Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism
- First Online:
- Cite this article as:
- Khan, H.Y., Zubair, H., Ullah, M.F. et al. Biometals (2011) 24: 1169. doi:10.1007/s10534-011-9475-9
- 212 Views
To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.
KeywordsCancer chemopreventionCopperEGCGGenisteinPolyphenolsProoxidant DNA breakage
Epidemiological studies have suggested that human consumption of fruits, vegetables and beverages such as green tea and red wine is associated with reduced risk of cardiovascular disease and certain types of cancers (Vainio and Weiderpress 2006; Park and Surh 2004). Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. Various classes of polyphenols are found in plants such as flavonoids, gallocatechins, tannins, curcuminoids, stilbenes such as resveratrol and anthocyanidins such as delphinidin. Of particular interest is the observation that the green tea polyphenol EGCG was found to induce internucleosomal DNA fragmentation in cancer cell lines such as human epidermoid carcinoma cells, human carcinoma keratinocytes, human prostate carcinoma cells and mouse lymphoma cells. However, such DNA fragmentation was not observed in normal human epidermal keratinocytes (Ahmed et al. 1997). Likewise, gallic acid showed cytotoxicity for a number of tumour cell lines but primary cultured rat hepatocytes and macrophages were found to be refractory to the cytotoxic effect (Inoue et al. 1994). Similar studies have shown that soy isoflavone genistein and red wine polyphenol resveratrol are able to induce apoptotic cell death in various cancer cell lines but not in normal cells (Chang et al. 2008; Clement et al. 1998). Moreover, Moiseeva et al. (2007) have reported that physiological concentrations of dietary phytochemicals including genistein results in reduced growth and induction of apoptosis in cancer cells.
Earlier studies in our laboratory have shown that flavonoids (Ahmad et al. 1992), tannic acid and its structural constituent gallic acid (Khan and Hadi 1998), curcumin (Ahsan and Hadi 1998), gallocatechins (Malik et al. 2003) and resveratrol (Ahmad et al. 2000) cause oxidative strand breakage in DNA either alone or in the presence of transition metal ions such as copper. Copper is an important metal ion present in chromatin and is closely associated with DNA bases, particularly guanine (Kagawa et al. 1991). It is also one of the most redox active of the various metal ions present in cells. Most of the copper present in human plasma is associated with ceruloplasmin, which has six tightly held copper atoms and a seventh, easily mobilized one (Swain and Gutteridge 1995). In a study by Satoh et al. (1997) copper was found to enhance the apoptosis-inducing activity of polyphenols, whereas iron was inhibitory. Although iron is considerably more abundant in biological systems, the major ions in the nucleus are copper and zinc (Bryan 1979).
Most of the plant polyphenols possess both antioxidant as well as prooxidant properties (Inoue et al. 1994; Ahmad et al. 1992) and we have earlier proposed that the prooxidant action of polyphenols may be an important mechanism of their anticancer and apoptosis inducing properties (Hadi et al. 2000). Some interesting studies have in fact suggested that increasing reactive oxygen species (ROS) generation over an established threshold by lowering antioxidant defenses may contribute to selective killing of cancer cells (Schumacker 2006; Trachootham et al. 2006). Such a mechanism for the cytotoxic action of polyphenolic compounds against cancer cells would involve mobilization of endogenous copper ions, possibly chromatin bound copper and the consequent prooxidant action.
According to our hypothesis, the preferential cytotoxicity of polyphenols toward cancer cells is explained by the fact that copper levels are significantly elevated in cancer cells. Indeed it has been shown that serum (Ebadi and Swanson 1998) and tissue (Yoshida et al. 1993; Nasulewis et al. 2004) concentrations of copper are greatly increased in various malignancies. As a further confirmation of our hypothesis, in this paper we show that oral administration of copper to rats leads to elevated copper levels in plasma as well as in lymphocytes. Further, when such lymphocytes are treated by various polyphenols, an increased level of cellular DNA degradation is observed.
Materials and methods
EGCG, genistein, resveratrol, neocuproine, bathocuproine, catalase, superoxide dismutase (SOD), agarose, low melting point agarose (LMPA), Histopaque 1077, RPMI 1640, phosphate buffered saline (PBS) Ca2+ and Mg2+ free, Triton X-100 and Trypan blue were purchased from Sigma (St. Louis, USA). All other chemicals used were of analytical grade. EGCG and resveratrol were dissolved in 3.0 mM cold NaOH before use as fresh stocks of 1.0 mM. Fresh solution of genistein was prepared as a stock of 1.0 mM in absolute methanol. Upon addition to reaction mixtures, in the presence of buffers mentioned and at the concentrations used, all the polyphenols used remained in solution. The volumes of stock solution added did not lead to any appreciable change in the pH of reaction mixtures.
Adult male Wistar rats weighing between 190 and 220 g were used in the study. The rats were housed in isopropelyene cages and acclimatized for a period of 1 week to laboratory conditions (23 ± 2°C and 60% humidity). They received a commercial standard diet (Ashirwad Industries, Chandigarh, India) and water ad libitum. After acclimatization, the rats were randomly divided into two equal groups and henceforth, identified as control and test groups. All the animal studies were carried out in compliance with the international practices for animal use according to the guidelines of Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India.
Cupric chloride was administered orally to rats from the test group in order to raise their intracellular status of copper. Rats from the control group were gavaged with only drinking water. Twelve hours post-gavaging, blood was drawn from control as well as copper-administered rats by cardiac puncture and kept in heparinised tubes.
Isolation of lymphocytes
Heparinized blood samples (2.0 ml) from both untreated rats and their copper-overloaded counterparts were diluted suitably in Ca2+ and Mg2+ free PBS. Lymphocytes were then isolated from the blood using Histopaque 1077 (Sigma) and the isolated cells (2 × 106) were subsequently suspended in RPMI 1640.
Viability assessment of lymphocytes
The lymphocytes were checked for their viability before the start and after the end of the reaction using Trypan Blue Exclusion test (Pool-Zoble et al. 1993). The viability of the cells was found to be almost 94%.
Measurement of copper in plasma and lymphocytes
3.9 ml of nitric acid (2.5%) was added to aliquots of 100 μl plasma/lymphocytes and vortexed. The solutions were then kept at 37°C for 5 h with regular shaking. The mixture was centrifuged at 3000 rpm for 5 min. Copper was measured in the clear supernatant by means of flame atomic absorption spectrophotometry (FAAS) (Varian Spectra 200 FS, Varian Inc, California, USA) (hollow cathode lamp, Flame type: Air acetylene; replicate 3; wavelength 324.8 nm) as described (US EPA 1994). Plasma and cellular copper levels of each animal were measured and a mean value was determined for the whole group.
Treatment of rat lymphocytes with polyphenols
Isolated lymphocytes were exposed to specified concentrations of polyphenols EGCG, genistein and resveratrol in a total reaction volume of 1.0 ml. Incubation was performed at 37°C for 1 h. In some experiments, lymphocytes were pre-incubated with various concentrations of different metal chelators prior to being treated with polyphenols. In another set of experiments, scavengers of ROS were added to the reaction mixture containing polyphenol at the final concentrations indicated. After incubation, the reaction mixture was centrifuged at 4000 rpm, the supernatant was discarded and pelleted lymphocytes were resuspended in 100 μl of PBS and processed further for Comet assay.
Evaluation of DNA breakage by comet assay
Comet assay was performed under alkaline conditions essentially according to the procedure of Singh et al. (1998) with slight modifications. Fully frosted microscopic slides precoated with 1.0% normal melting agarose at about 50°C (dissolved in Ca2+ and Mg2+ free PBS) were used. Around 10,000 cells were mixed with 75 μl of 2.0% LMPA to form a cell suspension and pipetted over the first layer and covered immediately by a coverslip. The agarose layer was allowed to solidify by placing the slides on a flat tray and keeping it on ice for 10 min. The coverslips were removed and a third layer of 0.5% LMPA (75 μl) was pipetted and coverslips placed over it and kept on ice for 5 min for proper solidification of layer. The coverslips were removed and the slides were immersed in cold lysing solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, and 1% Triton X-100 added just prior to use for a minimum of 1 h at 4°C. After lysis DNA was allowed to unwind for 30 min in alkaline electrophoretic solution consisting of 300 mM NaOH, 1 mM EDTA, pH > 13. Electrophoresis was performed at 4°C in a field strength of 0.7 V/cm and 300 mA current. The slides were then neutralized with cold 0.4 M Tris, pH 7.5, stained with 75 μl Ethidium Bromide (20 μg/ml) and covered with a coverslip. The slides were placed in a humidified chamber to prevent drying of the gel and analyzed the same day. Slides were scored using an image analysis system (Komet 5.5, Kinetic Imaging, Liverpool, UK) attached to a Olympus (CX41) fluorescent microscope and a COHU 4910 (equipped with a 510–560 nm excitation and 590 nm barrier filters) integrated CC camera. Comets were scored at ×100 magnification. Images from 50 cells (25 from each replicate slide) were analyzed. The parameter taken to assess lymphocytes DNA damage was tail length (migration of DNA from the nucleus, μm) and was automatically generated by Komet 5.5 image analysis system.
The statistical analysis was performed as described by Tice et al. (2000) and is expressed as mean ± SEM. A student’s t-test was used to examine statistically significant differences. Analysis of variance was performed using ANOVA. P values <0.05 were considered statistically significant.
Oral administration of cupric chloride to rats leads to elevated copper levels
Copper concentrations in plasma and lymphocytes of rats after different intervals of copper administration
Time interval between copper dosing and lymphocyte isolation (h)
Mean copper conc. in plasma (μg/ml ± SD)
Mean copper level in lymphocytes (μg/106 cells ± SD)
1.46 ± 0.09
8.96 ± 0.69
5.11 ± 0.34
31.28 ± 2.55
3.34 ± 0.21
18.64 ± 1.36
1.79 ± 0.05
10.56 ± 0.86
Cellular DNA breakage induced by polyphenols in lymphocytes isolated from rats with copper overload
Cellular DNA damage induced by different polyphenols in lymphocytes isolated from copper-administered rats as analysed by comet assay
Control group comet tail length (μm)
Copper administered group comet tail length (μm)
3.47 ± 0.22#
6.12 ± 0.47†
16.62 ± 0.93*
25.72 ± 1.59**
24.55 ± 1.64*
36.21 ± 2.34**
22.36 ± 1.86*
31.8 ± 1.97**
In all subsequent experiments, lymphocytes were isolated from rats 12 h after oral administration of copper. When isolated rat lymphocytes were treated with different polyphenols (Table 2), an invariably greater extent of DNA breakage was observed in the lymphocytes of copper-administered rats as compared to that of control group rats. Thus it is indicated that the elevated copper levels in copper-administered rats contributes to increased cellular DNA degradation. Further, the differences in the rate of DNA breakage by the three polyphenols tested is possibly due to differential cell membrane permeability and copper reducing efficiency of the polyphenols (Ahmad et al. 1992).
Effect of metal-specific sequestering agents on the polyphenol-induced DNA breakage in isolated rat lymphocytes with copper overload
Effect of scavengers of active oxygen species on polyphenol-induced DNA breakage in isolated rat lymphocytes with copper overload
Effect of scavengers of ROS on EGCG-induced DNA breakage in lymphocytes isolated from copper-administered rats
Comet tail length (μm)
2.41 ± 0.17#
+ EGCG (50 μM)
20.04 ± 0.81*
+ EGCG + SOD (100 μg/ml)
9.39 ± 0.36*
+ EGCG + Catalase (100 μg/ml)
10.96 ± 0.53*
+ EGCG + Thiourea (1 mM)
12.52 ± 0.78*
5.15 ± 0.30†
+ EGCG (50 μM)
34.73 ± 1.35**
+ EGCG + SOD (100 μg/ml)
13.38 ± 0.69**
+ EGCG + Catalase (100 μg/ml)
11.67 ± 0.74**
+ EGCG + Thiourea (1 mM)
15.66 ± 1.09**
The role of copper has been extensively studied in the etiology and growth of tumors (Brewer 2005; Goodman et al. 2004). Such studies were based on reports that copper distribution is altered in tumor bearing mice, rats and humans (Apelgot et al. 1986; Semczuk and Pomykalski 1973). Gupte and Mumper (2008) have reviewed several studies which indicate that both serum and tumor copper levels are significantly elevated in cancer patients compared to healthy subjects. Moreover, there are a number of studies that have focused on determining the levels of four important biological trace elements, namely copper, iron, zinc and selenium, in cancer patients. These studies showed that while iron, zinc and selenium concentrations were significantly lower in cancer patients, the copper levels were almost always found to be significantly elevated (up to two to three folds) compared to age-matched samples from normal tissue (Kuo et al. 2002; Zuo et al. 2006). However, the reason for an increased copper concentration in tumors is not clearly known.
We have earlier proposed that an important anticancer mechanism of plant-derived polyphenolic compounds could be the mobilization of endogenous copper ions and the consequent prooxidant action (Hadi et al. 2000). This is based on several lines of indirect evidence in literature and our own studies (Hadi et al. 2007). Using intact lymphocytes and isolated nuclei from these cells, we have established that plant polyphenols are able to mobilize chromatin bound copper leading to redox cycling of copper ions (Shamim et al. 2008). In the presence of molecular oxygen such a reaction leads to the formation of ROS such as hydroxyl radical, causing DNA cleavage. Further, we have suggested that the preferential cytotoxicity of plant polyphenols towards cancer cells is explained by the observation made several years earlier which showed that serum (Ebadi and Swanson 1998; Margalioth et al. 1987), tissue (Yoshida et al. 1993) and intracellular copper levels in cancer cells (Ebara et al. 2000) are significantly increased in various malignancies. Indeed, such levels have been described as a sensitive index of disease activity of several hematologic and non-hematologic malignancies (Pizzolo et al. 1978).
Since cancer cells contain elevated levels of copper, they may be more subject to electron transfer with polyphenols (Zheng et al. 2006) to generate ROS. In normal cells, there exists a balance between the free radical generation and the antioxidant system (Devi et al. 2000). However, it has been clearly documented that tumor cells are under persistent oxidative stress and have an altered antioxidant defense (Powis and Baker 1997; Pervaiz and Clement 2004) and thus further ROS stress in these malignant cells, surpassing a threshold level, could result in apoptosis (Gupte and Mumper 2008). These observations suggest that neoplastic cells may be more vulnerable to oxidative stress as they function with a heightened basal level of ROS owing to increased rate of growth and metabolism (Kong et al. 2000). Thus, in cancer cells, a further enhanced exposure to ROS, generated through the redox cycling of intracellular copper by polyphenols, can overwhelm the cells antioxidant capacity, leading to irreversible damage and apoptosis. On the other hand, normal non-malignant cells can better tolerate such an action due to their low basal ROS output and normal metabolic regulation, as also normal copper redox status. Hence, a disparity in the redox states of cancer cells and normal cells may provide a molecular basis for selective killing of cancer cells by the use of agents, like polyphenols, that can cause further ROS insults to the malignant cells. Therefore, we propose that this accounts for the preferential cytotoxicity of plant polyphenols toward cancer cells. Indeed we have recently shown that the polyphenol genistein mediated apoptotic cell death and cell proliferation inhibition in MDA MB 231 and MDA MB 468 breast cancer cell lines is inhibited by the copper chelator neocuproine, whereas iron and zinc chelators have little effect (Ullah et al. 2011).
In order to further substantiate our hypothesis, in this paper we have attempted to elevate copper levels in lymphocytes by administering cupric chloride to rats to a range similar to what has been reported in leukemic cancer patients (Carpentieri et al. 1986) [31.28 ± 2.55 μg Cu/106 cells (Table 1) vs. 52 ± 16 μg Cu/106 cells (in leukemia patients)]. It must be mentioned that copper itself is known to be cytotoxic and leads to cellular DNA cleavage. However as shown in the results the levels of copper achieved in lymphocytes on cupric chloride administration does not lead to a significant increase in comet tail lengths as compared to the untreated controls (Fig. 1). More importantly, the addition of polyphenols (EGCG and genistein) causes several fold increase in comet tail lengths, indicating that the contribution of copper alone to cellular DNA breakage is insignificant. Thus the above results taken together lead to the conclusion that the elevated copper levels in lymphocytes, such as those found in cancer patients could be an important factor in the anticancer mechanism of plant polyphenols.
In summary and in confirmation of our hypothesis, the following milestones have been achieved: (i) an in vitro reaction between plant polyphenols, Cu(II) and DNA leading to DNA cleavage has been characterised. Most polyphenols are capable of causing DNA breakage in this reaction (Ahsan and Hadi 1998; Ahmad et al. 2000; Rahman et al. 1989; Azam et al. 2004); (ii) as a further step we have shown that polyphenol-Cu(II) system is indeed capable of causing DNA degradation in a cellular system and this reaction could be of biological significance (Azmi et al. 2005); (iii) we have also shown that polyphenols are capable of mobilizing endogenous copper ions from cells leading to cellular DNA breakage (Azmi et al. 2006); (iv) we have further demonstrated that in the above oxidative cellular DNA breakage nuclear copper is mobilized (Shamim et al. 2008); (v) we have shown that polyphenol induced growth inhibition in breast cancer cell lines is inhibited by copper chelator to a significant extent whereas iron and zinc chelators are relatively ineffective (Ullah et al. 2011); (vi) finally, in this paper we have shown that oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by polyphenols. This substantiates the role of elevated copper levels in cancer tissue and cells in the anticancer mechanism of plant polyphenols.
The authors acknowledge the financial assistance provided by the University Grants Commission, New Delhi, under the DRS-II program and Junior Research Fellowship to HYK from CSIR, New Delhi.
Conflict of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.