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Venkatakrishnan and colleagues pointed out that phenacetin O-deethylase (POD)¹ activity in the presence of 10 µM fluvoxamine did not accurately reflect the low-affinity (i.e., non-CYP1A2) component of POD activity because fluvoxamine inhibits CYP2C19 and CYP2C9 as well as CYP1A2. We agree that fluvoxamine is not a specific CYP1A2 inhibitor. However, our experiments with pooled human liver microsomes revealed that fluvoxamine (10 µM) inhibited the POD activity to the same extent (about 25%) as anti-CYP1A2 antibodies (200 µl/mg protein) did, as shown in Figs. 3A and 4 of our article (Kobayashi et al., 1999). In addition, the inhibition of the POD activity in pooled human liver microsomes by fluvoxamine was similar to that by furafylline, a specific CYP1A2 inhibitor (Fig. 1). These results suggest that the inhibition of POD activity in human liver microsomes by fluvoxamine is mainly due to the inhibition of CYP1A2-mediated activity. Therefore, the POD activity in the presence of 10 µM fluvoxamine would accurately reflect the low-affinity component of POD activity in human liver microsomes.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effects of fluvoxamine () and furafylline () on POD activity in pooled human liver microsomes. The concentration of phenacetin was 500 µM. Each data point represents the mean of duplicate experiments. Furafylline (0.1-100 µM) was preincubated in the presence of an NADPH-generating system at 37°C for 30 min, and the reaction was initiated by the addition of a substrate dissolved in water. The data for fluvoxamine were cited from our recent reports (Kobayashi et al., 1999).

Venkatakrishnan et al. (1998) reported that CYP2C9 was active to phenacetin in a study using cytochrome P-450 (CYP) isoforms expressed in human B-lymphoblastoid cells. We also have confirmed this by using CYP2C9 expressed in human B-lymphoblastoid cells (Nakajima et al., 1999). However, as stated in our article (Kobayashi et al., 1999), CYP2C9 from baculovirus-infected insect cells did not exhibit significant POD activity. Yang et al. (1998) also reported that CYP2C9 expressed in HepG2 cells was not active to POD activity. This discrepancy might be due to the differences in the expression systems used in the studies (i.e., differences in the levels of NADPH-cytochrome P-450 reductase and cytochrome b₅, membrane composition, etc.). Therefore, we would like to emphasize that a study using not only a recombinant enzyme but also the combination of a chemical inhibitor, antibody, and correlation with a marker reaction is necessary to identify CYP isoforms involved in POD activity by human liver microsomes.

To re-analyze the CYP isoform(s) responsible for the low-affinity component of POD activity in human liver microsomes, we tested the effects of 10 µM furafylline, a more specific CYP1A2 inhibitor, 1 mM aniline, and 10 µM sulfaphenazole, alone and in combination, on POD activity in pooled human liver microsomes. As shown in Fig. 2, furafylline inhibited POD activity to 27.5 ± 0.6% of the control. The combination of furafylline and aniline inhibited POD activity to 9.0 ± 0.2% of the control. The extent of inhibition by the combination of furafylline and aniline was greater than that by the combination of furafylline and sulfaphenazole (to 20.4 ± 0.1% of the control). These results suggest that the main enzyme apart from CYP1A2 involved in POD activity at 500 µM of phenacetin in human liver microsomes is CYP2E1, not CYP2C9. Thus, we don't agree with Venkatakrishnan et al. that CYP2C9 is a principal low-affinity enzyme of POD activity.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effects of furafylline, aniline, and sulfaphenazole on POD activity in pooled human liver microsomes. The concentration of phenacetin was 500 µM. Each column represents the mean ± S.D. of three different experiments.

Venkatakrishnan et al. pointed out that the use of 1 mM phenacetin as the highest concentration of substrate was too low to determine the kinetic parameters of recombinant CYPs other than CYP1A1, CYP1A2, and CYP2E1. The use of higher substrate concentrations might enable the K_m values for recombinant CYP2C19, CYP2D6, and CYP3A4+b₅ to be estimated. However, recombinant CYP2C9 from baculovirus-infected insect cells did not exhibit sufficiently high POD activity to perform a kinetic study. On the other hand, recombinant CYP2E1 from baculovirus-infected insect cells showed significant POD activity with typical Michaelis-Menten kinetics, as stated in our article (Kobayashi et al., 1999). The mean K_m value of recombinant CYP2E1 was similar to that of the low-affinity component in pooled human liver microsomes (785 and 894 µM, respectively) when the same range of substrate concentrations (1 µM to 1 mM) were used. Therefore, our kinetic data support the speculation that CYP2E1 is a low-affinity enzyme of POD activity in human liver microsomes.

In their Letter to the Editor, Venkatakrishnan et al. stated that the low-affinity component of POD activity was isolated as an -naphthoflavone-inhibitable component in a panel of 12 human liver microsomes. We agree with the use of -naphthoflavone as a potent CYP1A2-specific inhibitor. However, -naphthoflavone is not only a potent CYP1A2-specific inhibitor but also an activator of CYP3A4 (Newton et al., 1995). Although Venkatakrishnan et al. (1998) reported that CYP3A4 failed to catalyze the POD activity, both recombinant CYP3A4 from baculovirus-infected insect cells and human B-lymphoblastoid cells exhibited significant POD activity in our study. POD activity was also observed in the reconstituted system of CYP3A4 (Nakajima et al., 1999). Therefore, it is thought that POD activity catalyzed by CYP3A4 is activated by -naphthoflavone in human liver microsomes. In fact, CYP3A4-mediated POD activity was enhanced at concentrations over 1 µM -naphthoflavone (Nakajima et al., 1999). Moreover, the extent of activation by -naphthoflavone was dependent on CYP3A4 levels in individual microsomes of human livers. Therefore, the data obtained from the study by Venkatakrishnan and associates would be inadequate because the addition of -naphthoflavone might confuse the contribution percentages of each CYP isoform to POD activity in intact microsomes from human livers.

Finally, we agree that fluvoxamine is not a specific inhibitor of CYP1A2. However, the inhibition of POD activity in human liver microsomes by fluvoxamine would be mainly due to the inhibition of CYP1A2-mediated activity, and the POD activity in the presence of 10 µM fluvoxamine would accurately reflect the low-affinity component of POD activity in human liver microsomes. The residual POD activity not inhibited by fluvoxamine was significantly correlated with immunoquantified CYP2E1 levels or chlorzoxazone 6-hydroxylase activity as described in our article (Kobayashi et al., 1999). Furthermore, our kinetic analysis of POD activity showed that the K_m value of CYP2E1 was similar to that of the low-affinity component of POD activity in human liver microsomes. Therefore, these observations show that CYP2E1 is a principal low-affinity enzyme of POD activity in human liver microsomes.