Original Papers
Protective Effects of Common Anthocyanidins against Genotoxic Damage Induced by Chemotherapeutic Drugs in Mice
Authors
Nidhi Khandelwal, Suresh K. Abraham
Affiliation
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Key words " anthocyanidins l " apoptosis l " anticancer drugs l " DNA damage l " micronucleus test l
Abstract
received revised accepted
April 1, 2014 July 23, 2014 August 18, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383050 Published online September 3, 2014 Planta Med 2014; 80: 1278–1283 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. S. K. Abraham School of Life Sciences Jawaharlal Nehru University New Delhi 110067 India Phone: + 91 98 95 06 63 96 [email protected]
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Experiments were performed to assess in mice the inhibitory effects of the anthocyanidins cyanidin, delphinidin, malvidin, and pelargonidin on genotoxic damage induced by the anticancer drugs cyclophosphamide, procarbazine, and cisplatin. Each anthocyanidin was administered 30 min before injecting the drug, and genotoxicity was assessed by measuring micronucleated polychromatic erythrocytes in bone marrow cells. In addition, we monitored the effect of anthocyanidins on apoptosis induced by cyclophosphamide and procarbazine. The results showed significant protective effects of cyanidin, delphinidin, malvidin, and pelargonidin against DNA damage induced by cyclophosphamide. With delphinidin and malvidin, a biphasic dose-response was observed for protection against cyclophosphamide. Dose-related reduction of genotoxicity was observed with pelargonidin against procarbazine. However with cyanidin, the medium dose of 2 mg/kg showed maximum protection against procarbazine. Cyanidin and pelargonidin significantly reduced the chromosomal damage induced by cisplatin. Furthermore, pre-treatment with
Introduction !
Anthocyanins constitute a very large and important group of water-soluble natural pigments, responsible for the orange, red, blue, and purple colors of flowers, fruits, and vegetables [1–4]. At present approximately 635 anthocyanins have been identified in nature [2]. The six most commonly found anthocyanidins (aglycone form of anthocyanins) are CYN (50%), PEL (12%), peonidin (12%), DEL (12%), petunidin (7 %), and MAL (7%) [1, 2]. The chemical structures of these anthocya" Fig. 1. Investigations nidins have been shown in l on the bioactivity of anthocyanins suggest that
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these anthocyanidins reduced the level of apoptosis induced by cyclophosphamide and procarbazine. In conclusion, this study shows that anthocyanidins can reduce the efficacy of anticancer drugs for inducing DNA damage and apoptosis.
Abbreviations !
CIS: CPH: CYN: DEL: DPPH: EGCG: MAL: MnPCEs: PCB: PCEs: PEL: PI: ROS:
cisplatin cyclophosphamide cyanidin delphinidin 2,2,diphenyl-1-picrylhydrazyl epigallocatechin gallate malvidin micronucleated polychromatic erythrocytes procarbazine polychromatic erythrocytes pelargonidin propidium iodide reactive oxygen species
Supporting information available online at http://www.thieme-connect.de/products
these polyphenols possess anti-inflammatory, anticarcinogenic, antigenotoxic, as well as preventive effects on cardiovascular diseases, obesity, and diabetes [1–4]. These health benefits of anthocyanins have been attributed mainly to their antioxidant activity/free radical scavenging property [2]. The different sources of dietary anthocyanin intake include fruits, vegetables, fruitbased processed food, fruit juices, and red wine [5]. In view of the wide spread consumption of anthocyanins and the known health benefits, we initiated this study to assess the role of these polyphenols as in vivo antigenotoxic agents. There are re-
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Fig. 1
Chemical structures of anthocyanidins.
ports which show that anthocyanidins exert in vitro protective effects against genotoxic damage [6, 7]. Our recent work demonstrated the in vivo antigenotoxic effects of the anthocyanidins CYN and PEL against the environmental carcinogens diepoxybutane and urethane [8]. Generation of ROS is important for activation of apoptosis needed to eliminate pre-neoplastic and neoplastic cells [9–12]. DNA lesions induced by genotoxic anticancer drugs can play an important role in causing cell death triggered by apoptosis [10]. Quenching of ROS by dietary antioxidants could possibly reduce the level of DNA damage and apoptosis, thereby affecting the efficacy of the anticancer drug [13, 14]. The above observations prompted us to initiate the present work with the main aim of probing whether or not anthocyanidins have an impact on DNA damage and apoptosis induced by anticancer drugs which are known to generate ROS or free radicals [11, 12]. In this study, the main focus was on the dose-response effect for induction of genotoxic damage following oral administration of the common anthocyanidins CYN, DEL, MAL, and PEL to mice before exposure to CPH/CIS/PCB. We also included experiments to evaluate the impact of anthocyanidin pre-treatment on the incidence of chemotherapeutic drug-induced apoptotic cells.
Results ! " Table 1 show the protective effects of the The data presented in l anthocyanidins PEL, CYN, MAL, and DEL against the genotoxic damage induced by CPH. PEL pre-treatment resulted in dose dependent response for protection against CPH, whereas the effect of CYN is not dose-dependent. Maximum protection is observed with the medium dose of CYN (2 mg/kg b. w.). The four doses of MAL showed significant reductions in CPH-induced genotoxicity. However, the protective effect of the lowest dose (0.25 mg/kg b. w.) is better than that of the higher doses. All three doses of DEL exerted significant protection against CPH-induced genotoxic damage. Pre-treatment with DEL did not show a dose-dependent antigenotoxic effect. The lowest test dose showed the maximum protection. " Fig. 2, there is significant reduction in PCB-inAs shown in l duced genotoxicity following pre-treatment with PEL or CYN. PEL exerted a dose-dependent anti-genotoxic effect against PCB, whereas the protective effect of CYN is not dose-dependent. The medium dose of CYN (2 mg/kg b. w.) shows a better protection than the other two doses. " Fig. 3 presents the anti-genotoxic effect of PEL and CYN against l the genomic damage induced by CIS. Both CYN and PEL show a significant reduction in the genotoxicity induced by CIS. The lower dose of CYN shows a better protection compared to the higher dose of PEL.
a
Pretreatment
Treatment
Mn PCEs/
PCEs/
Inhibition
and doseb
and doseb
2000 PCEsa
NCEs
(%)
Controlc PEL (20) CYN (4) MAL (2) DEL (1) Controlc EGCG (25) PEL (1) PEL (5) PEL (10) PEL (20) CYN (1) CYN (2) CYN (4) MAL (0.25) MAL (0.50) MAL (1) MAL (2) DEL (0.25) DEL (0.5) DEL (1)
Controlc Controlc Controlc Controlc Controlc CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50)
3.83 ± 1.16 5.00 ± 1.41 4.66 ± 1.75 5.33 ± 1.21 5.16 ± 1.16 76.11 ± 8.61 35.83 ± 4.70* 34.16 ± 6.85* 24.50 ± 7.39* 19.33 ± 3.07* 16.33 ± 5.27* 31.33 ± 2.42* 20.33 ± 4.13* 26.50 ± 3.72* 19.66 ± 5.39* 27.83 ± 6.79* 32.83 ± 7.08* 35.66 ± 4.32* 21.83 ± 4.40* 31.83 ± 6.36* 30.33 ± 5.78*
1.25 1.23 1.24 1.24 1.22 1.08 1.25 1.24 1.24 1.26 1.24 1.24 1.23 1.25 1.24 1.23 1.22 1.23 1.25 1.22 1.22
NA NA NA NA NA NA 52.93 55.11 67.80 74.60 78.54 58.83 73.28 65.18 74.16 63.43 56.86 53.14 71.31 58.17 60.14
Values are means ± SD from six mice; b Dose (mg/kg b. w.); c The control animals re-
ceived distilled water; * Significant when compared to mice treated with CPH alone (p < 0.05); % Inhibition was calculated as follows: effect of CPH alone effect of CPH þ anthocyanidin 100 effect of CPH alone
Apoptotic frequencies in bone marrow cells from control, CYN, PCB, CPH, PCB + CYN, and CPH + CYN treated groups were assessed by flow cytometry using annexin V/PI labeling (Fig. 1S, " Table 2 Supporting Information). The data are summarized in l which shows the percentage of early apoptotic and late apoptotic/necrotic cells. Treatment with DEL induced a significant increase in the frequency of early apoptotic and late apoptotic/necrotic cells when compared to the control. This effect was not observed with CYN, MAL, and PEL. Co-treatment of PCB/CPH with all the anthocyanidins resulted in a significant reduction in percentage of early apoptotic and late apoptotic/necrotic cells. The free radical scavenging activity of DEL, MAL, PEL, and CYN was assessed using the DPPH free radical scavenging technique. IC50 values obtained were: DEL = 0.16 mg/10 mL, CYN = 0.43 mg/ " Fig. 4). 10 mL, PEL = 0.67 mg/10 mL, and MAL = 0.89 mg/10 mL (l
Discussion !
The results obtained from our present investigation clearly indicate that the anthocyanidins CYN, DEL, MAL, and PEL can exert significant inhibitory effects on the chromosomal damage induced by the chemotherapeutic drugs CPH, CIS, and PCB. In addition, we observed a reduction in apoptosis when the animals are pre-treated with these anthocyanidins before exposure to CPH and PCB. For cancer chemotherapy, there is need for free radicals and double strand DNA damage to trigger apoptosis which would lead to death of the neoplastic cell [11, 12]. The protective effect exerted by anthocyanidins against DNA damage is likely to counteract the therapeutic efficacy of anticancer drugs. In this context, it should be emphasized that conflicting reports have appeared on the effects of anticancer drugs in combination with dietary
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Table 1 Protective effects of anthocyanidins pelargonidin, cyanidin, malvidin, and delphinidin against genotoxic damage induced by cyclophosphamide.
Original Papers
Fig. 2 Antigenotoxic effect of three doses of pelargonidin (2.5, 5.0, and 10.0 mg/kg b. w.) and cyanidin (1, 2, and 4 mg/kg b. w.) against procarbazine (50 mg/kg b. w.). EGCG (25 mg/kg) was used as positive control. Shown are means ± SD from six mice. * Significant when compared to the PCBtreated mice (p < 0.05).
antioxidants. For example, in transgenic mice developing brain tumors, depletion of antioxidants in the standard diet increases the apoptotic death of tumor cells [15]. Inhibition of CPH-induced tumor regression has been observed when the experimental animals received dietary curcumin, a well known antioxidant [16]. β-carotene increased the cytotoxicity of melphalan and CIS in tumor cell lines [17, 18], whereas the same dietary antioxidant reduced the genotoxicity of CPH in mice [19]. From the present dose-response studies, there is evidence for the efficacy of low doses of anthocyanidins as inhibitors of genotoxic damage. Both DEL and MAL have shown the best protection against CPH-induced chromosomal damage when the lowest test dose is administered, thereby suggesting a biphasic dose-response. The important role of low doses of these dietary antioxidants as effective chemoprotective agents against DNA damage induced by widely used antineoplastic drugs is evident from the present work.
Fig. 3 Effect of pelargonidin (5 mg/kg b. w.) and cyanidin (0.5 mg/kg b. w.) on cisplatin-induced micronucleus formation. EGCG (25 mg/kg) was used as positive control. Shown are means ± SD from six mice. * Significant when compared to the CIS-treated mice (p < 0.05).
The beneficial health effects of anthocyanin intake have been attributed to the free radical scavenging/antioxidant activity of this group of flavonoids. Anthocyanins are known for their ability to scavenge reactive oxygen species like superoxide, singlet oxygen, peroxide, hydrogen peroxide, and hydroxyl radical, and these effects have been observed in vitro in colon, liver, breast, endothelial, and leukemic cell cultures [4]. From in vitro studies, there is evidence for the role of anthocyanins in activation of antioxidant response element upstream of genes which code for glutathionerelated phase II antioxidant and detoxifying enzymes like glutathione reductase, glutathione peroxidase, and glutathione Stransferase [4]. Induction of apoptosis leading to cell death is an important step for the success of chemotherapy of cancer. When anthocyanidins
Table 2 Protective effects of delphinidin, cyanidin, malvidin, and pelargonidin against apoptosis induced by procarbazine and cyclophosphamide. Treatment and dose
% Live cells
% Damaged cells
% Early apoptotic cells
% Late apoptotic + necrotic
(mg/kg b. w.)
(no stain)
(PI positive)
(annexin V-FITC positive)
cells (PI + FITC positive)
Control DEL (1) CYN (1) MAL (1) PEL (1) PCB (50) PCB (50) + EGCG (25) PCB (50) + DEL (1) PCB (50) + CYN (1) PCB (50) + MAL (1) PCB (50) + PEL (1) CPH (50) CPH (50) + DEL(1) CPH (50) + CYN (1) CPH (50) + MAL (1) CPH (50) + PEL (1)
98.60 ± 0.18 97.19 ± 0.47 98.92 ± 0.35 98.51 ± 0.10 99.25 ± 0.06 90.26 ± 0.57 96.10 ± 0.58 96.20 ± 0.62 95.61 ± 0.77 94.86 ± 0.80 95.18 ± 1.32 91.26 ± 0.20 96.04 ± 0.76 97.68 ± 0.36 97.01 ± 0.41 96.66 ± 0.80
0.11 ± 0.04 0.17 ± 0.04 0.13 ± 0.04 0.13 ± 0.04 0.14 ± 0.02 1.25 ± 0.88 1.07 ± 0.50 1.17 ± 0.23 1.54 ± 0.43 1.17 ± 0.23 1.04 ± 0.63 0.87 ± 0.21 0.90 ± 0.41 1.09 ± 0.16 1.32 ± 0.52 0.61 ± 0.18
0.88 ± 0.18 1.80 ± 0.16** 0.67 ± 0.01 0.96 ± 0.16 0.59 ± 0.04 4.98 ± 1.43** 1.36 ± 0.34* 1.89 ± 0.30* 1.13 ± 0.48* 1.89 ± 0.30* 1.71 ± 0.29* 3.85 ± 0.44** 1.87 ± 0.32* 0.86 ± 0.13* 1.09 ± 0.15* 1.69 ± 0.40*
0.39 ± 0.03 1.16 ± 0.07** 0.46 ± 0.08 0.43 ± 0.01 0.39 ± 0.09 3.52 ± 0.66** 1.99 ± 0.33* 2.06 ± 0.44* 1.37 ± 0.58* 2.06 ± 0.59* 2.06 ± 0.53* 4.01 ± 0.85** 1.17 ± 0.17* 0.35 ± 0.14* 0.57 ± 0.07* 1.02 ± 0.30*
The above values are means ± SD; * Significantly different from PCB/CPH-treated group (p < 0.05); ** significantly different from control group (p < 0.05)
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granates, cherries, and plums [22]. Evaluation of anthocyanin absorption in rat and pig models revealed its presence in jejunum, stomach, kidney, liver, eyes, and brain [2]. Anthocyanins have the potential to cross the cell membrane and their presence has been detected in the interior of cells [23]. Orally administered anthocyanins are rapidly absorbed and eliminated. It has been estimated that < 1% of anthocyanin intake is absorbed and excreted in the urine [4]. This is evident from the short time of 0.25 h to reach Cmax for plasma [23]. It is excreted from the urine within 4–8 h [4]. Furthermore, increasing the dose does not lead to increase in anthocyanin absorption, thereby indicating that there is saturation of the absorption mechanism [23]. Our present investigation using the mouse model demonstrated that four of the most commonly consumed anthocyanidins can reduce the DNA damaging efficacy of the chemotherapeutic drugs CPH, CIS, and PCB. In addition, we observed that induction of apoptosis by CPH and PCB can be reduced by intake of these anthocyanidins.
Fig. 4 DPPH free radical scavenging by cyanidin, delphinidin, malvidin, and pelargonidin.
Materials and Methods scavenge free radicals and inhibit DNA damage, an anti-apoptotic effect can be expected. Our present work has shown a reduction in apoptotic cells in the bone marrow of mice pre-treated with the anthocyanidins CYN, DEL, MAL, and PEL before injecting CPH/PCB. In addition, our main findings have demonstrated the protective effects of these anthocyanidins against chemotherapeutic drug-induced chromosomal damage leading to formation of micronuclei. In order to assess the impact of anthocyanidins on the efficacy of chemotherapeutic drugs, there is need for information on intake of these dietary flavonoids from various food sources which include fruits, vegetables, wine, non-alcoholic beverages, cakes, and confectionery. The European Prospective Investigation into Cancer and Nutrition (EPIC) carried in twenty-seven centres covering ten countries in North and South Europe estimated that the mean value for individual anthocyanidin consumption ranges from 19.83 to 64.86 mg/day in men and 18.73 to 44.08 mg/day in women [5]. Turin in Italy registered the highest level of total anthocyanidin intake [5]. If these levels of anthocyanidin consumption are compared to the results of our study in which we observed significant reductions in genotoxic damage with low doses of 0.5 and 1.0 mg/kg b. w., it indicates that concurrent intake of foods and beverages containing these flavonoids during chemotherapy might affect the efficacy of antineoplastic drugs. Questions on the absorption and bioavailability of dietary phytochemicals are often raised when investigations are carried out to evaluate the in vivo antioxidant/antimutagenic/anticarcinogenic effects. When in vivo investigations are performed, factors like absorption, metabolism, and pharmacokinetics of distribution determine the dose of the ultimate reactive metabolite reaching different target tissues to exert a chemopreventive effect. In this context, we would like to highlight the fact that a human intervention study with anthocyanin-rich fruit juice showed a reduction in oxidative DNA damage and significant increase in reduced glutathione [20]. However, another investigation indicated that anthocyanins from cranberry juice had no effect on oxidative DNA damage or antioxidant status in healthy human volunteers [21]. In the human diet, there are many anthocyanin-containing fruits and vegetables like: red onion, egg plant, red radish, black bean, purple cabbage, purple sweet potato, black currant, grapes, pome-
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Chemicals CIS (CAS No. 15663-27-1; % purity ≥ 99%), CYN chloride (CAS No. 528-58-5; % purity ≥ 95%), CPH (CAS No. 6055-19-2; % purity ≥ 97%), DEL (CAS No. 528-53-0; % purity ≥ 95%), EGCG (CAS No. 989-51-5; % purity ≥ 95 %), MAL chloride (CAS No. 643-84-5; % purity ≥ 95%), and PEL chloride (CAS No. 134-04-3) were purchased from Sigma Chemical Company. PCB (CAS No. 366-70-1; % purity ≥ 95%) was a gift from Hoffmann-La Roche. FITC-annexin V apoptosis kit was purchased from BD Pharmigen (CAT No. 556547).
Animals All the experiments were carried out with male Swiss albino mice, which were 12–14 weeks old, weighing 30–36 g. These animals were bred in the University animal house and maintained at 25 ± 2 °C on the standard mouse diet and water ad libitum in accordance with CPCSEA, India guidelines. Approval for this work was obtained from the University Animal Ethical Committee (IAEC‑JNU 5/2011).
Pre-treatment PEL, CYN, MAL, and DEL were dissolved in double distilled water and administered to the experimental animals by gavage (10 mL/ kg b. w.). EGCG dissolved in double distilled water was used as positive control [24]. Negative control animals received the same volume of distilled water. Each treatment group consisted of six mice. The test doses of anthocyanidins were decided based on preliminary experiments and our previous work [8].
Genotoxin treatment Genotoxins were injected intraperitoneally 30 min after administration of the anthocyanidins. The control animals received the same volume (10 mL/kg b. w.) of distilled water.
Micronucleus assay Micronucleus test was carried out according to Schmid [25] to evaluate the genotoxic effects in the mouse bone marrow. The experimental animals were sacrificed after 27 h treatment, and bone marrow from both femurs were flushed into a centrifuge tube containing 1 mL fetal calf serum. The bone marrow sample
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Original Papers
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was collected in the form of fine suspension. This cell suspension was centrifuged at 2000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in a drop of serum before being used for preparing slides. Air-dried slides were stained with May-Grünwald and Giemsa stain. Six mice were used for each experimental point, and 2000 PCEs/animal were scored from a single slide to determine the frequency of MnPCEs. Special care was taken to ensure that the doses selected for the combination treatment did not lead to suppression of cell proliferation. This was done by monitoring the ratio of PCEs to NCEs (normochromatic erythrocytes). All the slides were scored by the same observer.
Acknowledgements !
This work was supported by the University Grants Commission (NETWORKING) and Department of Science and Technology (PURSE). N. K. thanks the University Grants Commission for the Meritorious Research Fellowship.
Conflict of Interest !
The authors state no conflict of interest.
References
DPPH free radical scavenging activity Antioxidant activities of PEL, CYN, MAL, and DEL were screened by performing the DPPH assay as described by Abraham and Khandelwal [26]. 25 µL of sample solution (different concentrations in distilled water) and 225 µL of DPPH (100 µM) in methanol were taken in a 96-well microplate and incubated at 37 °C for 15 min. The absorbance was measured at 517 nm by a microplate reader. Percent radical scavenging activity was determined by comparison with a methanol-containing control and calculated by the following equation: I (%) = 100(Ablank – Asample)/Ablank Where Ablank is the absorbance of the control reaction mixture excluding the test compounds and Asample is the absorbance of the reaction mixture with the tested compounds. IC50 values represent the concentrations of PEL, CYN, MAL, and DEL able to scavenge 50% of DPPH radicals and are expressed as means of three separate experiments.
Flow cytometry by annexin V-FITC/propidium iodide staining The mice for the above experiment were divided into the following groups: control, CPH (50 mg/kg), PCB (50 mg/kg), CPH (50 mg/ kg) + anthocyanidin (1 mg/kg), PCB (50 mg/kg) + anthocyanidin (1 mg/kg), and anthocyanidin (1 mg/kg) group. Anthocyanidins were administered by gavage. CPH (50 mg/kg)/PCB (50 mg/kg) was injected intraperitoneally. The marrow cells were harvested at 12 h after CPH/PCB administration (as described above), washed twice with pre-chilled PBS (4 °C) by centrifugation for 5 min and then resuspended for annexin V-FITC/PI staining assay (BD Pharmigen FITC annexin V apoptosis kit I). For the annexin VFITC/PI staining assay, the cell density was adjusted to 1 × 106 cells/mL. Approximately 1 × 105 cells were incubated in the dark with 5 µL annexin V and 5 µL PI solution for 15 min. Thereafter the suspension was analyzed by flow cytometry.
Statistical analysis Data are expressed as mean ± standard deviation. Studentʼs t-test was performed using Graph pad software for comparing the incidence of bone marrow micronuclei/incidence of apoptosis of two groups. The comparison was between the positive control and the group that received pre-treatment with anthocyanidin. Results were considered statistically significant at p < 0.05.
Supporting information The flow cytometric profile of mouse bone marrow cells stained with annexin V and PI is available as Supporting Information.
Khandelwal N, Abraham SK. Protective Effects of …
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Protective Effects of Common Anthocyanidins against Genotoxic Damage Induced by Chemotherapeutic Drugs in Mice
Authors
Nidhi Khandelwal, Suresh K. Abraham
Affiliation
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Key words " anthocyanidins l " apoptosis l " anticancer drugs l " DNA damage l " micronucleus test l
Abstract
received revised accepted
April 1, 2014 July 23, 2014 August 18, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383050 Published online September 3, 2014 Planta Med 2014; 80: 1278–1283 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. S. K. Abraham School of Life Sciences Jawaharlal Nehru University New Delhi 110067 India Phone: + 91 98 95 06 63 96 [email protected]
!
Experiments were performed to assess in mice the inhibitory effects of the anthocyanidins cyanidin, delphinidin, malvidin, and pelargonidin on genotoxic damage induced by the anticancer drugs cyclophosphamide, procarbazine, and cisplatin. Each anthocyanidin was administered 30 min before injecting the drug, and genotoxicity was assessed by measuring micronucleated polychromatic erythrocytes in bone marrow cells. In addition, we monitored the effect of anthocyanidins on apoptosis induced by cyclophosphamide and procarbazine. The results showed significant protective effects of cyanidin, delphinidin, malvidin, and pelargonidin against DNA damage induced by cyclophosphamide. With delphinidin and malvidin, a biphasic dose-response was observed for protection against cyclophosphamide. Dose-related reduction of genotoxicity was observed with pelargonidin against procarbazine. However with cyanidin, the medium dose of 2 mg/kg showed maximum protection against procarbazine. Cyanidin and pelargonidin significantly reduced the chromosomal damage induced by cisplatin. Furthermore, pre-treatment with
Introduction !
Anthocyanins constitute a very large and important group of water-soluble natural pigments, responsible for the orange, red, blue, and purple colors of flowers, fruits, and vegetables [1–4]. At present approximately 635 anthocyanins have been identified in nature [2]. The six most commonly found anthocyanidins (aglycone form of anthocyanins) are CYN (50%), PEL (12%), peonidin (12%), DEL (12%), petunidin (7 %), and MAL (7%) [1, 2]. The chemical structures of these anthocya" Fig. 1. Investigations nidins have been shown in l on the bioactivity of anthocyanins suggest that
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these anthocyanidins reduced the level of apoptosis induced by cyclophosphamide and procarbazine. In conclusion, this study shows that anthocyanidins can reduce the efficacy of anticancer drugs for inducing DNA damage and apoptosis.
Abbreviations !
CIS: CPH: CYN: DEL: DPPH: EGCG: MAL: MnPCEs: PCB: PCEs: PEL: PI: ROS:
cisplatin cyclophosphamide cyanidin delphinidin 2,2,diphenyl-1-picrylhydrazyl epigallocatechin gallate malvidin micronucleated polychromatic erythrocytes procarbazine polychromatic erythrocytes pelargonidin propidium iodide reactive oxygen species
Supporting information available online at http://www.thieme-connect.de/products
these polyphenols possess anti-inflammatory, anticarcinogenic, antigenotoxic, as well as preventive effects on cardiovascular diseases, obesity, and diabetes [1–4]. These health benefits of anthocyanins have been attributed mainly to their antioxidant activity/free radical scavenging property [2]. The different sources of dietary anthocyanin intake include fruits, vegetables, fruitbased processed food, fruit juices, and red wine [5]. In view of the wide spread consumption of anthocyanins and the known health benefits, we initiated this study to assess the role of these polyphenols as in vivo antigenotoxic agents. There are re-
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Fig. 1
Chemical structures of anthocyanidins.
ports which show that anthocyanidins exert in vitro protective effects against genotoxic damage [6, 7]. Our recent work demonstrated the in vivo antigenotoxic effects of the anthocyanidins CYN and PEL against the environmental carcinogens diepoxybutane and urethane [8]. Generation of ROS is important for activation of apoptosis needed to eliminate pre-neoplastic and neoplastic cells [9–12]. DNA lesions induced by genotoxic anticancer drugs can play an important role in causing cell death triggered by apoptosis [10]. Quenching of ROS by dietary antioxidants could possibly reduce the level of DNA damage and apoptosis, thereby affecting the efficacy of the anticancer drug [13, 14]. The above observations prompted us to initiate the present work with the main aim of probing whether or not anthocyanidins have an impact on DNA damage and apoptosis induced by anticancer drugs which are known to generate ROS or free radicals [11, 12]. In this study, the main focus was on the dose-response effect for induction of genotoxic damage following oral administration of the common anthocyanidins CYN, DEL, MAL, and PEL to mice before exposure to CPH/CIS/PCB. We also included experiments to evaluate the impact of anthocyanidin pre-treatment on the incidence of chemotherapeutic drug-induced apoptotic cells.
Results ! " Table 1 show the protective effects of the The data presented in l anthocyanidins PEL, CYN, MAL, and DEL against the genotoxic damage induced by CPH. PEL pre-treatment resulted in dose dependent response for protection against CPH, whereas the effect of CYN is not dose-dependent. Maximum protection is observed with the medium dose of CYN (2 mg/kg b. w.). The four doses of MAL showed significant reductions in CPH-induced genotoxicity. However, the protective effect of the lowest dose (0.25 mg/kg b. w.) is better than that of the higher doses. All three doses of DEL exerted significant protection against CPH-induced genotoxic damage. Pre-treatment with DEL did not show a dose-dependent antigenotoxic effect. The lowest test dose showed the maximum protection. " Fig. 2, there is significant reduction in PCB-inAs shown in l duced genotoxicity following pre-treatment with PEL or CYN. PEL exerted a dose-dependent anti-genotoxic effect against PCB, whereas the protective effect of CYN is not dose-dependent. The medium dose of CYN (2 mg/kg b. w.) shows a better protection than the other two doses. " Fig. 3 presents the anti-genotoxic effect of PEL and CYN against l the genomic damage induced by CIS. Both CYN and PEL show a significant reduction in the genotoxicity induced by CIS. The lower dose of CYN shows a better protection compared to the higher dose of PEL.
a
Pretreatment
Treatment
Mn PCEs/
PCEs/
Inhibition
and doseb
and doseb
2000 PCEsa
NCEs
(%)
Controlc PEL (20) CYN (4) MAL (2) DEL (1) Controlc EGCG (25) PEL (1) PEL (5) PEL (10) PEL (20) CYN (1) CYN (2) CYN (4) MAL (0.25) MAL (0.50) MAL (1) MAL (2) DEL (0.25) DEL (0.5) DEL (1)
Controlc Controlc Controlc Controlc Controlc CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50) CPH (50)
3.83 ± 1.16 5.00 ± 1.41 4.66 ± 1.75 5.33 ± 1.21 5.16 ± 1.16 76.11 ± 8.61 35.83 ± 4.70* 34.16 ± 6.85* 24.50 ± 7.39* 19.33 ± 3.07* 16.33 ± 5.27* 31.33 ± 2.42* 20.33 ± 4.13* 26.50 ± 3.72* 19.66 ± 5.39* 27.83 ± 6.79* 32.83 ± 7.08* 35.66 ± 4.32* 21.83 ± 4.40* 31.83 ± 6.36* 30.33 ± 5.78*
1.25 1.23 1.24 1.24 1.22 1.08 1.25 1.24 1.24 1.26 1.24 1.24 1.23 1.25 1.24 1.23 1.22 1.23 1.25 1.22 1.22
NA NA NA NA NA NA 52.93 55.11 67.80 74.60 78.54 58.83 73.28 65.18 74.16 63.43 56.86 53.14 71.31 58.17 60.14
Values are means ± SD from six mice; b Dose (mg/kg b. w.); c The control animals re-
ceived distilled water; * Significant when compared to mice treated with CPH alone (p < 0.05); % Inhibition was calculated as follows: effect of CPH alone effect of CPH þ anthocyanidin 100 effect of CPH alone
Apoptotic frequencies in bone marrow cells from control, CYN, PCB, CPH, PCB + CYN, and CPH + CYN treated groups were assessed by flow cytometry using annexin V/PI labeling (Fig. 1S, " Table 2 Supporting Information). The data are summarized in l which shows the percentage of early apoptotic and late apoptotic/necrotic cells. Treatment with DEL induced a significant increase in the frequency of early apoptotic and late apoptotic/necrotic cells when compared to the control. This effect was not observed with CYN, MAL, and PEL. Co-treatment of PCB/CPH with all the anthocyanidins resulted in a significant reduction in percentage of early apoptotic and late apoptotic/necrotic cells. The free radical scavenging activity of DEL, MAL, PEL, and CYN was assessed using the DPPH free radical scavenging technique. IC50 values obtained were: DEL = 0.16 mg/10 mL, CYN = 0.43 mg/ " Fig. 4). 10 mL, PEL = 0.67 mg/10 mL, and MAL = 0.89 mg/10 mL (l
Discussion !
The results obtained from our present investigation clearly indicate that the anthocyanidins CYN, DEL, MAL, and PEL can exert significant inhibitory effects on the chromosomal damage induced by the chemotherapeutic drugs CPH, CIS, and PCB. In addition, we observed a reduction in apoptosis when the animals are pre-treated with these anthocyanidins before exposure to CPH and PCB. For cancer chemotherapy, there is need for free radicals and double strand DNA damage to trigger apoptosis which would lead to death of the neoplastic cell [11, 12]. The protective effect exerted by anthocyanidins against DNA damage is likely to counteract the therapeutic efficacy of anticancer drugs. In this context, it should be emphasized that conflicting reports have appeared on the effects of anticancer drugs in combination with dietary
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Table 1 Protective effects of anthocyanidins pelargonidin, cyanidin, malvidin, and delphinidin against genotoxic damage induced by cyclophosphamide.
Original Papers
Fig. 2 Antigenotoxic effect of three doses of pelargonidin (2.5, 5.0, and 10.0 mg/kg b. w.) and cyanidin (1, 2, and 4 mg/kg b. w.) against procarbazine (50 mg/kg b. w.). EGCG (25 mg/kg) was used as positive control. Shown are means ± SD from six mice. * Significant when compared to the PCBtreated mice (p < 0.05).
antioxidants. For example, in transgenic mice developing brain tumors, depletion of antioxidants in the standard diet increases the apoptotic death of tumor cells [15]. Inhibition of CPH-induced tumor regression has been observed when the experimental animals received dietary curcumin, a well known antioxidant [16]. β-carotene increased the cytotoxicity of melphalan and CIS in tumor cell lines [17, 18], whereas the same dietary antioxidant reduced the genotoxicity of CPH in mice [19]. From the present dose-response studies, there is evidence for the efficacy of low doses of anthocyanidins as inhibitors of genotoxic damage. Both DEL and MAL have shown the best protection against CPH-induced chromosomal damage when the lowest test dose is administered, thereby suggesting a biphasic dose-response. The important role of low doses of these dietary antioxidants as effective chemoprotective agents against DNA damage induced by widely used antineoplastic drugs is evident from the present work.
Fig. 3 Effect of pelargonidin (5 mg/kg b. w.) and cyanidin (0.5 mg/kg b. w.) on cisplatin-induced micronucleus formation. EGCG (25 mg/kg) was used as positive control. Shown are means ± SD from six mice. * Significant when compared to the CIS-treated mice (p < 0.05).
The beneficial health effects of anthocyanin intake have been attributed to the free radical scavenging/antioxidant activity of this group of flavonoids. Anthocyanins are known for their ability to scavenge reactive oxygen species like superoxide, singlet oxygen, peroxide, hydrogen peroxide, and hydroxyl radical, and these effects have been observed in vitro in colon, liver, breast, endothelial, and leukemic cell cultures [4]. From in vitro studies, there is evidence for the role of anthocyanins in activation of antioxidant response element upstream of genes which code for glutathionerelated phase II antioxidant and detoxifying enzymes like glutathione reductase, glutathione peroxidase, and glutathione Stransferase [4]. Induction of apoptosis leading to cell death is an important step for the success of chemotherapy of cancer. When anthocyanidins
Table 2 Protective effects of delphinidin, cyanidin, malvidin, and pelargonidin against apoptosis induced by procarbazine and cyclophosphamide. Treatment and dose
% Live cells
% Damaged cells
% Early apoptotic cells
% Late apoptotic + necrotic
(mg/kg b. w.)
(no stain)
(PI positive)
(annexin V-FITC positive)
cells (PI + FITC positive)
Control DEL (1) CYN (1) MAL (1) PEL (1) PCB (50) PCB (50) + EGCG (25) PCB (50) + DEL (1) PCB (50) + CYN (1) PCB (50) + MAL (1) PCB (50) + PEL (1) CPH (50) CPH (50) + DEL(1) CPH (50) + CYN (1) CPH (50) + MAL (1) CPH (50) + PEL (1)
98.60 ± 0.18 97.19 ± 0.47 98.92 ± 0.35 98.51 ± 0.10 99.25 ± 0.06 90.26 ± 0.57 96.10 ± 0.58 96.20 ± 0.62 95.61 ± 0.77 94.86 ± 0.80 95.18 ± 1.32 91.26 ± 0.20 96.04 ± 0.76 97.68 ± 0.36 97.01 ± 0.41 96.66 ± 0.80
0.11 ± 0.04 0.17 ± 0.04 0.13 ± 0.04 0.13 ± 0.04 0.14 ± 0.02 1.25 ± 0.88 1.07 ± 0.50 1.17 ± 0.23 1.54 ± 0.43 1.17 ± 0.23 1.04 ± 0.63 0.87 ± 0.21 0.90 ± 0.41 1.09 ± 0.16 1.32 ± 0.52 0.61 ± 0.18
0.88 ± 0.18 1.80 ± 0.16** 0.67 ± 0.01 0.96 ± 0.16 0.59 ± 0.04 4.98 ± 1.43** 1.36 ± 0.34* 1.89 ± 0.30* 1.13 ± 0.48* 1.89 ± 0.30* 1.71 ± 0.29* 3.85 ± 0.44** 1.87 ± 0.32* 0.86 ± 0.13* 1.09 ± 0.15* 1.69 ± 0.40*
0.39 ± 0.03 1.16 ± 0.07** 0.46 ± 0.08 0.43 ± 0.01 0.39 ± 0.09 3.52 ± 0.66** 1.99 ± 0.33* 2.06 ± 0.44* 1.37 ± 0.58* 2.06 ± 0.59* 2.06 ± 0.53* 4.01 ± 0.85** 1.17 ± 0.17* 0.35 ± 0.14* 0.57 ± 0.07* 1.02 ± 0.30*
The above values are means ± SD; * Significantly different from PCB/CPH-treated group (p < 0.05); ** significantly different from control group (p < 0.05)
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granates, cherries, and plums [22]. Evaluation of anthocyanin absorption in rat and pig models revealed its presence in jejunum, stomach, kidney, liver, eyes, and brain [2]. Anthocyanins have the potential to cross the cell membrane and their presence has been detected in the interior of cells [23]. Orally administered anthocyanins are rapidly absorbed and eliminated. It has been estimated that < 1% of anthocyanin intake is absorbed and excreted in the urine [4]. This is evident from the short time of 0.25 h to reach Cmax for plasma [23]. It is excreted from the urine within 4–8 h [4]. Furthermore, increasing the dose does not lead to increase in anthocyanin absorption, thereby indicating that there is saturation of the absorption mechanism [23]. Our present investigation using the mouse model demonstrated that four of the most commonly consumed anthocyanidins can reduce the DNA damaging efficacy of the chemotherapeutic drugs CPH, CIS, and PCB. In addition, we observed that induction of apoptosis by CPH and PCB can be reduced by intake of these anthocyanidins.
Fig. 4 DPPH free radical scavenging by cyanidin, delphinidin, malvidin, and pelargonidin.
Materials and Methods scavenge free radicals and inhibit DNA damage, an anti-apoptotic effect can be expected. Our present work has shown a reduction in apoptotic cells in the bone marrow of mice pre-treated with the anthocyanidins CYN, DEL, MAL, and PEL before injecting CPH/PCB. In addition, our main findings have demonstrated the protective effects of these anthocyanidins against chemotherapeutic drug-induced chromosomal damage leading to formation of micronuclei. In order to assess the impact of anthocyanidins on the efficacy of chemotherapeutic drugs, there is need for information on intake of these dietary flavonoids from various food sources which include fruits, vegetables, wine, non-alcoholic beverages, cakes, and confectionery. The European Prospective Investigation into Cancer and Nutrition (EPIC) carried in twenty-seven centres covering ten countries in North and South Europe estimated that the mean value for individual anthocyanidin consumption ranges from 19.83 to 64.86 mg/day in men and 18.73 to 44.08 mg/day in women [5]. Turin in Italy registered the highest level of total anthocyanidin intake [5]. If these levels of anthocyanidin consumption are compared to the results of our study in which we observed significant reductions in genotoxic damage with low doses of 0.5 and 1.0 mg/kg b. w., it indicates that concurrent intake of foods and beverages containing these flavonoids during chemotherapy might affect the efficacy of antineoplastic drugs. Questions on the absorption and bioavailability of dietary phytochemicals are often raised when investigations are carried out to evaluate the in vivo antioxidant/antimutagenic/anticarcinogenic effects. When in vivo investigations are performed, factors like absorption, metabolism, and pharmacokinetics of distribution determine the dose of the ultimate reactive metabolite reaching different target tissues to exert a chemopreventive effect. In this context, we would like to highlight the fact that a human intervention study with anthocyanin-rich fruit juice showed a reduction in oxidative DNA damage and significant increase in reduced glutathione [20]. However, another investigation indicated that anthocyanins from cranberry juice had no effect on oxidative DNA damage or antioxidant status in healthy human volunteers [21]. In the human diet, there are many anthocyanin-containing fruits and vegetables like: red onion, egg plant, red radish, black bean, purple cabbage, purple sweet potato, black currant, grapes, pome-
!
Chemicals CIS (CAS No. 15663-27-1; % purity ≥ 99%), CYN chloride (CAS No. 528-58-5; % purity ≥ 95%), CPH (CAS No. 6055-19-2; % purity ≥ 97%), DEL (CAS No. 528-53-0; % purity ≥ 95%), EGCG (CAS No. 989-51-5; % purity ≥ 95 %), MAL chloride (CAS No. 643-84-5; % purity ≥ 95%), and PEL chloride (CAS No. 134-04-3) were purchased from Sigma Chemical Company. PCB (CAS No. 366-70-1; % purity ≥ 95%) was a gift from Hoffmann-La Roche. FITC-annexin V apoptosis kit was purchased from BD Pharmigen (CAT No. 556547).
Animals All the experiments were carried out with male Swiss albino mice, which were 12–14 weeks old, weighing 30–36 g. These animals were bred in the University animal house and maintained at 25 ± 2 °C on the standard mouse diet and water ad libitum in accordance with CPCSEA, India guidelines. Approval for this work was obtained from the University Animal Ethical Committee (IAEC‑JNU 5/2011).
Pre-treatment PEL, CYN, MAL, and DEL were dissolved in double distilled water and administered to the experimental animals by gavage (10 mL/ kg b. w.). EGCG dissolved in double distilled water was used as positive control [24]. Negative control animals received the same volume of distilled water. Each treatment group consisted of six mice. The test doses of anthocyanidins were decided based on preliminary experiments and our previous work [8].
Genotoxin treatment Genotoxins were injected intraperitoneally 30 min after administration of the anthocyanidins. The control animals received the same volume (10 mL/kg b. w.) of distilled water.
Micronucleus assay Micronucleus test was carried out according to Schmid [25] to evaluate the genotoxic effects in the mouse bone marrow. The experimental animals were sacrificed after 27 h treatment, and bone marrow from both femurs were flushed into a centrifuge tube containing 1 mL fetal calf serum. The bone marrow sample
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was collected in the form of fine suspension. This cell suspension was centrifuged at 2000 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in a drop of serum before being used for preparing slides. Air-dried slides were stained with May-Grünwald and Giemsa stain. Six mice were used for each experimental point, and 2000 PCEs/animal were scored from a single slide to determine the frequency of MnPCEs. Special care was taken to ensure that the doses selected for the combination treatment did not lead to suppression of cell proliferation. This was done by monitoring the ratio of PCEs to NCEs (normochromatic erythrocytes). All the slides were scored by the same observer.
Acknowledgements !
This work was supported by the University Grants Commission (NETWORKING) and Department of Science and Technology (PURSE). N. K. thanks the University Grants Commission for the Meritorious Research Fellowship.
Conflict of Interest !
The authors state no conflict of interest.
References
DPPH free radical scavenging activity Antioxidant activities of PEL, CYN, MAL, and DEL were screened by performing the DPPH assay as described by Abraham and Khandelwal [26]. 25 µL of sample solution (different concentrations in distilled water) and 225 µL of DPPH (100 µM) in methanol were taken in a 96-well microplate and incubated at 37 °C for 15 min. The absorbance was measured at 517 nm by a microplate reader. Percent radical scavenging activity was determined by comparison with a methanol-containing control and calculated by the following equation: I (%) = 100(Ablank – Asample)/Ablank Where Ablank is the absorbance of the control reaction mixture excluding the test compounds and Asample is the absorbance of the reaction mixture with the tested compounds. IC50 values represent the concentrations of PEL, CYN, MAL, and DEL able to scavenge 50% of DPPH radicals and are expressed as means of three separate experiments.
Flow cytometry by annexin V-FITC/propidium iodide staining The mice for the above experiment were divided into the following groups: control, CPH (50 mg/kg), PCB (50 mg/kg), CPH (50 mg/ kg) + anthocyanidin (1 mg/kg), PCB (50 mg/kg) + anthocyanidin (1 mg/kg), and anthocyanidin (1 mg/kg) group. Anthocyanidins were administered by gavage. CPH (50 mg/kg)/PCB (50 mg/kg) was injected intraperitoneally. The marrow cells were harvested at 12 h after CPH/PCB administration (as described above), washed twice with pre-chilled PBS (4 °C) by centrifugation for 5 min and then resuspended for annexin V-FITC/PI staining assay (BD Pharmigen FITC annexin V apoptosis kit I). For the annexin VFITC/PI staining assay, the cell density was adjusted to 1 × 106 cells/mL. Approximately 1 × 105 cells were incubated in the dark with 5 µL annexin V and 5 µL PI solution for 15 min. Thereafter the suspension was analyzed by flow cytometry.
Statistical analysis Data are expressed as mean ± standard deviation. Studentʼs t-test was performed using Graph pad software for comparing the incidence of bone marrow micronuclei/incidence of apoptosis of two groups. The comparison was between the positive control and the group that received pre-treatment with anthocyanidin. Results were considered statistically significant at p < 0.05.
Supporting information The flow cytometric profile of mouse bone marrow cells stained with annexin V and PI is available as Supporting Information.
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