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Abstract

Recent studies have proposed the use of low concentrations of phytochemicals and combinations of phytochemicals in chemoprevention to reduce cytotoxicity and simulate normal ingestion through diet. The purpose of the present study was to evaluate whether the DNA damage, chromosome instability, and oxidative stress induced by cisplatin (cDDP) are modulated by a combination of the natural pigments lutein (LT) and chlorophyll b (CLb). The protective effects observed for synergism between phytochemicals have not been completely investigated. The comet assay and micronucleus test were performed and the catalase activities and glutathione (GSH) concentrations were measured in the peripheral blood, bone marrow, liver, and kidney cells of mice. The comet assay and micronucleus test results revealed that the pigments LT and CLb were not genotoxic or mutagenic and that the pigments presented antigenotoxic and antimutagenic effects in the different cell types evaluated. This protective effect is likely related to antioxidant properties in peripheral blood cells through the prevention of cDDP-induced GSH depletion. Altogether our results show that the combination of LT and CLb, which are both usually present in the same foods, such as leafy green vegetables, can be used safely.

Keywords

Antioxidant activity diet genotoxicity mutagenicity

Introduction

Recently, a large number of studies have demonstrated the important role of natural phytochemicals from plants in the prevention of diseases related to DNA damage, such as cancer and atherosclerosis.^1

–4 Recent studies have proposed the use of these phytochemicals in low concentrations and in various combinations to simulate the intake of low quantities in the diet.^5,6 Chlorophyll b (CLb) and lutein (LT) are the phytochemicals with the potential to become effective chemopreventive agents.

LT is a carotenoid that is abundant in dark leafy green vegetables, such as spinach and kale,⁷ and it is the second most prevalent carotenoid in human serum.⁸ Most of the protective effects attributed to LT are related to its antioxidant activity, which has been previously described in vitro ^9
–11 and in vivo.^12
–14 CLb belongs to the class of tetrapyrrole derivatives, and CLb is composed of a porphyrin ring structure with a central nonreactive magnesium ion.¹⁵ Chlorophyll compounds possess a high antioxidant activity because the molecules can function as hydrogen donors,¹⁶ scavenge reactive species,¹⁷ and increase glutathione (GSH) levels.¹⁸

Our previous in vivo studies using the isolated phytochemicals LT and CLb showed the antioxidant capacity of both compounds and their protective effects regarding DNA damage.^12,18 However, considering that both compounds are naturally present in the same foods, such as green vegetables, the in vivo effects of these phytochemicals when combined remain unclear. Therefore, the present study was performed to confirm whether the beneficial effects observed in vivo with LT and CLb individually remain when both the pigments are combined. Thus, cisplatin (cDDP) was employed as damage inducer, and the protective effects of the combined pigments were evaluated through the comet assay, micronucleus test, catalase (CAT) activities, and GSH concentrations in mouse peripheral blood, bone marrow, liver, and kidney cells.

Materials and methods

Animals

The experimental protocols for this study were approved by the Local Ethics Committee for Animal Use of Campus Administrativo de Ribeirão Preto, Universidade de São Paulo, Brazil, Register No. 06.1.909.53.7.

The experiments were performed on female and male albino Swiss mice (Mus musculus) that were 7–8 weeks old and weighed approximately 30 g at the onset of the experiments. The animals were obtained from the Animal Center of Campus Administrativo de Ribeirão Preto at the Universidade de São Paulo (Brazil), and they were housed in polycarbonate cages with steel wire tops (four animals per cage) at standard room temperature (22 ± 2°C) and humidity (55 ± 10%) with a 12- h light/dark cycle. Standard food (Nuvilab®; Curitiba, Brazil) and water were provided ad libitum, according to the guidelines of the Canadian Council on Animal Care.¹⁹

Chemicals

The LT standard (purity between 95% and 99%) was donated by DSM Nutritional Products (Basel, Switzerland). cDDP (CAS: 15663-27-1) was purchased from Quiral Química do Brasil (Juiz de Fora, Brazil). Methyl methanesulfonate (MMS) (CAS: 66-27-3), CLb (CAS: 519-62-0), ethidium bromide (CAS: 1239-45-8), and trypan blue (CAS: 72-57-1) were purchased from Sigma-Aldrich (St Louis, Missouri, USA). All the other reagents were of analytical grade.

Dose selection and experimental design

Doses of CLb and LT were based on our previous studies where only the highest tested dose of isolated CLb showed protective effects.¹⁸ Therefore, the dosage of 0.5 mg/kg body weight (b.w.) of CLb and LT were combined in this study. The 0.5 mg/kg b.w. dosages reflect the recommended human daily intake for LT²⁰ and the amount of CL found in 200 g of vegetables, which are concentrations that have been correlated with a reduction in cancer risk.²¹ The cDDP dose (6 mg/kg b.w.) was selected based on the previous studies.^12,18,22

LT and CLb were diluted in mineral oil (MO), and the animals received approximately 0.2 mL of the mixture. To eliminate any interference of the solvent in the gain or loss of weight, b.w. was followed during the experiments and no difference was observed among the animals in the different groups evaluated (data not shown). The mice were divided into four groups (n = 8, four females and four males); I: MO (control group); II: LT + CLb; III: MO + cDDP (positive control group); and IV: LT + CLb + cDDP. The MO or LT + CLb groups were administered with their respective compounds daily by gavage for 13 consecutive days. On the 14th day, 0.9% saline (in I or II) or cDDP diluted in saline (in groups III and IV) was intraperitoneally administered. After 24 h (15th day), the mice were euthanized by decapitation under anesthesia with 10% chloral hydrate that was administered intraperitoneally (4 mL/kg b.w.).

Comet assay in mice peripheral blood, liver, and kidney cells

The comet assay (pH > 13) was conducted according to the protocols of Tice et al.²³ and Singh et al.²⁴ and followed the guidelines of Collins et al. and Hartmann et al.^25,26 Samples of liver and kidney were collected after euthanasia, and 0.2 g of each organ was placed in 1 mL of chilled Hank’s balanced salt solution in a petri dish. Then, the organs were sliced into fragments with a pair of scissors and filtered through two layers of gauze. Peripheral blood was collected from the caudal vein 4 h after the first treatment with MO or LT + CLb (T0) and 4 h after injection with saline or cDDP (T1) on the 14th day. A complementary experiment was performed with MMS (200 mg/kg b.w.) as a positive control for the comet assay. Trypan blue dye exclusion was used to determine the cell viability immediately before the comet assay, and the viability was above 90% for all the treatments.

Peripheral blood (10 µL) or the supernatant of the kidney or liver cell suspensions (40 µL) were mixed with 180 µL of low melting point agarose (0.5%) and spread onto microscope slides coated with normal melting point agarose (1.5%). The cells were covered with a coverslip and maintained at a temperature of 4°C for 5 min. The coverslips were removed from slides and immersed in freshly prepared lysis solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA), 10% dimethylsulfoxide, 1% Triton X-100, 10 mM Tris, and pH 10) for 60 min at 4°C. After lysis, the slides were placed in a horizontal electrophoresis unit containing 300 mM NaOH and 1 mM EDTA, pH > 13, and left for 20 min for the DNA to denature. Electrophoresis was run for 20 min at 1 V/cm (25 V and 300 mA). Subsequently, the slides were immersed in a neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 15 min.^12,18 After being dried at ambient temperature, the slides were fixed in ethanol for 5 min and stained with 30 µL of ethidium bromide (20 µL/mL) immediately before analysis. Image analysis was performed using fluorescence microscopy with a Diaphot 300 (Nikon, Tokyo, Japan) using a 510–560 nm filter and a 590 nm barrier. A total of 100 comets per animal were visually analyzed using Komet 6.0 software, and the results were expressed as a percentage of DNA in the tail (% tail DNA, the fraction of DNA in the tail divided by the amount of DNA in the cell multiplied by 100) and the tail moment (TM) is calculated as the product of the two values: the percentage of DNA in the comet tail and the tail length in micrometers.²⁷

Micronucleus test in mouse bone marrow and peripheral blood cells

Bone marrow was removed 24 h after saline or cDDP injection, which is in compliance with Organization for Economic Cooperation and Development (OECD) 474.²⁸ Briefly, the bone marrow was flushed out of the femurs into a centrifuge tube with fetal calf serum. The bone marrow cells were collected by centrifugation at 1000 r/min for 10 min, and the pellet was resuspended in 0.3 mL of supernatant (fetal calf serum) for the slide preparation. A drop of this suspension was smeared on a clean slide, air-dried, fixed in absolute methanol for 10 min, and stained in the following day with Giemsa (diluted with phosphate buffer, pH 6.8). Two thousand polychromatic erythrocytes (PCEs) were analyzed, and the number of micronucleated PCEs was recorded.²⁹ Cytotoxicity was measured using the percentage of PCEs among 500 erythrocytes (PCEs + normochromatic erythrocytes (NCEs)).

The micronucleus test on peripheral blood cells from the tail vein was performed according to the protocol described by Hayashi et al.³⁰ which uses slides prestained with acridine orange. Blood sampling was performed three times: T0, before the first treatment (first day); T1, 36 h after the first treatment; and T2, 24 h after the saline or cDDP injection (15th day). Each sample was placed at the center of a prestained slide and covered with a coverslip (24 × 50 mm). The slides were stored in the dark at −20°C until the cytological examination was performed. The cell preparations were examined under a fluorescence microscope (Zeiss) with a blue (488 nm) excitation filter and a yellow (515 nm) emission (barrier) filter using an immersion objective. A total of 1000 reticulocytes per treated animal were analyzed, and the proportion of micronucleated reticulocytes was counted. The percentage of reduction in micronucleated cells (%R) was calculated using the formula: $% R = ((m e a n i n A - m e a n i n B) / (m e a n i n A - m e a n i n C)) \times 100$ , where A is the group treated with cDDP (positive control), B is the group treated with different doses of CLb plus cDDP, and C represents the group treated with MO (control group).³¹

Evaluation of CAT activity and GSH concentrations in total blood

Total blood was collected immediately after euthanasia and was maintained at −80°C until the biochemical parameters could be evaluated. CAT activity was assayed according to the method of Aebi.³² The levels of hemoglobin (Hb) were analyzed in the total blood by measuring cyanmethemoglobin using Drabkin’s reagent.³³ CAT activity was expressed as κ/g Hb, in which κ is the rate constant: κ = (2•3/Δt) (log S ₁/S ₂), where Δt = t ₂–t ₁, which represents the measured time interval, and S ₁ and S ₂ = H₂O₂ concentrations at times t ₁ and t ₂, respectively. The constant κ can be used as a direct measure of CAT activity.

Reduced GSH concentrations were estimated using the method of Ellman.³⁴ Briefly, erythrocytes (0.3 mL) were hemolyzed using 10% Triton X-100 (0.1 mL) and precipitated with 0.2 mL of 20% trichloroacetic acid. After centrifugation at 5,000 r/min for 10 min, color was developed in the supernatant by adding 50 µL of 10 mM 5-5’-dithio-bis(2-nitrobenzoic acid), and the optical density was recorded at 412 nm using a standard α-cysteine curve at 0.01, 0.025, 0.05, 0.1, and 0.15 µM. The GSH content was expressed as micromoles per milliliter of erythrocytes.

Statistical analysis

There was no statistical difference between the results obtained for males and females for all endpoints analyzed. Therefore, the results of both sexes were grouped to improve the understanding of data.

All results were expressed as the means ± SD. The results of measurements of the antioxidant parameters, genotoxicity, mutagenicity, and the protective effects were compared using one-way analysis of variance and Tukey’s test (p < 0.05) in GraphPad Prism 5 Project software system (La Jolla, California, USA).

Results

Comet assay in peripheral blood, kidney, and liver cells

The results obtained in the comet assay are shown in Figure 1(a) and (b). The genotoxicity assessment of the combined pigments demonstrated that they were not genotoxic because the percentage of DNA in the tail obtained after treatment did not differ from the control group (group I) in peripheral blood, kidney, or liver cells (Figure 1(a)). Figure 1(b) shows that the same result was observed for the parameter TM.

Figure 1.

(a) Percentage of DNA in the tail (% tail DNA) and (b) TM of the peripheral blood (PB), liver, and kidney cells of mice. The mean ± SD for eight animals from each group are presented. PB(TO), blood samples collected 4 h after the first gavage; PB(T1), blood samples collected 4 h after i.p. injection of saline, MMS or cDDP on the 14th day. Liver and kidney cells were collected on the 15th day after euthanasia. *Different from the MO group; ^#Different from the MO + cDDP group. ANOVA and Tukey’s test with p < 0.05. TM: tail moment; i.p.: intraperitoneally; MO: mineral oil (control group); cDDP: cisplatin (6 mg/kg b.w.); LT: lutein (0.5 mg/kg b.w.); CLb: chlorophyll b (0.5 mg/kg b.w.); MMS: methyl methanesulfonate (200 mg/kg b.w.); ANOVA: analysis of variance.

Antigenotoxicity was evaluated when the combined pigments (LT + CLb) were associated with cDDP (group IV), and the pigments showed a significant protective effect in the three tissues evaluated. MMS was used to induce DNA migration in the positive control group because cDDP decreases the percentage of DNA in the tail relative to the negative control group. The decrease in DNA migration observed in group III (MO + cDDP) reflects the mechanism of action of cDDP linked to cross-link formation. The combined pigments are able to reduce the cross-link formation and maintain DNA migration similarly to the control group.

Micronucleus test in bone marrow and peripheral blood

The frequency of micronucleated cells in the bone marrow and in the peripheral blood cells of mice are shown in Figure 2. Oral treatment with MO or LT + CLb for 13 days followed by saline injection did not induce any mutagenic effects in both of the cell types evaluated. The mice that received the cDDP injection presented a significant increase in micronucleus frequency in bone marrow and at the last time point of treatment in peripheral blood (T2) compared with the control group.

Figure 2.

Frequency of micronucleated cells in peripheral blood (PB) and bone marrow cells. The plot shows the mean ± SD for eight animals (male and female) from each group. PB(T0), blood samples collected before the experiment; PB(T1), 36 h after the first treatment; PB(T2), 24 h after i.p. injection of saline or cDDP on the 15th day. Bone marrow was collected on the 15th day after euthanasia. *Different from the MO group; ^#Different from the MO + cDDP group. ANOVA and Tukey’s test with p < 0.05. i.p.: intraperitoneally; MO: mineral oil; cDDP: cisplatin (6 mg/kg b.w.); LT: lutein (0.5 mg/kg b.w.); CLb: chlorophyll b (0.5 mg/kg b.w.); ANOVA: analysis of variance.

Examination of the antimutagenic activity of the combined pigments revealed a significant decrease in the frequency of micronucleated cells relative to the MO + cDDP group with a reduction of 77.4% and 72.0% for peripheral blood cells and bone marrow, respectively. There was no statistical difference in the ratio of PCE/(PCE + NCE) detected, which indicated that none of the treatments induced cytotoxicity in bone marrow cells (Figure 3).

Figure 3.

PCE/PCE + NCE ratios based on the examination of 500 erythrocytes from the bone marrow cells. The mean ± SD for eight animals from each group are presented. No significant differences were observed among the tested groups. ANOVA and Tukey’s test with p < 0.05. MO: mineral oil (control group); cDDP: cisplatin (6 mg/kg b.w.); LT: lutein (0.5 mg/kg b.w.); CLb: chlorophyll b (0.5 mg/kg b.w.); PCE: polychromatic erythrocyte; NCE: normochromatic erythrocyte; ANOVA: analysis of variance.

Biochemical parameters of oxidative stress in peripheral blood: CAT and GSH

The results for the GSH concentrations and CAT activities in mouse peripheral blood are presented in Figure 4(a) and (b), respectively. The GSH concentration decreased in the MO + cDDP group and was maintained at levels similar to the control in the animals that received LT + CLb in addition to cDDP (group IV). The CAT activities did not present a statistical difference among all groups.

Figure 4.

Oxidative stress evaluation in total blood from mice after subacute treatment. (a) GSH levels and (b) CAT activity. The mean ± SD for eight animals (male and female) in each group are presented. *Different from the MO group. Statistical analysis performed using ANOVA and Tukey’s test with significance threshold of p < 0.05. GSH: glutathione; CAT: catalase; MO: mineral oil (control group); cDDP: cisplatin (6 mg/kg b.w.); LT: lutein (0.5 mg/kg b.w.); CLb: chlorophyll b (0.5 mg/kg b.w.); ANOVA: analysis of variance.

Discussion

The relative safety, low cost, and ease of oral bioavailability provide great potential for the use of phytochemicals for the early stages of cancer chemoprevention.³⁵ Primary prevention considers the inhibition of mutation and cancer initiation in extracellular and intracellular environments, including the inhibition of the uptake of mutagens and carcinogens as well as maintenance of the DNA structure and antioxidant activity.³⁶

Our previous studies showed that the individual application of LT and CLb at doses normally consumed in the diet (0.5 mg/kg b.w.) improved the cellular antioxidant status as well as prevented in vivo DNA damage and chromosome instability induced by the chemotherapeutic compound cDDP.^12,18 cDDP has two labile-binding sites that are capable of forming bifunctional DNA lesions.³⁷ The most reactive nucleophilic centers in DNA are guanine N7 atoms, therefore, guanine–guanine intrastrand and interstrand cross-links and DNA-protein cross-links are the most abundant lesions.³⁸ In addition, 1,2-intrastrand ApG (cross-links formed between adjacent adenine and guanine) and GpG (cross-links formed between adjacent guanines) cross-links are the major forms of DNA adducts, accounting for 85–90% of total lesions.³⁹

However, as LT and CLb are naturally present in many of the same foods, we hypothesized that these same pigments could exert protective effects in combination and that the compounds could act synergistically. The results of the present study demonstrate that using the two pigments together presented significant antigenotoxic and antimutagenic activities in different cell types of mice and improved the antioxidant status in peripheral blood cells.

The comet assay and micronucleus test results revealed that the LT and CLb pigments did not cause any toxic effects to DNA molecules when combined. In terms of the antigenotoxic and antimutagenic effects, the individual application of LT in our previous studies caused a 70.9% reduction in micronucleus frequency in peripheral blood cells and a 70.5% reduction in bone marrow cells. For CLb, the percentage reductions were much lower, approximately 31.3% in peripheral blood and 23.2% in bone marrow cells. When the pigments were combined in the present study, the percentages were slightly higher than for LT alone (77.4% in peripheral blood and 72.0% in bone marrow cells). These results showed that the antimutagenic power of LT remains when this carotenoid is combined with CLb, which predicts the same effect from whole vegetables.

The effects of combinations of compounds are scarce in the literature. Studies performed in our laboratory demonstrated that in vivo administration of curcumin and vitamin C showed protective effects only when individually administered,⁴⁰ and vitamins C and E were able to reduce chromosome aberrations induced by doxorubicin when combined in higher doses.⁴¹ A recent article by Abraham and coworkers⁶ showed that the individual applications of chlorogenic acid, pelargonidin, resveratrol, and epigallocatechin gallate are antimutagenic in vitro; however, they did not demonstrate any additive or synergistic effects when used in combination with phytochemicals.

The antigenotoxic and antimutagenic effects of phytochemicals are commonly related to their antioxidant activity,^6,42,43 and recent studies have highlighted an increased antioxidant capacity when natural compounds are combined.^44
–46 Here, the concentrations of an endogenous antioxidant, reduced GSH, were evaluated as a parameter of oxidative stress. cDDP reduced the GSH concentrations because GSH is able to bind to the platinum atom in cDDP molecules, which prevents binding to other cellular nucleophiles and subsequent damage to cellular components.⁴⁷ Mice that received the combination of LT and CLb as well as cDDP showed a significant increase in GSH concentrations relative to the group that received only cDDP. These data indicated that the combination of pigments was able to prevent the GSH depletion induced by cDDP. In addition, CAT activity was not altered among all treatments.

As LT is primarily found in green leafy vegetables, the abundant presence of CLb could interfere with the absorption of LT and the manifestation of its biological effects. However, our important results show that the LT and CLb pigments can be used safely in combination. In addition, these results indicate that CLb does not interfere with the protective effects of LT.

Footnotes

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgments

We are grateful to Mrs Joana D’arc Castania Darin (FCFRP-USP) for her technical assistance. We are also grateful to Prof Ilce Mara de Syllos Cólus for the donation of MMS. The authors also thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (no. 05/59552-6, 2008/06793-4 and 2010/05096-8), the Conselho Nacional para o Desenvolvimento Científico e Tecnológico (CNPq), and the Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES/DS).

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Effects of lutein and chlorophyll b on GSH depletion and DNA damage induced by cisplatin in vivo

Abstract

Keywords

Introduction

Materials and methods

Animals

Chemicals

Dose selection and experimental design

Comet assay in mice peripheral blood, liver, and kidney cells

Micronucleus test in mouse bone marrow and peripheral blood cells

Evaluation of CAT activity and GSH concentrations in total blood

Statistical analysis

Results

Comet assay in peripheral blood, kidney, and liver cells

Micronucleus test in bone marrow and peripheral blood

Biochemical parameters of oxidative stress in peripheral blood: CAT and GSH

Discussion

Footnotes

Conflict of interest

Acknowledgments

References