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Medicinal Chemistry, 2011, 7, 480-487
Relevance of Drug Metabolizing Enzyme Activity Modulation by Tea Polyphenols in the Inhibition of Esophageal Tumorigenesis Pius Maliakal1,*, Umesh T. Sankpal2, Riyaz Basha2, Cima Maliakal2, Andrea Ledford1 and Sompon Wanwimolruk3 1
MD Anderson Cancer Center Orlando, Orlando FL 32806, USA; 2MD Anderson Cancer Center Orlando, Cancer Research Institute, Orlando FL 32827, USA; 3University of Otago, Dunedin, New Zealand Abstract: Tea is a popular, socially accepted, drink that is enjoyed by millions of people. A growing body of evidence suggests that moderate consumption of tea may protect against several forms of cancer. It is also known that bioactivation of precarcinogens and detoxification of ultimate carcinogens are carried out mainly by drug metabolizing enzymes such as cytochrome P450 (CYP). The present study investigates the effect of tea consumption on modulating CYP and phase II conjugating enzymes, and their association in the chemopreventive effect against esophageal tumorigenesis using both in vitro and in vivo techniques. Female Wistar rats were given aqueous solutions (2% w/v) of six different teas, standard green tea extract (GTE) (0.5% w/v), and dandelion tea (2% w/v) as the sole source of fluid for two weeks prior to and during the entire period of tumor induction (12 weeks). Animals were gavaged with 0.5 mg/kg N-nitrosomethylbenzylamine (NMBA) twice weekly for 12 weeks for esophageal tumor induction and the activities of different CYP isoforms and phase II enzymes were determined in the liver microsomes or cytosols. GTE, green tea and Dandelion tea caused decrease in tumor multiplication, tumor size and tumor volume; however, none of these tea preparations altered tumor incidence. No appreciable changes in drug metabolizing enzyme activity were observed in the treatment groups. Thus, the modulations in the activities of CYP 1A1/ 1A2 and CYP2E enzymes, by pre-treatment with green and dandelion teas, observed in our earlier experiments, seem to be compensated by the tumor inducing agent, NMBA. The balance between phase I carcinogen-activating enzymes and phase II detoxifying enzymes could be important in determining the risk of developing chemically-induced cancer and the present study in conjunction with the previous observations suggest a possible role of drug metabolizing enzymes in the anticancer effect of tea.
Keywords: Green tea, P450, tumorigenesis, N-nitrosomethylbenzylamine.
INTRODUCTION The majority of adult cancers are carcinomas of epithelial origin with lung, esophagus, stomach, colon, and uterus as the primary sites which reflect a selective vulnerability of these tissues to carcinogenic insult as a result of frequent exposure to external environment. It is recognized that majority of all cancers are attributable to environmental risk factors, including chemicals, radiation, and viruses [1]. Almost one third of cancers are caused by dietary substances and the strategy of manipulation of diet is increasingly being recognized as a practical approach for cancer prevention. Next to water, green and black teas and coffee are major components of fluid intake in many parts of the world. Black and green tea are known to have beneficial attributes in lowering the risk of several diseases [2] and prevention of cancer through consumption of this widely-used beverage is receiving increasing attention [3]. Tea is an important dietary source of flavonols that have beneficial health effects due to their antioxidant properties. The inhibitory activity of tea and tea components against various stages of carcinogenesis has been demonstrated in
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many studies [4-7]. The antimutagenic and anti-carcinogenic effect of tea was reported in various laboratory testing systems and animal models using different carcinogens. Recent studies suggested that the cancer chemopreventive effect of green tea is mediated by its major polyphenolic constituent epigallocatechin gallate (EGCG) [8, 9]; however other studies postulated that caffeine could be the major inhibitory substance in both green tea and black tea [10, 11]. Inhibition of skin, stomach, colon and lung- carcinogenesis as well as the growth of human prostate and breast tumors by EGCG in athymic mice has been demonstrated [12, 13]. Nitrosamines constitute a large group of chemical carcinogens, which occur in the environment and can be formed in the body by nitrosation of secondary or tertiary amines [14]. Sufficiently high concentrations of nitrosamine could be generated in food stored with a preservative such as sodium nitrite [15]. Over 300 N-nitroso-compounds have been reported to be carcinogenic in more than 40 species. Almost every organ in one or more rodent species used for carcinogenesis testing has proven to be sensitive to one or more of the nitroso-compounds used. Some nitrosamines show remarkable organ specificity or organotropism dependent on chemical structure [16]. For example asymmetrical compounds, such as phenylmethylnitrosamine and benzylmethylnitrosamine, are highly specific for esophagus in the rat, and several compounds containing a N-butyl group induce bladder tumors predominantly or exclusively [17]. © 2011 Bentham Science Publishers Ltd.
Relevance of Drug Metabolizing Enzyme Activity
Tumors induced by nitroso-compounds are closely similar to their human counterparts. N-nitrosomethylbenzylamine (NMBA) is a potent esophageal carcinogen in animal species and is believed to be carcinogenic to humans [18]. There are a few studies where the anticancer properties of tea preparations have been elucidated against nitrosamineinduced tumors. Inhibition of nitrosation by tea preparations was demonstrated in vitro and in humans which may be an important factor in the prevention of certain cancers if the endogenously-formed N-nitroso-compounds are the causative factors [19]. Theaflavins in tea was shown to inhibit lung and esophageal carcinogenesis [20]. The present study investigated the inhibitory effect of tea preparations against NMBA-induced esophageal tumorigenesis. We previously reported the modulation of CYP and phase II enzymes in rats treated with various green tea solutions [21]. In that study hepatic CYP1A activity was found to be significantly increased in the rats pre-treated with tea solution. CYP1A1 is primarily involved in metabolism of polycyclic aromatic hydrocarbons, whereas CYP1A2 preferentially metabolizes heterocyclic amines, arylamines and aflatoxin B1. These isoforms may play critical roles in the activation of environmental carcinogens and an increase in the rat hepatic CYP1A activity with tea solutions suggested that the tea components may induce the activity of CYP1A1 in rat liver. These results are consistent with the work previously reported by other investigators [22]. Treatment of rats with green and black teas caused significant induction of CYP1A2, 1A1, 2B, and 4A1 [23]. The present study focuses on relating these changes in the enzyme modulation to the chemopreventive action of tea components against esophageal tumorigenesis in rats. Dandelion (Taraxacum Officinale) which belongs to the Compositae family is a common herb found in most gardens. The roots of this plant have been used in Chinese herbal medicine for the treatment of boils, sores, mastitis, lymphadenitis, inflammation of the eye, sore throat, lung and breast abscesses, acute appendicitis, jaundice, urinary tract infections, etc [24]. Since CYP2E is believed to play a key role in the bioactivation of nitrosamines it was anticipated that there is a negative association of the likelihood of tumorigenesis in rats given Dandelion tea extracts while being treated with a nitrosamine carcinogen, NMBA. We previously reported that treatment with dandelion tea modulated the hepatic drug metabolizing enzymes in rats [25]. In the present study we also included dandelion tea along with other green and black teas to investigate if it was effective in preventing esophageal tumorigenesis in rats and also to understand the relationship of its chemopreventive effect, if any, with its enzyme modulating capacity. Both green tea and black tea are derived from the same plant, Camelia Sinensis. Green tea is manufactured from fresh leaf while preventing oxidation of polyphenolic components. Black tea manufacturing is carried out to ensure a high degree of enzymatically-catalyzed aerobic oxidation of the polyphenols followed by heating and drying. This will result in varying amounts of polyphenols and also difference in the flavor and taste.
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MATERIALS AND METHODS Reagents and Chemicals N-methyl N-benzyl nitrosamine was purchased from Ash Stevens Inc, Detroit. All tea products were purchased from various healthcare shops and Tasman Tea Extracts in Nelson, New Zealand. (-)Epicatechin (EC) was purchased from Sigma Chemical Co (St Louis, MO, USA. (+)epicatechin (EC), (-)epicatechin gallate (ECG), (-)epigallocatechin (EGC) and (-)epigallocatechin gallate (EGCG) were kindly donated by Dr.Y. Hara, Mitsui Norin Company, Fujieda City, Japan. Treatment of Rats with Tea Solutions Female Wistar rats of 8-9 weeks, weighing 200-250 g were divided into 7 groups (n = 15 each) as follows: positive control, negative control, Green tea extract (GTE 0.5% w/v), New Zealand green tea, Dragon green tea, English Breakfast, and Dandelion herbal tea. Animals were housed individually in hanging steel wire cages at 24°C with a 12-hour light-dark cycle. The animals were fed with commercial rodent diet (R94, Reliance stock food company, Dunedin, New Zealand) and tap water ad libitum. Tea solutions (2% w/v) were prepared fresh each day. To prepare tea solutions, an appropriate amount of tea was weighed into a glass beaker into which the required volume of freshly boiled tap water was poured. Tea treatment was continued for 14 weeks. Induction of Esophageal Tumors After 2 weeks of pre-treatment with tea solutions, all rats except those in the negative control group were dosed through oral gavage with the NMBA solution (dissolved in Olive oil) at a dose of 0.5 mg/kg / twice weekly for 24 doses. Rats in the negative control group received only the corresponding volume of olive oil. At the end of 12 weeks treatment, animals were sacrificed; livers were removed immediately and stored at – 80oC until used. All the internal organs were thoroughly examined for any gross pathology. Whole esophagi were removed carefully and cut open longitudinally and pinned on cards. Esophageal tumor lesions greater than 0.5 mm in diameter were counted under a dissection microscope. Tumors were graded into 4 size ranges (< 0.5 mm, 0.5-1, 1-2, > 2 mm) while being counted. Determination of CYP and Phase II Enzyme Activities Hepatic microsomes prepared from rats in the experiment were used to determine activities of CYP1A1/ CYP1A2, CYP3A, CYP2D, CYP2E, glucuronyl transferase and glutathione-S-transferase enzymes using appropriate assay methods as previously reported. Liver microsomes and cytosols from rat livers were prepared by differential ultracentrifugation according to procedures described previously [26]. Microsomal protein and Cytochrome P450 (CYP) content was determined by the method of Lowry et al. [27].
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sample was performed using a mobile phase consisting of acetonitrile-HPLC water mixture (16:84, v/v) containing 1% glacial acetic acid and 60 mM diethylamine, finally adjusted to pH 3.0 with orthophosphoric acid. Determination of CYP2D Activity Microsomal incubations of midazolam were carried out as described by Wrighton and Ring [31] with slight modifications which included use of a C18 -Nucleosil (5 m, 4.6 mm I.D. x 15 cm) HPLC column (Phenomenex, Torrance, CA, USA) and mobile phase of 10 mM potassium phosphate buffer (pH 7.4)/acetonitrile/methanol (40:20:10, v/v/v) delivered at 1 ml/min with ultraviolet detection at 220 nm. Determination of Glucuronosyl Transferase Activity Microsomal glucuronosyl transferase activity was determined using two different substrates, i.e. p-nitrophenol and 2-aminophenol. UDP-glucuronyl transferase activity towards p-nitrophenol was determined using the spectrophotometric method described by Luquita et al. (1994) with slight modifications. p-Nitrophenol (800 μM) was used as a substrate for the assay. A membrane perturbant Triton –X 100 (0.05 mg/protein) and a -glucuronidase inhibitor, i.e. D-saccharic acid lactone (0.5 mM) were systematically incorporated in to the reaction medium. The UDP-glucuronyl transferase activity towards 2-aminophenol was determined using a method described earlier [32]. Determination of Glutathione-S-transferase Activity Fig. (1). (a) Esophageal sections from a rat in the positive control group showing (a) papilloma and (b) basal cell hyperplasia (X 40).
Determination of CYP1A1/CYP1A2 Activity Phenacetin-O-deethylase activity (CYP1A1/1A2) of liver microsomes was determined using HPLC by measurement of the formation of metabolite, namely, paracetamol from phenacetin as previously described [28].The incubation mixtures (1 ml) comprised the substrate, phenacetin (5 M) for 1A2 or 300 M for 1A1, 0.2 mg/ml of rat liver microsomal protein and 1 mM NADPH in phosphate buffer (0.067 M, pH 7.4). Determination of CYP3A Activity CYP3A activity was determined from the metabolite formation of quinine 3-hydroxyquinine from qunine by CYP3A using a reversed HPLC method previously described [29]. The fluorescence detector was set at excitation and emission wavelengths of 350 and 450 nm, respectively. Chromatograms were recorded on a Hitachi D-2500 integrator (Hitachi, Kyoto, Japan). Chromatographic separations were carried out at room temperature. Determination of CYP2E Activity CYP2E1 activity was determined by method described by Tassaneeyakul [30] with some modifications in HPLC column used. The HPLC column was 150 mm x 4.6 mm ID packed with a reversed phase C18 material (5 μm Nucleosil®, Macherey–Nagel, Duren, Germany). Analysis of the
Hepatic cytosolic glutathione–S-transferase activity was determined using a spectrophotometric method [33]. This procedure was based on the enzyme-catalyzed condensation of glutathione with the model substrate 1-chloro-2,4dinitrobenzene. Statistical Analysis The SPSS package was used for all statistical analysis. Results are expressed as mean ± standard deviation. Differences in tumor multiplicities among various groups were tested for significance using one way analysis of variance (ANOVA). The Bonferroni procedure for comparison was used and, in all cases, p < 0.05 was considered as the minimum level of statistical significance. Tumor incidence was compared by non-parametric test using the SPSS package. Differences in the enzymatic activities between all the groups were compared using one way ANOVA. RESULTS 14 weeks of tea treatment and 12 weeks of carcinogen administration did not influence the growth rate of rats compared to the negative control group receiving only the vehicle solvent. Neither tea treatment nor NMBA ingestion affected liver weights of rats by the end of 14 weeks. Hepatic CYP content of rats in the positive control group was slightly decreased, when compared to that of negative control. However this was not statistically significant. There were no significant differences in CYP content in the tea-treated rats as compared to the negative control.
Relevance of Drug Metabolizing Enzyme Activity
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Table 1. Effect of Treatment with Tea on Tumor Incidence in Rats Group
No. of Rats
No. of Rats Surviving
No. of Rats with Tumor
Tumor Incidence (%)
Negative Control
15
15
0
0.0
Positive Control
15
15
14
93.3
Green Tea Extract
15
15
12
80.0
New Zealand green tea
15
15
11
73.3
Dragon green tea
15
15
13
86.7
English Breakfast tea
15
14
14
100.0
Dandelion tea
15
15
11
73.3
Table 2. Effect of Treatment with tea on Tumor Multiplicity in Rats Group
Small Tumors (1mm)
Total Tumors /Rat
Tumor Volume (mm3)
Negative Control
Nil
Nil
Nil
Nil
Positive Control
7.9 ± 3.3
2.1 ± 1.4
10.1 ± 4.2
52.5 ± 31.9
Green Tea Extract
5.0 ± 3.4
0.7 ± 1.1 *
5.7 ± 4.4 *
14.8 ± 21.9 *
New Zealand green tea
4.7 ± 3.4
0.6 ± 0.8 *
5.3 ± 4.0 *
15.1 ± 20.5 *
Dragon green tea
6.5 ± 3.3
1.7 ± 1.4
8.2 ± 4.1
37.9 ± 35.0
Eng. breakfast tea
7.0 ± 2.8
1.2 ± 1.3
8.3 ± 3.5
31.8 ± 28.2
Dandelion tea
5.1 ± 3.9
0.53 ± 0.9 *
5.6 ± 4.7 *
14.3 ± 21.9 *
Results are expressed as Mean ± s.d * significantly different from positive control (p < 0.05)
Tumor Incidence The number of rats which developed tumor lesions in their esophagi was 14, 12, 11, 13, 14, and 11 in the positive control, Green Tea Extract, New Zealand green tea, Dragon green tea, English Breakfast tea, and Dandelion tea groups, respectively. These figures corresponded to 93.3%, 80%, 73.3%, 86.7%, 100% and 73.3% incidence of tumor respectively in the above groups (Table 1). However, these were not of any statistical significance in comparison to the positive control (p > 0.05). Rats in the negative control group, which received only the vehicles (water and olive oil), did not develop tumors. One rat in the English breakfast black tea group had to be euthanized 10 weeks after the first NMBA administration as its health worsened with difficulties in breathing and feedings. All other 104 rats survived the entire period of experiment. Tumor Analysis Tumor lesions in the esophagi were seen as irregular, multiple, flat and exophytic swellings arising from the esophageal epithelium, mostly ovoid in shape and oriented along the axis of esophagus. No ulceration was noted in any part of the esophagus. Sections of esophagi were histologically examined for any presence or absence of hyperkeratosis, hyperplasia, benign papilloma, and dysplasia, carcinoma
in situ and invasive carcinoma. Most of the abnormal esophagi showed hyperkeratosis and papillomata. There was basal cell hyperplasia and dysplasia while none of them had carcinoma in situ or invasive carcinoma. Effect on Tumor Development The positive control group showed the maximum number of tumors / rat with an average of 10.1 ± 4.2 followed by English Breakfast (8.3 ± 3.5), Dragon tea (8.2 ± 4.1), Green Tea Extract (5.7 ± 4.4), Dandelion tea (5.6 ± 4.7) and New Zealand green tea (5.3 ± 4) groups respectively (Table 2) This decrease in tumor multiplicity exhibited in rats receiving tea solutions or Green Tea Extract was statistically significant in Green Tea Extract, New Zealand green tea and Dandelion tea (p < 0.05) groups. The number of larger tumors (>1mm diameter) showed the following descending order: Positive control (2.1 ± 1.4) > Dragon tea (1.7 ± 1.4) > English Breakfast tea (1.2 ± 1.2 > Green Tea Extract (0.73 ± 1.1) > New Zealand green tea (0.6 ± 0.8) > Dandelion tea (0.53 ± 0.92) (Table 3). All the treatment groups also demonstrated a decreasing trend in the number of newly formed smaller tumors (with diameters less than 1mm). The apparent tumor volume of the tumors were calculated using the largest diameter of the tumor lesions and cal-
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Table 3. Effect of Tea Consumption on CYP and Phase II Enzyme Activities in NMBA-induced Tumor Rats
Group
CYP1A1 (nmol/mg/min)
CYP1A2 (nmol/mg/min)
CYP2E (nmol/mg/min)
CYP3A (nmol/mg/min)
UDPGT (nmol/mg/min)
GST (nmol/mg/min)
Negative Control
0.70 ± 0.16
0.08 ± 0.02
0.67 ± 0.09
0.18±0.05
5.09 ± 2.7
0.21 ± 0.03
Positive Control
0.57 ± 0.13 *
0.06 ± 0.01 *
0.49 ± 0.10 *
0.14 ± 0.04 *
3.69 ± 1.97
0.19 ± 0.03
Green Tea Extract
0.73 ± 0.13
0.07±0.01
0.64 ± 0.10
0.16 ± 0.08
5.36 ± 0.92
0.21 ± 0.03
New Zealand GT
0.77 ± 0.15
0.07 ± 0.01
0.61 ± 0.14
0.15 ± 0.03
5.20 ± 1.79
0.22 ± 0.03
Dragon GT
0.66 ± 0.14
0.07 ± 0.02
0.61 ± 0.11
0.17 ± 0.04
5.76 ± 2.49
0.21 ± 0.03
Eng Breakfast BT
0.71 ± 0.14
0.07 ± 0.01
0.61 ± 0.12
0.17 ± 0.05
5.02 ± 4.58
0.21 ± 0.04
Dandelion Herbal
0.69 ± 0.12
0.07 ± 0.01
0.60 ± 0.11
0.17 ± 0.04
5.27 ± 2.27
0.22 ± 0.03
* significantly different from negative control (p < 0.05) Abbreviation: UDPGT = UDP-glucuronyl transferase activity toward p-nitrophenol, GST = Glutathione-S-transferase.
culating the apparent volume as 4/3 r3 where r is the radius. There was a statistically significant reduction (p < 0.05) in tumor volumes in three groups, namely GTE, New Zealand green tea and Dandelion tea. CYP1A1/ 1A2 The activity of phenacetin-O-deethylase (low affinity, high Km isozyme CYP1A1) was decreased significantly (p < 0.05) in the positive control group when compared to negative control group; i.e 81 % of negative control group. In the other tea-treated groups this activity did not show any significant change. The positive control group also showed a significant decrease (p
