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Introduction

Human cytochrome P450 (CYP) 2A6 and mouse CYP2A5 share 82% of their amino acid sequences, and belong to the cytochrome 2A family of enzymes (1,2). The two enzymes are mainly expressed in the liver and are involved in the metabolization of a series of xenobiotics including nicotine and methoxyflurane, and in activating a variety of precarcinogens such as afltoxin B1, tobacco-specific nitrosamines, N-nitrosodiethylamine, 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and N-nitrosonornicotine (3–5). Human CYP2A6 has received more attention, as it is a key enzyme with respect to the biotransformation of nicotine into inactive cotinine and the activation of tobacco-specific nitrosamines (5). Thus, the deliberate inhibition of CYP2A6 activity is a potential strategy in chemoprevention, since CYP2A6 inhibition has been found to prolong the biological lifetime of nicotine, therefore decreasing cigarette consumption (6). CYP2A6 has also been found to reduce the number of active carcinogens transferred from tobacco (7).

Celery (Apium graveolens) belongs to the umbelliferae family and is widely used in food and for medicinal purposes. Studies have found that celery extracts can exert beneficial effects, including antioxidant, hypoglycemic, hypolipidemic and anti-platelet aggregation effects (8,9). A previous study demonstrated that the juice extracted from Apium graveolen and Petroselinum sativum extended the analgesic effect of aminopyrine and paracetamol, suggesting that the juice inhibited CYP activity in the liver, including that of aminopyrine N-demethylase (10). Whether or not other CYP enzymes are inhibited remains unclear. Coumarin 7-hydroxylation is catalyzed by CYP2A enzymes and is thus considered to be a specific indicator for the presence of CYP2A enzymes, such as CYP2A5 in mice and CYP2A6 and CYP2A13 in humans (11). Notably, celery extract contains a number of coumarin derivatives including furocoumarins and pyranocoumarins. The present study therefore postulates that celery extract may inhibit CYP2A5/6-mediated coumarin 7-hydrolase activity according to a structure-activity association. To the best of our knowledge, there have been few studies on the inhibitory effects and mechanisms of celery extract on CYP2A5/6. Therefore, the objectives of the present study are to assess the inhibition potency of celery extract on CYP2A5/6 activity using mouse and human liver microsomes, and to analyze the inhibition mechanisms of celery extract using time- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent, ultracentrifugation tests. Finally, the present study will clarify whether or not furocoumarin 5-methoxypsoralen (bergapten), present in celery extract, is the predominant inhibitor of CYP2A5/6.

Materials and methods

Chemicals and reagents

Coumarin, 4-methylumbelliferyl and NADPH were purchased from Sigma-Aldrich (EMD Millipore, Billerica, MA, USA). The 7-hydroxycoumarin was purchased from Aladdin Shanghai Biochemical Tech Co. Ltd. (Shanghai, China). Human liver microsomes were purchased from the Research Institute for Liver Diseases Co. Ltd. (Shanghai, China). Ultra-pure water was prepared with Milli-Q (EMD Millipore). High performance liquid chromatography (HPLC)-grade acetonitrile was obtained from Tedia (Fairfield, OH, USA). All other reagents were of analytical grade or above and commercially available.

Celery extract preparation

In total, 15 g fresh celery was used to make celery powder by pulverizeration. Celery powder (4 g) was extracted with 60 ml petroleum ether at 40°C following saturation for three times. The extracts were filtered and evaporated resulting in a volume of ~5 ml, and finally metered to 10 ml with petroleum ether. The final liquid extract was stored at 4°C prior to use.

Animal experiments

All animal care and experimental protocols were approved by the Animal Center of Chongqing Medical University (Chongqing, China). Male Swiss mice (15–25 g, age 7 weeks) were purchased from the Animal Center of Chongqing Medical University. The mice were housed in a temperature-controlled room, with free access to rodent chow and water and a 12:12 h light-dark cycle. Subsequent to a one-week acclimation period, celery liquid extract (0.2 mg/l; n=5) or solvent (n=5) was orally administered twice to the mice, as previously described by Jakovljevic et al (10). The mice were sacrificed 2 h after the last administration, and their livers were harvested. The liver microsomes were then prepared and the CYP2A5 activity was determined as described below.

Mouse liver microsomes preparation

Mouse liver microsomes were prepared from male Swiss mice (15–25 g, age 6–8 weeks) as described by Pinto et al (12). The protein concentrations of microsomal samples were determined by the Lowry method (13).

Assay of coumarin 7-hydroxylation in liver microsomes

A coumarin 7-hydroxylation assay was performed as described by Aitio (14) with certain modifications. The procedure was as follows. The incubation mixture contained mouse (0.4 mg/l) or human liver microsomes (0.1 mg/l; a kind gift from Dr Guo of Chongqing Medical University), coumarin (100 mmol/l) and 100 mM potassium phosphate buffer (pH 7.4) creating a total volume of 180 µl. Subsequent to 3 min pre-incubation, the reaction was initiated by the addition of 20 µl of 10 mM NADPH and performed for either 20 min (mouse liver microsomes) or 15 min (human liver microsomes) at 37°C, and terminated by the addition of 200 µl of 100 nM 4-methylumbelliferyl, an internal standard, in ice-cold acetonitrile. The resulting samples were then centrifuged at 10,000 × g for 15 min. The product formed, 7-hydroxycoumarin (umbelliferone), was quantified by HPLC, as previously described by Farooq et al (15). Protein concentration and incubation time were optimized and product formation was linear under the aforementioned conditions.

Microsomal CYP2A5/6 inactivation assay

To examine the concentration-dependence and the value of the concentration of the inhibitor where binding is reduced by half (IC50) of CYP2A5/6 inactivation, celery extracts of increasing concentrations were pre-incubated with the incubation mixture at 37°C for 3 min, and the same pre-incubation mixture was added to the NADPH solution to start the reaction. The reaction was performed as aforementioned to determine the level of residual coumarin 7-hydroxylase activity.

To test the time-dependence of CYP2A5 inactivation, celery extracts of increasing concentrations (0, 200, 400, 600, 800 and 1,000 µg/ml) were pre-incubated with the liver microsomes at 37°C for the selected time points. At selected time points (0, 3, 6 and 9 min), 20 µl of mixture was added to the NADPH solution to start the reaction. The reaction was performed as aforementioned to determine the residual coumarin 7-hydroxylase activity.

To investigate the dependence of CYP2A5/6 inactivation on NADPH, celery extract was pre-incubated with the mouse or human liver microsomes and 100 mM of potassium phosphate buffer (pH 7.4) at 37°C for 30 min in the presence or absence of NADPH. The reaction was then initiated via the addition of coumarin and NADPH and performed as aforementioned to determine the level of residual coumarin 7-hydroxylase activity.

IC50 shift assay

The IC50 shift assay was performed as described by Perloff et al (16) with certain modifications. Celery extract of increasing concentration was pre-incubated with the human liver microsomes, NADPH and potassium phosphate buffer for 0 or 30 min. Subsequent to pre-incubation, the pre-incubation mixtures were then transferred to the secondary incubations containing coumarin and NADPH to initiate the reaction. This transferal took place at approximately the Km, which is the concentration of substrate required to produce 50% of the Vmax (maximum velocity or rate at which the enzyme catalyzed the reaction). The reaction was performed as aforementioned to determine the level of residual coumarin 7-hydroxylase activity and the value of IC50.

Ultracentrifugation test

The ultracentrifugation test was performed as described by Lee et al (17) with certain modifications. To evaluate the reversibility of the drug-enzyme interaction, the celery extract was incubated with the mouse liver microsomes and a potassium phosphate buffer (pH 7.4) at 37°C for 30 min. The microsomes were re-isolated by ultracentrifugation of the pre-incubation mixtures at 80,000 × g for 60 min at 4°C. The microsomes were washed twice with 0.1 M potassium phosphate buffer (pH 7.4). The residual CYP2A5 activity was then determined as aforementioned.

Data analysis

Data is expressed as the mean ± standard error of the mean. All tests were performed at least 3 times. The enzyme kinetic and the Lineweaver-Burk plot analyses were performed with GraphPad Prism 5.0 (GraphPad software, Inc., La Jolla, CA, USA). Statistical significance was calculated using a one-tailed Student t-test, or a one-way analysis of variance and Dunnet's test as a post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Celery extract inhibits CYP2A5/6 activity

The potential inhibitory effect of celery extract on CYP2A5/6 activity was investigated in the mouse and human liver microsomes, using coumarin as a specific probe. The Km and Vmax values for the coumarin 7-hydroxylation of CYP2A5 were 1.4 µM and 15.3 pM umbelliferone/min/mg microsomal protein. The Km and Vmax values for CYP2A6 were 1.9 µM and 48.3 pM umbelliferone/min/mg microsomal protein. When celery extract was added to the incubation mixture at varying concentrations, coumarin 7-hydroxylation was concentration-dependently inhibited with IC50 values of 345.1 and 888.7 µg/ml, respectively, for CYP2A5 and CYP2A6 (Fig. 1A and B). The animal experiment verified that celery extract significantly decreased the CYP2A5 activity by 16% (P=0.039) (Fig. 1C). Therefore, celery extract was found to be an inhibitor of CYP2A5/6.

Figure 1. Inhibitory effects of celery extract on mouse CYP2A5 and human CYP2A6 activities. (A) Celery extract concentration-dependently inactivated CYP2A5 and (B) CYP2A6 in the mouse and human liver microsomes, respectively. (C) Celery extract significantly decreased the mouse CYP2A5 activity in vivo. Data is expressed as the mean ± standard deviation (n=3). *P<0.05. CYP, cytochrome P450.

Celery extract reversibly inhibits mouse CYP2A5 but irreversibly inhibits human CYP2A6

The present study used kinetic inhibition studies to investigate the inhibition modes of the celery extract on CYP2A5/6. Time-dependent inhibition of CYP2A5 with celery extract was not observed (Fig. 2A). Celery extract samples that were pre-incubated in the presence and absence of NADPH demonstrated the same levels of CYP2A5 activity (Fig. 2B), suggesting that CYP2A5 inactivation occurs independently of NADPH. The ultracentrifugation test demonstrated that CYP2A5 activity returned to a normal level if the incubation mixture was ultracentrifuged at 80,000 × g for 60 min, indicating reversible non-covalent binding of the inhibitor to CYP2A5, and the reversible inhibition of celery extract to CYP2A5 (Fig. 2C). The aforementioned results suggest that celery extract is a reversible inhibitor for CYP2A5. The Lineweaver-Burk plot demonstrates that the lines intersect on the second quadrant, indicating that the inhibition of CYP2A5 was mixed competitive/noncompetitive, with a Ki value of 266.4 µg/ml (Fig. 2D).

Figure 2. Reversible inhibition of CYP2A5 by celery extract in mouse liver microsomes. (A) Time-independent inhibition of celery extract on CYP2A5. (B) NADPH-independent inhibition of celery extract on CYP2A5. (C) CYP2A5 activity was recovered by ultracentrifugation. (D) Lineweaver-Burk and its second plot for the inhibition of CYP2A5 activity by different concentrations of celery extract in the mouse liver microsomes. The inhibition of celery extract on CYP2A5 activity is best described as a competitive/noncompetitive mechanism. Data is expressed as the mean ± standard deviation (n=3). *P<0.05. CYP, cytochrome P450; NADPH, nicotinamide adenine dinucleotide phosphate.

By contrast, time-dependent inhibition of CYP2A6 by celery extract was observed, since a 3.4-fold shift for CYP2A6 was observed in the IC50-shift experiments (Fig. 3A). Samples that were pre-incubated with celery extract in the presence of NADPH exhibited less activity compared with those incubated in the absence of NADPH (P=0.019; Fig. 3B), suggesting the dependence of CYP2A6 inactivation on NADPH. Therefore, the aforementioned results revealed that the inactivation of CYP2A6 activity by celery extract is dependent on celery concentration, time and NADPH, indicating that celery extract is an irreversible inhibitor for CYP2A6. To characterize the irreversible inhibition of CYP2A6 by celery extract, the Ki and Kinact values were determined. The Ki and Kinact values were 1,018 µg/ml and 0.3/min, respectively (Fig. 3C).

Figure 3. Irreversible inhibition of CYP2A6 by celery extract in human liver microsomes. (A) A 3.4-fold IC50 shift showed time-dependent inactivation of CYP2A6 by celery extract. (B) NADPH-dependent inhibition of celery extract on CYP2A6. (C) Pre-incubation time- and concentration-dependent inhibition of CYP2A6 by celery extract in the human liver microsomes. The inactivation rate (kobs) initially observed was determined by linear regression analysis of the natural logarithm of the percentage remaining activity vs. pre-incubation time. The Ki and Kinact values were determined using nonlinear regression analysis of the kobs value vs. celery extract concentration. Data is expressed as the mean ± standard deviation (n=3). *P<0.05. CYP, cytochrome P450; NADPH, nicotinamide adenine dinucleotide phosphate; IC50, value of the concentration of the inhibitor where binding is reduced by half; Ki, measure of the tightness of bonds between an enzyme and its inhibitor; Kinact, maximal rate of enzyme inactivation at a saturating concentration of inhibitor.

5-methoxypsoralen reduces less CYP2A5/6 activity compared with celery extract

To explore whether 5-methoxypsoralen, the main component present in celery extract, played a critical role in the inhibition of CYP2A5/6 activity, the present study analyzed the inhibitory effects of 5-methoxypsoralen. Fig. 4 demonstrates that 5-methoxypsoralen (0.3 µM) significantly decreased CYP2A5/6 activity (P=0.037), but to a lesser extent compared with celery extract (400 µg/ml) did, compared with the control (P=0.031). The results suggest that other coumarin derivatives, besides 5-methoxypsoralen, also contribute to the inhibition of CYP2A5/6 activity.

Figure 4. 5-methoxypsoralen reduced (A) CYP2A5 and (B) CYP2A6 activity by less than celery extract in the mouse and human liver microsomes, respectively. Data is expressed as the mean ± standard deviation (n=3). *P<0.05 vs. control; **P<0.05 vs. celery extract. CYP, cytochrome P450.

Discussion

The present study revealed that celery extract inhibited mouse CYP2A5- and human CYP2A6-mediated coumarin 7-hydroxylase activity via different mechanisms. To explore whether celery extract played a concentration-dependent role on the level of CYP2A5/6 activity, the present study used 3 different concentrations of celery extract. The results demonstrate that celery extract is an inhibitor of CYP2A5/6 (Fig. 1A). For CYP2A5, celery extract behaves as a reversible (mixed competitive and noncompetitive) inhibitor, but for CYP2A6, celery extract behaves as an irreversible inhibitor. In addition, it appears that celery extract is a more potent inhibitor of CYP2A5 compared with CYP2A6, since the IC50 value for CYP2A5 is ~2-times smaller than that of CYP2A6. A previous study also identified that 8-methoxypsoralen, a furocoumarin present in celery extract, is a more potent inactivator for CYP2A5 compared with for CYP2A6 in vitro, with IC50 values 1.0 and 5.4 µM, respectively (18).

These differences may be associated with the size of mouse CYP2A5 and human CYP2A6 active sites. The majority of compounds studied, including furocoumarins, metyrapone, miconazole and lactone derivatives, have been found to be stronger suppressors of CYP2A5 activity than of CYP2A6 activity (18–20). Previous studies demonstrated that larger compounds were more powerful inhibitors of CYP2A5 activity than of CYP2A6 activity, suggesting that CYP2A5 has a larger active site compared with CYP2A6 (19,21). Therefore, the compounds in celery extract are more likely to get into, and interact with, the active site of CYP2A5. These differences may be due to amino acid changes in the active sites of these enzymes. A single mutation, whereby phenylalanine 209 is substituted by leucine in the substrate-binding site, changes the specificity of CYP2A5 from coumarin to steroid hydroxylation (19,22). Therefore, similar amino acid sequences are shared in CYP2A5/6 enzymes, but small amino acid changes between active sites affect their substrate-recognition ability. The present study supports previous studies that revealed that CYP2A5 and CYP2A6 have different substrates and inhibitor specificities (21,23).

In addition, the present study demonstrated that the coumarin derivative 5-methoxypsoralen in celery extract markedly inhibited CYP2A5/6 activity, but to a lesser extent than celery extract did, suggesting that other coumarin derivatives besides 5-methoxypsoralen also contribute to the inactivation of CYP2A5/6 activity. A coumarin derivative that may be present in celery extract was considered to be the furocoumarin 8-methoxypsoralen, since it is the only well-characterized inhibitor for CYP2A enzymes including CYP2A5 and CYP2A6 and as it also demonstrated distinct inhibition modes for CYP2A5 and CYP2A6 (24–26).

Pilot studies suggested that inactive variations of the CYP2A6 gene have been revealed to markedly increase the bioavailability of nicotine and thus decrease cigarette consumption, and reduce the risk of developing lung cancer due to smoking (27–29). Therefore, CYP2A6 inhibitors may act as promising adjunct drugs in smoking cessation therapy. Whether celery extract, as a CYP2A6 inhibitor, could affect nicotine kinetics in vivo and therefore that affects smoking behaviors must be investigated in another study.

In conclusion, the present study suggests that celery extract inhibits mouse CYP2A5 and human CYP2A6 via different mechanisms, suggesting that the mouse model is not applicable for future studies involving the inhibition of CYP2A enzymes by celery extract. Future studies must be performed to identify which components of celery extract play the main role in the inhibition of CYP2A enzyme activity, and to investigate whether the deliberate inhibition of CYP2A6 by celery extract can modulate smoking behavior and therefore reduce the risk of lung cancer.

Acknowledgements

This study was supported by the National Nature Science Foundation of China (grant no. 81370403) and Specialized Research Fund for the Doctoral Program of Higher Education (grant no. 20125503110008).

References