Imad Hamdan Paper

Current Drug Metabolism, 2007, 8, 9-15

9

Effects of Dietary Broccoli on Human in Vivo Caffeine Metabolism: A Pilot Study on a Group of Jordanian Volunteers Nancy Hakooz* and Imad Hamdan Faculty of Pharmacy, University of Jordan, Amman 11942 Jordan Abstract: Objectives: Induction or inhibition of cytochrome P450 (CYP) enzyme activities, enzymes that activate or detoxify xenobiotics, is one mechanism by which vegetables may alter cancer risk. As the effect of food on CYP enzyme activities have not been studied in the Jordanian population, we examined the effect of supplementing the diet with broccoli on CYP1A2 and CYP2A6 activities. Methods: Five men and five women, non-smokers, consumed a standard diet of broccoli (500 g) for 6 days. Enzyme activities were determined by measuring urinary metabolite ratios after a 100 mg caffeine tablet on the seventh day. Results: The mean CYP1A2 activity for men (21.1 ±3.2) was significantly lower than that for women (27.6 ±1.6) before the consumption of broccoli (P <0.05). These activities were significantly induced in both men (52.5 ±6.6) and women (36.6 ±8.4) after a standard diet of broccoli (P <0.005). Similarly, the mean value of CYP2A6 activity for men was 0.061 ±0.040 and for women, 0.144 ±0.039 before consumption of broccoli, which were significantly different (P <0.05). The activity of CYP2A6 was induced in both groups significantly after broccoli consumption (P <0.05). The mean value for men was 0.193 ±0.02 and for women, 0.214 ±0.064. Conclusion: Our study on a group of Jordanians confirmed the well-established observation that broccoli induces CYP1A2 activity. This study also demonstrates the effect of gender and broccoli consumption on CYP2A6 activity in Jordanians.

Key Words: Broccoli, CYP1A2, CYP2A6, Caffeine, In vivo, Urine metabolic ratio, Jordanian population. INTRODUCTION In epidemiological studies, it was shown that intake of cruciferous plants is inversely associated with kidney, prostate, bladder, colon, rectum and lung cancer risk. Cruciferae comprise a large number of vegetables such as red cabbage, white cabbage, cauliflower, Savoy cabbage, Brussels sprouts and broccoli [1]. The most important principles of chemoprevention by cruciferous vegetables are their ability to influence phase 1 and phase 2 biotransformation enzyme activities. The cruciferous vegetables contain a specific group of constituents, the glucosinolates. It has been suggested that isothiocyanates function as cancer chemopreventive agents by strongly inducing glutathione S-transferases or by modifying various CYP xenobiotic metabolizing enzymes [2]. Apart from the glucosinolates, cruciferous vegetables contain other potentially protective constituents such as flavonoids, polyphenols, vitamins, fibers and pigments [1, 3]. The amount of glucosinolate present in vegetables is highly variable, depending on the compound, the plant and the method of analysis concentrations of up to several hundred micromoles per 100 g have been reported [4].

used today are metabolized by CYP1A2 isoenzyme. Cruciferous vegetables containing indole-3-carbinol can induce this enzyme [5]. The human CYP2A6 enzyme metabolizes several xenobiotic compounds of clinical or toxicological importance. CYP2A6 has been detected in liver where it constitutes about 4% of total CYP content [5]. This enzyme is subject to marked interindividual variability and it was suggested that there are ethnic-related differences in the expression of CYP2A6 protein, polymorphism has been suggested to occur in <2% of the population [5]. Several reports about these genetic variants have been published in different ethnically diverse populations [6-8]. However, very little is known about the effect of food on the expression of CYP2A6.

CYP1A family of enzymes is responsible for the metabolic activation of some known procarcinogenic environmental chemicals and toxins [5]. This family contains CYP1A1 and CYP1A2. Approximately 15% of all drugs

Relatively selective substrate probes in vivo are available for several major CYP isoforms involved in oxidative drug metabolism. Caffeine (1,3,7- trimethylxanthine) is a substrate that can be used as a probe to test CYP1A2 activity in vivo in humans [9, 10]. The N3- demethylation of caffeine to paraxanthine (1,7-dimethylxanthine (17X)) accounts about 80% or 90% of the total caffeine metabolism in humans, and the reaction is completely controlled by the activity of CYP1A2. Because of its specificity, this reaction is used to measure the activity of the 1A2 enzyme in vivo in humans by determining the ratios of caffeine urinary metabolites [10].

*Address correspondence to this author at the Faculty of Pharmacy, University of Jordan, Amman 11942 Jordan; Tel: +962-6 5355000 ext. 2643; Fax: +962-6 5339649; E-mail: [email protected]

The main metabolic routes of caffeine are illustrated in Fig. (1). Caffeine is extensively metabolized in man and at least 17 metabolites have been identified. CYP1A2 is solely responsible for the caffeine N3-demethylation to 17X. The

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10 Current Drug Metabolism, 2007, Vol. 8, No. 1

Hakooz and Hamdan

O

N N

N N O Caffeine

CYP1A2 O O

OH

N

O CYP2A6

N

N

CH3

NH

NAT2 CH O

NH

NH O

O 17U

NH

N

N

N

NH O

O

N

AFMU

17X

Non enzymatic decomposition

CYP1A2

H O

O

N O

NH

CH3

N

N NH

N

1X

O

NH2 NH

O

AAMU XO

H O

OH

N N

N NH O 1U

Fig. (1). Biotransformation pathway for caffeine. Caffeine, 1,3,7 trimethylxanthine (1,3,7X); 1,7-dimethylxanthine (17X); 1,7-dimethylurate (1,7U); 1-methylxanthine (1X); 1-methylurate (1U); 5-acetylamino-6-formylamino-3-methyluracil (AFMU); 5-acetylamino-6-amino-3methyluracil (AAMU); N-acetyltransferase (NAT); xanthine oxidase (XO).

metabolite 17X is further metabolized by three reactions; 8hydroxylation by CYP2A6 to 1,7-dimethylurate (17U), 7demethyltion by CYP1A2 to form 10-methylxanthine (1X) and, thirdly, the formation of the open ring product 5acetylamino-6-formylamino-3-methyluracil (AFMU) which is catalyzed by N-acetyltransferase (NAT). AFMU is unstable product that may be deformylated nonenzymatically to 5acetylamino-6-amino-3-methyluracil (AAMU). Part of 1X is metabolized to 1-methylurate (1U) by xanthine oxidase (XO) [11, 12].

Several urinary metabolic ratios (MR) have been proposed to assess CYP1A2 activity. Sinués et al. [13] studied five of the MRs reported in literature to select the most sensitive MR/timing schedule to discriminate non exposed and exposed to inducers of CYP1A2 activity. The authors recommended the use of (AFMU + 1X + 1U)/17U which was proposed by Campbell et al. [14] in the period of 4-5 hours after caffeine intake. Conversely, they concluded that there are no differences between several of the other MR reported in literature in detecting induction of CYP1A2 if urine sam-

Effects of Dietary Broccoli on Human in Vivo Caffeine Metabolism

ples are collected 4-5 hours after ingesting caffeine [13]. Molar ratios of the different caffeine metabolites have been used also to assess the metabolizer phenotypes of NAT and CYP2A6 [11]. According to Rostami-Hodjegan et al. [15] NAT activity has been assessed from the ratio of AFMU/ (AFMU + 1X + 1U) because the rate of conversion of 1X to 1U does not influence the measurements. CYP2A6 MR can be calculated from the ratio 17U/(17U+17X+1U+1X+ AFMU) [16]. Jordan is a developing Middle Eastern country of 5.3 million people that is undergoing rapid modernization. Jordanians are Arabs, except for a few small communities of Circassians, Armenians, and Kurds that have adapted to Arab culture and represent less than 1% of the population. Genetically, the Jordanian population has been characterized as being heterogeneous, and this reflects ancient and recent admixture with neighboring populations, and important human migratory trends throughout history [17]. Few studies on drug metabolism have been conducted on the Jordanian population. These studies examined the N-acetylation phenotyping using dapsone [18], acetylator phenotypes in Jordanian diabetics [19], S-mephenytoin hydroxylation which is considered as a standard CYP2C19 phenotyping probe [20, 21] and genetic polymorphism of CYP2D6 [22, 23] and CYP2A6 activities in a Jordanian population [24]. Jordanian population has not been screened for CYP activities or the effect of food on these activities. In the present study we examined the effects of dietary broccoli on CYP1A2 and CYP2A6 activities in a group of Jordanian volunteers by assessing the molar concentration ratios of urinary caffeine metabolites.

Current Drug Metabolism, 2007, Vol. 8, No. 1

11

tion containers that were labeled with broccoli. 100 mg ascorbic acid was added to 15 ml urine samples and then samples were immediately stored at -20°C until analysis. Volunteers The study started with 17 healthy non-smoking volunteers, eight men and nine women. The objectives of this study were explained to the volunteers. The informed consent was obtained from each volunteer. Seven volunteers dropped out after the beginning of the study. The ten volunteers that completed the study, five men and five women, had a mean age 28.3 ± 9 years (range 21-45) and a mean weight 69.2 ± 11.8 kg (53-90). None of the selected volunteers suffered from any food allergies or intolerance. They were devoid of any long-term drug therapy. Volunteers were instructed to avoid alcohol intake, passive smoking and cabbage and other cruciferous vegetables during the period of the experiment. In addition, each volunteer was given written instructions regarding the investigation and a 24-hour urine collection container. Broccoli Broccoli was purchased from the local market. It was supplied fresh. The volunteers were given instructions on how to eat the broccoli. Broccoli was eaten raw together with salad and any dressing or it was gently prepared by the volunteers in one of the following ways: steamed for a maximum of 3 minutes, microwaved for 2-3 minutes, boiled for 2-3 minutes (the boiling water should also be ingested) or an ingredient in a casserole. Analysis of Urinary Caffeine Metabolites

MATERIALS AND METHODS Chemicals All chemicals used were of the highest available commercial purity. Caffeine (100 mg) tablets were a kind gift from Jordan Sweden Medical and Sterilization Company (JOSWE), Naur, Jordan. AFMU was generously donated by Nestlé Research Center of Lausanne (Lausanne, Switzerland). The metabolites: 1-methylxanthine (1X), (1,7-dimethylurate (17U), 1,7-dimethylxanthine (17X) were from Sigma (St. Louis, USA). Ascorbic acid was from ICN Pharmaceuticals Inc. (Irvine, USA). Tetrahydrfuran and acetonitrile were HPLC grade and obtained from Merck (Darmstadt, Germany). Protocol Caffeine (100 mg) tablet was administered at day 1, 30 minutes prior to breakfast and after 12 hours caffeine abstention. After 5 hours of administrating the caffeine tablet volunteers drained their bladder and delivered a urine sample that was labeled a negative control. Urine samples were collected in a sterile 24-hour urine collection containers. 100 mg ascorbic acid was added to 15 ml urine samples and then samples were immediately stored at -20°C until analysis. A standard diet of broccoli (500 g) was consumed over the next six days (day 2-day 7). On day 8 after an overnight fast and 12 hour caffeine abstention, 100 mg tablet of caffeine was administered 30 minutes prior to breakfast. After 5 hours urine samples were collected in sterile 24-hour urine collec-

The metabolites of caffeine (1,7-dimethylurate (17U), 1,7-dimethylxanthine (17X), 1-methylxanthine (1X), 5acetylamino-6-amino-3-methyluracil (AFMU)) and caffeine (1,3,7-trimethylxanthine (137X)) were analyzed using a previously published method [11]. Nevertheless, the method was validated thoroughly in light of the FDA guidelines for validation of bioanalytical methods [25]. Briefly the selectivity of the method was established through obtaining chromatograms for each of the standard metabolites to be assayed as well as mixture of them and a sample of blank urine. Linearity and precision of the method were also established. The technique of standard addition was employed in order to increase the specificity of the method. The final procedure was briefly as follows: the pH of a 5 ml urine sample was adjusted to pH 3.5 and kept in ice, then 250 μl of the sample is transferred to a 10 ml tube and mixed with 250 μl of internal standard solution (150 μg/ml paracetamol) and 120 mg of ammonium sulfate. The mixture was vortexed for 2 minutes and 6 ml of chloroform:isopropanol (4:1) mixture were added before the mixture was centrifuged for 5 minutes at 1000g. The organic phase was removed to a new tube and evaporated under a stream of nitrogen at 40ºC and reconstituted with 1 ml 0.05% acetic acid from which a 20 μl aliquot were injected onto HPLC system. Chromatographic Conditions The chromatographic system employed was a MerckHitachi L-7100pump, l-7420 UV/VIS detector and D-7500

12 Current Drug Metabolism, 2007, Vol. 8, No. 1

integrator. Separation of metabolites was achieved on Wakosil C18 column (250 x 4.6mm, particle size 5 um) from SGE at a flow rate of 1ml/min with a mobile phase of acetic acid (33%): tetrahydrohuran: acetonitrile: distilled water (1:2.5:44:952.5). All chromatograms were monitored at 280 nm. RESULTS Caffeine and four of its metabolites (AFMU, 1X, 17U and 17X) were determined in urine using a previously published method [11]. To minimize any potential effect of urine flow on caffeine MRs, urine volume was also adjusted for in the study. The collected urine samples from volunteers before and after consumption of broccoli were analyzed for caffeine and its metabolites (AFMU, 1X, 17U and 17X) and their molar concentrations were determined as outlined in experimental section. The estimated concentrations of the examined metabolites were employed to calculate the metabolic ratios for the enzymes: CYP1A2 ((17U+17X)/137X) and CYP2A6 (17U/(AFMU+1X+17X+17U)). Table 1 summarizes the obtained metabolic ratios of caffeine before and after consumption of broccoli.

Hakooz and Hamdan

Table1.

Estimated Metabolic Ratios for CYP1A2 and CYP2A6 for the Assayed Caffeine Metabolites in Collected Urine Samples Before and After the Consumption of Broccoli CYP1A2

Volunteer No.

CYP2A6

Gender Before

After

Before

After

1

W

28.6

51.2

0.152

0.225

2

W

28.0

30.7

0.100

0.236

3

W

24.9

33.4

0.198

0.252

4

W

28.8

32.1

0.159

0.256

5

W

28.0

35.7

0.110

0.101

27.6

36.6

0.144

0.214

1.6

8.4

0.039

0.064

5.6

22.9

27.4

30.1

Average SD

W

RSD 6

M

20.3

59.7

0.037

0.182

7

M

18.4

55.0

0.130

0.223

CYP1A2 MR

8

M

26.2

56.7

0.030

0.195

As shown in Table 1, before consumption of broccoli, the mean value of CYP1A2 activities (as assessed by the urinary ratio of (17U+17X)/137X) for women was 27.6 ±1.6 (n=5). The mean value for men was 21.1 ±3.2 (n=5). The values showed individual differences of 1.2-fold for women to 1.5fold for men. The CYP1A2 activities of women and men were significantly different (P=0.006). After 6 days consumption of broccoli, the mean value for women (36.6 ±8.4) was significantly lower than the mean value for CYP1A2 activity in men (52.5 ±6.6, P=0.005). Fig. (2) represent the CYP1A2 MR before and after consumption of broccoli for women and men.

9

M

18.8

44.3

0.047

0.170

10

M

22.1

46.8

0.061

0.197

21.1

52.5

0.061

0.193

3.2

6.6

0.04

0.02

15.1

12.6

65.9

10.34

Average SD RSD

M

Th The data in Table 1 and in Fig. (2) indicate the CYP1A2 is induced to higher levels in men as compared to women. The range of induction of CYP1A2 activity for

Fig. (2). The individual values of CYP1A2 MR determined in women (1-5) and men (6-10) before and after consumption of broccoli.

Effects of Dietary Broccoli on Human in Vivo Caffeine Metabolism

Current Drug Metabolism, 2007, Vol. 8, No. 1

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women was between 10%-79%, showing 8-fold individual difference, and the mean value of induction was 32% ±28. The range for men was between 112%-199%, showing 1.8fold individual difference, and the mean value of induction was 152% ±42.4. In both groups, women and men, the induction of CYP1A2 after consumption of broccoli was statistically significant (P=0.03 for women and P=0.002 for men).

were determined using urinary caffeine MR before and after 6 days of ingesting a diet rich with broccoli (500 g/day). The aim of this study is to investigate if consumption of broccoli by a group of Jordanian volunteers will have similar effects on drug metabolism activities as it has been reported.

The variability of the analytical method itself was tested using standard concentration (quality control samples) at three concentrations levels (low, medium and high). In general the obtained results were within the recommendations of the FDA guidelines [25] for validation of the bioanalytical methods (average RSD at lowest concentration was 18.5%). Thus the results suggest a genuine variability in metabolic activities of the different enzymes within the study sample.

One group of vegetables that has been widely regarded as potentially cancer protective are vegetables of the Cruciferae family. Cruciferous vegetables are the major source of glucosinolates in the diet that distinguishes them from other vegetables. The mechanisms underlying the anticarcinogenicity of brassica vegetables were reviewed by Verhoeven et al. [26]. Most evidence concerning anticarcinogenic effects of glucosinolate hydrolysis products and brassica vegetables has come from studies in animals. Because of interspecies differences, and since the dose of both the anticarcinogenic and the carcinogenic compound used in animal studies often greatly exceeds the estimated level of the compound in a normal human diet, it is difficult to extrapolate results from animal studies to humans. Therefore, more studies are required in which normal consumption levels are used to find out whether the glucosinolate hydrolysis products and brassica vegetables also exert their protective effects in humans.

CYP2A6 MR CYP2A6 MR was measured using the urinary metabolic ratio of 17U/(AFMU+1X+17X+17U). The results are presented in Table 1 and in Fig. (3). The range of CYP2A6 activity for women before consumption of broccoli was between 0.100-0.198, showing 2-fold individual differences, and mean value was 0.144 ±0.039. The range for men was between 0.030-0.130, a 4-fold individual differences, and mean value was 0.061 ±0.040. The CYP2A6 MRs for women and men before consumption of broccoli were significantly different (P=0.006). After the consumption of broccoli the mean values for women (0.214 ±0.064) and men (0.193 ±0.02) were not significantly different (P=0.3). There was a significant increase in the mean values of both women (P=0.02) and men (P=0.0002) when compared before and after consumption of broccoli. This finding indicates that CYP2A6 activity was more inducible by broccoli consumption in men (up to 550%) compared to women (up to 135%). DISCUSSION Ten healthy non-smoking Jordanian volunteers participated in this study. The activities of CYP1A2 and CYP2A6

CYP1A2 Activity

Various urinary ratios for caffeine phenotyping have been proposed [10, 14, 16, 27, 28], but shortcomings have been demonstrated for all the proposed urinary MRs. The MR of caffeine was estimated in this study based on the recommendations of Butler et al. [10]. Although other MR have been applied, the ratio of (17X + 17U)/137X was used in this study because of the availability of the metabolites. This MR was identified from pharmacokinetic analyses of 12 subjects as being better correlated with the rate constant for caffeine 3-demethylation than other previously suggested ratios [10]. The interval of 4-5 hours after the intake of caffeine has been proposed by Butler et al. [10] to minimize the impact of 17X conversion to secondary metabolites that allowed the detec-

Fig. (3). The individual values of CYP2A6 MR determined in women (1-5) and men (6-10) before and after consumption of broccoli.

14 Current Drug Metabolism, 2007, Vol. 8, No. 1

tion of CYP1A2 induction. This finding is in agreement with that reported by Sinués et al. [13]. Chung et al. [29] have stated that the urinary MR of (17X + 17U)/137X have been used widely by different groups to study the effects of environmental and genetic factors on CYP1A2 activity. This MR have been used by the same group [29] to study the effect of age and smoking on in vivo CYP1A2 activity in Koreans. Earlier studies on the effect of gender on CYP1A2 activity by use of the caffeine MR have shown that the enzyme activity in women was lower than that of men [11, 30-32]. However, other studies reported that the enzyme activity was not influenced by gender in adults [33, 29, 34]. The results obtained in this study with Jordanian volunteers showed that CYP1A2 activity was higher in women compared to men (Table 1 and Fig. (2)). Similar findings were reported by Carrillo and Benitez [28]. The CYP1A2 MRs reported in this study are similar to those reported by Chung et al. [29] when the same formula for calculating the MR was used to evaluate CYP1A2 activity (the range was between 2.1-29.3 but no significant difference was seen between men and women). A polymorphic distribution of CYP1A2 activity, as measured by the ratio of caffeine metabolites, has been observed, but the genetic basis for this polymorphism has not yet been identified. The activity of CYP1A2 is highly inducible, and differences in exposure to inducers may be more important in the phenotypic expression of the enzyme than genetic differences [35]. CYP1A2 activity was reported to increase by 12-19% after ingesting 500 g of broccoli [34, 3638]. Whereas Lampe et al. [33] has reported 50% increase when the diet was supplemented with brassica vegetables for 6-days. In this study the increase in CYP1A2 activity was 10-79% for women and 112-199% for men. This induction of CYP1A2 after consumption of broccoli was statistically significant (P=0.03 for women and P=0.002 for men). The higher degree of induction found in the Jordanians may be due to the species of broccoli that was consumed. Variation of the relative content of glucosinolates may occur between Brassica species, cultivators, between the plant parts within the same plant and during plant development throughout the plant cycle [39, 40]. Conversely, the way by which the broccoli was consumed might be another factor. The higher degree of induction for men (152% ±42.4, n=5) compared to women (32% ±28) might explain the increased incidence of bladder cancer in men. This finding was explained by Chung et al. [29] by greater occupational exposure to these procarcinogens in men rather than the higher CYP1A2 activity observed in men. CYP2A6 Activity Very little is known about the effect of food on the expression of CYP2A6. In our study, we used the caffeine urinary MR proposed by Tang et al. [16]. This MR is one of the commonly used MR by different groups [11, 41]. In this study, the CYP2A6 activity was found to be significantly different in women compared to men (P=0.006). The mean value was 0.144 ±0.039 for women and 0.061 ±0.040 for men. Similar findings were reported by Krul and Hageman [11]. Our study has reported a significant increase in the mean values of CYP2A6 activity after a 6-day consumption of broccoli in both women (P=0.02) and men (P=0.0002).

Hakooz and Hamdan

The effect of food on CYP2A6 activity has not been studied before, the findings of this study are indicating an effect of broccoli on CYP2A6 activity. This needs further investigating before a solid conclusion can be reached as very little is known about the effect of food on the expression of CYP2A6. This enzyme is subject to marked interindividual variability and it was suggested that there are ethnic-related differences in the expression of CYP2A6 protein [6-8]. Polymorphism of CYP2A6 was found in the Jordanian population using Coumarin 7-hydroxylation pathway but no further investigations in the activity of this enzyme were preformed [24]. In conclusion, this study on Jordanians confirmed the well-established observation that broccoli induces CYP1A2 activity. By using the caffeine MRs in urine, the study have also shown changes in CYP2A6 activities after consumption of broccoli. The effects of gender and broccoli consumption have been shown on CYP2A6 activity, which has not been demonstrated previously to the best of our knowledge. Finally, this study indicates higher inducibility of CYP1A2 activities in men compared to women. These finding need to be confirmed by using larger number of volunteers under controlled dietary conditions. ACKNOWLEDGEMENT The Deanship of Academic Research at the University of Jordan for the financial support. REFERENCES [1]

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Received: May 31, 2006

Revised: July 20, 2006

Accepted: July 24, 2006

Current Drug Metabolism, 2007, Vol. 8, No. 1 [30] [31] [32] [33]

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