Citrus Limonoids and Flavonoids: Enhancement of Phase II

Chapter 10

Citrus Limonoids and Flavonoids: Enhancement of Phase II Detoxification Enzymes and Their Potential in Chemoprevention

Downloaded by UNIV OF ARIZONA on June 12, 2013 | http://pubs.acs.org Publication Date: June 9, 2006 | doi: 10.1021/bk-2006-0936.ch010

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Hassan Ahmad1,2, Jiaxing Li2, Maria Poison , Kimberly Mackie , Walter Quiroga , and Bhimanagouda S. Patil 1

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Department of Chemistry, University of Texas-Pan American, 1201 West University Drive, Edinburg, TX 78539 Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Mail Code 2119, Texas A&M University, College Station, TX 77843

Enhancement of the detoxification pathway for the elimination of toxic electrophiles by the Phase-II enzymes (Glutathione S— transferase, GST; and NAD(P)H:quinine reductase, QR) is one means of cellular protection that may be anticarcinogenic. Effects of citrus limonoids (nomilin, limonin, aglycone mixtures) and flavonoids (hesperidin, naringin, and crude flavonoids mixture) were investigated in various tissues of mice. Three doses of 20 mg of citrus limonoids and flavonoids were given to 10-week-old female A/JOlaHsd mice over a period of one week. Limonoid aglycones were more effective inducers of GST activity than their respective glucosides. Nomilin and an aglycone mixture were the most potent inducers of GST. Citrus flavonoids showed the most significant induction of GST and QR in the stomach. The GST and QR induction by the limonoids and flavonoids appeared to be tissue-specific and related to the structures of the compounds. The enhanced level of phase-II enzymes by citrus flavonoids suggests their importance in chemoprevention.

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© 2006 American Chemical Society

In Potential Health Benefits of Citrus; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction For over a quarter century, a consensus supported by a growing number of epidemiologic studies suggests that consumption of diets rich in fruits and vegetables is associated with lower risks of developing various types of malignant cancers (1-6). Multiple mechanisms are undoubtedly involved in the protective effects of diets rich in fruits and vegetables. It is difficult to identify the specific, individual contributions from various components of the diet to overall cancer risk reduction, and the issue is only confounded by the potential synergism between the constituents. Nonetheless, there is much evidence that specific compounds from fruits and vegetables such as flavonoids, carotenoids and limonoids among others protect against chemical carcinogenesis. A compelling body of evidence now indicates that substantial protection against chemical carcinogenesis can be achieved by non-nutrient components of vegetables and fruits capable of inducing enzymes that are involved in carcinogen metabolism. Although the anticarcinogenic effects of many nonnutrient phytochemicals are not entirely understood, an enhanced detoxification of carcinogenic electrophiles by Phase-II detoxification enzymes, such as glutathione S-transferase (GST) and NAD(P)H:quinone reductase (QR), appears to be the single most important component of the mechanisms responsible for their anticarcinogenic activity (7-9). Both of these phase II enzymes are highly inducible in mammals (10,11). Thus, induction of Phase II enzymes by naturally occurring or synthetic agents represents a promising strategy for cancer prevention. GSTs are ubiquitous enzymes that protect cells by detoxification of carcinogens by catalytic and non-catalytic binding mechanisms. GSTs catalyze the conjugation of tripeptide glutathione (GSH) to a wide variety of endogenous and exogenous chemicals with electrophilic functional groups. These include the products of oxidative stress, pollutants and carcinogens, which upon conjugation with GSH yield water soluble thioesters (12-15). A multitude of GST isoenzymes belonging to various gene families are reported to be present in mammalian tissues (16-18). The most abundant cytosolic mammalian GST isoenzymes are grouped into four major classes, a, ji, n, and 8 (18-20). This classification is based on substrate specificity, primary structure, kinetics, and immunological properties of the isoenzymes (18-20). A large number of carcinogens are known to be metabolized by GST (16,21,22). Numerous studies strongly implicate the induction of GST with enhanced detoxification of carcinogens. For example, l,2-dithiol-3-thione has an anticarcinogenic effect against aflatoxin B induced hepatocarcinogenesis in rats. This is believed to be due to induction of GSTs which exhibit a 4 to 6-fold increase in the specific r



132 activity of "aflatoxin-GSH conjugation" (23). More recent studies, which focus on the search for biomarkers of cancer risk and prevention, strongly suggest that individuals deficient in protection rendered by Phase-II detoxification enzymes, especially GSTs, are at an increased risk of cancer (24-28). Quinonides are formed as intermediates during Phase-I metabolism of various carcinogens. QR mediates the two-electron reduction of quinones leading to the formation of relatively stable hydroquinones, through redoxcycling. This shields against their ability to generate oxidative stress. Induction of QR, therefore, is considered important in diminishing the toxicity of quinone metabolites. The hydroquinone intermediates that are produced by QR are susceptible to conjugation with glucuronic acid by UDP-glucuronosyl transferases to produce water soluble glucoronyl conjugates (29,30). It is, therefore, believed that the induction of NAD(P)H:quinone reductase is also an important component in the overall mechanisms by which various Phase-II enzyme-inducing agents protect from chemical carcinogenesis, especially those mediated by quinonide (31-34). Phase I enzymes catalyze reactions largely through oxidation and reduction. Phase II enzymes promote conjugation of toxic Phase I enzyme-products with endogenous ligands such as GSH, amino and glucuronic acids. This sequence of reactions usually leads to more soluble and easily excretable products. Monofunctional inducers are, therefore, more desirable and preferred as the agents for achieving chemoprevention (24,31,32). Chemoprotection can be achieved by preferentially raising the levels of Phase II enzymes, and many inducers of this type are already present in the human diet. Numerous studies have shown that administration of phenolic antioxidants, e.g. flavonoids, produce marked elevations of GSTs and other Phase II enzymes in many tissues and cell lines (35-39). Limonoids are a group of triterpenoids. Their general structure contains a furan ring attached to CI, oxygen-containing functional groups, a 14,15 epoxide group and an oxymethylene at C19 (See Figure 1). In citrus, limonin and nomilin are the most prevalent limonoids. Citrus limonoids are known to possess anticarcinogenic activity in various organs such as the forestomach (40), lung (41), colon (42), oral cavity (43-45), skin (46) and breast cancer cell lines in vitro (47). Our recent studies have demonstrated the differential inhibition of the proliferation of human cancer cells by citrus limonoids (48). In another recent investigation, limonoid glucosides at micromolar concentrations are lethal to neuroblastoma cells in culture (49). In another study, we have demonstrated that citrus flavonoids have stronger antioxidant activity compared to limonoids (50). Since citrus is one of the major fruits of the human diet, evaluating the potential of phytochemicals in chemoprevention from citrus fruits is of particular interest. Here, we report the effect of citrus flavonoids and limonoids on the induction of phase II enzymes (Fig. 1).



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Figure 1. Structures of two citrus limonoids: (a) limonin (b) nomilin and (c) a general flavanone: Hesperidin 3 '=OH, Naringin 3 '=H; Hesperidin 4 '=OMe, Naringin 4'=0H


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Materials and Methods Most of the reagents used in this study were obtained from Sigma Chemicals Co. St. Louis, MO. The citrus flavonoids and limonoids were isolated by using established procedures (51-53). The structures of the purified compounds were analyzed and verified with HPLC-ESI/MS.


Treatment of Animals Female Albino A/JOlaHsd mice, 5-6 weeks old, were purchased from Harlan Sprague Dawley, Inc., Indianapolis, IN. Guidelines approved by the Institutional Review Committee for animal use and care were followed. The animals were acclimatized at our animal care facility for two weeks prior to start of the experiments. The animals were housed under controlled lightning conditions (12 hours light and 12 hours dark cycle) and were fed semi-purified diet (AIN-76, ICN Laboratories, Cleveland, OH) and water ad libitum. The control and experimental groups, each containing five mice, were formed. The experimental groups received three doses of citrus flavonoids, limonoids, or mixtures in corn oil, administered by gavage every other day, while the control mice received only appropriate amounts of corn oil. Each dose consisted of 20 mg of flavonoid, limonoid or mixtures dissolved in 0.25 mL of corn oil. The animals were sacrificed by cervical dislocation forty-eight hours after the last dose. The organs were harvested and rinsed with cold phosphate-buffered saline. A small amount of excised tissue from each group was used for enzyme activity assays, while the remaining tissues were frozen at -20°C until used. Details of these procedures are published elsewhere (54).

Sample preparation and Enzyme Activity Organ homogenates (10% (w/v)) were prepared from each group in 10 mM phosphate buffer, pH 7.0, containing 1.4 mM /?-mercaptoethanol. The homogenization was carried out with a Potter-Elvehjem type homogenizer. The homogenates were centrifuged at 22,000 x g for 45 min in a Beckman T30 Avanti centrifuge. GST activity against l-chloro-2,4-dinitrobenzene (CDNB) was determined as described by Habig et al (55) and QR activity against 2,6dichlorophenolindophenol (DCPIP) was determined as described by Benson et al (56). The Beckman-DU640 UV/Visible spectrophotometer equipped with enzyme kinetics software was programmed to calculate the enzyme units. One unit of enzyme activity is defined as the amount of enzyme that used 1 /miole of


135 substrate per minute at 25°C. Protein concentration was determined by the dye binding method of Bradford (57) using bovine serum albumin as a standard. Resutls are presented as means ± S.D. of n=4-5 per group and the student's t-test was used to assess the significance of the data.

Results and Discussion


Effect of Flavonoids on GST Activity GST activity was significantly (p

Citrus Limonoids and Flavonoids: Enhancement of Phase II

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