Potent preventive action of curcumin on radiation-induced initiation of mammary tumorigenesis in rats
Hiroshi Inano1,6,
Makoto Onoda1,
Naoshi Inafuku2,
Megumi Kubota2,
Yasuhiro Kamada2,5,
Toshihiko Osawa3,
Hisae Kobayashi4 and
Katsumi Wakabayashi4
1 First Research Group, National Institute of Radiological Sciences, 9-1 Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555,
2 Ryukyu Bio-Resource Development Co. Ltd, 606-2 Toyohara, Motobu-cho 905-0204, Okinawa,
3 Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya-shi 464-8601 and
4 Institute for Molecular and Cellular Regulation, Gunma University, Showa-machi, Maebashi-shi 371-8512, Japan
 |
Abstract
|
---|
This investigation evaluated the preventive effect of curcumin on radiation-induced tumor initiation in rat mammary glands. Fifty-four female rats were mated and then divided into two groups at day 11 of pregnancy. As the control group, 27 rats were fed a basal diet during the experimental period. As the experimental group, 27 rats were fed a diet containing 1% curcumin between day 11 of pregnancy and parturition (day 23 of pregnancy). All rats of both groups received whole body irradiation with 1.5 Gy
-rays from a 60Co source at day 20 of pregnancy and were then implanted with a diethylstilbestrol pellet 1 month after weaning. A high incidence (70.3%) of mammary tumorigenesis was observed in the control group. The tumor incidence (18.5%) was significantly reduced in the rats fed curcumin during the initiation stage. The appearance of the first palpable tumor was delayed by 6 months in the curcumin-fed group and the average latent period until the appearance of mammary tumors was 2.5 months longer in the curcumin-fed group than in the control group. By histological examination, the proportion of adenocarcinoma (16.7%) in total tumors in the curcumin-fed rats was found to be decreased to half that (32.1%) in the control group. Compared with the control rats, the body weight of rats in the experimental group was decreased slightly by administration of the curcumin diet from day 11 of pregnancy, in spite of a similar intake of diet, but had recovered to the level of the control by the end of the experiment. At the time of irradiation, curcumin did not have any effect on organ weight or on the development and differentiation of mammary glands of pregnant rats. In addition, the serum concentrations of fatty acids, thiobarbituric acid-reactive substances and ovarian and pituitary hormones, except LH, remained at the control level. Also, no change in litter size and body weight of pups born from curcumin-fed rats indicated no toxicity of curcumin. These results suggest that curcumin does not have any side-effects and is an effective agent for chemoprevention acting at the radiation-induced initiation stage of mammary tumorigenesis.
Abbreviations: curcumin, 1,7-bis(4'-hydroxy-3'-methoxyphenyl)-1,6-heptadiene-3,5-dione; DES, diethylstilbesterol; DMBA, 7,12-dimethylbenz [a]anthracene; ER, estrogen receptor; FSH, follicle-stimulating hormone; IL, interleukin; iNOS, inducible nitric oxide synthase; I
B, inhibitory protein
B; LH, luteinizing hormone; LPS, lipopolysaccharide; MDA, malondialdehyde; NF-
B, nuclear factor
B; resveratrol, 3,5,4'-trihydroxy-trans-stilbene; TBARS, thiobarbituric acid-reactive substances; WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid.
 |
Introduction
|
---|
Tumor initiation by radiation in mammary glands is dependent upon cell stage, because estrogen is a direct or indirect sensitizer for tumor initiation by radiation (1,2). Previous studies in our laboratory have demonstrated that administration of aminothiols, such as S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) and cysteamine, prior to irradiation has a potent preventive effect at the initiation stage of mammary tumorigenesis (3). The protection against radiation offered by WR-2721 (4) and cysteamine (5) is considered to be due to the scavenging of free radicals produced by the interaction of biological molecules and radiation. WR-2721 and cysteamine are toxic at effective doses (6,7), therefore, we have undertaken an evaluation of less toxic phytochemicals whose anti-oxidant properties may make the potential chemopreventive agents for radiation-induced mammary tumorigenesis. A recent study has indicated that phytochemicals with anti-oxidant and anti-inflammatory properties can inhibit tumor initiation and promotion in mouse skin (8). 1,7-Bis(4'-hydroxy-3'-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin), a major pigment in turmeric obtained from the powdered rhizomes of Curcuma longa L., possesses both anti- inflammatory and anti-oxidant properties (9) and has no toxicity (10). In the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor model, when rats were fed a diet containing 1% curcumin prior to dosing with the chemical carcinogen, the incidence of animals with tumors was not significantly altered (11). Because radiation is the only proven relevant human breast carcinogen, we have attempted to evaluate the preventive effects of curcumin on radiation-induced initiation of mammary tumorigenesis and on estrogen-induced tumor promotion in rat mammary glands initiated with radiation in our research series. Our previous study suggested that when administered orally for a long period, curcumin has potent preventive activity during tumor promotion in radiation-initiated mammary tumorigenesis (12). In the present study we have carried out further investigations of the chemopreventive effects of dietary curcumin on radiation-induced initiation.
 |
Materials and methods
|
---|
Materials
Diethylstilbestrol (DES), cholesterol and sulfatase were purchased from Sigma (St Louis, MO). ß-Glucuronidase was purchased from Wako Pure Chemical Industries (Osaka, Japan). Pellets were prepared in a medical Silastic tube (Dow Corning, Midland, MI) and were filled with 3 mg DES mixed with 27 mg cholesterol. Curcumin, commonly used in food as a coloring agent, was obtained from Aldrich Chemical Co. (Milwaukee, WI). Diet containing 1% (w/w) curcumin was prepared in biscuit form by Funabashi Farm (Chiba, Japan). A basal diet (MB-1) of the same form was used for the control experiments. The major components of MB-1 are as follows: total carbohydrate, 54.1%; protein, 24.6%; fat, 4%; fiber, 3.8%; moisture, 7.7%; ash, 5.8%. [2,4,6,7-3H]Estradiol-17ß (sp. act. 4 TBq/mmol) was purchased from Du Pont/NEN Research Products (Boston, MA).
Animals and treatment
The rats used in the present study were treated and handled according to the Recommendations for Handling of Laboratory Animals for Biomedical Research compiled by the Committee on the Safety and Handling Regulations for Laboratory Animal Experiments in our Institute. Wistar-MS rats from a stock colony of Nippon SLC Co. (Hamamatsu, Japan) were kept at 23 ± 1°C in a controlled environment (14 h light/10 h dark). They received water and food ad libitum. For experiments on the prevention of mammary tumors, 54 female rats, 2.5 months old, were mated and then randomized into two groups of 27 rats each at day 11 of pregnancy (the presence of a vaginal plug denoting day 1). The control rats were fed a basal diet (MB-1) throughout the experimental period, received whole body irradiation with 1.5 Gy
-rays (0.15 Gy/min) from a 60Co source at day 20 of pregnancy and were then implanted with a DES pellet at 1 month after weaning (Figure 1
). The experimental group rats were fed the diet containing 1% curcumin between day 11 of pregnancy and parturition (day 23 of pregnancy) and implanted with a DES pellet at 1 month after termination of nursing. The pellets were replaced every 8 weeks. The rate of release of DES from the pellet was 0.38 ± 0.01 µg/day (13). The rats were examined for palpable mammary tumors for 1 year starting from the date of pellet implantation. When mammary tumors >2 cm in diameter were detected, the rats were killed by CO2 asphyxiation and the tumors were removed for further observation. Each mammary tumor was fixed in 10% neutral buffered formalin for histopathological examination. The remaining rats were killed 1 year after administration of the DES pellet and were autopsied to ascertain whether they had any non-palpable mammary tumors and pituitary tumors. Tumor incidence was calculated from the number of rats that developed tumors within 1 year. Iball's index of mammary tumors was calculated as follows: the ratio of incidence (%) to the average latency period in daysx100 (14). For studies on the morphological and biochemical effects of the treatment with curcumin, separate experiments were carried out for which 20 pregnant rats were divided into two groups of 10 rats each at day 11 of pregnancy. Control rats were fed the basal diet and the curcumin group the diet containing 1% curcumin. At day 20 of pregnancy, corresponding to the time of irradiation for tumor initiation described above, six rats in each group were killed for biochemical and morphological studies. The remaining four dams in each group bore pups at full-term gestation. Body weights of dams and newborn pups were measured after parturition.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1. Experimental schedule in this study. Open bar, control diet (MB-1); closed bar, diet containing 1% curcumin; closed arrowhead, whole body irradiation with 1.5 Gy -rays at day 20 of pregnancy for tumor initiation; open arrowhead, implantation with DES pellet for tumor promotion; M, months old.
|
|
Assays
A blood sample, collected from each rat by cardiocentesis under anesthesia, was allowed to clot and was centrifuged to obtain serum. The sera were immediately frozen and stored at 80°C until the assay was started. Concentrations of prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined with NIDDK radioimmunoassy kits (the National Hormone and Pituitary Program, Rockville, MD). The serum concentrations of estradiol-17ß and progesterone were assayed with commercially available radioimmunoassay kits. For assays of total curcuminoids (free form plus conjugates), serum was incubated with 10 mM McIlvaine buffer (pH 5.0) containing 20% ascorbic acid, 0.17% EDTA, 500 U ß-glucuronidase and 40 U sulfatase at 37°C for 60 min (15). Curcumin and its metabolites were extracted with ethylacetate and then analyzed by HPLC with a multiwavelength detector on a Develsoil ODS-HG-5 column (4.6x250 mm; Nomura Chemical Co., Seto, Japan) eluted with a mixture of acetonitrile:water (1:1 v/v) containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The chromatogram was monitored at a wavelength of 430 nm for detection of curcumin and at 280 nm for tetrahydrocurcumin (16). Fatty acids were extracted from serum with hexane and were treated with 14% trifluorobenzene dissolved in methanol: methanol:benzene (35:35:30 v/v/v) for 10 min in boiling water for esterification. The methyl esters of fatty acids were analyzed by gas chromatography with a hydrogen flame ionization detector (17). For assay of lipid peroxidation products, the serum was mixed with 20% trichloroacetic acid and 0.67% thiobarbituric acid and heated for 15 min in boiling water. The concentration of thiobarbituric acid-reactive substances (TBARS) extracted with n-butanol was estimated by absorption at 530 nm. TBARS were expressed as malondialdehyde (MDA) equivalents, using freshly produced MDA as the standard prepared from 1,1,3,3-tetramethoxypropane with HCl (18). The estrogen receptor (ER) in mammary glands was analyzed by the dextran-coated charcol method, using [2,4,6,7-3H]estradiol-17ß (19) as the radioligand. Maximum binding sites for the receptor were determined by a Scatchard plot analysis (20).
Histological examination
The removed mammary glands and mammary tumors were immediately fixed in 10% formalin neutralized with 0.1 M phosphate buffer (pH 7.2). Paraffin sections (4 µm thick) were prepared and stained with hematoxylin and eosin. The tumors were classified as fibroadenoma or adenocarcinoma according to the criteria for the classification of mammary tumors (21).
Statistical analysis
Statistical analyses were conducted using the
2 test for incidence of mammary tumors and for the proportion of adenocarcinoma and fibroadenoma and Student's t-test for the level of significance of the difference between two mean values of body weight, organ weight, hormone and fatty acid concentrations, latent period and multiplicity. The cumulative proportions of rats with tumors (incidence curves) were calculated by the product-limit method where rats which died or were killed without mammary tumors were included and the difference between groups was tested for statistical significance by the MantelCox test. The analyses were performed using StatView-J4.5 software (Abacus Concepts, Berkeley, CA). P values <5% were considered significant.
 |
Results
|
---|
Development of irradiation-induced mammary tumors in rats fed curcumin
When the control rats received whole body irradiation with 1.5 Gy
-rays, a tumor initiator, at day 20 of pregnancy and then were implanted with DES, a tumor promoter, after weaning, a high incidence (70.3%) of tumorigenesis of the mammary glands was observed. The tumor incidence (18.5%) in the rats fed curcumin during the initiation stage was about 25% (P < 0.0001) of that in the control rats (Table I
). The administration of dietary curcumin during initiation by irradiation significantly decreased the cumulative incidence curve of the mammary tumors for the 1 year period (P < 0.0001), compared with the control diet group (Figure 2
). The appearance of the first palpable tumors was delayed by 6 months in the curcumin-fed group, compared with the control group. Also, the average latent period until the appearance of total mammary tumors was delayed by ~2.5 months in the curcumin-fed group (11.2 ± 0.2 months) compared with the control group (8.7 ± 0.4 months) (P < 0.005). There were no significant differences (P = 0.495) between the two groups in the number of mammary tumors per tumor-bearing rat. Iball's index for overall development of mammary tumors in the curcumin-fed rats was 20% of that in the control group. Histological examination was performed for all of the mammary tumors that developed in the control and curcumin-fed rats. The proportion of adenocarcinoma and fibroadenoma in total tumors was 32.1 and 67.9%, respectively, in rats fed the control diet (Table I
). In the curcumin-fed group, the proportion (16.7%) of adenocarcinoma was decreased to 50% of that in the control group. Conversely, the proportion (83.3%) of fibroadenoma was 1.2-fold higher than that in the control group. However, no significant difference (P = 0.450) in the proportion of adenocarcinoma and fibroadenoma was observed between the two groups with the
2 test.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2. Cumulative incidence of development of mammary tumors in irradiated rats treated with DES. Solid and dotted lines represent the control diet and the curcumin diet groups, respectively. Statistical evaluation of the cumulative proportion data (overall incidence curves) by the MantelCox test yielded P < 0.0001, indicating a significant difference between the control diet and curcumin-containing diet groups during the observation period.
|
|
Biological effects at the time of initiation of curcumin administration during pregnancy
Body weight of dams was decreased to 91% of that observed in rats fed the control diet by administration of the curcumin diet from day 11 of pregnancy, in spite of a similar intake of diet throughout the experiment. No change in weight of liver (P = 0.262), adrenal gland (P = 0.378) or pituitary gland (P = 0.079) of dams at day 20 of pregnancy was observed between the control and curcumin-fed groups (Table II
). The litter size of curcumin-fed rats was comparable with that of the rats fed the control diet. In addition, the body weight of fetuses at day 20 of pregnancy was increased slightly by the administration of curcumin, but no significant difference was observed (P = 0.109). Also, the body weight (5.6 ± 0.1 g) of pups (1 day-old) born to curcumin-fed dams was the same as that of pups of the dams fed the control diet (5.6 ± 0.1 g).
Biochemical effects at the time of initiation of curcumin administration during pregnancy
Serum concentrations of ovarian and pituitary hormones were measured 10 days after the start of the administration of dietary curcumin, the time corresponding to initiation with radiation. No significant differences in estradiol-17ß (P = 0.677) and progesterone (P = 0.223) concentrations were observed between the two groups (Table III
). In addition, curcumin did not have any effect on the concentrations of prolactin (P = 0.502) and FSH (P = 0.883). However, the concentration of LH in the rats fed the curcumin diet was increased to 1.8-fold of that observed in rats fed the control diet (P < 0.05). No significant difference (P = 0.592) in TBARS was observed on administration of curcumin for 10 days. The serum concentration of curcumin in rats fed the curcumin diet was below the level detectable by HPLC (4 ng/ml). The tetrahydrocurcumin concentration was 39 ± 10 ng/ml in the curcumin-fed rats.
View this table:
[in this window]
[in a new window]
|
Table III. Serum concentrations of hormones, TBARS and curcumin at the time of irradiation (day 20 of pregnancy) in the rats fed curcumin
|
|
Histological observations of and number of ER in mammary glands at the time of initiation
Whole mounts of inguinal mammary glands corresponding to the time of irradiation were prepared to examine the effects of curcumin on development and differentiation of the glands in pregnant rats. On day 20 of pregnancy, mammary glands of rats fed the control diet showed many alveolar buds with branched lactiferous ducts (Figure 3a
). The whole mounts showed that the mammary glands in pregnant rats fed the curcumin diet exhibited the same development as the glands of control rats (Figure 3b
). On histological examination, no significant differences were observed in the population of parenchymal cells (the glandular epithelium) in the glands of pregnant rats fed the curcumin diet compared with those in control rats (Figure 3c and d
). These observations at the time of irradiation were consistent with the finding of no significant differences in the number of ER in the mammary glands of control (10.9 ± 1.0 fmol/mg protein) and curcumin-fed rats (10.0 ± 0.5 fmol/mg protein) (P = 0.466).

View larger version (142K):
[in this window]
[in a new window]
|
Fig. 3. Whole mount and histological observations in inguinal mammary glands of rats. (a and c) Control rats fed the basal diet (MB-1); (b and d) rats fed the diet containing 1% curcumin. Scale bars: a and b, 5 mm; c and d, 1 mm.
|
|
Effect of curcumin on the fatty acid profile in serum at the time of initiation
The serum concentrations of fatty acids were measured 10 days after the start of administration of dietary curcumin, corresponding to the day of irradiation. No significant change was observed in the concentrations of the fatty acids assayed (Table IV
). With regard to unsaturated fatty acids, the serum concentrations of linoleic acid (P = 0.487), linolenic acid (P = 0.628) and arachidonic acid (P = 0.093) were slightly decreased by the administration of dietary curcumin. However, the concentrations of eicosapentaenoic acid (P = 0.912) and docosahexanoic acid (P = 0.505) were weakly increased in rats fed the curcumin diet. Ratios of total saturated fatty acids to total unsaturated fatty acids (S/U) and of total saturated fatty acids to total polyunsaturated fatty acids (S/PU) were not significantly altered by 10 days administration of curcumin.
Biological effects at the end of the curcumin treatment experiment
The final body weights and organ weights are summarized in Table V
. No significant changes in body weight were observed between the control and curcumin-fed groups (P = 0.253). Treatment with curcumin during initiation by radiation decreased the liver weight (P < 0.01) and increased the uterus weight (P < 0.05) significantly. When the 27 control rats were autopsied, eight (29.6%) were found to have developed pituitary tumors; the pituitary tumor incidence (18.5%) in the curcumin-fed rats was two-thirds of that in the control rats (P = 0.340) (Table I
). No change in weight of normal pituitary glands (P=0.389) and pituitary glands with tumors (P = 0.493) was observed between the control and curcumin-fed groups.
View this table:
[in this window]
[in a new window]
|
Table V. Biological effects at the end of the experiment (17 months old) of curcumin treatment during the initiation stage
|
|
 |
Discussion
|
---|
Explanations of the cytotoxic effects of radiation have previously emphasized the involvement of reactive oxygen species such as the superoxide anion (O2·) and the hydroxyl radical (·OH) (22,23). McLennan et al. have reported that O2· may be non-toxic, but it is a precursor in the formation of ·OH, which is the most toxic radical resulting from radiation (24). The involvement of oxygen-derived free radicals in the carcinogenic process correlates well with the protective effects of free radical scavengers, as seen by inhibition of the development of radiation-induced mammary tumors by administration of WR-2721 prior to irradiation (3,25). A recent study indicated that reactive oxygen radical species generated by radiation increased the frequency of a tandem CC
TT double substitution in the DNA strand (26).
Chemoprevention is a rapidly growing field in cancer research which focuses on inhibiting and delaying the onset of carcinogenesis. A large number of natural products have been evaluated as potential chemopreventive agents (27,28). Recent studies on components of plants indicated that phenolic compounds with anti-oxidant and/or anti-inflammatory properties can inhibit tumor initiation and promotion in mouse skin (8). Most of the natural anti-oxidants have either a phenolic group or a ß-diketone group (29,30). Curcumin is a unique compound, having both phenolic and ß-diketone functional groups, and would be expected to have remarkable anti-oxidant and free radical scavenging activities (31,32). Curcumin not only exhibits the above properties, but also enhances the activities of anti-oxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase (33). Furthermore, curcumin is a potent inhibitor of oxygen radical-generating enzymes such as cyclooxygenase-2 (34,35).
The formation of chromosomal aberrations (36) and micronucleated polychromatic erythrocytes (37) caused by whole body exposure to
-rays were significantly inhibited by oral administration of curcumin. Also, curcumin suppressed lipid peroxidation in rats irradiated with
-rays (38,39). Earlier studies from our laboratory demonstrated a marked preventive effect of curcumin on DES-dependent promotion in radiation-initiated mammary tumorigenesis (12). The data presented herein indicate that administration of curcumin for 12 days, i.e. 9 days before and 3 days after irradiation, also markedly reduced radiation-induced initiation in mammary tumorigenesis in rats. Curcumin has been shown to display anti-initiation activities, as indicated by its ability to prevent tumorigeneses induced in the colon by azoxymethane (40), mouth by 4-nitroquinoline-1-oxide (41), skin by benzo[a]pyrene (42) and duodenum by N-ethyl-N'-nitro-N-nitrosoguanidine (43). It was suggested that many chemical carcinogens act by forming free radicals (4446). We would suggest that one possible mechanism of the anti-initiation activity of curcumin is the scavenging of free radicals produced by a variety of chemical carcinogens or radiation as tumor initiator at target sites. However, dietary curcumin did not lower the cumulative incidence or affect tumor multiplicity in the initiation stage of DMBA-induced mammary tumorigenesis (11,47). The reason why no protective effect of curcumin was observed in the chemical carcinogenesis of mammary glands is still not known.
Nitric oxide plays a key role in physiological as well as pathological processes, including inflammation and cancer. The enhancement of NO production by irradiation was attributed to high levels of expression of inducible nitric oxide synthase (iNOS) (48). Excessive production of NO by activated iNOS may result in the formation of toxic intermediates, such as peroxynitrite (ONOO) and N2O3, causing tissue damage and genotoxicity (49,50), and thus has potential carcinogenic effects (51). In immunohistochemical experiments, iNOS expression was apparently increased in the basal layers of alveoli and lactiferous ducts of the mammary glands treated with lipopolysaccharide (LPS) as an inflammatory agent and this increase was reflected in an enhancement of NO production (52). Furthermore, NO production by LPS-stimulated mammary glands was significantly decreased in the presence of curcumin, as was the amount of a 122 kDa iNOS (53). On the other hand, 3,5,4'-trihydroxy-trans-stilbene (resveratrol), a phytopolyphenol isolated from the seeds and skins of grapes, inhibited the expression of LPS-induced iNOS (54) and decreased LPS-stimulated NO production (55). Mgbonyebi et al. (56) have reported that resveratrol is a potential chemopreventive agent for both ER-positive and ER-negative breast cancers. Also, formation of azoxymethane-induced colonic aberrant crypt foci was significantly suppressed in the presence of an iNOS-specific inhibitor, S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (57). These findings suggest that suppression of iNOS activity by curcumin in the mammary gland of irradiated rats helps to prevent radiation-induced tumor initiation.
The transcription factor nuclear factor
B (NF-
B) has been implicated in the inducible expression of a variety of genes involved in inflammatory and immune responses (58). Singh and Aggarwal have reported that curcumin inhibits the NF-
B activation pathway at a step before inhibitory protein
B (I
B)
phosphorylation (59). Recently, Jobin et al. (60) have reported that interleukin (IL)-1ß-mediated expression of the adhesion molecule, intercellular adhesion molecule-1, and the chemokine IL-8 were reduced by blockade of transcriptional activation cascades, such as cytokine-induced NF-
B DNA binding activity, RelA nuclear translocation, I
B
degradation, I
B Ser32 phosphorylation and I
B kinase activity, by curcumin. Their results suggest that curcumin blocks a signal upstream of the NF-
B-inducing kinase, but below the junction of the IL-1ß signal pathways. NF-
B is activated by radiation (61,62). Activation of NF-
B may be particularly important for cell survival in response to oxidative stress induced by radiation, but it has been shown recently that inhibition of NF-
B activation enhances radiation-induced apoptosis (63,64). We would suggest that another possible mechanism of the chemopreventive activity of curcumin for mammary tumorigenesis is elimination of radiation-initiated tumor origin cells from the mammary gland by apoptosis.
At the time corresponding to initiation with radiation, no detectable serum curcumin was observed in rats fed the curcumin diet. It was shown that curcumin administered orally was metabolized to tetrahydrocurcumin during absorption through the intestine (65,66). In fact, tetrahydrocurcumin was detected in serum of rats fed the diet containing curcumin for 9 days in the present study. Tetrahydrocurcumin exhibited a significant inhibitory effect on O2· generation induced by 12-O-tetradecanoylphorbol-13-acetate (67) and on lipid peroxidation of erythrocyte membrane ghosts induced by t-butylhydroperoxide compared with curcumin (16,68). Also, feeding of a diet containing tetrahydrocurcumin resulted in a significant repression of 1,2-dimethylhydrazine-induced formation of aberrant crypt foci, which are regarded as a precursor lesion for colon cancer (69). The results obtained from the present study thus suggest that tetrahydrocurcumin has potential as a chemopreventive agent for radiation-initiated mammary tumorigenesis.
Finally, Wahlström and Blennow (70) found no apparent toxic effects of curcumin at doses of up to 5 g/kg body wt in rats when given orally. In the present study, pregnant rats consumed 18.2 ± 0.3 g of diet containing 1% curcumin/day, which corresponds to 0.67 g curcumin/kg/day, for 12 days. Body weight in pregnant rats fed the curcumin diet was reduced to 91% of that observed in rats fed the control diet. The reduction was significant, but would be too low to indicate a toxic action of curcumin. Therefore, it is likely that a reduction in body weight occurs in curcumin-fed rats having a decreased concentration of serum triglycerides (12). Curcumin did not have any adverse effects on growth or teratogenesis of fetuses nor on organ weight or serum concentrations of hormones of dams, suggesting no toxic effect when administered orally. Lack of a mutagenic effect of curcumin was also reported in the presence or absence of a rat hepatic microsomal activation system in the Ames test with Salmonella typhimurium (71).
In conclusion, radiation-induced initiation of mammary tumorigenesis was markedly inhibited by administration of dietary curcumin. Oral administration of curcumin did not produce any side-effects on endocrinological and physiological status. These results raise the possibility of clinical application of curcumin in the management of radio-diagnosis to diminish tissue damage by radiation.
 |
Notes
|
---|
5 Present address: Okinawa Industrial Technology Center, 12-2 Suzaki, Gushikawa-shi 904-2234, Okinawa, Japan 
6 To whom correspondence should be addressed Email: inano{at}nirs.go.jp 
 |
Acknowledgments
|
---|
We thank Mrs M.Takahashi for excellent assistance in the care of the animals. This work was partly supported by a grant from the Special Program for Bioregulation of the National Institute of Radiological Sciences.
 |
References
|
---|
-
Inano,H., Yamanouchi,H., Suzuki,K., Onoda,M. and Wakabayashi,K. (1995) Estradiol-17ß as an initiation modifier for radiation-induced mammary tumorigenesis of rats ovariectomized before puberty. Carcinogenesis, 16, 18711877.[Abstract]
-
Yamanouchi,H., Ishii-Ohba,H., Suzuki,K., Onoda,M., Wakabayashi,K. and Inano,H. (1995) Relationship between stages of mammary development and sensitivity to
-ray irradiation in mammary tumorigenesis in rats. Int. J. Cancer, 60, 230234.[ISI][Medline]
-
Inano,H., Onoda,M., Suzuki,K., Kobayashi,H. and Wakabayashi,K. (2000) Inhibitory effects of WR-2721 and cysteamine on tumor initiation in mammary glands of pregnant rats by radiation. Radiat. Res., 153, 6874.[ISI][Medline]
-
Littlefield,L.G., Joiner,E.E., Colyer,S.P., Sallam,F. and Frome,E.L. (1993) Concentration-dependent protection against X-ray-induced chromosome aberrations in human lymphocytes by the aminothiol WR-1065. Radiat. Res., 133, 8893.[ISI][Medline]
-
Henderson,B.W. and Miller,A.C. (1986) Effects of scavengers of reactive oxygen and radical species on cell survival following photodynamic treatment in vitro; comparison to ionizing radiation. Radiat. Res., 108, 196205.[ISI][Medline]
-
Kligerman,M.M., Glover,D.J., Turrisi,A.T., Norfleet,A.L., Yuhas,J.M., Coia,L.R., Simone,C., Glick,J.H. and Goodman,R.L. (1984) Toxicity of WR-2721 administration in single and multiple doses. Int. J. Radiat. Oncol. Biol. Phys., 10, 17711776.
-
Devi,P.U., Navalkha,P.L., Kumar,S., Kumar,A., Jagetia,G.C., Surana,M., Gupta,S. and Pareek,B.P. (1984) Studies on toxic effect of WR-2721 in mouse. Indian J. Med. Sci., 38, 6569.[Medline]
-
Huang,M.-T., Smart,R.C., Wong,C.-Q. and Conney,A.H. (1988) Inhibitory effect of curcumin, chlorogenic acid, caffeic acid and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res., 48, 59415946.[Abstract]
-
Ammon,H.P.T. and Wahl,M.A. (1991) Pharmacology of Curcuma longa. Planta Med., 57, 17.[ISI][Medline]
-
Shankar,T.N., Shantha,N.V., Ramesh,H.P., Murthy,I.A. and Murthy,V.S. (1980) Toxicity studies on turmeric (Curcuma longa): acute toxicity studies in rats, guinea pig and monkeys. Indian J. Exp. Biol., 18, 7375.[ISI][Medline]
-
Pereira,M.A., Grubbs,C.J., Barnes,L.H., Li,H., Olson,G.R., Eto,I., Jukiana,M., Whitaker,L.M., Kelloff,G.J., Steele,V.E. and Lubet,R.A. (1996) Effects of the phytochemicals, curcumin and quercetin, upon azoxymethane-induced colon cancer and 7,12-dimethylbenz[a]anthracene-induced mammary cancer in rats. Carcinogenesis, 17, 13051311.[Abstract]
-
Inano,H., Onoda,M., Inafuku,N., Kubota,M., Kamada,Y., Osawa,T., Kobayashi,H. and Wakabayashi,K. (1999) Chemoprevention by curcumin during the promotion stage of tumorigenesis of mammary glands in rats irradiated with
-rays. Carcinogenesis, 20, 10111018.[Abstract/Free Full Text]
-
Inano,H., Suzuki,K., Ishii-Ohba,H., Yamanouchi,H., Takahashi,M. and Wakabayashi,K. (1993) Promotive effects of diethylstilbestrol, its metabolite (Z,Z-dienestrol) and a stereoisomer of the metabolite (E,E-dienestrol) in tumorigenesis of rat mammary glands pregnancy-dependently initiated with radiation. Carcinogenesis, 14, 21572163.[Abstract]
-
Iball,J. (1939) The relative potency of carcinogenic compounds. Am. J. Cancer, 35, 188190,
-
Lee,M.-J., Wang,Z.-Y., Li,H., Chen,L., Sun,Y., Gobbo,S., Balentine,D.A. and Yang,C.S. (1995) Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol. Biomarkers Prev., 4, 393399.[Abstract]
-
Sugiyama,Y., Kawakishi,S. and Osawa,T. (1996) Involvement of ß-diketone moiety in the antioxidative mechanism of tetrahydrocurcumin. Biochem. Pharmacol., 52, 519525.[ISI][Medline]
-
Ozawa,A., Takayanagi,K., Fujita,T., Hirai,A., Hamazaki,A., Terano,T., Tamura,Y. and Kumagai,A. (1982) Determination of long chain fatty acids in human total plasma lipids using gas chromatography. Bunseki Kagaku, 31, 8791.[ISI]
-
Buege,J.A. and Aust,S.D. (1978) Microsomal lipid peroxidation. Methods Enzymol., 52, 302310.[Medline]
-
Johnson,R.B., Nakamura,R.M. and Libby,R.M. (1975) Simplified Scatchard plot assay for estrogen receptor in human breast cancer. Clin. Chem., 21, 17251730.[Abstract/Free Full Text]
-
Scatchard,G. (1949) The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci., 51, 660672.[ISI]
-
Russo,J., Gusterson,B.A., Rogers,A.E., Russo,I.H., Wellings,S.R. and van Zwieten,M.-J. (1990) Comparative study of human and rat mammary tumorigenesis. Lab. Invest., 62, 244278.[ISI][Medline]
-
Buc-Calderon,P., Defresne,M.P., Barvais,C. and Roberfroid,M. (1989) N-acyl dehydroalanines protect from radiation toxicity and inhibit radiation carcinogenesis in mice. Carcinogenesis, 10, 16411644.[Abstract]
-
Riley,P.A. (1994) Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int. J. Radiat. Biol.,65, 2733.[ISI][Medline]
-
McLennan,G., Oberley,L.W. and Autor,A.P. (1980) The role of oxygen-derived free radicals in radiation-induced damage and death of nondividing eucaryotic cells. Radiat. Res., 84, 122132.[ISI][Medline]
-
Inano,H., Onoda,M., Suzuki,K., Kobayashi,H. and Wakabayashi,K. (2000) Prevention of radiation-induced mammary tumors in rats by combined use of WR-2721 and tamoxifen. Int. J. Radiat. Biol., in press.
-
Reid,T.M. and Loeb,L.A. (1993) Tandem double CC TT mutations are produced by reactive oxygen species. Proc. Natl Acad. Sci. USA, 90, 39053907.
-
Sharma,S., Stutzman,J.D., Kelloff,G.J. and Steele,V.E. (1994) Screening of potential chemopreventive agents using biochemical markers of carcinogenesis. Cancer Res., 54, 58485855.[Abstract]
-
Ohigashi,H., Murakami,A. and Koshimizu,K. (1996) An approach to functional food: cancer preventive potential of vegetables and fruits and their active constituents. Nutr. Rev., 54, S24S28.[ISI][Medline]
-
Masuda,T. and Jitoe,A. (1994) Antioxidative and antiinflammatory compounds from tropical gingers: isolation, structure determination and activities of cassumunins A, B and C, new complex curcuminoids from Zingiber cassumunar. J. Agric. Food Chem., 42, 18501856.[ISI]
-
Subramanian,M., Sreejayan, Rao,M.N.A., Devasagayan,T.P.A. and Singh,B.B. (1994) Diminution of singlet oxygen-induced DNA damage by curcumin and related antioxidants. Mutat. Res., 311, 249255.[ISI][Medline]
-
Reddy,A.C.P. and Lokesh,B.R. (1994) Studies on the inhibitory effects of curcumin and euganol on the formation of reactive oxygen species and the oxidation of ferrous iron. Mol. Cell. Biochem., 137, 18.[ISI][Medline]
-
Toda,S., Miyase,T., Arichi,H., Tanizawa,H. and Takino,Y. (1985) Natural antioxidants III. Antioxidative components isolated from rhizome of Curcuma longa L. Chem. Pharm. Bull., 33, 17251728.[ISI][Medline]
-
Reddy,A.C.P. and Lokesh,B.P. (1994) Effect of dietary turmeric (Curcuma longa) on iron-induced lipid peroxidation in rat liver. Food Chem. Toxicol., 32, 279283.[ISI][Medline]
-
Huang,M.T., Lysz,T., Ferraro,T., Abidi,T.F., Laskin,J.D. and Conney,A.H. (1991) Inhibitory effect of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis. Cancer Res., 51, 813819.[Abstract]
-
Zhang,F., Altorki,N.K., Mester,J.R., Subbaramaiah,K. and Dannenberg,A.J. (1999) Crucumin inhibits cyclooxygenase-2 transcription in bile acid- and phorbol ester-treated human gastrointestinal epithelial cells. Carcinogenesis, 20, 445451.[Abstract/Free Full Text]
-
Thresiamma,K.C., George,J. and Kuttan,R. (1998) Protective effect of curcumin, ellagic acid and bixin on radiation induced genotoxicity. J. Exp. Clin. Cancer Res., 17, 431434.[ISI][Medline]
-
Abraham,S.K., Sarma,L. and Kesavan,P.C. (1993) Protective effects of chlorogenic acid, curcumin and ß-carotene against
-radiation-induced in vivo chromosomal damage. Mutat. Res., 303, 109112.[ISI][Medline]
-
Nishigaki,I., Kuttan,R., Oku,H., Ashoori,F., Abe,H. and Yagi,K. (1992) Suppressive effect of curcumin on lipid peroxidation induced in rats by carbon tetrachloride or 60Co-irradiation. J. Clin. Biochem. Nutr., 13, 2329.[ISI]
-
Sreejayan,N., Rao,M.N.A., Priyadarsini,K.I. and Devasagayan,T.P.A. (1997) Inhibition of radiation-induced lipid peroxidation by curcumin. Int. J. Pharmaceut., 151, 127130.[ISI]
-
Rao,C.V., Simi,B. and Reddy,B.S. (1993) Inhibition by dietary curcumin of azoxymethane-induced ornithine decarboxylase, tyrosine protein kinase, arachidonic acid metabolism and aberrant crypt foci formation in the rat colon. Carcinogenesis, 14, 22192225.[Abstract]
-
Tanaka,T., Makita,H., Ohnishi,M., Hirose,H., Wang,A., Mori,H., Satoh,K., Hara,A. and Ogawa,H. (1994) Chemoprevention of 4-nitroquinoline-1-oxide-induced oral carcinogenesis by dietary curcumin and hesperidin: comparison with the protective effect of ß-carotene. Cancer Res., 54, 46534659.[Abstract]
-
Huang,M.-T., Wang,Z.Y., Georgiadis,C.A., Laskin,J.D. and Conney,A.H. (1992) Inhibitory effects of curcumin on tumor initiation by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene. Carcinogenesis, 13, 21832186.[Abstract]
-
Huang,M.-T., Lou,Y.-R., Ma,W., Newmark,H.L., Reuhl,K.R. and Conney,A.H. (1994) Inhibitory effects of dietary curcumin on forestomach, duodenal and colon carcinogenesis in mice. Cancer Res., 54, 58415847.[Abstract]
-
Nagata,C., Inomata,M., Kodama,M. and Tagashira,Y. (1968) Electron spin resonance study on the interaction between the chemical carcinogens and tissue components III. Determination of the structure of the free radical produced either by stirring 3,4-benzopyrene with albumin or incubating with liver homogenates. Gann, 59, 289298.[ISI][Medline]
-
Nagata,V., Nakadate,M., Ioki,Y. and Imamura,A. (1972) Electron spin resonance study on the free radical production from N-methyl-N'-nitro-N-nitrosoguanidine. Gann, 63, 471481.[ISI][Medline]
-
Varnes,M.E. and Biaglow,J.E. (1979) Interactions of the carcinogen 4-nitroquinoline-1-oxide with the non-protein thiols of mammalian cells. Cancer Res., 39, 29602965.[Abstract]
-
Singletary,K., MacDonald,C., Iovinellr,M., Fisher,C. and Wallig,M. (1998) Effect of the ß-diketones diferuloylmethane (curcumin) and dibenzoylmethane on rats mammary DNA adducts and tumors induced by 7,12-dimethylbenz[a]anthracene. Carcinogenesis, 19, 10391043.[Abstract]
-
Ibuki,Y. and Goto,R. (1997) Enhancement of NO production from resident peritoneal macrophages by in vitro gamma-irradiation and its relationship to reactive oxygen intermediates. Free Radic. Biol. Med., 22, 10291035.[ISI][Medline]
-
Tamir,S. and Tannenbaum,S.R. (1996) The role of nitric oxide (NO) in the carcinogenic process. Biochim. Biophys. Acta, 1288, F31F36.[ISI][Medline]
-
Wink,D.A., Vodovotz,Y., Laval,J., Laval,F., Dewhirst,M. and Mitchell,J.B. (1998) The multifaceted roles of nitric oxide in cancer. Carcinogenesis, 19, 711721.[Abstract]
-
Ohshima,H. and Bartsch,H. (1994) Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat. Res., 305, 253264.[ISI][Medline]
-
Onoda,M. and Inano,H. (1998) Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland. J. Histochem. Cytochem., 46, 12691278.[Abstract/Free Full Text]
-
Onoda,M. and Inano,H. (2000) Effect of curcumin on the production of nitric oxide by cultured mammary gland. Arch. Biochem. Biophys. B Nitric Oxide Biol. Chem., in press.
-
Jang,M. and Pezzuto,J.M. (1999) Cancer chemopreventive activity of resveratrol. Drug. Exp. Clin. Res., 25, 6577.[ISI]
-
Wadsworth,T.L. and Koop,D.R. (1999) Effects of the wine polyphenols quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages. Biochem. Pharmacol., 57, 941949.[ISI][Medline]
-
Mgbonyebi,O.P., Russo,J. and Russo,I.H. (1998) Antiproliferative effect of synthetic resveratrol on human breast epithelial cells. Int. J. Oncol., 12, 865869.[ISI][Medline]
-
Rao,C.V., Kawamori,T., Hamid,R. and Reddy,B.S. (1999) Chemoprevention of colonic aberrant crypt foci by an inducible nitric oxide synthase-selective inhibitor. Carcinogenesis, 20, 641644.[Abstract/Free Full Text]
-
Barnes,P.J. and Karin,M. (1997) Nuclear factor-
B, a pivotal transcription factor in chronic inflammatory diseases. New Engl. J. Med., 336, 10661071.[Free Full Text]
-
Singh,S. and Aggarwal,B.B. (1995) Activation of transcription factor NF-
B is suppressed by curcumin (diferuloylmethane). J. Biol. Chem., 270, 2499525000.[Abstract/Free Full Text]
-
Jobin,C., Bradham,C.A., Russo,M.P., Juma,B., Narula,A.S., Brenner,D.A. and Sartor,R.B. (1999) Curcumin blocks cytokine-mediated NF-
B activation and proinflammatory gene expression by inhibiting inhibitory factor I
B kinase activity. J. Immunol., 163, 34743483.[Abstract/Free Full Text]
-
Weichsellaum,R.R., Hallahan,D., Fuks,Z. and Kufe,D. (1994) Radiation induction of immediate early genes: effectors of the radiation-stress response. Int. J. Radiat. Oncol. Biol. Phys., 30, 229234.[ISI][Medline]
-
Zhou,D., Brown,S.A., Yu,T., Chen,G., Barve,S., Kang,B.C. and Tompson,J.S. (1999) A high dose of ionizing radiation induces tissue-specific activation of nuclear factor-
B in vivo. Radiat. Res., 151, 703709.[ISI][Medline]
-
Wang,C.-Y., Mayo,M.W. and Baldwin,A.S. (1996) TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-
B. Science, 274, 784787.[Abstract/Free Full Text]
-
Yamagishi,N., Miyakoshi,J. and Takebe,H. (1997) Enhanced radiosensitivity by inhibition of nuclear factor
B activation in human malignant glioma cells. Int. J. Radiat. Biol., 72, 157162.[ISI][Medline]
-
Holder,G.M., Plummer,J.L. and Ryan,A.J. (1978) The metabolism and excretion of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) in the rat. Xenobiotica, 8, 761768.[ISI][Medline]
-
Ravindranath,V. and Chandrasekhara,N. (1981) In vitro studies on the intestinal absorption of curcumin in rats. Toxicology, 20, 251257.[ISI][Medline]
-
Nakamura,Y., Ohto,Y., Murakami,A., Osawa,T. and Ohigashi,H. (1998) Inhibitory effects of curcumin and tetrahydrocurcuminoids on the tumor promoter-induced reactive oxygen species generation in leukocytes in vitro and in vivo. Jpn. J. Cancer Res., 89, 361370.[ISI][Medline]
-
Osawa,T., Sugiyama,Y., Inayoshi,M. and Kawakishi,S. (1995) Antioxidative activity of tetrahydrocurcuminoids. Biosci. Biotechnol. Biochem., 59, 16091612.[ISI][Medline]
-
Kim,J.M., Araki,S., Kim,D.J., Park,C.B., Takatuka,N., Baba-Toriyama,H., Ota,T., Nir,Z., Khachik,F., Shimidzu,N., Tanaka,Y., Osawa,T., Uraji,T., Murakoshi,M., Nishino,H. and Tsuda,H. (1998) Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation. Carcinogenesis, 19, 8185.[Abstract]
-
Wahlström,B. and Blennow,G. (1978) A study on the fate of curcumin in the rats. Acta Pharmacol. Toxicol., 43, 8692.[Medline]
-
Jensen,N.J. (1982) Lack of mutagenic effect of turmeric oleoresin and curcumin in the Salmonella/mammalian microsome test. Mutat. Res., 105, 393396.[ISI][Medline]
Received March 24, 2000;
revised June 26, 2000;
accepted June 30, 2000.