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Genet. Mol. Biol. vol.22 n.3 São Paulo Sept. 1999
Effects of turmeric and its active principle, curcumin, on bleomycin-induced chromosome aberrations in Chinese hamster ovary cells
Maria Cristina P. Araújo1,2, Francisca da Luz Dias2, Sergio N. Kronka3 and Catarina S. Takahashi2,4
1Departamento de Genética, Universidade Federal de Pernambuco, PE, Brasil. 2Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, USP, Av. Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brasil. Send correspondence to M.C.P.A. 3Departamento de Ciências Exatas, UNESP, Jaboticabal, SP, Brasil. 4Departamento Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP, Ribeirão Preto, SP, Brasil.
Naturally occurring antioxidants have been extensively studied for their capacity to protect organisms and cells from oxidative damage. Many plant constituents including turmeric and curcumin appear to be potent antimutagens and antioxidants. The effects of turmeric and curcumin on chromosomal aberration frequencies induced by the radiomimetic agent bleomycin (BLM) were investigated in Chinese hamster ovary (CHO) cells. Three concentrations of each drug, turmeric (100, 250 and 500 mg/ml) and curcumin (2.5, 5 and 10 mg/ml), were combined with BLM (10 mg/ml) in CHO cells treated during the G1/S, S or G2/S phases of the cell cycle. Neither turmeric nor curcumin prevented BLM-induced chromosomal damage in any phases of the cell cycle. Conversely, a potentiation of the clastogenicity of BLM by curcumin was clearly observed in cells treated during the S and G2/S phases. Curcumin was also clastogenic by itself at 10 µg/ml in two protocols used. However, the exact mechanism by which curcumin produced clastogenic and potentiating effects remains unknown.
Recently a variety of compounds that possess antimutagenic properties has been detected in vegetables and spices, and evidence is accumulating that their dietary intake decreases the risk of cancer and other malignant diseases in human (Kada et al., 1986). Antioxidant compounds, especially phenols such as gallic acid, tannins, curcumin, ellagic acid and eugenol, are of considerable interest from the viewpoint of dietary antioxidant supplementation (Halliwell, 1994).
Turmeric, a spice obtained from the rhizome of Curcuma longa Linn (Zingiberaceae), has been regularly used for its coloring, flavoring and medicinal properties (Ammon and Wahl, 1991). Studies suggest that turmeric is a potent antimutagenic in vivo against carcinogens such as benzo[a]pyrene and methylcholanthrene (Polasa et al., 1991), and it is effective in inhibiting the formation and excretion of urinary mutagens in smokers (Polasa et al., 1992).
Curcumin, the active principle of turmeric, is commonly used as a coloring agent in foods, drugs and cosmetics, and has a wide range of effects. Curcumin is known to act as an antioxidant, antimutagen and anticarcinogen (Sharma, 1976; Kunchandy and Rao, 1990; Nagabhushan and Bhide, 1992; Rao et al., 1995; Anto et al., 1996). Curcumin also has therapeutic properties for some human diseases (Srimal and Dhawan, 1973; Srivastava et al., 1985).
Bleomycin (BLM) is an antibiotic and radiomimetic glycopeptide that is routinely used in cancer chemotherapy as an antineoplastic agent. BLM is mutagenic in diverse genetic assays (Povirk and Austin, 1991) and is thought to exert its genotoxic effects through free radical production and the induction of oxidative damage to DNA (Lown and Sim, 1977; Cunningham et al., 1984; Sikic, 1986).
The aim of the present study was to investigate the action of the antioxidants turmeric and curcumin on chromosomal damage induced by the oxidative agent BLM, by determining their effect on the frequency of BLM-induced chromosomal aberrations in Chinese hamster ovary (CHO) cells.
MATERIAL AND METHODS
Turmeric was donated by Quest International Company (Brazil), curcumin (diferuloylmethane; C.I. 75300) was purchased from Sigma Chemical Co. (USA), and bleomycin sulfate was obtained from Hospital das Clínicas, USP, Brazil (BlenoxaneÒ; Bristol-Myers Squibb, Brazil). Turmeric and BLM were dissolved in distilled water and curcumin was dissolved in dimethyl sulfoxide (DMSO; Sigma), just before each treatment. The final concentration of DMSO in the medium was 0.5%. Concentrations of BLM (10 mg/ml), turmeric (100, 250 and 500 mg/ml) and curcumin (2.5, 5 and 10mg/ml) were chosen based on results obtained in pilot experiments (data not shown).
Cell culture and treatments
Chinese hamster ovary cells (CHO-9 cell line) were kindly supplied by Prof. A.T. Natarajan (University of Leiden, The Netherlands). Cells were maintained as monolayers growing at 37°C in 25-cm2 flasks (Corning) containing HAM-F10 (Sigma) plus DMEM (Dulbecco's modified Eagle's medium; Sigma), supplemented with 10% newborn bovine serum (Cultilab), antibiotics (0.1 mg/ml streptomycin and 0.06 mg/ml penicillin), and 2.38 mg/ml HEPES (Sigma). Cells were subcultured two or three times a week. The experiments were performed with cells at six to 12 passages.
Cells were seeded at a density of 106 cells/flask and then treated with either turmeric or curcumin, that remained in the culture until cell harvesting, 1 h before BLM addition. BLM treatment lasted 30 min. After this, cells were washed twice with PBS and refed complete medium plus either turmeric or curcumin. Exponentially growing CHO cells have a doubling time of 12-14 h (Preston et al. 1987; Cozzi et al., 1993). In this work, total culture time was 14 h. Cells were harvested three, eight and 12 h after initiation of BLM treatment. According to Preston et al. (1981), cells fixed three, eight and 12 h after the beginning of treatment are, respectively, in the late G2/S, middle S and early G1/S phases of the cell cycle at the moment they are treated. Untreated controls were handled identically, with the same changes in medium. Colcemide (100 ml at a concentration of 5 mg/ml) was added to the culture medium 2 h before harvesting. Each experiment was repeated three times.
Cells were harvested by the method of Moorhead et al. (1960), with modifications (1% sodium citrate hypotonic solution, methanol/acetic acid 3:1 fixative). The air-dried chromosome preparations were stained with 3% Giemsa, diluted in phosphate buffer. Only well-spread metaphases presenting 21 ± 1 chromosomes were analyzed. Three hundred metaphases (100 in each experiment) were analyzed per treatment in order to determine the frequencies of chromosomal aberrations (blind test). The mitotic index was obtained by counting the number of mitotic cells in 3,000 cells per treatment (1,000 in each experiment).
Data concerning number of abnormal metaphases and total number of chromosomal aberrations, including gaps, and mitotic index were statistically analyzed by analysis of variance, with calculations of the F-statistic and respective P values. In all cases in which P < 0.05 the mean values of each treatment were compared by the Tukey test, with calculation of the minimum significant difference for P = 0.05.
Frequencies of abnormal metaphases, chromosomal aberrations and mitotic indices in CHO cells treated with turmeric combined or not with BLM during G1/S, S or G2/S phases of the cell cycle were determined (Tables I, II and III). Mitotic indices observed in cultures treated with turmeric were not significantly different from those found in untreated controls, except when 500 µg/ml turmeric was associated with BLM at the G1/S phase (Table I). Turmeric was not clastogenic in any protocol tested. Although a statistically significant difference was not detected when compared with BLM alone, there was a tendency for the highest concentration of turmeric combined with BLM to increase the frequency of abnormal metaphases and chromosomal aberrations in cells treated at G1/S phase. On the other hand, turmeric did not show any significant effect on bleomycin-induced aberrations.
A 30-min pulse treatment with BLM at 10 mg/ml to CHO cells produced a marked induction of chromosome aberrations. BLM at this concentration was also used as a clastogenic agent by other authors (Salvadori et al., 1994; Anderson et al., 1994). In this paper, the main aberration types after BLM exposure at G1/S were isochromatid breaks and dicentrics, and in the case of treatment at S and G2/S phases, chromatid breaks, gaps and exchanges were the most frequent.
The effects of curcumin on the frequencies of abnormal metaphases, chromosomal aberrations and mitotic indices induced by BLM in cells treated during G1/S, S or G2/S phases were determined (Tables IV, V and VI). Curcumin at 10 µg/ml was clastogenic but not cytotoxic. The chromosomal damage induced by 10 µg/ml curcumin was statistically significant when compared with the untreated control, and chromatid breaks and gaps were the most frequent alterations observed (Tables IV and V). A cytotoxic effect was observed when curcumin at 2.5 and 10 µg/ml was combined with BLM at S phase (Table V) and curcumin at 10 mg/ml combined with BLM at G2/S phase (Table VI). When compared with BLM alone plus curcumin control, curcumin at 5 and 10 mg/ml potentiated the clastogenicity of BLM at S (Table V) and in all three concentrations tested in G2/S (Table VI). These effects were observed for both abnormal metaphases and chromosome aberrations. The number of abnormal metaphases increased 109 and 83% in cultures treated at S phase by BLM plus curcumin at 5 or 10 µg/ml, respectively, and 41, 60 and 115% in cultures treated at G2/S phase by BLM plus curcumin at 2.5, 5 or 10 µg/ml, respectively. The total number of chromosome aberrations increased 239 and 229% in cultures treated at S phase by BLM plus curcumin at 5 or 10 µg/ml, respectively, and 153 and 359% in cultures treated at G2/S by BLM plus curcumin at 5 or 10 µg/ml, respectively. These values were calculated by comparing those observed for the association BLM + curcumin with the expected values (sum of the effects caused by each agent alone). The main aberration types observed in cells treated with curcumin combined with BLM, in which a synergistic effect had occurred, were chromatid breaks and gaps.
The CHO cell system has been used to evaluate mutagenesis and antimutagenesis activities of different compounds (Cozzi et al., 1991; Musk et al., 1997). Several natural compounds and antioxidant agents, such as vanillin (Sasaki et al., 1987a,b, 1990), garlic acid (Knasmüller et al., 1989), humic acid (Cozzi et al., 1993), fatty acids (Sasaki et al., 1994), squalene (Fan et al., 1996) and Tochu tea (Nakamura et al., 1997), have shown antimutagenic properties against chromosomal damage in CHO cells.
As oxidative damage in biological systems is considered to cause aging, degenerative diseases and cancer, particular attention has been focused on the possibility of modulating these effects through the use of free-radical scavengers to minimize cellular injury. Curcumin is a natural antioxidant derived from turmeric, which has therapeutic properties and anticancer effects. These beneficial effects of turmeric have been postulated to be due to curcumin (Reddy and Lokesh, 1994).
In the present work, turmeric was not clastogenic by itself whereas curcumin increased both the number of abnormal metaphases and the frequency of chromosomal aberrations at the highest concentration in cells treated for 13 and 9 h. As the cell cultures were treated with curcumin during 13, 9 and 4 h, respectively for cells treated by BLM in G1/S, S and G2/S phases, the curcumin-induced damage was higher in the 13-h treatment compared with the 9-h treatment, and was not statistically significant in the 4-h treatment compared with untreated controls (Tables IV, V and VI).
Turmeric was found to be non-mutagenic in mice and rats (Vijayalaxmi, 1980) and in the Ames test (Nagabhushan et al., 1987). However, it induced chromosomal damage in plant systems (Abraham et al., 1976) and was weakly clastogenic in acutely treated mice (Mukhopadhyay et al., 1998). Turmeric can also antagonize the mutagenicity of other substances (Shalini and Srinivas, 1990; Srinivas and Shalini, 1991; Mukundan et al., 1993). According to our results turmeric did not reduce the BLM-induced chromosomal aberrations; instead, it showed a slight tendency to increase the chromosomal damage induced by BLM at G1/S phase (Table I).
Some authors have noticed that curcumin is non-mutagenic in CHO cells (Au and Hsu, 1979), Ames test (Nagabhushan et al., 1987) and mice (Abraham et al., 1993). Nevertheless, our data clearly indicate that curcumin at 10 µg/ml is clastogenic when present in the culture for more than 9 h. Clastogenic effects of curcumin were also observed by others. Mukhopadhyay et al. (1998) reported a slight increase in the number of chromosomal aberrations in acutely treated mice by curcumin. Giri et al. (1990) observed a significant increase in SCE and a weak increase in the frequency of chromosomal aberrations in mice and rats treated acute and chronically with curcumin. Ishidate Jr. et al. (1984), using a Chinese hamster fibroblast cell line, noticed that curcumin was clastogenic at 30 mg/ml. We have observed that curcumin is cytotoxic at concentrations higher than 20 mg/ml (data not shown).
Curcumin significantly reduced the frequencies of micronucleated polychromatic erythrocytes in mice exposed to g-radiation (Abraham et al., 1993), and it was also indicated as an antimutagen against environmental mutagens in vitro and an antitumor drug in vivo (Nagabhushan et al., 1987; Nagabhushan and Bhide, 1992). However, curcumin and turmeric could not inhibit cyclophosphamide- or mitomycin-C-induced chromosomal aberrations in mice (Mukhopadhyay et al., 1998). According to our data, curcumin increased significantly the frequencies of both abnormal metaphases and chromosomal aberrations induced by BLM at S and G2/S phases when compared with BLM plus curcumin controls. The potentiating influence of curcumin on the clastogenicity of BLM was reproducible in three independent experiments. A potentiation effect with curcumin was also observed by Sahu and Washington (1992). They demonstrated the pro-oxidant properties of ascorbic acid and curcumin on quercetin-induced nuclear damage in presence of iron and copper. Therefore, according to these authors ascorbic acid and curcumin may have a dual role in carcinogenesis.
A potentiation effect of known antioxidant compounds combined with clastogenic compounds has also been reported by others. Chromosome aberrations induced by alkylating agents in cultured Chinese hamster cells were enhanced in the presence of s-vanillin, an isomer of vanillin (Matsumura et al., 1993). Aruoma (1993) found a pro-oxidant action of vitamin E in an assay for DNA damage with BLM and iron. Anderson et al. (1994) showed that ascorbic acid produced exacerbating effects using Comet assay with human lymphocytes when it was combined with BLM. Cozzi et al. (1997) observed that ascorbic acid increased BLM-induced aberrations in CHO cells, whereas b-carotene had no effect on chromosomal aberrations induced by BLM. Conversely, Salvadori et al. (1994) observed that b-carotene enhanced the clastogenicity induced by BLM in CHO cells.
Since BLM is a radiomimetic compound, the effects of some radioprotectors on its genetic activity have been evaluated. Radioprotectors, such as DMSO, 2-[(aminopropyl)amino] ethanethiol (WR-1065) (Hoffman et al., 1993) and cysteamine (Hoffman and Littlefield, 1995), potentiated the clastogenic activity of BLM in Go human lymphocytes. In contrast, WR-1065 has been reported to protect against the effects of BLM in cultured Chinese hamster cells (Nagy and Grdina, 1986).
BLM acts by generating oxygen radical species and it can be destroyed by the reactive oxygen species produced by itself (Scheulen et al., 1981; Lopez-Larraza et al., 1990). Salvadori et al. (1994) suggested that the presence of a scavenger, such as ascorbic acid and b-carotene, could cancel or reduce this detoxification. As curcumin is also a free-radical scavenger (Kunchandy and Rao, 1990), a similar mechanism may explain the potentiating effect observed. However, we have observed that curcumin also enhances g-radiation-induced chromosomal aberrations in CHO cells (Araújo et al., 1999), and since curcumin was clastogenic by itself, another mechanism could be involved.
Chromosomal aberrations are believed to result from unrepaired and misrepaired DNA lesions, and inhibitors of DNA repair can induce an enhancement of the DNA damage caused by many kinds of compounds (Kihlman and Natarajan, 1984; Noviello et al., 1994). Curcumin increased the BLM-induced chromosomal damage at S and G2/S phases, but not in G1/S. These data suggest that curcumin could inhibit a repair mechanism operating during S and G2/S phases in CHO cells.
Kuo et al. (1996) have noticed that curcumin induces apoptotic cell death in promyelocytic leukemia HL-60 cells through free-radical generation. Although the exact mechanism by which curcumin induces reactive oxygen species remains to be elucidated, Kunchandy and Rao (1990) reported the ability of curcumin at low concentrations to generate hydroxyl radical through the Fenton reaction by reducing Fe3+ to Fe2+, as well as possessing scavenging properties at higher levels. On the other hand, it has been postulated that BLM interacts with molecular oxygen and Fe2+ to produce superoxide anion and other oxygen metabolites (Sausville et al., 1978).
Potentiation of BLM by curcumin could also depend on its pro-oxidant activity. According to Sahu and Washington (1992), the antioxidant curcumin, like ascorbic acid, can become a pro-oxidant agent depending on the redox state of the biological environment. Therefore, the clastogenic and potentiating effects of curcumin found in the present work could be explained by the fact that curcumin would act as a pro-oxidant agent at the highest concentrations tested under the conditions of the present report. Possibly, in other conditions curcumin acts as an antioxidant agent.
On the basis of these data, we may conclude that turmeric and curcumin did not protect against BLM-induced chromosomal aberrations; conversely, curcumin exhibited a clastogenic and potentiating effect. However, the mechanism by which curcumin acts remains to be investigated, and further studies are necessary to clarify this point.
The authors thank Ana Maria R. Romero for preparing bleomycin, and Mr. Luiz A. Costa Jr. and Miss Sueli A. Neves for technical assistance. This investigation was supported by CAPES, FAPESP and CNPq. Publication supported by FAPESP.
Antioxidantes de ocorrência natural têm sido exaustivamente estudados quanto a sua capacidade de proteger organimos e células contra danos oxidativos. Muitos constituintes das plantas, incluindo cúrcuma e curcumina, parecem ser potentes antimutágenos e antioxidantes. Os efeitos de cúrcuma e curcumina na freqüência de aberrações cromossômicas induzidas pelo agente radiomimético bleomicina (BLM) foram investigados em células do ovário de hamster chinês (CHO). Três concentrações de cada droga, cúrcuma (100, 250 e 500 mg/ml) e curcumina (2,5, 5,0 e 10 mg/ml), foram combinadas com BLM (10 mg/ml) em células CHO tratadas durante as fases G1/S, S ou G2/S do ciclo celular. Nem cúrcuma nem curcumina evitaram o dano cromossômico induzido pela BLM em fase alguma do ciclo celular. Ao contrário, a potenciação da clastogenicidade da BLM pelo curcumina foi nitidamente observada em células tratadas durante as fases S e G2/S. A curcumina também se mostrou clastogênica na dose de 10 mg/ml nos protocolos de tratamento de 9 e 13 h. Contudo, o mecanismo exato pelo qual a curcumina produziu efeitos potenciadores e clastogênicos permanece desconhecido.
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(Received January 6, 1998)