Introduction

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There have been numerous reports related to the organization of cytochrome P450 and its redox partners in the microsomal membrane. Many of these studies have examined the interaction between NADPH-cytochrome P450 reductase and P450¹ (Bernhardt et al., 1988; Nadler and Strobel, 1988; Shimizu et al., 1991; Voznesensky and Schenkman, 1992, 1994; Shen and Strobel, 1994; Cvrk and Strobel, 2001) as well as cytochrome b₅ and P450 (Yamazaki et al., 1995, 1996; Bridges et al., 1998; Schenkman and Jansson, 1999). These reports have largely focused on the points of interaction between reductase and P450 or reductase and cytochrome b₅. Recently, the interactions among multiple P450 enzymes and reductase were examined. The goal of these studies was to determine whether the presence of one P450 enzyme could influence the catalytic properties of another P450 (Cawley et al., 1995). Using mixtures of CYP2B4, CYP1A2, and NADPH-cytochrome P450 reductase reconstituted into dilauroylphosphatidylcholine liposomes, the addition of a second P450 enzyme dramatically alters the activity of the original P450 enzyme in the mixed reconstituted system. This effect is dependent on the substrate present. For example, CYP2B4-dependent 7-pentoxyresorufin-O-dealkylation (PROD) is dramatically inhibited by the addition of CYP1A2. This inhibitory response of CYP2B4-dependent PROD is more pronounced at subsaturating reductase, exhibiting an 80% inhibition but only showing a 20% inhibition at saturating reductase. On the other hand, CYP2B4-dependent benzphetamine dealkylation shows a much weaker inhibitory response at subsaturating reductase and appears to be roughly additive when reductase levels are not limiting. Other substrates actually produce a synergistic stimulation of monooxygenase activities in mixed reconstituted systems. CYP1A2-selective 7-ethoxyresorufin-O-dealkylation (EROD) is stimulated by the addition of a second P450 enzyme (CYP2B4). Under these conditions, the rate is greater than the sum of the rates of the simple binary systems. Synergism of testosterone 6-hydroxylation has also been reported in mixed reconstituted systems containing reductase, CYP3A4, and CYP1A2 (Yamazaki et al., 1997). Taken together, the interaction among these enzymes is consistent with the formation of heteromeric P450-P450 complexes, which have an altered ability to associate with reductase (Backes et al., 1998).

Although the interaction among reductase and these P450 enzymes has been established in reconstituted systems, it is possible that these enzymes do not respond in a similar manner in the more natural environment of the microsomal membrane. The purpose of this study was to provide evidence for similar interactions among P450 enzymes in microsomal preparations. This was accomplished by treating rabbits with phenobarbital (PB), -naphthoflavone (NF), or PB + NF to induce CYP2B4, CYP1A2, or both CYP2B4 and CYP1A2 and comparing benzphetamine and 7-pentoxyresorufin (7-PR) metabolism with each of these microsomal preparations.

	Experimental Procedures

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Materials. The substrates, 7-ethoxyresorufin and 7-PR, were purchased from Molecular Probes, Inc. (Eugene, OR). PB and NF were obtained from Mallinckrodt (St. Louis, MO) and Sigma (St. Louis, MO), respectively. Benzphetamine was a gift from Upjohn (Kalamazoo, MI). Antibody to CYP2B4 (chicken anti-rabbit) was a gift from Dr. John B. Schenkman (University Connecticut Health Center, Farmington, Connecticut). Anti-rat CYP1A1/1A2 monoclonal antibody was purchased from Oxford Biomedical Research (Oxford, MI). Purified recombinant CYP2B4 was a gift from Dr. Lucy Waskell (University of Michigan, Ann Arbor, MI). All other chemicals were reagent quality and were purchased from Sigma.

Methods. Rabbits, divided into three groups, were treated with PB, NF, or both PB + NF to enrich the microsomal preparations with CYP2B4, CYP1A2, and both CYP2B4 and CYP1A2, respectively. PB was administered by daily intraperitoneal injection for 3 days at a dose of 80 mg/kg. Rabbits were euthanized 24 h after the final injection. NF was administered as a single injection (40 mg/kg), and the rabbits were killed 72 h afterward. Rabbits treated with both PB and NF were given injections with both inducers on day 1, followed by PB injections on the second and third days. Rabbits in this group were euthanized 24 h after the final PB injection.

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	Results

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Reconstituted Systems Containing CYP2B4, CYP1A2, and NADPH-cytochrome P450 reductase. In previous studies with reconstituted systems, the ability of one P450 enzyme to influence the catalytic properties of another P450 was established. In these studies, the addition of CYP1A2 to a reconstituted system containing reductase and CYP2B4 exhibited an almost stoichiometric inhibition of PROD (Cawley et al., 1995). This effect was not observed with all substratesbenzphetamine demethylation did not exhibit this response. Furthermore, the amount of inhibition was dependent on the reductase-P450 ratiothe inhibition of PROD was much greater at subsaturating reductase when compared with that found at higher reductase-P450 ratios. The inhibition of CYP2B4-dependent activities in mixed reconstituted systems can be observed in Fig. 1, using recombinant CYP2B4 rather than the CYP2B4 purified from rabbit liver used in earlier studies (Cawley et al., 1995; Backes et al., 1998). Generally, the activities observed with recombinant CYP2B4 (Fig. 1) and CYP2B4 purified from rabbit liver were similar (Backes et al., 1998). Additionally, similar interactions among reductase, CYP1A2, and CYP2B4 were obtained with both preparations of CYP2B4.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Interactions among CYP2B4, CYP1A2, and reductase on the metabolism of different substrates. Metabolism of several substrates was examined in complex reconstituted systems containing both CYP1A2 and recombinant CYP2B4 and compared with the results found in simple reconstituted systems containing a single P450 enzyme. Reconstituted systems contained the following components. The CYP2B4 reconstituted system contained 16 µM DLPC, 0.05 µM CYP2B4, and either 0.025 or 0.075 µM reductase. The CYP1A2 reconstituted system contained 16 µM DLPC, 0.05 µM CYP1A2, and either 0.025 or 0.075 µM reductase. The mixed reconstituted system contained 16 µM DLPC, 0.05 µM CYP2B4, 0.05 µM CYP1A2, and either 0.05 or 0.15 µM reductase. In separate experiments, an increase of the DLPC concentration of the mixed reconstituted system to 32 µM did not obviate the inhibition of PROD or the synergism of EROD. The P450 concentrations were 0.05 µM CYP2B4 for the CYP2B4 reconstituted system, 0.05 µM CYP1A2 for the CYP1A2 system, and 0.05 µM both CYP2B4 and CYP1A2 for the mixed reconstituted system. Groups represent the mean ± S.E. for four determinations. Those marked with asterisks are significantly different from the sum of the rates of the simple reconstituted systems (*, p < 0.05; **, p < 0.01; ***, p < 0.001). A, effect of CYP1A2 on CYP2B4-dependent benzphetamine metabolism; B, effect of CYP1A2 on CYP2B4-dependent PROD; C, effect of CYP2B4 on CYP1A2-dependent EROD.

Are the Interactions among P450 Enzymes and Reductase also Observed in Microsomal Preparations? To determine whether similar interactions can be found with microsomal preparations, groups of rabbits were treated with PB, NF, or both PB and NF. Table 1 shows the quantification of overall P450, NADPH-cytochrome c reductase, and immunoreactive CYP1A2 and CYP2B4 levels. NADPH-cytochrome P450 reductase levels were estimated from cytochrome c reductase activities. These levels were elevated in PB-treated rabbits compared with both the NF- and the PB + NF-treated groups. Interestingly, NF (in the PB + NF-treated group) appeared to block the elevation in NADPH-cytochrome P450 reductase elicited by PB alone. Overall, P450 levels in the PB + NF group were higher than those found in NF- and PB-treated rabbits. Although P450 levels appeared to be higher in the PB versus the NF-treated group, the difference did not rise to the level of significance.

Microsomal studies: examination of the behavior of CYP2B4-selective substrates. Benzphetamine demethylation and PROD, both CYP2B4-selective substrates, were examined in each group of microsomal preparations (Fig. 2A). Benzphetamine demethylation was lowest in the NF-treated group and was more than 3-fold higher in PB-treated rabbit liver microsomes. This activity was also elevated in the PB + NF group, being 80% that found in the PB group. These results suggest that the elevated levels of CYP1A2 in the PB + NF group only marginally affected benzphetamine demethylation.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Effect of PB, NF, and PB + NF treatment on microsomal benzphetamine demethylation and PROD. Rabbits were treated with PB, NF, or PB plus NF to induce CYP2B4, CYP1A2, or both enzymes, respectively. Microsomal metabolism of benzphetamine and 7-pentoxyresorufin were then determined. Both substrates are predominantly metabolized by CYP2B4. Groups represent the mean ± S.E. for four determinations. Those marked with asterisks are significantly different from the PB-treated group (*, p < 0.05; **, p < 0.01; ***, p < 0.001). A, microsomal benzphetamine demethylation and PROD expressed as a function of protein concentration; B, benzphetamine demethylation and PROD expressed as a function of the amount of NADPH-cytochrome c reductase activity.

Microsomal studies: examination of the behavior of CYP1A2-selective substrates. When the CYP1A2-selective substrate 7-ethoxyresorufin was examined, the results showed an elevation of EROD activity in the NF-treated groups (Fig. 3A). There was no difference between the PB- and PB + NF-treated groups. This was particularly surprising because of the apparent elevation of immunoreactive CYP1A2 protein in the group treated with both compounds (Table 1). The lack of a difference between these groups may be, at least in part, a reflection of the 50% higher levels of reductase in the PB-treated group. When normalized to the amount of reductase, EROD in the NF-treated group was elevated compared with that treated with PB alone (Fig. 3B); however, this activity was smaller than anticipated based on CYP1A2 immunoreactive protein levels (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of PB, NF, and PB + NF treatment on microsomal EROD. Rabbit were treated with either PB or NF or PB plus NF, as indicated under Methods and in the legend to Fig. 2. EROD is predominantly metabolized by CYP1A2. Groups represent the mean ± S.E. for four determinations. Those marked with asterisks are significantly different from the PB-treated group (*, p < 0.05). A, microsomal EROD expressed as a function of protein concentration; B, EROD expressed as a function of the amount of NADPH-cytochrome c reductase activity.

	Discussion

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Despite the knowledge for more than 30 years that P450s exist in multiple forms, little is known about how these heme proteins interact and how they are organized within the endoplasmic reticulum. There have been several attempts to examine these potential interactions. Because multiple P450s exist in the presence of limiting concentrations of NADPH-cytochrome P450 reductase, the potential for different P450 enzymes to compete for the available reductase would be a logical assumption. Evidence for such interactions has been acquired. Early studies by West and Lu (1972) demonstrated that P448 and P450 would compete at limiting reductase. More recently, human CYP2A6 and CYP2E1, expressed in baculovirus-infected Sf9 cells, were shown to compete for reductase in a similar manner (Tan et al., 1997). These reports are consistent with each P450 having its own affinity for reductase and the ensuing competition between different P450s being dependent not only on the relative concentrations of the different P450 enzymes but also on the relative affinities of the P450s for reductase. These interactions can be described by the following species: 1A2 + 2B4 + OR  1A2-OR + 2B4-OR (model 1), where the amount of each complex is dependent on the relative concentration of the component proteins and the affinities of reductase for each of the P450 enzymes. As an additional complication to this model, it is possible for the affinities of specific P450 enzymes for reductase to be affected by a particular substrate or effector molecule.

Although simple competition between P450s for limiting reductase is an expected outcome that has support in the literature, several studies suggest that, in some cases, specific interactions among multiple P450s and reductase (aside from the competition described above) may occur. The nearly stoichiometric inhibition of PROD in the reductase-CYP1A2-CYP2B4 system suggests that something more than simple competition for reductase is involved (Cawley et al., 1995). Subsequent data showed that in the reductase-CYP1A2-CYP2B4 system, the results were consistent with the formation of a CYP2B4-CYP1A2 complex that was able to tightly bind reductase at the CYP1A2 binding site (Backes et al., 1998). The details of such a model include: OR + 2B4 + 1A2  OR-2B4 + OR-1A2 + OR-1A2-2B4 + 1A2-2B4-OR + OR-1A2-2B4-OR (model 2). There are five potential functional complexes to this model: two binary complexes (OR-2B4 and OR-1A2), two ternary complexes (OR-1A2-2B4 + 1A2-2B4-OR), and a quaternary complex (OR-1A2-2B4-OR). The unique feature of model 2 is that substrate is able to facilitate the formation of heteromeric P450 complexes with altered function.

The concept of the formation of heteromeric P450 complexes is supported by the results obtained with the CYP1A2-preferring substrate 7-ethoxyresorufin in which the addition of CYP2B4 synergistically stimulated this CYP1A2-dependent activity at subsaturating reductase concentrations (Backes et al., 1998). Synergism has also been reported for mixed reconstituted systems containing reductase, CYP3A4, and CYP1A2 (Yamazaki et al., 1997). In this report, there was a 2-fold stimulation of testosterone 6-hydroxylation by the addition of CYP1A2 at saturating reductase to a reconstituted system containing reductase and CYP3A4. A similar synergistic response was observed in mixed reconstituted systems containing reductase, CYP1A2, and recombinant CYP2B4 at a 1.5:1 [reductase]/[P450] (Fig. 1, A and C). These types of interactions can be most readily explained through the formation of heteromeric complexes in the mixed reconstituted systems (Backes et al., 1998).

The major advantage to examining the interaction of P450s in reconstituted systems is that the concentrations of the components and the lipid environment can be carefully regulated. A potential drawback to studies in reconstituted systems is that the proteins may not interact in the same manner as observed in the more natural environment of the microsomal membranes where the cellular machinery is used to incorporate the proteins. On the other hand, measurement of these inhibitory responses is confounded by the complexity of the microsomal systems. Not only do these systems contain a much larger number of P450 enzymes, other proteins, such as cytochrome b₅, heme oxygenase, and squalene monooxygenase, are present, each capable of interacting with the reductase. The larger number of components and the inability to carefully control their concentrations, makes it much more difficult to systematically examine the interactions of P450 proteins in microsomes. Therefore, it is important to determine whether the results obtained in microsomes are consistent with those observed in the reconstituted systems.

In an effort to detect these interactions in microsomal preparations, we took advantage of the fact that the addition of CYP1A2 to reconstituted systems containing reductase and CYP2B4 led to a large degree of inhibition of one CYP2B4 activity (PROD), whereas another CYP2B4 activity (benzphetamine demethylation) was only marginally affected. We tried to mimic the CYP2B4-, CYP1A2-, and CYP1A2 + CYP2B4-containing reconstituted systems by induction of these P450s in rabbit liver with PB, NF, and PB + NF, respectively. The results clearly showed that when CYP2B4 was preferentially induced (PB treatment), both PROD and bzp were elevated. However, when both CYP2B4 and CYP1A2 were induced (PB + NF treatment), only bzp activity remained elevatedPROD was dramatically decreased. These results are consistent with the results obtained in reconstituted systems. The synergism of EROD at subsaturating reductase in the mixed reconstituted system had a much smaller effect than that of PROD. Consequently, we were not able to detect analogous changes in rabbit liver microsomes treated with both PB + NF.

There are several potential complications to such experiments. First, in each of the treatment groups, CYP1A2 is present. Consequently, a relative decrease in PROD would be expected in each of the treatments groups. However, this CYP2B4 activity should be decreased to a greater extent in the presence of higher levels of CYP1A2as we were able to detect in the PB + NF-treated group (Backes et al., 1998). Second, in the microsomal studies, some of the metabolism of a substrate may be catalyzed by P450 enzymes other than CYP2B4 and CYP1A2. This would be expected to either diminish or completely mask the inhibition/synergism predicted by the defined reconstituted systems. The fact that we were able to detect inhibition of PROD in the microsomal preparations from PB + NF rabbits is most likely due to the large magnitude of the inhibition by CYP1A2. It is possible that the relative differences in PROD and benzphetamine metabolism in the PB and PB + NF groups could be due to differential induction of P450 enzymes other than CYP2B4 and CYP1A2. However, this condition would require the induction of a form of P450 in PB rabbits that specifically induces PROD metabolism (and not benzphetamine). Furthermore, induction of this form of P450 would then have to be inhibited in rabbits treated with both PB + NF. Although possible, this condition would require that CYP2B4 catalyze no more than 20% of the total PROD metabolism in the microsomal preparations (the fractional difference between PB and PB + NF microsomes). In separate experiments, antibody to CYP2B4 caused a 64% inhibition of PROD in PB-treated rabbits (not shown) and is not consistent with this alternative explanation. Finally, although the current data indicates that the CYP1A2 and CYP2B4 form a complex in the presence of 7-pentoxyresorufin, the data does not give us direct information on the organization of single P450s in the binary reconstituted systems. It is possible that homodimers (or even higher order multimers) exist both in reconstituted systems and in microsomes. The use of kinetic data in these studies allows us to focus on alterations that influence formation of functional complexes.

In conclusion, the results demonstrate that the interactions among CYP2B4, CYP1A2, and reductase observed in defined reconstituted systems are also found in microsomal preparations. These results support the idea that specific interactions among various P450 enzymes are possible. Although these studies have focused on the interaction of only two P450 enzymes, evidence from the literature suggests that the interactions described in this article are by no means unique and can have a considerable impact on the metabolism and toxicity of foreign compounds.