boulton et al., 1997; Paterson et al., 1998; Berry, 1999). The goal of this study was to determine whether R-2HMP and R-2HPA inhibited the activity of CYP2B1, as has been shown for R-deprenyl. In addition, an examination of the effects of these propargylamines on the activities of CY1A2, a normally abundant isoform, and CYP1A1, a minor but inducible isoform, was undertaken. Other propargylamines, pargyline, desmethyldeprenyl, as well as the S-enantiomers of deprenyl, 2HMP, and 2HPA were included for comparison.

	Experimental Procedures

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Materials. -Nicotinamide adenine dinucleotide phosphate, reduced form, tetrasodium (NADPH), glycerol; sucrose, disodium EDTA, Trizma base, -naphthoflavone (ANF), -naphthoflavone (BNF), dimethyl sulfoxide (DMSO), resorufin, 7-pentoxyresorufin, proadifen, metyrapone, and pargyline were purchased from Sigma-Aldrich Canada Ltd. (Ontario, Canada). 7-Ethoxyresorufin was purchased from Molecular Probes (Eugene, OR). (R)- and (S)-Deprenyl and (R,S)-desmethyldeprenyl were obtained from Sigma/RBI (Natick, MA). R-2HMP, S-2HMP, R-2HPA, S-2HPA, (R)-N-2-heptyl-N-methylamine (R-2HMA), (R)-N-2-heptylamine (R-2HA), and (R)-N-2-heptylaminopropionic acid (R-2HPcAc) were synthesized by Dr. B. Davis (Durden et al., 2000). The chemical purity of these compounds was determined by elemental analysis, NMR, and mass spectrometry to be 99%, except for R-2HPcAc, which was 95% pure. Disposable semimicro cuvettes were purchased from VWR Canada, Edmonton, AB, Canada.

Drug Treatment of Rats. Female Wistar rats (body weight, 225-250 g; Charles River, Montreal, PQ, Canada) were injected i.p. with vehicle (saline or corn oil); phenobarbital (PB), 75 mg/kg in saline; or BNF in corn oil, 15 mg/kg i.p. once daily for four consecutive days. Phenobarbital at this dosage regimen increases the content of rat liver CYP2B1/2 20 or more times above control (Lubet et al., 1985; Masubuchi et al., 1995). BNF at the dosage selected increases the levels of CYP1A1 (Burke et al., 1985, 1994; Oinonen et al., 1994) and is less toxic than methylcholanthrene, another CYP1A1 inducer (Zhao and Shichi, 1995). All procedures were approved by the Animal Care Committee of the University of Saskatchewan and were in accordance with the guidelines of the Canadian Council on Animal Care.

Preparation of Rat Liver Microsomes. Rat liver was rinsed with ice-cold 0.15 M KCl/0.2 M sucrose/10 mM EDTA (pH 7-7.2) (Nims et al., 1994), finely sliced, and homogenized (1 g to 3 ml of solution) in this ice-cold solution using a Polytron homogenizer at setting 6 with 20- to 30-s bursts, and centrifuged for 30 min at 9000g at 4°C. The supernatant was centrifuged at 100,000g for 1 h at 4°C in a Beckman L7-65 ultracentrifuge; the microsomal pellet was washed in the aforementioned solution and recentrifuged at 100,000g for 1 h at 4°C. The final microsomal pellet was suspended in ice-cold 50 mM Tris buffer, pH 7.5, containing 20% glycerol and 1 mM EDTA, partitioned into aliquots, and stored at 70°C (Hopkins et al., 1992).

CYP1A2, CYP1A1, and CYP2B1 Assays. The O-deethylation of 7-ethoxyresorufin to 7-hydroxyresorufin by liver microsomes from control and from BNF-pretreated rats can be used as a measure of CYP1A2 and CYP1A1 activity, respectively (Burke et al., 1985; Lubet et al., 1985).

Inhibition Studies. Microsomes were preincubated with known CYP450 inhibitors or with other drugs in the presence of substrate for 3 min prior to the addition of NADPH to initiate the enzyme assay (Roberts et al., 1995; Beebe et al., 1996). Drugs were dissolved in the Tris assay buffer, except for ANF, which was solubilized in DMSO. DMSO itself inhibits CYP450 activity (Burke et al., 1985); therefore, the ANF samples were compared with control samples with 5 µl of DMSO added. Known inhibitors were initially tested to confirm the selectivity of the assays, then other drugs at an initial concentration of 100 µM were tested for their ability to inhibit rat liver microsomal CYP1A2, 1A1, or 2B1 activities.

Inactivation Experiments. Microsomes were preincubated with R-deprenyl, R-2HMP, R-2HPA, S-2HPA, or R-2HA in the presence of NADPH for 0, 0.5, 1, 2, 3, 4, or 5 min. Following the various preincubation time periods, 7-pentoxyresorufin was added and fluorescence readings taken after a 10-min incubation. For comparison, proadifen, which is not a mechanism-based inactivator, was run as a negative control. The natural logarithm of the percentage of activity remaining values was plotted against the time of preincubation at each concentration of these drugs (Roberts et al., 1995; Beebe et al., 1996; Sharma et al., 1996).

Membrane Filtration Experiments. To assess the reversibility of the inactivation of microsomal CYP2B1 by the selected propargylamines, these drugs were preincubated with liver microsomes in the presence of NADPH, and then the samples were filtered by centrifugation at 14,000g through a Microcon YM-30 (Millipore Corporation, Bedford, MA), a filter device fitted with a YM-30 cellulose membrane with a molecular cut-off of 30,000. The retentate, typically a 40- to 50-µl volume, containing the recovered CYP2B1 protein (Roberts et al., 1998) was added to a 1-ml incubation mixture containing NADPH. 7-Pentoxyresorufin was added and fluorescence readings taken following a 5-min incubation. Control samples consisted of liver microsome samples that received the same pretreatment and that were incubated at the same time as the YM-30-filtered samples but were centrifuged in a polyethylene tube (i.e., no YM-30 membrane filtration).

Protein Determination. The protein concentrations of the microsomal preparations were determined (Lowry et al., 1951). The amount of liver microsomal protein used per assay from control rats was typically 250 to 280 µg, 200 to 250 µg from PB-treated rats, and 30 to 40 µg from BNF-treated rats.

Statistical Analyses. The percentage of activities remaining in samples incubated with inhibitors was calculated using the control samples run at the same time as the inhibitor. To determine whether the reductions in the percentage of activity remaining in the presence of the tested compounds were significant, the activities of the inhibitors were compared with control samples using one-way analysis of variance and by determining significant differences with Scheffé's test (Winer, 1962).

	Results

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Confirmation of CYP1A2, 1A1, and 2B1 Enzyme Activity Assays. All three P450 assays were linear up to 10-min incubation. With a 10-min incubation, the CYP1A2 assay was linear up to 400 µg of protein, the CYP1A1 assay was linear up to 50 µg of protein, and the CYP2B1 assay was linear up 380 µg of protein. The CYP1A2 activity of liver microsomes obtained from vehicle-treated rats typically was 0.3 ± 0.02 nmol/min/mg of protein. CYP1A1 activity of liver microsomes from BNF-treated rats was 4.0 ± 0.1 nmol/min/mg of protein and CYP2B1 activity of microsomes from PB-treated rats was 0.7 ± 0.03 nmol/min/mg of protein.

Effects of Propargylamines and Related Compounds on CYP1A2, 1A1, and 2B1 Activity. The effects of several propargylamines and structurally similar compounds (Fig. 1) all at a concentration of 100 µM were assessed (Table 1). None of the compounds affected CYP1A1 activity except for desmethyldeprenyl, in which case a slight but significant decrease was observed. The propargylamine compounds (R-deprenyl, desmethyldeprenyl, pargyline, R-2HMP, and R-2HPA) significantly reduced CYP1A2 and CYP2B1 activities compared with control. About 50 to 60% CYP1A2 activity remained in the presence of these compounds; however, only about 10% or less of CYP2B1 activity remained in the presence of these propargyl drugs.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Chemical structures of the selected propargylamines and related compounds.

Inhibition of Microsomal CYP2B1 by Propargylamines. Because the propargylamines were found to have their greatest effect on CYP2B1 activity, this enzyme was selected for further study. Different concentrations of R-deprenyl, R-2HMP, and R-2HPA were incubated with microsomes for 10 min to determine their IC₅₀ values for the inhibition of CYP2B1 activity. Graphical analyses estimated the IC₅₀ values to be 2.6 µM for R-deprenyl, 8.5 µM for R-2HMP, and 3.6 µM for R-2HPA (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Percentage of rat liver microsomal CYP2B1 activity remaining in the presence of various concentrations of R-deprenyl, R-2HMP, or R-2HPA. Values are shown as mean ± S.E.M., n = 6. IC50 values were derived mathematically from the equations describing the best linear fit of the individual graphs.

Effects of Stereochemistry on Inhibition of CYP2B1 by Propargylamines. A comparison of the ability of (R)- and (S)-enantiomers of deprenyl, 2HMP, and 2HPA at concentrations of 10 µM to decrease the activity of microsomal CYP2B1 activity was undertaken. With all three propargylamines, the percentage of activity remaining in the presence of the (R)-isoform of the compounds was less than that remaining with the corresponding (S)-isoforms (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of increasing time of incubation on the liver microsomal CYP2B1 activity in the presence of 10 µM R- and S-deprenyl, 10 µM R- and S-2HMP, and 10 µM R- and S-2HPA. Values are log percentage of CYP2B1 activity remaining, mean ± S.E.M., n = 6.

Effect of NADPH Preincubation Time and Drug Concentration on CYP2B1 Inactivation. The effect of inhibitor concentration and the length of the preincubation time with NADPH on the ability of R-deprenyl, R-2HMP, R-2HPA, S-2HPA, and R-2HA to inactivate CYP2B1 activity was examined next. For comparison, proadifen was included as a negative control. With proadifen (2.5 and 5 µM), the ln percentage of CYP2B1 activity remaining values increased with increasing time of preincubation (data not shown); in contrast, with R-deprenyl, R-2HMP, R-2HPA, and S-2HPA, the ln percentage of activity remaining values decreased in a concentration-dependent manner with increasing length of preincubation time (Fig. 4). However, with 50 µM R-2HA, the ln percentage of activity remaining values did not decrease significantly as the preincubation time increased (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Effects of increasing preincubation time with NADPH on the inhibition of CYP2B1 by different concentrations of R-deprenyl, R-2HMP, R-2HPA, and S-2HPA. Values are the natural log of the percentage of CYP2B1 activity remaining, mean ± S.E.M., n = 6.

Estimation of Kinetic Constants for Inactivation of Microsomal CYP2B1 Activity. The slopes (k_obs) of the individual lines in Fig. 4 were obtained by linear regression analysis, and then the reciprocals of the slopes and the reciprocals of the drug concentrations were plotted to derive the kinetic constants (Fig. 5). From the equation describing the best fit for each propargylamine, the inactivation constants k_inact and K_I were calculated to be 1.3 µM and 0.32 min¹ for R-deprenyl, 0.8 µM and 0.08 min¹ for R-2HMP, 0.5 µM and 0.36 min¹ for R-2HPA, and 0.24 µM and 0.18 min¹ for S-2HPA.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Double reciprocal plots of the rate of inactivation (kobs) of microsomal CYP2B1 activity as a function of the concentrations of R-deprenyl, R-2HMP, R-2HPA, or S-2HPA. Kinetic constants were derived from the equations (shown in inset into the graphs) describing the best linear fit. Values shown are means, n = 6.

Membrane Filtration Studies. To assess the reversibility of the inactivation of microsomal CYP2B1 activity by 3.2 µM concentrations of R-deprenyl, R-2HMP, R-2HPA, or S-2HPA, each of these drugs was preincubated with the microsomes and then filtered through a membrane that retains molecules greater than 30,000 mol. wt. For comparison, 100 µM R-2HA was included. It can be seen in Fig. 6 that filtering of the microsomal samples preincubated with each of the four propargyl compounds did not diminish significantly the inactivation of CYP2B1 activity by the propargylamines, but the effects of R-2HA were almost completely reversed by YM-30 filtration.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Investigation of the reversibility of the inactivation of microsomal CYP2B1 activity by the propargylamines of interest. Microsomes were preincubated with R-deprenyl, R-2HMP, R-2HPA, or S-2HPA (3.2 µM) or with 100 µM R-2HA in the presence of NADPH. Then the samples were either filtered via centrifugation through a cellulose membrane with a molecular weight cut-off of 30,000 (YM-30) or they were simply centrifuged (no YM-30) prior to addition of substrate and incubation for a 5-min time period. Values are percentage of CYP2B1 activity remaining, mean ± S.E.M., n = 6.

	Discussion

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Generally, it is accepted that CYP2B1 activity can be assayed with high selectivity by using 7-pentoxyresorufin as the substrate and microsomes obtained from phenobarbital-pretreated rats (Burke et al., 1985, 1994; Lubet et al., 1985). Similarly, CYP1A1 activity is assayed predominantly over CYP1A2 using liver microsomes from BNF-pretreated rats with 7-ethoxyresorufin as the substrate (Burke et al., 1985), whereas CYP1A2 activity is detected with uninduced (vehicle-treated) rat liver microsomes. The pattern of inhibition by proadifen, ANF, and metyrapone confirmed the selectivity of the assays (data not shown). ANF and metyrapone are selective inhibitors of CYP1A2 and CYP2B, respectively (Barham et al., 1994; Halpert et al., 1994).

Our results confirm a prior report that showed that R-deprenyl inhibits purified CYP2B1 but not CYP1A1 (Sharma et al., 1996). In this article, we show that several other propargylamines, (R,S)-desmethyldeprenyl, R-2HMP, R-2HPA, and pargyline also inhibited the activity of rat liver microsomal CYP2B1; furthermore, we found that these drugs inhibited microsomal CYP1A2 activity (Table 1). Structurally similar compounds lacking the propargyl function (Fig. 1) did not reduce CYP1A2 and CYP1A1 activities, but significant, although smaller, reductions in CYP2B1 activity were observed with R-2HMA and R-2HA (Table 1).

Using IC₅₀ values as a measure of the efficacy of inhibition of microsomal CYP2B1 activity, it was found that the two alkyl compounds, R-2HMP and R-2HPA, were similar to R-deprenyl (Fig. 2). It appears that the alkyl side chain was comparable to the phenyl group of deprenyl. Further studies are in progress to determine the effect of the length and chirality of the alkyl side chain on inhibition of CYP2B1 by such compounds.

An examination of the time course of the inhibition of CYP2B1 activity by 10 µM concentrations of the (R)- and (S)-enantiomers of deprenyl, 2HMP and 2HPA, revealed that the (R)-enantiomers inhibited CYP2B1 activity to a greater extent than the respective (S)-compounds (Fig. 3). Using R- and S-2HPA, this was further explored by determining inactivation constants for these two compounds.

Two criteria that can be used to define a particular drug as a mechanism-based inactivator are that the drug-induced CYP450 inhibition is dependent on the length of the preincubation time with NADPH and on the concentration of inactivator (Roberts et al., 1995; Beebe et al., 1996; Sharma et al., 1996). R-Deprenyl has been reported previously to be a mechanism-based inactivator of purified CYP2B1 (Sharma et al., 1996). By these criteria, R-deprenyl, R-2HMP, R-2HPA, and S-2HPA were mechanism-based inactivators since their inactivation of microsomal CYP2B1 activity increased with increasing time of preincubation in a concentration-dependent manner (Fig. 4). In contrast to the propargylamines, R-2HA, which inhibited CYP2B1 activity (Table 1) but lacks the propargyl group, did not fit these criteria. Its inactivation of CYP2B1 was not affected by varying the NADPH preincubation time.

The k_inact and K_I values, derived from Fig. 5, found here (1.4 µM and 0.32 min¹) for the inactivation by R-deprenyl are similar to those reported for purified CYP2B1 (1.1 µM and 0.23 min¹) (Sharma et al., 1996). The K_I values for R-2HMP, R-2HPA, and S-2HPA were 0.8, 0.5, and 0.24 µM, respectively. The maximal rates of inactivation, k_inact, were similar for R-deprenyl and R-2HPA (0.32 and 0.36 min¹) and for R-2HMP and S-2HPA were somewhat lower (0.08 and 0.18 min¹). The rate of inactivation of S-2HPA was less than that of R-2HPA. We have shown that R-2HMP is metabolized in the rat to R-2HPA (Durden et al., 2000). Perhaps this demethylation step by the liver microsomes accounts for the differences in the k_inact constants of R-2HPA and R-2HMP. As yet, it is not known whether the demethylation is involved in the formation of a metabolite intermediate complex or whether S-2HMP is demethylated. Further studies are necessary to determine this.

To determine whether the effects of the four propargylamines of interest, R-deprenyl, R-2HMP, R-2HPA, and S-2HPA, inactivated CYP2B1 activity irreversibly, microsomes were preincubated with 3.2 µM concentrations of these drugs and then spun through a cellulose membrane with a molecular cut-off of 30,000. The propargyl compounds with molecular weights of about 200 would pass through the filter, unless they were bound to the enzyme. The percentage of inhibition of CYP2B1 activity of these samples was not significantly different from microsomes that had not been spun through a membrane (Fig. 6); thus, it appears that these drugs inactivate the enzyme irreversibly because separating the protein from the inactivator made no difference. In contrast, the percentage of inhibition of CYP2B1 activity by R-2HA was almost completely reversed by filtration through the YM-30 membrane (Fig. 6).

R-Deprenyl, R-2HMP, and R-2HPA have been reported to reduce apoptotic cell death in a variety of systems (Ansari et al., 1993; Tatton et al., 1996; Boulton et al., 1997; Paterson et al., 1998; Berry, 1999). One may speculate that these compounds might act, at least partially, by inhibiting the actions of CYP450 enzymes, such as CYP2B1, 1A2, or others, and by doing so, decrease the production of ROS, which in turn decreases ROS-initiated apoptotic cell death. Because, however, the (S)-enantiomers of these drugs are not antiapoptotic (Paterson et al., 1998; Berry, 1999), but were inhibitors of microsomal CYP2B1 activity, inactivation of CYP450 enzymes cannot be the sole mechanism whereby R-deprenyl, R-2HMP, and R-2HPA interfere with apoptotic cell death.

In summary, the results presented here demonstrated that a number of propargylamines (pargyline, R-deprenyl, desmethyldeprenyl, R-2HMP, R-2HPA, and S-2HPA) inhibited the rat liver microsomal activities of CYP1A2 and CYP2B1, but not CYP1A1. All the tested propargylamines were more effective inhibitors of liver microsomal CYP2B1 activity than of CYP1A2. The inhibition of CYP2B1 activity exhibited some preference for the (R)- over the (S)-enantiomers of deprenyl, 2HMP, and 2HPA. This is the first report concerning the inhibition by the alkylpropargylamines, 2HMP and 2HPA, of CYP450 enzyme activity. Furthermore, it appears that R-2HMP, R-2HPA, and S-2HPA, like R-deprenyl, are mechanism-based inhibitors of CYP2B1 activity.