Food Phytochemicals for Cancer Prevention I - American Chemical

Chapter 16

Inhibition of Chemically Induced Carcinogenesis by Citrus Limonoids 1,2

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Luke Κ. T. Lam , Jilun Zhang , Shin Hasegawa , and Herman A. J . Schut

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 9, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch016

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Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455 L K T Laboratories Inc., Minneapolis, MN 55413 Fruit and Vegetable Chemistry Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 263 South Chester Avenue, Pasadena, CA 91106 Department of Pathology, Medical College of Ohio, Toledo, OH 43614 2

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Limonin and nomilin are two of the bitter principles found in citrus fruits such as lemon, lime, orange and grapefruit. Both citrus limon oids have been found to induce increased activity of the detoxifying enzyme glutathione S-transferase. The increased enzyme activity was correlated with the ability of these compounds to inhibit chemically induced carcinogenesis in laboratory animals. Admini stration of nomilin by gavage to ICR/Ha mice reduced the incidence and number of forestomach tumors per mouse induced by benzo[a]pyrene (BP). Addition of nomilin and limonin to the diet at various concentrations inhibited BP-induced lung tumor formation in A / J mice. The inhibition of lung tumors was correlated with inhibition of the formation of BP-DNA adducts in the lung. Topical application of the limonoids was found to inhibit both the initiation and the promotion phases of carcinogenesis in the skin of SENCAR mice. Nomilin appeared to be more effective during the initiation stage while limonin was more potent as an inhibitor during the promotion phase of carcinogenesis. These findings suggest citrus limonoids may be useful as cancer chemopreventive agents. Citrus limonoids are one of the two main classes of compounds responsible for the bitter taste in citrus fruits. The most prevalent limonoids are limonin and nomilin. They are present in Rutaceous plants, which include the commonly eaten fruits lemon, lime, orange, and grapefruit (7). The bitterness due to limonin is of major concern and economic impact (2). The characteristic of this class of compounds is a furan moiety substituted at the 3 position (3). The point of substitution is the D-ring lactone of a highly oxygenated triterpene. Recently, it has been determined that citrus limonoids have certain biological activities that may allow their use as chemo preventive agents. 0097-6156/94/0546-0209$06.00/0 © 1994 American Chemical Society

Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES


Glutathione 5-transferase Induction Many furan-containing natural products have been shown to induce the detoxifying enzyme system, glutathione S-transferase (GST) (4-7). The GST system is a major detoxifying enzyme system that catalyzes the conjugation of glutathione with electrophiles, including activated carcinogens (8). The conjugates are generally less reactive and more water soluble, facilitating their excretion. A n increase of GST activity by a substance, therefore, usually enhances the protection mechanism against the noxious effects of xenobiotics, including carcinogens. Many chemicals that are GST inducers have been found to inhibit chemically induced carcinogenesis. The structures of the naturally occurring furanoids that have been found to induce GST activity range from the simple 2-alkyl substituted compound, 2-nheptyl furan, and the sulfur analog, 2-n-butyl thiophene, formed during the roasting of meat (7), to the more complex molecules such as kahweol and cafestol, isolated from green coffee beans (4), and salannin, identified in the seed of the neem tree of India (9). The presence of the furan moiety appears to be essential for enzyme induction. When the furan ring in cafestol is saturated by hydrogénation, its activity as GST enzyme inducer is lost (5). In the case of limonin and nomilin, the triterpene structure also plays a part in the relative GST inducing activity of these compounds. The activity of limonin was found to be much less than that of nomilin. The A ring in limonin is orthogonal to the plane of the molecule. On the other hand, if the A or A ' ring is open such that the A ring is no longer above the plane of the molecule, as in the case of ichangin and isoobacunoic acid, the inducing activity is higher than that of limonin (6). Table I shows the effects of limonin and nomilin on GST activity in the liver and small intestinal mucosa of mice. Butylated hydroxyanisole, a known inducer of GST (10), was used as a positive marker in this experiment. The test compounds were either dissolved or suspended in cottonseed oil and were given by gavage at the indicated doses once every other day over a period of seven days. On the eighth day the animals were sacrificed and the tissues removed. GST assay was performed using the universal substrate chlorodinitrobenzene (CDNB) (77). B H A in this experiment induced GST activity to three times the control level, which is comparable to previous observations at this dose level (10). Limonin was found to be inactive in the liver. Nomilin, on the other hand, was quite active even at the low dose of 5 mg. The relative inducing activity in the small bowel mucosa was higher than that in the liver for both limonin and nomilin. No appreciable elevation of GST activity was detected in the forestomach. Inhibition of Benzo[a]pyrene-induced Forestomach Tumors The high GST inducing activity of the limonoids suggested that they might be chemopreventive agents. The first experiment to test their effectiveness was carried out with female ICR mice. The limonoids were given by gavage three times a week at two dose levels for 4 weeks. Two additional administrations before and one after the carcinogen were given. The carcinogen, benzo[a]pyrene (BP), was given at 1 mg/animal twice a week on the days when the inhibitors were not given. Eighteen weeks after the first dose of BP, the animals were sacrificed. The forestomach was excised and the tumors were counted. The tumors were confirmed histopathologically.



16. LAM ET AL.

Inhibition of Chemically Induced Carcinogenesis

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Table II shows a significant decrease in tumor incidence as a result of B H A and nomilin treatment. B H A was used as an inhibitor control because it has been found to reduce forestomach tumor formation in similar experiments. The level of inhibition by B H A in this experiment was similar to that observed previously (72). Nomilin given at 10 mg/dose was found to reduce the number of tumors/animal by greater than 50%. Limonin under these conditions was somewhat less effective (13) . These results indicated that the inhibitory effects of limonin and nomilin follow the same trend as their ability to induce GST activity. Nomilin, a much better inducer of GST than limonin, was more active as an inhibitor of carcinogenesis than limonin. The inability of limonoids to elevate GST activity in the forestomach did not appear to be an important factor in the inhibition of carcinogenesis in this target tissue. A significant enhancement of GST activity in the major detoxifying center, namely the liver, appeared to be positively correlated with the inhibitory potential of limonoids not only in the portal of entry but also at a remote site such as the lung. Inhibition of Benzo[a]pyrene-induced Lung Tumors Besides forestomach tumors, BP also induces lung tumors in laboratory animals (14) . In general, A strain mice are used to test inhibitors that may inhibit B P induced lung tumor formation. In this experiment, the inhibition of BP-induced lung tumorigenesis by dietary limonoids in A/J mice was determined (75). Limonin and nomilin at various concentrations were added to the diet of A/J mice before and during BP administration. The carcinogen was given by gavage at 1 mg/animal twice a week for 4 weeks. Three days after the last dose of BP, the animals were returned to normal lab chow diet. The experiment was terminated 18 weeks after the first dose of BP. The results are shown in Table III. The weight gain of the animals at the end of the experiment was similar for all experimental groups. Under these experimental conditions, 100% of the animals developed pulmonary adenoma with the exception of the 0.27% nomilin group, which had 83% incidence, which was not statistically different from the control group. The number of tumors per animal, however, was significantly reduced for both the limonin and nomilin groups. The reduction was dose dependent. At 0.5%, the limonin group had 6.1 tumors /animal compared to 11.9 in the control group, a 50% reduction. At 0.25%, there were 8.1 tumors/animal, a 32% reduction, and at 0.125% no reduction was obtained. The dosages used for nomilin were half those of limonin because we anticipated it would be more active judging from the previous forestomach tumor experiment (13). At 0.27%, 2.9 tumors/animals were found in this group, compared to 11.9 in the control group, which is an inhibition of 75%. In the 0.135% group, 5.7 tumors/animal were found (a 52% reduction) and no protection was obtained at 0.068%, the lowest dose. At 0.27%, it is estimated that the animals were consuming approximately 10 mg nomilin/day. At this level a small reduction of forestomach tumor was also found which was similar to that found in the previous experiment using ICR mice. Like the inhibition of forestomach tumors, the protection against lung carcinogenesis by limonoids was positively correlated with the induction of GST activity.


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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES

Table I. Effects of Limonin, Nomilin and Β HA on the Activity of GlutathioneS-transferase in the Tissues of Female ICR/Ha Mice

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Compound (dose in mg) Control (—)

13

—

1.15 ±0.19

Forestomach Small Intestine Mucosa GST Test/ GST Test/ activity control activity control 15

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0.33 ± 0.04

—

0.66 ±0.08

—

B H A (7.5)

5.24 ±0.60*** 4.55

1.08 ±0.03*** 3.25

0.84 ±0.18

1.29

Limonin (5) Limonin (10)

1.29 ±0.35 1.24 ±0.23

1.12 1.08

0.45 ± 0.08* 0.45 ± 0.09*

1.33 1.36

0.69 ±0.13 0.60 ±0.12

1.05 0.92

Nomilin (5) Nomilin (10)

2.86 ±0.60** 3.96 ±0.81**

2.48 3.44

1.00 ±0.03*** 3.00 1.39 ±0.15*** 4.17

0.70 ±0.16 0.76 ±0.15

1.07 1.16

C


Liver GST Test/ activity control

SOURCE: Reprinted with permission from ref. 13. Copyright 1989. Significantly different from control, *p

Food Phytochemicals for Cancer Prevention I - American Chemical

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