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The potential effect of excessive coffee consumption on nicotine metabolism: CYP2A6 inhibition by caffeic acid and quercetin

The potential effect of excessive coffee consumption on nicotine metabolism: CYP2A6 inhibition by... Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn021 ......................................................................................................................................................................................................................................... Research article The potential effect of excessive coffee consumption on nicotine metabolism: CYP2A6 inhibition by caffeic acid and quercetin Gary M. Woodward* Faculty of Sciences, Staffordshire University, College Road, Stoke on Trent, Staffordshire ST4 2DE, UK. * Corresponding author: Diet and health group, Biomedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, NR4 7TJ, UK. Tel: þ44 (0)7746144623. Email: g.woodward@uea.ac.uk Supervisor: Dr Susan Bird, Faculty of Sciences, Staffordshire University, College Road, Stoke on Trent, Staffordshire ST4 2DE, UK. ........................................................................................................................................................................................................................................ Over the past decade interest in the biochemical and biological properties of polyphenols has grown considerably, as epidemiological evidence for their beneficial effects on health continues to increase. Dietary polyphenol antioxidants are reported to have many ‘health promoting’ properties, including anti-inflammatory, vasoprotection, anti-cancer and anti-obesity effects. However, their absorption and metabolism are as yet not fully elucidated, particularly with regard to their interactions with other metabolized compounds, such as nicotine. Epidemiological studies have shown that coffee-drinking cigarette smokers are more likely to smoke less in comparison to non-coffee drinking cigarette smokers. Nicotine is primarily metabolized by the hepatic cytochrome P450 enzyme CYP2A6 and may be inhibited by the metal chelating properties of polyphenols via haem interactions with their hydroxyl groups. To model nicotine metabolism, bovine liver microsomes, shown to suitably represent human CYP2A6 metabolism, were isolated by calcium precipitation and differential centrifugation. Fluorometric analysis of 7-hydroxycoumarin, the CYP2A6 metabolite of coumarin and known probe for CYP2A6 activity, was used to model nicotine metabolism in vitro and to quantify the degree of CYP2A6 inhibition imposed by caffeic acid and quercetin. It was found that both caffeic acid and quercetin, major polyphenolic constituents of caffeine containing beverages, sig- nificantly inhibited CYP2A6 activity in vitro by 37.9% (P, 0.05) and 48.2% (P, 0.05), respectively. Thus, this study demonstrates the first reported biochemical evidence in support of previous epidemiological observations, where it is suggested that polyphenol interaction with CYP2A6 prolongs the pharmacological effects of nicotine by decreasing its rate of elimination. Key words: polyphenols, CYP2A6, caffeic acid, quercetin, nicotine. ........................................................................................................................................................................................................................................ include anti-inflammatory, vasoprotection and anti-obesity Introduction effects. In addition, phenolic antioxidants commonly con- Polyphenols are the most abundant antioxidants in the diet, sumed in our diets are being proposed as preventative treat- 3, 7– 11 with a total dietary intake that exceeds that of vitamin C, ments against chronic human diseases. 1, 2 vitamin E and carotenoids. Their main dietary sources However, if these food-based antioxidant approaches are include fruits and plant-derived beverages such as fruit to be employed as prevention or treatments for chronic dis- juices, tea, coffee and red wine. Vegetables, cereals, choco- eases, clarification of their little known metabolism must late and dry legumes also contribute to the total polyphenol be achieved, particularly given the negative findings from 12, 13 intake. These dietary nutraceuticals have been shown to con- studies of antioxidant supplementation and the contro- tribute significantly to the prevention of disease, where a versy that exists within the field regarding the bioavailability recent study by Mink et al. revealed that flavonoids found and bioactivity of many phytochemicals. Furthermore, little in fruits and vegetables are strongly associated with research has been conducted regarding the interactions reduced cardiovascular disease mortality. These health pro- between dietary antioxidants and other environmental pro- moting effects are thought to be due to bioactivities that moters of oxidation, such as cigarettes. ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 98 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Cigarette smoking, the foremost form of nicotine addiction, continues to be one of the world’s most serious public health problems and is considered to be the major risk factor of ischae- mic heart disease, lung cancer and chronic obstructive pulmonary disease. A key factor in reducing the pharmaco- logical and addictive effects of nicotine would be to improve its removal by the drug metabolizing pathways of the liver. However, interactions with dietary components, particularly polyphenol antioxidants, may hinder its removal. Of the nico- tine absorbed through smoking, 80% is metabolized in Figure 2. Structure of quercetin. the liver by the cytochrome P450 enzyme CYP2A6 via 16–18 C-oxidation to cotinine. CYP2A6, therefore, plays a sodium cacodylate, glucose-6-phosphate, acid molybdate, pivotal role in the removal of nicotine in humans. The relation- sodium dodecyl sulphate, ascorbic acid, sodium hydroxide, ship between nicotine and polyphenol metabolism is indicated copper sulphate, sodium potassium tartrate, sodium carbon- in a review by Swanson et al. who stated that 86.4% of ciga- ate, Folin reagent, bovine serum albumin, coumarin, caffeic rette smokers consume coffee compared with 77.2% of non- acid, quercetin, nicotinamide adenine dinucleotide phos- smokers, and also in a study by Kozlowski demonstrating phate (NADP), perchloric acid and 7-hydroxycoumarin that coffee-drinking cigarette smokers smoked more when were purchased from Sigma Aldrich Co. (UK). Bovine liver they ingested almost no caffeine than when they ingested an samples were supplied to Staffordshire University by a local amount of caffeine, in the form of coffee, ranging from 75 to abattoir. 300 mg. Caffeic acid and quercetin (Figs 1 and 2) represent major antioxidant constituents within caffeine containing bev- 21–23 Liver microsome isolation erages and are potential CYP2A6 inhibitors. Thus, in the present study, the CYP2A6 inhibition potential of caffeic acid Whole bovine livers were delivered within 4 h of animal and quercetin was investigated, where it was hypothesized death and placed directly on ice. Liver microsome isolation that CYP2A6 inhibitionisimposedbycaffeicacid and quercetin was performed using modification to the methods stated by 24 25 and it is this interaction responsible for the epidemiological Ernster et al. and Hamilton et al. Liver tissue samples observation that coffee-drinking cigarette smokers smoke less of 80–100 g were cut from the whole bovine liver, rinsed than non-coffee-drinking cigarette smokers. with 0.25 M sucrose in 25 mM potassium phosphate buffer (pH 7.4) and minced using a scalpel blade. Minced liver samples were transferred to a homogenization tube in 20– Materials and methods 25 g portions, and homogenized for 2–3 min with 10 ml 0.25 M sucrose in 25 mM potassium phosphate buffer (pH Materials and reagents 7.4) per portion. The homogenate was centrifuged at Potassium dihydrogen phosphate, di-potassium monohydro- 1000g for 10 min at 48C using a Sigma Howe 3K30 centri- gen phosphate, sucrose, calcium chloride, sodium cholate, fuge with Sigma 12156-H rotor. Following careful aspiration of the thin floating lipid layer, the remaining supernatant was then transferred to another centrifuge tube and the pellet dis- carded. The supernatant was centrifuged at 12 000g for 15 min at 48C. Again, the thin floating lipid layer was care- fully aspirated and the supernatant retained. This super- natant fraction was called the post-mitochondrial fraction (PMF). A volume of 8 mM calcium chloride, 7.5 times the volume of the PMF was added dropwise to the PMF with constant stirring and left to stir for 15 min at 48C. This mixture was then centrifuged at 8000g for 10 min at 48C. The supernatant was removed and the pellet re-suspended in 0.25 M sucrose in 25 mM potassium phosphate buffer (pH 7.4) at a volume of 0.3 ml for each gram of original tissue. The suspension was vortexed and treated with 0.5% (w/v) sodium cholate, at a volume one-tenth the final suspension volume, for 20 min at 48C. This suspension, called the microsome isolation, was stored at 2808C until Figure 1. Caffeic acid (I) is naturally found as an ester with quinic acid called Chlorogenic acid (II). necessary. ......................................................................................................................................................................................................................................... 99 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Protein determination made up to a final volume of 160 ml with 160 mM sodium cacodylate. 1.6 ml Burchell’s reagent was added to the stan- The protein assays were performed, in duplicate, using the dard curve solutions and incubated as stated earlier. method developed by Lowry et al. The microsome iso- lation was diluted 1:100 with 0.5 M sodium hydroxide and CYP2A6 activity assay 0.2, 0.4, 0.6, 0.8 and 1 ml of the diluted microsome isolation The CYP2A6 activity assay was performed, in duplicate, was made up to a final volume of 1 ml, respectively, with using a modification to the methods stated by Creaven 0.5 M sodium hydroxide. A blank was prepared using 1 ml 29 30 31 et al., Koenings et al. and Ngui et al. Initially, 0.5 M sodium hydroxide. To all diluted microsome isolation 25 mM coumarin in 25 mM potassium phosphate buffer samples was added 5 ml of copper reagent (containing 2% (pH 7.4) was pre-incubated with 2 ml microsomal isolation (w/v) sodium carbonate in 0.1 M sodium hydroxide, 1% for 3 min at 378C. The reaction was initiated by the addition (w/v) copper sulphate and 2% (w/v) sodium potassium tart- of an NADPH generation system consisting of 10 mM rate, prepared at a ratio 100:1:1 by volume, respectively). glucose-6-phosphate and 0.5 mM NADP (final incubation These mixtures were then vortexed and allowed to stand volume, 6 ml). After 10 min at 378C, the incubation was for 10 min at room temperature. Finally, 0.5 ml 1 N Folin quenched with the addition of 100 ml 6 N perchloric acid, reagent was added to all the samples and left to stand set on ice for 2 min and centrifuged at 1000g for 10 min for 30 min at room temperature subsequent to vortexing. using a Sigma Howe 3K30 centrifuge with Sigma 12156-H Absorbance was read at 750 nm on a Cecil CE1010 spectro- rotor. Controls (blanks) were prepared with the addition of photometer after zeroing on the blank. Protein concentration the NADPH generation system after the incubation period. was estimated by direct interpolation from a standard curve. Fluorescence was recorded on a Kontron SFM 25 fluorom- A standard curve was constructed using a stock solution eter at excitation and emission wavelengths of 351 and of bovine serum albumin (BSA) at a concentration of 454 nm, respectively, zeroed against the blank. An additional 100 mg/ml in 0.5 M sodium hydroxide. BSA stock solutions CYP2A6 activity assay was performed with the incubation at volumes 0, 0.2, 0.4, 0.6, 0.8 and 1 ml were made up to a time increased to 15 min (substrate control) to ensure the final volume of 1 ml with 0.5 M sodium hydroxide (equival- assay substrates were not depleted during incubation. ent to 0, 20, 40, 60, 80 and 100 mg protein/ml, respectively). The amount of metabolite (7-hydroxycoumarin) formed The standard curve solutions were then processed as by the CYP2A6 activity assays were quantified with a stan- described earlier. dard curve generated from known amounts (0–100 ng/ml) of 7-hydroxycoumarin in 25 mM potassium phosphate Glucose-6-phosphatase assay buffer (pH 7.4) and 100 ml 6 N perchloric acid, made up This assay was performed, in duplicate, using a modification to a final volume of 6 ml with distilled water. to the methods stated by Taussky and Shorr and Nordlie CYP2A6 inhibition assay and Arion. Initially, 50 ml 20 mM glucose-6-phosphate and 20 ml 160 mM sodium cacodylate were pre-incubated This assay was performed, in duplicate, following the for 10 min at 308C. The incubation reaction was initiated method described earlier with the addition of 25 mM with the addition of 90 ml microsome isolation and incu- caffeic acid and quercetin, respectively, to the incubation bated for 20 min at 308C. The incubation reaction was mixture. stopped with the addition of 1.6 ml Burchell’s reagent con- Statistical analysis sisting of 0.42% (w/v) acid molybdate, 5% (w/v) sodium dodecyl sulphate and 10% (w/v) ascorbic acid prepared at Repeated measures analysis of variance was applied as a the ratio 6:2:1 by volume, respectively. The incubation three factor test to determine between-treatment effects fol- mixture was then placed on ice for 15 min and re-incubated lowed by Turkey HSD and Bonferroni post-hoc tests using for 20 min at 478C. The absorbance was measured on a Cecil Windows SPSS (version 15) software. CE1010 spectrophotometer at 820 nm only after the incu- bation mixtures had stabilized to room temperature. In Results and discussion control samples glucose-6-phosphate was added after the 20 min incubation period. Organic phosphate concentration Many animal species have been shown to possess CYP2A6 was estimated by direct interpolation from a standard curve like activity, where CYP2A6 has been shown to be the as described in what follows. For samples with an absor- major coumarin 7-hydroxylase in bovine liver with activities 32, 33 bance exceeding the range of the standard curve, a 1:1 similar to that of human liver. Thus, bovine liver dilution was performed and the absorbance re-measured. samples were used to model human CYP2A6 coumarin A standard curve was constructed using 0.5 mM potass- 7-hydroxylation within this study. CYP2A6 is the major, if 34, 35 ium di-hydrogen phosphate at volumes 0–150 ml (final phos- not the only, coumarin 7-hydroxylase in humans, phate concentrations 0, 20, 25, 40, 50, 75 nM, respectively), where coumarin is almost exclusively metabolized into ......................................................................................................................................................................................................................................... 100 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... 7-hydroxycoumarin (68–92% of the dose). As coumarin is This equated to a CYP2A6 activity inhibition of 37.9 and almost exclusively metabolized by CYP2A6 to produce 48.2% by caffeic acid and quercetin, respectively, at a con- 7-hydroxycoumarin, a highly fluorescent compound, it was centration of 25 mM. While the maximum plasma concen- an ideal probe and marker for CYP2A6 activity and inhi- tration of caffeic acid is reported to be 3.8 mM following bition studies. Thus, within this study coumarin had been 200 ml of oral coffee consumption, excessive consumption substituted for nicotine as an alternative CYP2A6 substrate may significantly increase this plasma concentration. 21 39 to model in vitro nicotine hydroxylation. Studies by Olthof et al. and Rechner et al. have shown Each microsomal isolation procedure yielded a 30 ml that between 0.3 and 5% of ingested chlorogenic and microsomal isolation mixture. Using the Lowry protein caffeic acid may be excreted in the urine. Thus, with coffee determination method it was shown that microsome iso- beverages containing up to 478 mg/ml chlorogenic acid, it lations yielded a protein concentration of 10.89 mg.ml is possible that hepatic concentrations of caffeic acid may (SD+ 3.11, n ¼ 7). This protein concentration provided be considerably increased with excessive coffee consump- only an estimate for the quantity of microsomes isolated tion, particularly since the polar phenolic acids may be and did not quantify CYP2A6 content. However, for the subject to enterohepatic circulation and accumulation purpose of this study it was not necessary to quantify within the gallbladder. Therefore, while the concentrations CYP2A6 content as protein concentration provided sufficient of phenolic acid within this study exceed reported physio- means by which subsequent assays could be standardized logical levels, a higher concentration was selected as an and compared with previously reported data. Microsome initial trial to determine inhibition potential and in response isolations showed glucose-6-phosphatase activities of 0.44 to previously reported data, in which concentrations of 21 21 41 pmole.mg protein .min (SD+ 0.1, n ¼ 6). Although chlorogenic acid exceeded 30 mM. Further studies should this activity was lower than previously reported glucose-6- be conducted to assess inhibition at physiological concen- 36 – 38 phosphatase activities in rats, it was still significantly tration. However, this study provides a positive indication higher than control samples (P, 0.05). Thus, the liver for the inhibitory potential of these polyphenols on nicotine microsome isolations demonstrated evidence for the presence metabolism. of rough endoplasmic reticulum (RER) membrane enzymes, Little previously reported data was found regarding the such as CYP2A6, as glucose-6-phosphatase is an RER inhibitory effects of caffeic acid on phase I metabolizing membrane-bound protein. enzymes and no data regarding CYP2A6 inhibition, although The bovine liver samples showed a mean rate of coumarin a study by Teel and Huynh showed that caffeic acid did sig- 21 21 7-hydroxylation of 48.66 pmole.mg protein .min (SD+ nificantly inhibit the activities of cytochrome P450 enzymes 28.38, n ¼ 6) under uninhibited conditions in comparisons benzyloxyresorufin O-dealkylase (BOD) and methoxyresoru- to a mean rate of 30.33 (SD+ 13.22, n ¼ 6) and 25.19 fin O-demethylase (MROD) by 46 and 40%, respectively. 21 21 41 (SD+ 11.36, n ¼ 6) pmole.mg protein .min in the A study by Obach evaluated the inhibitory effects of chloro- presence of caffeic acid and quercetin, respectively (Fig. 3), genic acid on five CYP enzymes (CYP1A2, CYP2C9, with caffeic acid and quercetin demonstrating signifi- CYP2C19, CYP2D6 and CYP3A4) and found no CYP inhi- cant inhibition of coumarin 7-hydroxylation (P, 0.05). bition by chlorogenic acid at concentrations 30 mM. The average plasma concentration of quercetin achieved by dietary means is reported to be 1.46 mM, however, an equal concentration as caffeic acid was selected in this study to allow comparison to caffeic acid and previously reported data. Thus, CYP2A6 inhibition by quercetin showed similar results to that of caffeic acid. This compares favourably with previously reported data, showing that quercetin inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 by 50% at concentrations of 7.5, 47, .100, 24 and 22 mM, respectively. No studies were found regarding quercetin inhibition of CYP2A6. Within this study it was shown that both quercetin and caffeic acid inhibited CYP2A6 hydroxylation activity, although the nature of this interaction was not assessed. A study by Obach showed that quercetin demonstrates non-competitive inhibition of CYP1A2 and a study by Figure 3. CYP2A6 activity and inhibition. The addition of caffeic acid or Yang et al. suggests that phenolic compounds with free quercetin to incubation reactions marginally lowered the mean rate of cou- marin 7-hydroxylation. Results expressed as mean+ SD (n ¼ 6). Asterisk ( ) hydroxyl groups on the benzene ring are capable of inhibit- indicated significance from control (P, 0.05). ing hydroxylation reactions. This is a possible validation ......................................................................................................................................................................................................................................... 101 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... for the characteristics observed by both caffeic acid and that these findings are concurrent with previously reported quercetin within this and other studies. Additionally, as data. Consequently it is suggested that caffeic acid and quer- caffeic acid and quercetin showed no significant difference cetin are plausible inhibitors of nicotine metabolism. This in their inhibitory effects (P . 0.05), it may be suggested study provides the first biochemical evidence in support of that their inhibitory effect occurs via similar or generic epidemiological observations of smoking behaviour and mechanisms. Thus it may be hypothesized that caffeic acid suggests that the caffeic acid, quercetin and similar polyphe- and quercetin are intermediates of CYP2A6 that form a nolic structures present in caffeine containing beverages are complex via interactions between the adjacent hydroxyl responsible for coffee-drinking cigarette smokers smoking groups of the polyphenolic structures and the ferric portion less than non-coffee drinking cigarette smokers. However, of CYP2A6’s active site, effectively chelating the ferric as nicotine metabolism was modelled by CYP2A6 coumarin portion of CYP2A6. Absorbance changes may thus be uti- hydroxylation activity in vitro, these results should be viewed lized in further studies to quantitatively describe caffeic as preliminary findings in justification of further study. acid and quercetin complexing (chelation) to the ferric proto- porphyrin IX prosthetic group of CYP2A6 and other cyto- Acknowledgements chrome P450s. By determination of the apparent spectral dissociation constant and the maximal spectral change eli- The author acknowledges the following scientists and staff of cited by the inhibitor, formally similar to the K and V m max the Faculty of Sciences, Staffordshire University, for the use values described by Michaelis–Menton kinetics, a measure of their expertise and facilities during this investigation: of inhibitor affinity to cytochrome P450 may be determined. Dr Robert Manning and Dr Sue Bird for their continued In addition, it is suggested that further experimentation to support and advice, Dr Stephen Merry for his guidance establish Lineweaver–Burk kinetics of this inhibitory reac- and input and Audra Jones for obtaining the bovine liver tion be conducted to assess competitive or non-competitive samples. inhibition. Although only a model system for nicotine metabolism, this study provides some evidence in support of the obser- Funding 20 19 vations by Kozlowski and Swanson et al., linking The present study was supported by the Faculty of Sciences, coffee drinking with cigarette smoking. Furthermore, it is Staffordshire University, UK. probable that drinking caffeine containing beverages increases the nicotine ‘hit’ received by coffee-drinking ciga- rette smokers by increasing the body’s exposure to nicotine. References However, evidence suggests that most phenolic compounds 44 1. Manach C, Scalbert A, Morand C et al. 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Biochem J 96: 390–398. ........................................................................................................................................................................................................................................ Submitted on 20 September 2007; accepted on 28 January 2008; advance access publication 2 May 2008 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

The potential effect of excessive coffee consumption on nicotine metabolism: CYP2A6 inhibition by caffeic acid and quercetin

Bioscience Horizons , Volume 1 (2) – Jun 2, 2008

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Abstract

Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn021 ......................................................................................................................................................................................................................................... Research article The potential effect of excessive coffee consumption on nicotine metabolism: CYP2A6 inhibition by caffeic acid and quercetin Gary M. Woodward* Faculty of Sciences, Staffordshire University, College Road, Stoke on Trent, Staffordshire ST4 2DE, UK. * Corresponding author: Diet and health group, Biomedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, NR4 7TJ, UK. Tel: þ44 (0)7746144623. Email: g.woodward@uea.ac.uk Supervisor: Dr Susan Bird, Faculty of Sciences, Staffordshire University, College Road, Stoke on Trent, Staffordshire ST4 2DE, UK. ........................................................................................................................................................................................................................................ Over the past decade interest in the biochemical and biological properties of polyphenols has grown considerably, as epidemiological evidence for their beneficial effects on health continues to increase. Dietary polyphenol antioxidants are reported to have many ‘health promoting’ properties, including anti-inflammatory, vasoprotection, anti-cancer and anti-obesity effects. However, their absorption and metabolism are as yet not fully elucidated, particularly with regard to their interactions with other metabolized compounds, such as nicotine. Epidemiological studies have shown that coffee-drinking cigarette smokers are more likely to smoke less in comparison to non-coffee drinking cigarette smokers. Nicotine is primarily metabolized by the hepatic cytochrome P450 enzyme CYP2A6 and may be inhibited by the metal chelating properties of polyphenols via haem interactions with their hydroxyl groups. To model nicotine metabolism, bovine liver microsomes, shown to suitably represent human CYP2A6 metabolism, were isolated by calcium precipitation and differential centrifugation. Fluorometric analysis of 7-hydroxycoumarin, the CYP2A6 metabolite of coumarin and known probe for CYP2A6 activity, was used to model nicotine metabolism in vitro and to quantify the degree of CYP2A6 inhibition imposed by caffeic acid and quercetin. It was found that both caffeic acid and quercetin, major polyphenolic constituents of caffeine containing beverages, sig- nificantly inhibited CYP2A6 activity in vitro by 37.9% (P, 0.05) and 48.2% (P, 0.05), respectively. Thus, this study demonstrates the first reported biochemical evidence in support of previous epidemiological observations, where it is suggested that polyphenol interaction with CYP2A6 prolongs the pharmacological effects of nicotine by decreasing its rate of elimination. Key words: polyphenols, CYP2A6, caffeic acid, quercetin, nicotine. ........................................................................................................................................................................................................................................ include anti-inflammatory, vasoprotection and anti-obesity Introduction effects. In addition, phenolic antioxidants commonly con- Polyphenols are the most abundant antioxidants in the diet, sumed in our diets are being proposed as preventative treat- 3, 7– 11 with a total dietary intake that exceeds that of vitamin C, ments against chronic human diseases. 1, 2 vitamin E and carotenoids. Their main dietary sources However, if these food-based antioxidant approaches are include fruits and plant-derived beverages such as fruit to be employed as prevention or treatments for chronic dis- juices, tea, coffee and red wine. Vegetables, cereals, choco- eases, clarification of their little known metabolism must late and dry legumes also contribute to the total polyphenol be achieved, particularly given the negative findings from 12, 13 intake. These dietary nutraceuticals have been shown to con- studies of antioxidant supplementation and the contro- tribute significantly to the prevention of disease, where a versy that exists within the field regarding the bioavailability recent study by Mink et al. revealed that flavonoids found and bioactivity of many phytochemicals. Furthermore, little in fruits and vegetables are strongly associated with research has been conducted regarding the interactions reduced cardiovascular disease mortality. These health pro- between dietary antioxidants and other environmental pro- moting effects are thought to be due to bioactivities that moters of oxidation, such as cigarettes. ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 98 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Cigarette smoking, the foremost form of nicotine addiction, continues to be one of the world’s most serious public health problems and is considered to be the major risk factor of ischae- mic heart disease, lung cancer and chronic obstructive pulmonary disease. A key factor in reducing the pharmaco- logical and addictive effects of nicotine would be to improve its removal by the drug metabolizing pathways of the liver. However, interactions with dietary components, particularly polyphenol antioxidants, may hinder its removal. Of the nico- tine absorbed through smoking, 80% is metabolized in Figure 2. Structure of quercetin. the liver by the cytochrome P450 enzyme CYP2A6 via 16–18 C-oxidation to cotinine. CYP2A6, therefore, plays a sodium cacodylate, glucose-6-phosphate, acid molybdate, pivotal role in the removal of nicotine in humans. The relation- sodium dodecyl sulphate, ascorbic acid, sodium hydroxide, ship between nicotine and polyphenol metabolism is indicated copper sulphate, sodium potassium tartrate, sodium carbon- in a review by Swanson et al. who stated that 86.4% of ciga- ate, Folin reagent, bovine serum albumin, coumarin, caffeic rette smokers consume coffee compared with 77.2% of non- acid, quercetin, nicotinamide adenine dinucleotide phos- smokers, and also in a study by Kozlowski demonstrating phate (NADP), perchloric acid and 7-hydroxycoumarin that coffee-drinking cigarette smokers smoked more when were purchased from Sigma Aldrich Co. (UK). Bovine liver they ingested almost no caffeine than when they ingested an samples were supplied to Staffordshire University by a local amount of caffeine, in the form of coffee, ranging from 75 to abattoir. 300 mg. Caffeic acid and quercetin (Figs 1 and 2) represent major antioxidant constituents within caffeine containing bev- 21–23 Liver microsome isolation erages and are potential CYP2A6 inhibitors. Thus, in the present study, the CYP2A6 inhibition potential of caffeic acid Whole bovine livers were delivered within 4 h of animal and quercetin was investigated, where it was hypothesized death and placed directly on ice. Liver microsome isolation that CYP2A6 inhibitionisimposedbycaffeicacid and quercetin was performed using modification to the methods stated by 24 25 and it is this interaction responsible for the epidemiological Ernster et al. and Hamilton et al. Liver tissue samples observation that coffee-drinking cigarette smokers smoke less of 80–100 g were cut from the whole bovine liver, rinsed than non-coffee-drinking cigarette smokers. with 0.25 M sucrose in 25 mM potassium phosphate buffer (pH 7.4) and minced using a scalpel blade. Minced liver samples were transferred to a homogenization tube in 20– Materials and methods 25 g portions, and homogenized for 2–3 min with 10 ml 0.25 M sucrose in 25 mM potassium phosphate buffer (pH Materials and reagents 7.4) per portion. The homogenate was centrifuged at Potassium dihydrogen phosphate, di-potassium monohydro- 1000g for 10 min at 48C using a Sigma Howe 3K30 centri- gen phosphate, sucrose, calcium chloride, sodium cholate, fuge with Sigma 12156-H rotor. Following careful aspiration of the thin floating lipid layer, the remaining supernatant was then transferred to another centrifuge tube and the pellet dis- carded. The supernatant was centrifuged at 12 000g for 15 min at 48C. Again, the thin floating lipid layer was care- fully aspirated and the supernatant retained. This super- natant fraction was called the post-mitochondrial fraction (PMF). A volume of 8 mM calcium chloride, 7.5 times the volume of the PMF was added dropwise to the PMF with constant stirring and left to stir for 15 min at 48C. This mixture was then centrifuged at 8000g for 10 min at 48C. The supernatant was removed and the pellet re-suspended in 0.25 M sucrose in 25 mM potassium phosphate buffer (pH 7.4) at a volume of 0.3 ml for each gram of original tissue. The suspension was vortexed and treated with 0.5% (w/v) sodium cholate, at a volume one-tenth the final suspension volume, for 20 min at 48C. This suspension, called the microsome isolation, was stored at 2808C until Figure 1. Caffeic acid (I) is naturally found as an ester with quinic acid called Chlorogenic acid (II). necessary. ......................................................................................................................................................................................................................................... 99 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Protein determination made up to a final volume of 160 ml with 160 mM sodium cacodylate. 1.6 ml Burchell’s reagent was added to the stan- The protein assays were performed, in duplicate, using the dard curve solutions and incubated as stated earlier. method developed by Lowry et al. The microsome iso- lation was diluted 1:100 with 0.5 M sodium hydroxide and CYP2A6 activity assay 0.2, 0.4, 0.6, 0.8 and 1 ml of the diluted microsome isolation The CYP2A6 activity assay was performed, in duplicate, was made up to a final volume of 1 ml, respectively, with using a modification to the methods stated by Creaven 0.5 M sodium hydroxide. A blank was prepared using 1 ml 29 30 31 et al., Koenings et al. and Ngui et al. Initially, 0.5 M sodium hydroxide. To all diluted microsome isolation 25 mM coumarin in 25 mM potassium phosphate buffer samples was added 5 ml of copper reagent (containing 2% (pH 7.4) was pre-incubated with 2 ml microsomal isolation (w/v) sodium carbonate in 0.1 M sodium hydroxide, 1% for 3 min at 378C. The reaction was initiated by the addition (w/v) copper sulphate and 2% (w/v) sodium potassium tart- of an NADPH generation system consisting of 10 mM rate, prepared at a ratio 100:1:1 by volume, respectively). glucose-6-phosphate and 0.5 mM NADP (final incubation These mixtures were then vortexed and allowed to stand volume, 6 ml). After 10 min at 378C, the incubation was for 10 min at room temperature. Finally, 0.5 ml 1 N Folin quenched with the addition of 100 ml 6 N perchloric acid, reagent was added to all the samples and left to stand set on ice for 2 min and centrifuged at 1000g for 10 min for 30 min at room temperature subsequent to vortexing. using a Sigma Howe 3K30 centrifuge with Sigma 12156-H Absorbance was read at 750 nm on a Cecil CE1010 spectro- rotor. Controls (blanks) were prepared with the addition of photometer after zeroing on the blank. Protein concentration the NADPH generation system after the incubation period. was estimated by direct interpolation from a standard curve. Fluorescence was recorded on a Kontron SFM 25 fluorom- A standard curve was constructed using a stock solution eter at excitation and emission wavelengths of 351 and of bovine serum albumin (BSA) at a concentration of 454 nm, respectively, zeroed against the blank. An additional 100 mg/ml in 0.5 M sodium hydroxide. BSA stock solutions CYP2A6 activity assay was performed with the incubation at volumes 0, 0.2, 0.4, 0.6, 0.8 and 1 ml were made up to a time increased to 15 min (substrate control) to ensure the final volume of 1 ml with 0.5 M sodium hydroxide (equival- assay substrates were not depleted during incubation. ent to 0, 20, 40, 60, 80 and 100 mg protein/ml, respectively). The amount of metabolite (7-hydroxycoumarin) formed The standard curve solutions were then processed as by the CYP2A6 activity assays were quantified with a stan- described earlier. dard curve generated from known amounts (0–100 ng/ml) of 7-hydroxycoumarin in 25 mM potassium phosphate Glucose-6-phosphatase assay buffer (pH 7.4) and 100 ml 6 N perchloric acid, made up This assay was performed, in duplicate, using a modification to a final volume of 6 ml with distilled water. to the methods stated by Taussky and Shorr and Nordlie CYP2A6 inhibition assay and Arion. Initially, 50 ml 20 mM glucose-6-phosphate and 20 ml 160 mM sodium cacodylate were pre-incubated This assay was performed, in duplicate, following the for 10 min at 308C. The incubation reaction was initiated method described earlier with the addition of 25 mM with the addition of 90 ml microsome isolation and incu- caffeic acid and quercetin, respectively, to the incubation bated for 20 min at 308C. The incubation reaction was mixture. stopped with the addition of 1.6 ml Burchell’s reagent con- Statistical analysis sisting of 0.42% (w/v) acid molybdate, 5% (w/v) sodium dodecyl sulphate and 10% (w/v) ascorbic acid prepared at Repeated measures analysis of variance was applied as a the ratio 6:2:1 by volume, respectively. The incubation three factor test to determine between-treatment effects fol- mixture was then placed on ice for 15 min and re-incubated lowed by Turkey HSD and Bonferroni post-hoc tests using for 20 min at 478C. The absorbance was measured on a Cecil Windows SPSS (version 15) software. CE1010 spectrophotometer at 820 nm only after the incu- bation mixtures had stabilized to room temperature. In Results and discussion control samples glucose-6-phosphate was added after the 20 min incubation period. Organic phosphate concentration Many animal species have been shown to possess CYP2A6 was estimated by direct interpolation from a standard curve like activity, where CYP2A6 has been shown to be the as described in what follows. For samples with an absor- major coumarin 7-hydroxylase in bovine liver with activities 32, 33 bance exceeding the range of the standard curve, a 1:1 similar to that of human liver. Thus, bovine liver dilution was performed and the absorbance re-measured. samples were used to model human CYP2A6 coumarin A standard curve was constructed using 0.5 mM potass- 7-hydroxylation within this study. CYP2A6 is the major, if 34, 35 ium di-hydrogen phosphate at volumes 0–150 ml (final phos- not the only, coumarin 7-hydroxylase in humans, phate concentrations 0, 20, 25, 40, 50, 75 nM, respectively), where coumarin is almost exclusively metabolized into ......................................................................................................................................................................................................................................... 100 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... 7-hydroxycoumarin (68–92% of the dose). As coumarin is This equated to a CYP2A6 activity inhibition of 37.9 and almost exclusively metabolized by CYP2A6 to produce 48.2% by caffeic acid and quercetin, respectively, at a con- 7-hydroxycoumarin, a highly fluorescent compound, it was centration of 25 mM. While the maximum plasma concen- an ideal probe and marker for CYP2A6 activity and inhi- tration of caffeic acid is reported to be 3.8 mM following bition studies. Thus, within this study coumarin had been 200 ml of oral coffee consumption, excessive consumption substituted for nicotine as an alternative CYP2A6 substrate may significantly increase this plasma concentration. 21 39 to model in vitro nicotine hydroxylation. Studies by Olthof et al. and Rechner et al. have shown Each microsomal isolation procedure yielded a 30 ml that between 0.3 and 5% of ingested chlorogenic and microsomal isolation mixture. Using the Lowry protein caffeic acid may be excreted in the urine. Thus, with coffee determination method it was shown that microsome iso- beverages containing up to 478 mg/ml chlorogenic acid, it lations yielded a protein concentration of 10.89 mg.ml is possible that hepatic concentrations of caffeic acid may (SD+ 3.11, n ¼ 7). This protein concentration provided be considerably increased with excessive coffee consump- only an estimate for the quantity of microsomes isolated tion, particularly since the polar phenolic acids may be and did not quantify CYP2A6 content. However, for the subject to enterohepatic circulation and accumulation purpose of this study it was not necessary to quantify within the gallbladder. Therefore, while the concentrations CYP2A6 content as protein concentration provided sufficient of phenolic acid within this study exceed reported physio- means by which subsequent assays could be standardized logical levels, a higher concentration was selected as an and compared with previously reported data. Microsome initial trial to determine inhibition potential and in response isolations showed glucose-6-phosphatase activities of 0.44 to previously reported data, in which concentrations of 21 21 41 pmole.mg protein .min (SD+ 0.1, n ¼ 6). Although chlorogenic acid exceeded 30 mM. Further studies should this activity was lower than previously reported glucose-6- be conducted to assess inhibition at physiological concen- 36 – 38 phosphatase activities in rats, it was still significantly tration. However, this study provides a positive indication higher than control samples (P, 0.05). Thus, the liver for the inhibitory potential of these polyphenols on nicotine microsome isolations demonstrated evidence for the presence metabolism. of rough endoplasmic reticulum (RER) membrane enzymes, Little previously reported data was found regarding the such as CYP2A6, as glucose-6-phosphatase is an RER inhibitory effects of caffeic acid on phase I metabolizing membrane-bound protein. enzymes and no data regarding CYP2A6 inhibition, although The bovine liver samples showed a mean rate of coumarin a study by Teel and Huynh showed that caffeic acid did sig- 21 21 7-hydroxylation of 48.66 pmole.mg protein .min (SD+ nificantly inhibit the activities of cytochrome P450 enzymes 28.38, n ¼ 6) under uninhibited conditions in comparisons benzyloxyresorufin O-dealkylase (BOD) and methoxyresoru- to a mean rate of 30.33 (SD+ 13.22, n ¼ 6) and 25.19 fin O-demethylase (MROD) by 46 and 40%, respectively. 21 21 41 (SD+ 11.36, n ¼ 6) pmole.mg protein .min in the A study by Obach evaluated the inhibitory effects of chloro- presence of caffeic acid and quercetin, respectively (Fig. 3), genic acid on five CYP enzymes (CYP1A2, CYP2C9, with caffeic acid and quercetin demonstrating signifi- CYP2C19, CYP2D6 and CYP3A4) and found no CYP inhi- cant inhibition of coumarin 7-hydroxylation (P, 0.05). bition by chlorogenic acid at concentrations 30 mM. The average plasma concentration of quercetin achieved by dietary means is reported to be 1.46 mM, however, an equal concentration as caffeic acid was selected in this study to allow comparison to caffeic acid and previously reported data. Thus, CYP2A6 inhibition by quercetin showed similar results to that of caffeic acid. This compares favourably with previously reported data, showing that quercetin inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 by 50% at concentrations of 7.5, 47, .100, 24 and 22 mM, respectively. No studies were found regarding quercetin inhibition of CYP2A6. Within this study it was shown that both quercetin and caffeic acid inhibited CYP2A6 hydroxylation activity, although the nature of this interaction was not assessed. A study by Obach showed that quercetin demonstrates non-competitive inhibition of CYP1A2 and a study by Figure 3. CYP2A6 activity and inhibition. The addition of caffeic acid or Yang et al. suggests that phenolic compounds with free quercetin to incubation reactions marginally lowered the mean rate of cou- marin 7-hydroxylation. Results expressed as mean+ SD (n ¼ 6). Asterisk ( ) hydroxyl groups on the benzene ring are capable of inhibit- indicated significance from control (P, 0.05). ing hydroxylation reactions. This is a possible validation ......................................................................................................................................................................................................................................... 101 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... for the characteristics observed by both caffeic acid and that these findings are concurrent with previously reported quercetin within this and other studies. Additionally, as data. Consequently it is suggested that caffeic acid and quer- caffeic acid and quercetin showed no significant difference cetin are plausible inhibitors of nicotine metabolism. This in their inhibitory effects (P . 0.05), it may be suggested study provides the first biochemical evidence in support of that their inhibitory effect occurs via similar or generic epidemiological observations of smoking behaviour and mechanisms. Thus it may be hypothesized that caffeic acid suggests that the caffeic acid, quercetin and similar polyphe- and quercetin are intermediates of CYP2A6 that form a nolic structures present in caffeine containing beverages are complex via interactions between the adjacent hydroxyl responsible for coffee-drinking cigarette smokers smoking groups of the polyphenolic structures and the ferric portion less than non-coffee drinking cigarette smokers. However, of CYP2A6’s active site, effectively chelating the ferric as nicotine metabolism was modelled by CYP2A6 coumarin portion of CYP2A6. Absorbance changes may thus be uti- hydroxylation activity in vitro, these results should be viewed lized in further studies to quantitatively describe caffeic as preliminary findings in justification of further study. acid and quercetin complexing (chelation) to the ferric proto- porphyrin IX prosthetic group of CYP2A6 and other cyto- Acknowledgements chrome P450s. By determination of the apparent spectral dissociation constant and the maximal spectral change eli- The author acknowledges the following scientists and staff of cited by the inhibitor, formally similar to the K and V m max the Faculty of Sciences, Staffordshire University, for the use values described by Michaelis–Menton kinetics, a measure of their expertise and facilities during this investigation: of inhibitor affinity to cytochrome P450 may be determined. Dr Robert Manning and Dr Sue Bird for their continued In addition, it is suggested that further experimentation to support and advice, Dr Stephen Merry for his guidance establish Lineweaver–Burk kinetics of this inhibitory reac- and input and Audra Jones for obtaining the bovine liver tion be conducted to assess competitive or non-competitive samples. inhibition. Although only a model system for nicotine metabolism, this study provides some evidence in support of the obser- Funding 20 19 vations by Kozlowski and Swanson et al., linking The present study was supported by the Faculty of Sciences, coffee drinking with cigarette smoking. Furthermore, it is Staffordshire University, UK. probable that drinking caffeine containing beverages increases the nicotine ‘hit’ received by coffee-drinking ciga- rette smokers by increasing the body’s exposure to nicotine. References However, evidence suggests that most phenolic compounds 44 1. Manach C, Scalbert A, Morand C et al. 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Biochem J 96: 390–398. ........................................................................................................................................................................................................................................ Submitted on 20 September 2007; accepted on 28 January 2008; advance access publication 2 May 2008 .........................................................................................................................................................................................................................................

Journal

Bioscience HorizonsOxford University Press

Published: Jun 2, 2008

Keywords: Key words polyphenols CYP2A6 caffeic acid quercetin nicotine

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