Diastereospecific kinetics of nicotine N'-oxidation in

x e n o b io t ic a

, 1998, v o l . 28, n o . 2, 127± 135

D iastereospeci® c kinetics of nicotine N « -oxidation in rat liver m icrosom es M . NAK AJIMA ‹ *, K. IWAT A ‹ , T. YOSHIDA Œ , T. YAM AM OTO ‹ and Y. KURO IWA ‹ Department of ‹ Clinical Pharmacy and Œ Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, Tokyo 142, Japan Received 3 June 1997 1. In kinetic studies, both Eadie± Hofstee plots for cis- and trans-nicotine-1 « -N-oxide formation from nicotine in rat liver microsomes were linear. For the formation of cis- and trans-nicotine-1 « -N-oxide, the apparent K were 0± 240³ 0± 069 and 1± 524³ 0± 951 mm m respectively. Corresponding V were 1± 52³ 0 ± 48 and 1± 19³ 0 ± 74 nmol} mg } min respectmax ively. 2. The formation of cis-nicotine-1 « -N-oxide was greater than the formation of transnicotine-1 « -N-oxide in rat liver microsomes and the intrinsic clearance of cis-nicotine-1 « N-oxide formation was 8 ± 1-fold greater than that of trans-nicotine-1 « -N-oxide formation. 3. The formation of both cis- and trans-nicotine-1 « -N-oxide in rat liver microsomes was inhibited by the addition of 1-(1-naphthyl)-2-thioure a or by heat-treatme nt of microsomes. 2-Diethylaminoethyl-2, 2-diphenylvalerate (SKF525A) and carbon monoxide did not aå ect these activities even at high concentrations. 4. Formations of cis- and trans-nicotine-1 « -N-oxide correlated signi® cantly with each other (r ¯ 0± 862, p ! 0± 01). These results suggested that the same ¯ avin-containing monooxygenase (FMO) isoform is responsible for the formation of cis- and trans-nicotine1« -N-oxide in rat liver.

In trodu ction

In most mammalian species, nicotine is rapidly and extensively metabolized primarily in the liver (Gorrod and Jenner 1975, Nakayama 1988). Although Coxidation (i.e. cotinine formation) is the major pathway of nicotine metabolism, the formation of N-oxide derivatives of nicotine constitutes an important route of nicotine biotransformation (Sepkovic et al. 1986). N icotine-1 « -N-oxide, the main N-oxide metabolite of nicotine, was ® rst isolated and identi® ed from the reaction mixture after incubation of a 9000 g supernatant of rabbit liver with nicotine (Papadopoulos and Kintzios 1963, Papadopoulos 1964). N icotine-1 « -N-oxide has been identi® ed in humans and experimental animals in vivo (Beckett et al. 1971, Thompson et al. 1985), as well as in vitro hepatic preparations from several mammalian species (Kyerematen et al. 1990). The N-oxidation of nicotine has received speci® c attention because of the interesting stereoselectivity in the diastereoisomeric product formation (Jenner et al. 1973a, b). Beckett et al. (1973) assigned the cis- and trans-nicotine-1 « -N-oxide structure and showed the NM R spectra. The separation of cis- and trans-nicotine-1 « -N-oxide has been ® rstly reported by Booth and Boyland (1970) using a paper chromatographic method. It was reported that ¯ avin-containing monooxygenase (FM O) appears to be the major enzyme responsible for nicotine-1 « -N-oxide formation in mammals (Damani * Author for correspondence at Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920, Japan. 0049± 8254} 98 $12± 00 ’

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et al. 1988, Nakayama 1988). In addition, some isoforms of cytochrome P450 are also capable of the formation of nicotine-1 « -N-oxide (Park et al. 1993). However, no kinetic studies have yet been carried out with hepatic microsomes in any species. In the current study the stereoselective formation of nicotine-1« -N-oxide diastereoisomers and its kinetic analyses in rat liver microsomes have been investigated.

M aterials and m eth ods Chemicals (S)-nicotine was purchased from Sigma Chemical Co. (St Louis, MO, USA). NADP + , glucose 6phosphate and glucose 6-phosphate dehydrogenase were from Oriental Yeast (Tokyo, Japan). Other chemicals were of the highest grade commercially available. Analytical and preparative TLC were done with 10¬ 20-cm FUNACEL SF cellulose thin layer plate (Funakoshi, Tokyo, Japan) and with 20¬ 20-cm, 1000-l m cellulose microcrystalline PK2F (Whatman, Clifton, NJ, USA) respectively. Synthesis of nicotine-1 « -N-oxide Nicotine-1 « -N-oxide was synthesized by a method previously described (Taylor and Boyer 1959). The diastereomers of nicotine-1 « -N-oxide were separated on preparative TLC with 1-butanol } 2propanol } ammonium hydroxide (2 : 1 : 1, v} v} v) as described previously (Cashman et al. 1992). The purities of each diastereomer have been con® rmed by HPLC. The structure of the each puri® ed synthetic diastereomer was con® rmed by " H-NM R. " H-NMR spectra were recorded on a FT NMR SYSTEM, JNM-LA500 (JEOL, Tokyo, Japan). These spectra (data not shown) were almost identical with a previous report (Cashman et al. 1992). Thus, the synthesized nicotine-1 « -N-oxide diastereomers were used for authentic standards.

Preparation of rat liver microsomes Male Sprague-Dawley rats (6 weeks old, 200± 250 g) were supplied from Saitama Experimental Animals Supply Co. Ltd (Saitama, Japan). Liver microsomes were prepared from the untreated rat as described previously (Kamataki and Kitagawa 1974) and were stored at ® 80 ° C until use. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. In vitro formation of nicotine-1 « -N-oxide diastereomers A typical incubation mixture (® nal volume, 0± 5 ml) consisted of 50 mm potassium phosphate buå er (pH 8 ± 4), NADPH-generating system (0 ± 5 mm NADP + , 5 mm glucose 6-phosphate, 5 mm MgCl , # 1 U } ml glucose 6-phosphate dehydrogenase), 0± 5 mg } ml microsomal protein. We selected the incubation condition at pH 8± 4 because it has been reported to be the optimum pH for FMO (Gold and Ziegler 1973). The reaction was initiated by the addition of nicotine following a 2-min preincubation at 37 ° C. The reaction was terminated with 0 ± 5 ml ice-cold acetone followed by addition of caå eine (50 ng) as an internal standard. After removal of protein by centrifugation, the reaction mixtures were extracted with 4 ml 2-propanol } CH Cl (1 : 2, v} v) after saturation with solid Na CO and centrifuged at 1000 g for # # # $ 10 min to separate the aqueous and organic fractions. The organic fraction was evaporated under a gentle stream of nitrogen at 40 ° C and the residue redissolved in 50 l l of the mobile phase and a 20 l l portion of the samples was subjected to HPLC. HPLC analyses were performed using a DG-980-50 degasser, PU-980 intelligent pump (both Jasco, Tokyo, Japan), AS-8010 autosampler (Tosoh, Tokyo, Japan), 807-IT integrator (Jasco) equipped with a Capcell Pak C18 UG120 column (4 ± 6¬ 250 mm, 5 l m ; Shiseido, Tokyo, Japan). The eluent was monitored at 260 nm using a UV-970 intelligent UV } VIS detector (Jasco). The mobile phase was 7± 5 % acetonitrile containing 0± 01 % acetic acid and 1 mm sodium heptane sulphonate. The ¯ ow rate was 1± 0 ml } min and the column temperature was 35 ° C. The quantitation of the metabolite was performed by comparing HPLC peak heights to those of authentic standards with reference to an internal standard (caå eine).

Kinetic analysis The kinetic studies were performed using liver microsomes prepared from four diå erent rats with a nicotine concentration ranging from 0± 05 to 1 mm . Eadie± Hofstee plots were constructed for determination of the presence of a mono- or a biphasic model. The maximum velocity (V ) and max Michaelis± Menten constant (K ) were evaluated by non-linear regression analyses of untransform ed m kinetic data.

Kinetics of nicotine N « -oxidation in rat liver

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Figure 1. Representative HPLC chromatograms of formation of cis- and trans-nicotine-1 « -N-oxide from nicotine in rat liver microsomes. The standard mixture spiked with cis- and trans-nicotine1« -N-oxide, and caå eine as an internal standard extracted under the same conditions as described for samples (A) ; the incubation mixture of rat liver microsomes without (B) and with (C) nicotine. Peak : 1, caå eine ; 2, cis-nicotine-1 « -N-oxide ; 3, trans-nicotine-1 « -N-oxide ; 4, nicotine.

Inhibition analysis The eå ects of the cytochrome P450-speci® c inhibitor, 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride (SKF525A) or a substrate of FMO, 1-(1-naphthyl)-2-thiourea, on cis- and trans-nicotine1« -N-oxide formation were determined at a 0 ± 1 or 1 mm nicotine concentrations respectively. The incubation mixture including the chemical inhibitor (1 mm ) was pre-incubated for 2 min before the reaction was initiated by addition of the substrate. Separately, microsomes were bubbled with carbon monoxide for 2 min or heat-treated at 45 ° C for 5 min.

Statistical analyses Correlation between cis- and trans-nicotine-1 « -N-oxide formation in liver microsomes from ten diå erent rats at 0± 1 mm nicotine concentration was determined by Pearson’ s product moment method (Pearson 1895). Values were expressed as mean³ SD throughout the text. Statistical analysis was performed by Student’ s t-test for paired samples. Diå erences were considered signi® cant when p ! 0± 05.

R esu lts

Representative HPLC chromatograms of formation of cis- and trans-nicotine-1 « N-oxide from nicotine in rat liver microsomes are shown in ® gure 1. Retention times for caå eine, cis-nicotine-1 « -N-oxide, nicotine and trans-nicotine-1 « -N-oxide were 16 ± 0, 20 ± 5, 23 ± 0 and 28 ± 0 m in respectively. Cis- and trans-nicotine-1« -N-oxide formation at a 0 ± 1 m m nicotine concentration increased linearly with an incubation time up to 30 min in the presence of 0 ± 5 mg } ml microsomal protein (® gure 2A) and with 1 ± 0 mg } ml microsomal protein with 20-min incubation (® gure 2B). Unless speci® ed, an incubation time of 20 min and 0 ± 5 mg } ml microsomal protein were employed to ensure initial rate conditions for the formation of nicotine-1« -N-oxide. The calibration graphs were obtained with standards extracted under the same conditions as described for the samples (® gure 1A). The stability of cis- and transnicotine-1 « -N-oxide during incubation was determined. As shown in ® gure 3, cis-

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Figure 2. Formation of cis- and trans-nicotine-1 « -N-oxide from nicotine as a function of incubation time or the amount of microsomal protein. Incubations were carried out for varying times (A) or with varying amounts of rat liver microsomes (B). D , cis-nicotine-1 « -N-oxide formation ; E , trans-nicotine-1 « -N-oxide formation. Data are the mean of duplicate determination.

Figure 3. Eå ects of incubation time on the stability of cis- and trans-nicotine-1 « -N-oxide. Each value represents the HPLC peak± height ratio to an internal standard. D , cis-nicotine-1 « -N-oxide ; E , trans-nicotine-1 « -N-oxide. Data are the mean of duplicate determination.

Figure 4. Typical Eadie± Hofstee plots for cis- and trans-nicotine-1 « -N-oxide formation in rat liver microsomes. D , cis-nicotine-1 « -N-oxide formation ; E , trans-nicotine-1 « -N-oxide formation. Data are the mean of duplicate determination.

Kinetics of nicotine N « -oxidation in rat liver Table 1.

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Kinetic parameters for nicotine-1 « -N-oxide formation in rat liver microsomes. K m (mm )

cis-nicotine-1 « -N « oxide formation trans-nicotine-1 « -N « oxide formation

V max (nmol } mg } min)

V } K max m ( l l } mg } min)

0± 240³

0± 069

1 ± 52³

0± 48

6± 41³

1± 27

1± 524³

0± 951

1 ± 19³

0± 74

0± 79³

0± 03

Kinetic analyses were determined by nicotine concentrations between 0± 05 and 1 mm . All values represent mean³ SD (n ¯ 4).

Figure 5. Eå ects of FMO or cytochrome P450 inhibitors, carbon monoxide or heat-treatment of microsomes on the formation of cis- and trans-nicotine-1 « -N-oxide by rat liver microsomes. The formation of cis- and trans-nicotine-1 « -N-oxide by rat liver microsomes were determined at 0± 1 mm (A) or 1 mm (B) nicotine concentration. The concentration of the chemical inhibitor was 1 mm . Microsomes were bubbled with carbon monoxide for 2 min or treated at 45 ° C for 5 min. Each column represents the percentage of the control (mean³ SD, n ¯ 4). * , cis-nicotine-1 « -Noxide formation ; 7 , trans-nicotine-1 « -N-oxide formation. ND, not detected. * p ! 0± 05 and ** p ! 0± 01 compared with control.

and trans-nicotine-1« -N-oxide in the incubation m ixture were stable up to 90 min. In addition, when one diastereomer was incubated, the diastereomer was stable and the other diastereomer was not detected. Furthermore, no reduction to nicotine was observed (data not shown). The kinetic studies for cis- and trans-nicotine-1 « -N-oxide formation in rat liver microsomes were determined with 0 ± 05± 1 m m nicotine concentrations. Figure 4 shows typical Eadie± Hofstee plots and both plots for cis- and trans-nicotine-1 « -Noxide formation in rat liver microsomes were linear. The apparent K m for cis- and trans-nicotine-1 « -N-oxide formation from the four samples were 0 ± 240³ 0 ± 069 and 1 ± 524³ 0 ± 951 m m respectively. The Vmax were 1 ± 52³ 0 ± 48 and 1 ± 19³ 0 ± 74 nmol} mg } min respectively (table 1). The intrinsic clearance (Vmax } K m ) of cis-nicotine-1 « N-oxide formation was 8 ± 1-fold greater than that of trans-nicotine-1« -N-oxide formation. In rat liver microsomes, cis-nicotine-1 « -N-oxide form ation was greater than trans-nicotine-1 « -N-oxide formation. The eå ects of FM O or cytochrome P450 inhibitors, treatment of m icrosomes with carbon monoxide or heat-treatment on the formation of cis- and trans-nicotine1 « -N-oxide were investigated at low (0 ± 1 m m ) and high (1 m m ) substrate concentrations. The formations of cis- and trans-nicotine-1 « -N-oxide in rat liver micro-

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Figure 6.


Correlation between cis- and trans-nicotine-1 « -N-oxide formation in microsomes from ten rat livers at a 0± 1 mm nicotine concentration.

somes were inhibited by 1-(1-naphthyl)-2-thiourea or heat treatment of microsomes. The addition of SKF525A (2-diethylaminoethyl-2, 2-diphenylvalerate) to the incubation mixture nor carbon monoxide aå ect the formation of both cis- and transnicotine-1 « -N-oxide in rat liver m icrosomes even at a high substrate concentrations (® gure 5). In the correlation analysis, the formations of cis- and trans-nicotine-1« -N-oxide in ten rat liver microsomes signi® cantly correlated each other at a 0 ± 1 m m concentration, as shown in ® gure 6 (r ¯ 0 ± 862, p ! 0 ± 01).

D iscussion

The HPLC method achieved a good separation of cis- and trans-nicotine-1 « -Noxide as shown in ® gure 1. Com pared with the incubation mixture of rat liver microsomes without nicotine, all chromatograms were free from endogenous compound interference. Nicotine eluted later than cis-nicotine-1 « -N-oxide and earlier than trans-nicotine-1 « -N-oxide. However, since almost all nicotine volatilized under the nitrogen stream, nicotine did not interfere with both cis- and transnicotine-1 « -N-oxide, as shown in ® gure 1C. In contrast with nicotine, it was con® rmed (data not shown) that nicotine-1 « -N-oxide was not volatile under the nitrogen stream. It has been reported that the reduction of nicotine-1 « -N-oxide to nicotine could occur in vitro under anaerobic conditions (Dajani et al. 1972) and in vivo in the gastrointestinal tract (Jenner et al. 1973c). However, the reduction of neither cis- nor trans-nicotine-1« -N-oxide was observed during the incubation and extraction procedures under aerobic conditions in this study. In addition, no detectable diastereomeric conversion of either cis- and trans-nicotine-1« -N-oxide was observed on incubation. This result is in agreement with the report of Cashman et al. (1992). Therefore, the precise activities for nicotine-1 « -N -oxide formation in rat liver microsomes were determined reproducibly in the current study. The formation ratios of cis- } trans-nicotine-1 « -N-oxide in rat liver m icrosomes were previously reported to be about 1 ± 1 at a 5 m m nicotine concentration (Jenner et al. 1973). In the current study, the formation of cis-nicotine-1 « -N-oxide was greater than the formation of trans-nicotine-1 « -N-oxide in rat liver microsomes, over the


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range of nicotine concentrations investigated (0 ± 05± 1 m m ). However, the formation ratio of cis- } trans-nicotine-1 « -N-oxide was dependent on the nicotine concentration. The higher the nicotine concentration was, the lower the formation ratio of cis} trans-nicotine-1 « -N-oxide was, since the K m for trans-nicotine-1« -N-oxide formation was higher than that for cis-nicotine-1 « -N -oxide formation. Therefore, the intrinsic clearance would be an adequate indicator of the formation ratio. As a result of the kinetic analysis in this study, it was shown that the intrinsic clearance of cisnicotine-1 « -N-oxide formation was 8 ± 1-fold greater than that of trans-nicotine-1 « N-oxide formation. The eå ects of the inhibitors of FMO or cytochrome P450 were determined for investigation of the enzymes responsible for the formation of cis- and trans-nicotine1 « -N-oxide in rat liver microsomes. The lack of the eå ects of SKF525A on nicotine1 « -N-oxide form ation coincided with the previous work using guinea pig 10,000 g hepatic preparations (Gorrod et al. 1971). The inhibition study suggested that the contribution of cytochrome P450 to nicotine N-oxidation in the rat liver was negligible and the enzymes responsible for these activities were FM Os. In a preliminary experiment, the activities of cis- and trans-nicotine-1 « -N-oxide formation at pH 7 ± 4, a physiological condition, were lower than that at pH 8 ± 4. Because the condition at pH 8 ± 4 was reported to be optimum for FMO (Gold and Ziegler 1973), the formation of nicotine-1 « -N -oxide determined at pH 8 ± 4 might be maximum, as described herein. In mammals, FM O has been reported to be responsible for the formation of nicotine-1 « -N-oxide (Damani et al. 1988, Nakayama 1988). The mammalian FM O represents a multigene family and FM O1, FMO3, FMO4 and FMO5 have been detected in human liver, and FM O2 is present in lung microsomes of the rabbit and guinea pig (Hines et al. 1994). In contrast, only FM O1 have been reported in rat liver (Itoh et al. 1993). It was reported that FM O1 and FM O3 produced nicotine1 « -N-oxide exclusively with stereoselectivity in the pig and human liver respectively (Park et al. 1993, Cashman et al. 1995). However, it is not clear which FMO isoform is responsible for the formation of nicotine-1« -N-oxide diastereomers in rat liver. The correlation of the form ation of cis- and trans-nicotine-1« -N-oxide, as shown in ® gure 6, suggest that the same FM O isoform m ight be responsible for both cis- and trans-nicotine-1 « -N-oxide formation, although this remains to be veri® ed. Species diå erences in stereoselectivity of diastereoisomeric nicotine-1« -N-oxide formation have also been reported. In rodents, cis-nicotine-1 « -N-oxide formation was reported to be preferred over trans-nicotine-1 « -N-oxide formation and the formation ratio of cis- and trans-nicotine-1 « -N-oxide ranged from 1 ± 1 to 10 (Jenner et al. 1973b). On the other hand, in monkey and human liver, only trans-nicotine-1 « N-oxide was form ed. (Cashman et al. 1992, Park et al. 1993). Park et al. (1993) also reported that the formation ratio of cis- } trans-nicotine-1 « -N-oxide was 40 } 60 and 0 } 100 in pig liver FMO1 and human liver FM O3 respectively. Therefore, it is possible that the species diå erences in stereoselectivity of diastereomeric nicotine1 « -N-oxides formation are due to the diå erence of FM O isoform responsible for metabolite formation. If the same isoform is involved in diastereomeric nicotine-1 « N-oxide formation in several species, the species diå erences of stereoselectivity might be due to the diå erences of the primary structure of the FM O isoform in these species. In summary, the kinetic properties of diastereoisomeric formation of nicotine1 « -N-oxide in rat liver microsomes have been established. W e conclude that the

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same FMO form may be involved in the formation of both diastereomers of nicotine-1 « -N-oxide in the rat liver, but the speci® c FM O isoform which is responsible remains to be established.

A ckn ow ledgem ent

This work was supported in part by a grant from The Smoking Research Foundation, Japan.

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