Modifying effects of dietary capsaicin and rotenone on 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

Takuji Tanaka1,3, Hiroyuki Kohno1, Keiko Sakata2, Yasuhiro Yamada2, Yoshinobu Hirose2, Shigeyuki Sugie2 and Hideki Mori2

1 Department of Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 and
2 Department of Pathology, 40 Tsukasa-machi, Gifu 500-8705, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of dietary administration of capsaicin and rotenone on 4-nitroquinoline 1-oxide (4-NQO)-induced tongue tumorigenesis were investigated in male F344 rats. In pilot studies, gavage with capsaicin and rotenone elevated the phase II enzymes glutathione S-transferase (GST) and quinone reductase (QR), in the liver and tongue. Also, a 10 week period of feeding of 500 p.p.m. capsaicin or rotenone together with 4-NQO exposure inhibited the occurrence of tongue dysplasia. Subsequently, a long-term study was conducted to test the protective effects of both compounds on 4-NQO-induced tongue carcinogenesis. One group was treated with 4-NQO alone (20 p.p.m. in drinking water for 8 weeks) and four other groups received the carcinogen treatment plus diets containing 500 p.p.m. test compounds for 10 weeks (initiation phase) or for 28 weeks (post-initiation phase). At the termination of the study (38 weeks), feeding of rotenone during the initiation phase, but not during the post-initiation phase, was found to significantly reduce the incidence of tongue squamous cell carcinoma (53% vs. 16%, 70% reduction, P b=e 0.0250) and severe dysplasia (80% vs. 42%, 70% reduction, P = 0.028). Capsaicin feeding during either the initiation or promotion phase and rotenone feeding during the promotion phase also reduced the frequency of tongue carcinoma without statistical significance. The treatment with two compounds especially rotenone lowered cell proliferation activity in the tongue, elevated phase II enzymes’ activities of the liver and tongue, and increased the apoptotic index of tongue carcinoma. Although our results suggest that rotenone feeding during the initiation stage prevented 4-NQO-induced tongue carcinoma, chronic intravenous exposure of rotenone reproduces several features of human Parkinson’s disease in rats (Nat. Neurosci., 3, 1301–1306, 2000), suggesting that additional studies to confirm the safety of rotenone are warranted.

Abbreviations: AP-1, activator protein 1; CDNB, 1-chloro-2,4-dinitrobenzene; COX, cyclooxygenase; DCNB, 1,2-dichloro-4-nitrobenzene; DMBA, 7,12-dimethylbenz[a]anthracene; GST, glutathione S-transferase; NF-{kappa}B, nuclear factor-kappa B; 4-NQO, 4-nitroquinoline 1-oxide; ODC, ornithine decarboxylase; PCNA, proliferating cell nuclear antigen; QR, quinone reductase; ssDNA, single stranded DNA


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oral and pharyngeal cancers account for <2% of all cancer-related deaths and >2% of all new cancer diagnoses (1). Risk factors including lifestyle, viruses, dietary, and genetic changes are associated with oral cancer (2,3). Oral cancer progresses from hyperplastic epithelial lesions through dysplasia to invasive carcinoma. The concept of `field cancerization’ with molecular alterations can be applied to oral cavity tumorigenesis (4). Despite recent surgical advances, the survival of patients with oral carcinoma remains poor: 30–40% of patients with oral carcinoma have 5 years’ survival rate (5). This short survival time might be caused, mostly, due to late detection of this malignancy. Public awareness of oral carcinoma compared with other cancers is low and this may contribute to delay in diagnosis (6). Also, significant improvement in treatment and prognosis will depend on more detailed understanding of the multi-step process leading to cancer development (7). In this context, active primary prevention such as chemoprevention against carcinoma development is now important. A number of studies are currently directed at identifying possible chemopreventive agents (8). A number of possible chemopreventive agents that are present in edible plants, including fruits and vegetables, against tongue carcinogenesis have been reported (9). Such chemopreventors possess anti-proliferation, anti-inflammatory, and antioxidant effects.

The most frequently used animal models in oral cancer research studies have been the hamster buccal pouch model, rat, and less frequently, mouse (7,10–13). A fat-soluble 7,12-dimethylbenz[a]anthracene (DMBA) and a water-soluble 4-nitroquinoline 1-oxide (4-NQO) are the most frequently used carcinogens in these studies. One of the most important routes of oral exposure to carcinogens is through food and liquid containing different levels of water-soluble carcinogens. Since 4-NQO is water-soluble, it is well suited in examining the role of xenobiotics in experimental oral carcinogenesis. Topical application of 4-NQO and administration of 4-NQO in drinking water both induce premalignant and malignant transformation in the rat oral cavity. This results in papilloma and invasive squamous cell carcinoma, resembling the clinical and histologic changes observed in these neoplasms in humans (11,14,15). Cell proliferation plays an important role in multistage carcinogenesis with multiple genetic changes (16). Therefore, control of cell proliferation is important for cancer prevention (17).

Capsaicin (8-methyl-n-vanillyl-6-nonenamide, Figure 1aGo) is a homovanillic acid derivative and the principal pungent and irritating constituent of Capsicum fruits (Solanaceae) such as red pepper (Capsicum annuum L.). The content of capsaicin in C.annuum is ~0.02% in fresh fruit and 0.5–1.0% in dried ripe fruit (18). Capsaicin is consumed as a food additive throughout the world, particularly in South-east Asia and Latin-American countries. In Korea, average daily per capita consumption of capsaicin may reach 50 mg (19). Excessive intake of hot peppers containing capsaicin may be a risk factor for several gastrointestinal lesions including gastric ulcer and cancer. However, some studies suggested that capsaicin may have a beneficial effect on human peptic ulcer and certain types of cancer (18,20,21). In animal carcinogenesis, capsaicin could inhibit cancer development in multiple organs such as stomach, lung, and liver (18).



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Fig. 1. Chemical structures of (a) capsaicin and (b) rotenone. Note the vanillyl-like moiety on each molecule, which is essential for biological activity of capsaicin and rotenone.

 
A naturally occurring pesticide derived from Derris and Lonchorcarpus species root and bark, rotenone (Figure lbGo) is relatively harmless for mammals, especially after oral administration (22). Rotenoids including rotenone and deguelin are active ingredients of botanical insecticides used for at least 150 years to control crop pests. They have been used even longer as fish poisons by native tribes to obtain food in South America and East Africa and more recently in fish management to achieve the desired balance of species. The acute toxicity of rotenone to insects, fish, and mammals is attributable to inhibition of mitochondrial NADH:ubiquinone oxidoreductase activity as the primary target. Rotenoids are known not only as toxicants but also as candidate chemopreventive agents against mouse liver tumors (23), rat mammary tumors (24), and mouse skin tumors (25). Also, rotenoids could inhibit cell proliferation induced by a peroxisome proliferator in mouse liver. Deguelin and its derivatives can inhibit phorbol ester-induced ornithine decarboxylase (ODC) activity (26,27). We have recently found that capsaicin and rotenone could inhibit chemically induced colon tumorigenesis in rats (28).

Apoptosis induction is one of the important characteristics of candidate cancer chemopreventive agents (29). Capsaicin and rotenone have structural similarity of the vanillyl-like moiety essential for biological activity (Figure 1Go) (30). Both compounds have been shown to induce apoptosis (31,32). Therefore, in the present study, three experiments were conducted to investigate the modifying effects of capsaicin and rotenone on tongue tumorigenesis induced by 4-NQO in rats. In the first, in vivo short-term pilot experiments were done to assess the effects of capsaicin and rotenone on the development of tongue dysplasia induced by 4-NQO and on the activities of phase II detoxifying enzymes glutathione S-transferase (GST) and quinone reductase (QR) in the liver and tongue, since compounds that elevate phase II enzymes’ activities are known to be possible chemopreventive agents against cancer development (33,34). Subsequently, a long-term bioassay was performed to confirm and evaluate the preventive effects of dietary capsaicin and rotenone on 4-NQO-induced tongue carcinogenesis in rats. The effects of dietary capsaicin and rotenone on the cell proliferation activity of tongue carcinoma were also assessed by measuring proliferating cell nuclear antigen (PCNA)-positive index for cell proliferation activity and by counting single stranded DNA (ssDNA) for apoptotic nuclei. In addition, polyamine levels of tongue mucosa were assayed since polyamine metabolism is one of the targets for cancer chemoprevention (35).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals, diets, and chemicals
A total of 170 male F344 rats, 4 weeks old (Shizuoka Laboratory Animal Center, Shizuoka, Japan) were used. All animals were housed in wire cages (three or four rats per cage) with free access to drinking water and basal diet (CE-2; CLEA Japan, Tokyo, Japan), under controlled conditions of humidity (50 ± 10%), lighting (12 h light/dark cycle) and temperature (23 ± 2°C). They were quarantined for 7 days and randomized into experimental and control groups for three experiments (dysplasia bioassay, GST and QR assay, and a long-term bioassay). 4-NQO (CAS 56-57-5; 98% pure) was obtained from Wako Pure Chemical Ind. (Osaka, Japan). 4-NQO solution (20 p.p.m.) was given to rats to induce tongue preneoplastic and neoplastic lesions. Powdered CE-2 diet (342.2 cal/100 g) was used as the basal diet throughout the study. Capsaicin and rotenone purchased from SIGMA-ALDRICH Chemical Co., Tokyo, Japan were mixed in the powdered basal diet CE-2 at a concentration of 500 p.p.m. (w/w). These experimental diets were prepared on a weekly basis and stored in a cold room (<4°C) until use.

Pilot studies
Thirty rats aged 5 weeks were used to test the modifying effect of capsaicin or rotenone on the development of tongue dysplasia induced by 4-NQO (a dysplasia bioassay). Animals were given 4-NQO (20 p.p.m. in drinking water) with or without capsaicin or rotenone (500 p.p.m.) in diet for 10 weeks. Remaining animals (five rats) served as untreated control. All rats were killed 10 weeks after the start, and the incidence and multiplicity of tongue dysplasia was determined on hematoxylin and eosin-stained sections. In the next pilot study, 40 male rats aged 5 weeks were used for a GST and QR assay. They were gavaged with capsaicin or rotenone at a dose level of 0, 40, 200, or 400 mg/kg body weight in 0.5 ml of 5% gum arabic (SIGMA-ALDRICH Chemical Co.) for 5 consecutive days. All rats were killed by decapitation 30 min after the last gavage. At killing, the livers and tongues were excised immediately. The livers were perfused with PBS (pH 7.4) to remove blood and minced into small pieces. The tongue was slit longitudinally and washed with PBS (pH 7.4). The tongue mucosa was collected by scraping the mucosal surface using a stainless steel disposable microtome knife (S35, Feather Safety Razor Co., Seki, Japan). Aliquots of minced liver and of mucosal scrapings were processed for cytosolic fraction. The activities of GST and QR were determined using 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) for GST and NADH/menadione for QR as substrates, as described previously (36–38). All spectrographic assays were based on absorption at 340 nm and all samples were measured in triplicate. One unit of enzyme activity is defined as the amount of enzyme catalyzing the conversion of 1 µmol of substrate to product per min at 25°C. Cytosolic protein concentrations were determined by the Bradford method (39) using bovine serum albumin as the standard.

A long-term bioassay
For the long-term study, a total of 105 rats were randomly divided into eight groups (Figure 2Go). Groups 1–5 received 4-NQO solution (20 p.p.m.) as drinking water for 8 weeks. Rats in groups 2 and 3 were fed diets containing 500 p.p.m. capsaicin and rotenone for 10 weeks, respectively, commencing one week before the 4-NQO exposure. Groups 4 and 5 were fed the diet mixed with 500 p.p.m. capsaicin and rotenone, respectively, for 28 weeks of the post-initiation phase, starting 1 week after the cessation of 4-NQO treatment. Groups 6 and 7 did not receive 4-NQO and were fed diets mixed with 500 p.p.m. capsaicin and rotenone, respectively, for the duration of the study (38 weeks). Group 8 served as an untreated control. All rats were carefully observed daily, weighed weekly until they reached 14 weeks of age and every 4 weeks thereafter. Consumption of the experimental diets was also recorded to estimate the intake of test compounds. The experiment was terminated 38 weeks after the commencement and all animals were killed by an ether overdose to assess the incidences of tongue tumors and preneoplasms. Other organs were also carefully inspected for pathological lesions. The tongues of all rats were longitudinally cut into halves for histological immunohistochemical or biochemical examinations. Squamous cell carcinomas were stained for immunohistochemistry of PCNA and ssDNA and the tongues without macroscopic lesions were used for assays of tissue polyamine content and GST and QR activities. For histological examination, tissues and gross lesions were fixed in 10% buffered formalin, embedded in paraffin blocks and stained with hematoxylin and eosin. Tongue lesions (hyperplasia, dysplasia and neoplasms) were diagnosed according to the criteria described by Kramer et al. (40).



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Fig. 2. Experimental protocol of a long-term bioassay.

 
PCNA and ssDNA immunohistochemistry
For the determination of PCNA-incorporated nuclei, the PCNA-immunohistochemistry was performed according to the method described by Watanabe et al. (41). Apoptotic index was also evaluated by immunohistochemistry for ssDNA (41). This immunohistochemistry was done using a stain system kit DAKO LSAB 2 kit/HRP (DAKO Japan, Kyoto, Japan). A mouse monoclonal antibody against PCNA (1:50 dilution; PC10, DAKO Japan) or a rabbit polyclonal antibody against ssDNA (1:300 dilution; DAKO Japan) was applied to the sections according to the manufacturer’s protocol. All incubation steps were carried out for 15 min at 37°C. The chromogen used was 3,3'-diaminobenzidine tetrahydrochloride. Slides were lightly counterstained with Meyer’s hematoxylin for 1 min, dehydrated, and coverslipped. Negative controls were incubated by substituting the primary antibodies with non-immune mouse or rabbit serum. Slides were subsequently reviewed in a blinded fashion by the study pathologist (T.T.). The PCNA and apoptotic indices of squamous cell carcinomas in groups 1–5 and normal appearing squamous epithelium of groups 6–8 (three rats of each group) were determined by counting the number of positive nuclei among at least 200 cell nuclei in the lesion, and were indicated as percentages.

Polyamine levels
Scraped tongue mucosa was stored at –70°C until measured. Proteins were extracted from the mucosa, and then tissue polyamine levels in the extraction were determined by the methods described by Koide et al. (42).

Statistical methods
One-factor ANOVA, the Kruskal–Wallis test or Fisher’s exact probability test was used for statistical analyses. A value of P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pilot studies
At the end of a dysplasia bioassay, treatment of capsaicin did not affect body weight gain. The mean body weights of rats given 4-NQO + 500 p.p.m. rotenone (208 ± 9 g, P < 0.001) and those given 500 p.p.m. rotenone alone (204 ± 21 g, P < 0.001) were significantly lower than that of 4-NQO alone (293 ± 10 g) and untreated controls (281 ±10 g), respectively. The mean multiplicity (no./cm) of tongue dysplasia in rats treated with 4-NQO + 500 p.p.m. capsaicin (0.32 ± 0.10) was significantly smaller than that of 4-NQO alone (P < 0.05). The incidence and mean multiplicity (no./cm) of tongue dysplasia in rats given 4-NQO + 500 p.p.m. rotenone (20%, P = 0.0238 and 0.27 ± 0.17, P < 0.05) were significantly lower than that in rats treated with 4-NQO alone (100% and 0.48 ± 0.11). In a GST and QR assay, when compared with rats gavaged with solvent alone, dosing of 40, 200, and 400 mg/kg body weight of capsaicin significantly elevated liver GST (GST-CDNB) activity 1.10-fold (P < 0.02), 1.51-fold (P < 0.001), and 1.67-fold (P < 0.001), respectively, and also increased liver QR activity 1.13-fold (P < 0.05), 1.22-fold (P < 0.005), and 1.37-fold (P < 0.02), respectively. Capsaicin administration at 200 and 400 mg/kg body weight exerted a 1.46-fold (P < 0.002) increase and a 1.71-fold (P < 0.002) increase of liver GST-DCNB activity, respectively. Similarly, gavage with 200 and 400 mg/kg body weight of rotenone caused a 1.21-fold increase (P < 0.02) and a 1.37-fold (P < 0.002) increase in liver GST-CDNB activity, respectively. The treatments also increased liver QR activity 1.11-fold (P < 0.05) and by 1.33-fold (P < 0.002), respectively, when compared with those of rats given solvent alone. Dosing of 400 mg/kg body weight of rotenone resulted in 1.63-fold increase (P < 0.02) in liver GST-DCNB activity compared to that of rats given solvent alone. As for tongue GST and QR activities, gavage with 400 mg/kg body weight of capsaicin caused a 1.47 increase in GST-CDNB activity (P < 0.001), but did not significantly affect QR activity. Gavage with 400 mg/kg body weight of rotenone resulted in 1.26-fold increase (P < 0.02) in tongue GST-CDNB activity and in 1.24-fold increase (P < 0.002) in tongue QR activity compared with that in rats gavaged with solvent alone.

General observation in the long-term bioassay
In the long-term bioassay, the daily food intake of groups 2 (4-NQO + 500 p.p.m. capsaicin), 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), 5 (4-NQO -> 500 p.p.m. rotenone), 6 (500 p.p.m. capsaicin), and 7 (500 p.p.m. rotenone) did not significantly differ from that of groups 1 (4-NQO alone) and 8 (untreated), which were fed the basal diet without capsaicin and rotenone (data not shown). In this study, dietary administration of the two test compounds did not cause any clinical signs of toxicity, low survival or poor clinical condition. At the end of the study (week 38), the mean body weights of groups 2 (4-NQO + 500 p.p.m. capsaicin), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone) were significantly smaller than that of group 1 (4-NQO alone) (P < 0.002, P < 0.005 or P < 0.02) as shown in Table IGo. The mean liver weight of group l (4-NQO alone) was significantly greater than that of group 8 (untreated) (P < 0.001). The values of groups 4 (4-NQO -> 500 p.p.m. capsaicin) and 5 (4-NQO -> 500 p.p.m. rotenone) were significantly smaller than group 1 (4-NQO alone) (P < 0.002 or P < 0.001). The mean relative liver weight of group 1 (4-NQO alone) was significantly greater than group 8 (untreated) (P < 0.002). The value of group 5 (4-NQO -> 500 p.p.m. rotenone) was significantly lower than that of group 1 (4-NQO alone) (P < 0.02).


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Table I. Body, liver and relative liver weights of rats treated with 4-NQO and/or 500 p.p.m. capsaicin or rotenone at the end of the study (266 days of growth)
 
Incidence and multiplicity of tongue neoplasms and preneoplasms
Macroscopically, most tongue tumors developed in the posterior portion of the tongue root in rats of group 1 (4-NQO alone), 2 (4-NQO + 500 p.p.m. capsaicin), 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone). They were histologically diagnosed as squamous cell carcinoma or papilloma, with a higher incidence of carcinoma. Animals of groups 6 (500 p.p.m. capsaicin), 7 (500 p.p.m. rotenone), and 8 (untreated) did not have neoplasms in any organs examined. The incidence and multiplicity of tongue neoplasms are shown in Tables IIGo and III, respectively. 4-NQO exposure induced 53% (eight of 15 rats) incidence of tongue squamous cell carcinomas with a multiplicity of 0.53 ± 0.50. The incidences of tongue carcinoma and papilloma in groups 2 (4-NQO + 500 p.p.m. capsaicin), 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone) were smaller than that of group 1 (4-NQO alone) and statistical significance was noted between groups 3 (4-NQO + 500 p.p.m. rotenone) and 1 (P = 0.0250 for carcinoma and P = 0.0461 for papilloma). Total incidence of tongue neoplasms (carcinoma and papilloma) in groups 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone) were significantly smaller than those of group 1 (4-NQO alone) (P = 0.0243, P = 0.0407, and P = 0.0226, respectively). As shown in Table III, the multiplicity of tongue carcinoma in groups 2 (4-NQO + 500 p.p.m. capsaicin), 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone) were smaller than group 1, with a statistical significance between groups 2 (4-NQO + 500 p.p.m. capsaicin) or 5 (4-NQO -> 500 p.p.m. rotenone) and 1 (4-NQO alone) (P < 0.02 and P < 0.05). The value of tongue papilloma in group 3 (4-NQO + 500 p.p.m. rotenone) was significantly smaller than group l (4-NQO alone) (P < 0.05). The multiplicities of tongue tumors (carcinoma + papilloma) in groups 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone) were significantly lower than group 1 (P < 0.01 or P < 0.05). As indicated in Table IVGo, tongue preneoplastic lesions such as squamous cell hyperplasia and dysplasia developed in rats given 4-NQO. The incidences of dysplasia (total incidence of various degrees of dysplasia) and severe dysplasia in group 3 (4-NQO + 500 p.p.m. rotenone) were significantly smaller than group 1 (4-NQO alone) (P = 0.020 and P = 0.028, respectively).


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Table II. Incidence of tongue tumors in rats treated with 4-NQO and/or 500 p.p.m. capsaicin or rotenone
 

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Table IV. Incidence of tongue preneoplastic lesions in rats treated with 4-NQO and/or 500 p.p.m. capsaicin or rotenone
 
PCNA-labeling index and apoptotic index
The data on PCNA-labeling index and apoptotic index in the tongue squamous cell carcinoma are indicated in Table VGo. The mean PCNA-labeling indices of groups 2 (4-NQO + 500 p.p.m. capsaicin) and 5 (4-NQO -> 500 p.p.m. rotenone) were lower than group 1 (4-NQO alone), but the differences did not reach statistical significance. Apoptotic indices evaluated by counting positive nuclei for ssDNA in the tongue squamous cell carcinoma of rats given 4-NQO and capsaicin or rotenone were greater than those treated with 4-NQO alone, but the differences were not statistically significant.


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Table V. PCNA-labeling index and ssDNA-positive index in the tongue squamous carcinoma and squamous epithelium of rats treated with 4-NQO and/or 500 p.p.m. capsaicin or rotenone
 
Polyamine content of the tongue at the end of the study
The results of polyamine assay in tongue mucosa are summarized in Table VIGo. 4-NQO exposure increased total polyamine level (diamine + spermidine + spermine) when compared with group 8, without statistical significance. These values in groups 2 (4-NQO + 500 p.p.m. capsaicin), 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone) were smaller than group 1 and the difference between groups 1 (4-NQO alone) and 3 (4-NQO + 500 p.p.m. rotenone) was statistically significant (P < 0.05).


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Table VI. Tongue polyamine levels of rats treated with 4-NQO and/or 500 p.p.m. capsaicin or rotenone
 
Tongue GST and QR activities at the end of the long-term bioassay
As summarized in Table VIIGo, GST-CDNB activity of the tongue mucosa at the termination of the long-term bioassay did not significantly differ among the groups. GST-DCNB activity in group 5 (4-NQO -> 500 p.p.m. rotenone) was significantly higher than group 1 (4-NQO alone) (P < 0.01). The value of groups 6 (500 p.p.m. capsaicin) was significantly lower than group 8 (untreated) (P < 0.05) and that in group 7 (500 p.p.m. rotenone) was significantly higher than group 8 (untreated) (P < 0.02). As for tongue QR activity, the value in group 1 was significantly greater than group 8 (P < 0.005). When compared with group 1, the values of groups 3 (4-NQO + 500 p.p.m. rotenone), 4 (4-NQO -> 500 p.p.m. capsaicin), and 5 (4-NQO -> 500 p.p.m. rotenone) were significantly smaller (P < 0.05 or P < 0.02).


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Table VII. GST and QR activities in the tongue of rats treated with 4-NQO and/or 500 p.p.m. capsaicin or rotenone
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In pilot studies both test compounds capsaicin and rotenone inhibited tongue preneoplastic lesions dysplasia induced by 4-NQO and elevated phase II enzymes GST and QR in the liver and tongue. These results suggested that the two chemicals may suppress chemically induced tongue carcinogenesis, although rotenone feeding caused retardation of body weight gain. Subsequent long-term experiments confirmed the results of pilot studies. The data on the incidence of tongue neoplasms indicated that rotenone feeding together with or after 4-NQO exposure and capsaicin feeding after 4-NQO administration could inhibit tongue carcinogenesis, the suppression of dietary rotenone during the initiation phase being more effective. Recently we observed similar cancer preventive effects of capsaicin and rotenone in an animal model of colon carcinogenesis (28). Our data suggest that capsaicin and rotenone are possible new dietary preventive agents against tongue cancer development.

The effects of dietary capsaicin and rotenone on 4-NQO-induced tongue dysplasia indicate that this short-term bioassay may be useful for screening agents for chemoprevention of tongue tumorigenesis, as reported (43). The lesion has been suggested to be the premalignancy of chemically induced tongue cancer (9). However, it would probably be prudent to use tumor incidence as the end point for definitive investigations since the lesions are reversible. Our results suggest that capsaicin and rotenone may have blocking and suppressing effects, respectively on 4-NQO-induced tongue tumorigenesis.

Several dietary factors are known to modulate carcinogenesis in humans and rodents (44,45). Also, dietary restriction can modulate both spontaneous and induced carcinogenesis (46,47). In the dysplasia bioassay, feeding of rotenone caused retardation of body weight gain. This may reflect the development of tongue dysplasia. However, in the long-term bioassay retardation of body weight gain by feeding of rotenone during either the initiation or promotion phase was slight. Therefore, chemopreventive effects of capsaicin and rotenone observed in this study may not be due to alteration of body weight gain.

Published reports indicate that capsaicin affects xenobiotic metabolism. Capsaicin inhibits the in vitro metabolism and binding to DNA of aflatoxin B1 (48). When capsaicin was administered orally to hamsters, it inhibited the in vitro {alpha}-carbon hydroxylation of a tobacco-specific carcinogenic nitrosamine 4-(methylnitroszunino)-1-(3-pyridy1)-1-butanone by lung microsomes (49). These may suggest that capsaicin inhibits biotransformation of certain carcinogens by modifying activities of phase I as well as phase II enzymes in liver and other organs. In fact, capsaicin could inhibit P450 2El isoform (50). In this study, we did not measure phase I enzymes, but gavage with capsaicin and rotenone elevated GST and QR in the liver and tongue. Thus, chemopreventive ability observed in the current study may be due to modulation of phase II enzymes (GST and QR). In the long-term bioassay, both test compounds reduced polyamine content of tongue mucosa, suggesting that modulation of cell proliferation may also contribute its chemopreventive ability, although inhibition in PCNA-labeling index and apoptotic index of tongue squamous cell carcinomas were not statistically significant. Recently, inhibitory effects of capsaicin on tumor promoter-induced activation of two well-defined eukaryotic transcription factors, nuclear factor-kappa B (NF-{kappa}B) and activator protein 1 (AP-1) were reported (51). In addition, capsaicin was found to inhibit the expression of cyclooxygenase (COX)-2, which plays an important role in rat tongue tumorigenesis (52) and oxidative gastric injury (53). It would be interesting to investigate the effects of capsaicin on the expression of these factors involved in carcinogenesis with or without inflammation.

In the current study, suppressing effects of rotenone on 4-NQO-induced tongue carcinogenesis was relatively greater than its blocking effects. The mechanism(s) by which rotenone exerts its inhibitory action are not known, but feeding of rotenone reduced PCNA-labeling index, and polyamine level in tongue mucosa. These results may indicate that modulation of cell proliferation by feeding of rotenone accounts in part for its chemopreventive action. Previous reports (26,27) also indicated that inhibition of ODC, which is essential for polyamine biosynthesis and growth in tumor cells (35), by rotenone treatment is responsible for its chemopreventive action. Rotenone can induce apoptosis and G2/M cell cycle arrest in human B cell lymphoma cell line PW (31,32). In this study, feeding of rotenone increased apoptotic index of squamous cell carcinomas, although the increase did not reach statistical significance. Our results suggest possible cancer chemopreventive ability of rotenone, but chronic exposure of rotenone to rats caused nigrostriatal dopaminergic degeneration which resembles human Parkinson’s disease (54). Therefore, chronic toxicity studies of rotenone, especially at a low dose should be done prior to its use as a chemopreventive agent in humans.

In conclusion, the results of our study demonstrate the inhibitory effects of dietary capsaicin and rotenone on 4-NQO-induced tongue carcinogenesis in rats. Further experiments, including pre-clinical efficacy and mechanistic studies, are warranted to fully evaluate these natural compounds for their cancer preventive properties and to understand their mode of action. One advantage of these compounds as chemopreventive agents in human trials is that, unlike synthetic chemopreventive agents, they are naturally occurring compounds that are produced endogenously in plants.


    Notes
 
3 To whom correspondence should be addressed Email: takutt{at}kanazawa-med.ac.jp Back


    Acknowledgments
 
This study was supported in part by the Grant-in Aid for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan; the Grant-in-Aid for Cancer Research (13–15) from the Ministry of Health, Labour and Welfare of Japan; the Grants-in Aid for Scientific Research (no. 13671986) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the grant (H20004) for the Project Research from High-Technology Center of Kanazawa Medical University; and the grant Research (S200l-2) for Promoted Research from Kanazawa Medical University.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 24, 2002; revised May 20, 2002; accepted May 21, 2002.