Acetylsalicylic acid exhibits anticlastogenic effects on cultured human lymphocytes exposed to doxorubicin

Mutation Research 626 (2007) 155–161

Acetylsalicylic acid exhibits anticlastogenic effects on cultured human lymphocytes exposed to doxorubicin Lusˆania Maria Greggi Antunes a,b,∗ , Rafaela de Barros e Lima Bueno b , Francisca da Luz Dias b , Maria de Lourdes Pires Bianchi a a

Depto de An´alises Cl´ınicas, Toxicol´ogicas e Bromatol´ogicas, Faculdade de Ciˆencias Farmacˆeuticas de Ribeir˜ao Preto, Universidade de S˜ao Paulo, S˜ao Paulo, Brazil b Depto de Ciˆ encias Biol´ogicas, Universidade Federal do Triˆangulo Mineiro, Minas Gerais, Brazil Received 4 August 2006; received in revised form 20 September 2006; accepted 29 September 2006 Available online 13 November 2006

Abstract Acetylsalicylic acid (ASA) is a non-steroidal anti-inflammatory drug (NSAID) with many pharmacological properties, such as anti-inflammatory, antipyretic and analgesic. Many studies have suggested the possible efficiency of ASA and other NSAIDs in preventing cancer. ASA could also have antimutagenic and antioxidant properties. The aim of this study was to investigate the possible clastogenic and anticlastogenic effects of different concentrations of ASA on doxorubicin-induced chromosomal aberrations in human lymphocytes. Human blood samples were obtained from six healthy, non-smoking volunteers; and the chromosomal aberration assay was carried out using conventional techniques. The parameters analyzed were mitotic index, total number of chromosomal aberrations and percentage of aberrant metaphases. The concentrations of ASA (25, 50 or 100 ␮g/mL) tested in combination with DXR (0.2 ␮g/mL) were established on the basis of the results of the mitotic index. The treatment with ASA alone was neither cytotoxic nor clastogenic (p > 0.01). In lymphocyte cultures treated with different combinations of ASA and DXR, a significant decrease in the total number of chromosome aberrations was observed compared with DXR alone (p < 0.01). This protective effect of ASA on DXR-induced chromosomal damage was obtained for all combinations, and it was most evident when ASA was at 25.0 ␮g/mL. In our experiments, ASA may have acted as an antioxidant and inhibited the chromosomal damage induced by the free radicals generated by DXR. The identification of compounds that could counteract the free radicals produced by doxorubicin could be of possible benefits against the potential harmful effects of anthracyclines. The results of this study show that there is a relevant need for more investigations in order to elucidate the mechanisms underlying the anticlastogenic effect of ASA. © 2006 Elsevier B.V. All rights reserved. Keywords: Acetylsalicylic acid; Anticlastogenicity; Antimutagenesis; Doxorubicin; Lymphocytes; Chromosome aberrations

1. Introduction There is a growing body of epidemiological evidence and experimental studies supporting that a high con-

∗ Corresponding author at: Av. do Caf´ e s/n, Ribeir˜ao Preto, SP 14040-903, Brazil. Tel.: + 55 16 3602 4294; fax: + 55 16 3602 4725. E-mail address: [email protected] (L.M.G. Antunes).

1383-5718/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2006.09.009

sumption of fruits and vegetables is capable of inhibiting, retarding, or reversing the multiple steps of the carcinogenic process, and also that their compounds can present antimutagenic, anticarcinogenic and antioxidant properties [1,2]. More recently, there is strong evidence from in vitro studies and clinical trials that other compounds, such as salicylic acid, widely found in plants, spices, vegetables, and beverages and non-steroidal antiinflammatory drugs (NSAIDs) can modulate oxidative

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stress and contribute to decrease the risk of several cancers [3–5]. Acetylsalicylic acid (ASA) is a non-steroidal antiinflammatory drug (NSAID) with many pharmacological properties, such as anti-inflammatory, antipyretic, and analgesic. ASA is also a synthetic analogue structurally related to the salicylate family of compounds, salicylic acid being its main metabolite. Many studies have suggested the possible efficiency of ASA and other NSAIDs in preventing cancer, and particularly in reducing the mortality rate due to colorectal cancer and bowel adenomas [4,6]. The suppressive and protective effects of ASA as an anti-cancer drug might be partially ascribed to its antioxidants properties. The ability of ASA to inhibit reactive oxygen species-mediated DNA damage was investigated. The inhibition of oxidative stress by ASA was concentration-dependent in in vitro assays, and it was proposed that the antioxidant activity of ASA may contribute for cancer chemoprotection in humans [7]. Since anti-inflammatory drugs can reduce oxygen radical species, such as superoxide anion, hydroxyl radical and hydrogen peroxide produced by inflammatory cells such as neutrophils, they have been suggested to be effective antioxidant and protective agents. The antimutagenic and antioxidant properties of NSAIDs have been investigated. Suppressive effects of ASA on peripheral blood and bone marrow micronucleus induced by the antitumor drug mitomycin C in mice was reported by Niikawa et al. [8]. Recently, celecoxib, an antiinflammatory drug, significantly inhibited doxorubicininduced mutagenicity in Salmonella typhimurium strains TA98 [9]. Although there is evidence indicating that ASA and other NSAIDs can be potentially antimutagenic against drugs that generate free radicals, there is still little on this subject in literature.

The aim of this study was to investigate the possible clastogenic and anticlastogenic effects of different concentrations of ASA on doxorubicin-induced chromosomal aberrations in human lymphocytes. To our knowledge, this is the first study carried out to investigate the anticlastogenic effects of ASA on cultured human lymphocytes exposed to the widely used antineoplastic agent doxorubicin. Determining the cytotoxic and clastogenic effects of this frequently used analgesic in human cells is important not only to establish its safety, but also to assess the possible hazards to human health when it is combined with other chemical agents, such as the antiinflammatory and chemotherapy drugs used in cancer therapy. 2. Materials and methods 2.1. Chemicals Doxorubicin (DXR, Rubidox® , CAS no. 25316-40-9) was a gift from Laborat´orio Qu´ımico Farmacˆeutico Bergamo LTDA (Brazil) and in order to obtain the desired final concentration, DXR was dissolved in distilled water just before each experiment. Acetylsalicylic acid (ASA, Melhoral® , CAS no. 50-78-2) was purchased in the local market. The concentrations used in the present study were obtained by dissolving ASA in distilled water and dimethyl sulfoxide (DMSO; final concentration 0.4%) from Merck & Co. Inc. All experiments were done in minimal indirect light. All other chemicals and solvents used were of analytical grade. 2.2. Cell cultures and treatments The chromosomal aberration assay was carried out using conventional techniques [10]. Human blood samples were obtained by venipuncture from six healthy, non-smoking vol-

Table 1 Mitotic index, total of chromosomal aberrations and percentage of aberrant metaphases in human lymphocytes treated with different concentrations of acetylsalisylic acid (ASA) and respective controls Treatments (␮g/mL)

MI (%)

Ctg

Ctb

Chb

Ex

Total of chromosome aberrations

Percentage of aberrant metaphases

Untreated control DMSO 0.4%

7.6 5.8

0 0

0 0

0 0

0 0

0 0

0 0

ASA 1.0 5.0 10.0 50.0 75.0 100.0 150.0

6.2 8.1 6.8 6.9 5.0 4.9 4.1

0 0 0 0 0 0 0

0 0 0 0 0 0 1

0 0 0 1 0 0 1

0 0 0 0 0 0 0

0 0 0 1 0 0 2

0 0 0 1.0 0 0 2.0

One hundred cells were analyzed per treatment and 1000 cells were analyzed for mitotic index (MI). Ctg, chromatid gaps; Ctb, chromatid breaks; Chb, chromosomal breaks; Ex, exchange figures.

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2.3. Statistical analysis

unteers, three females and three males aging from 21 to 37 years with their signed informed consent. The study was approved by the Universidade Federal do Triˆangulo Mineiro Ethics Committee (protocol no. 501). Heparinized total blood (0.5 mL) was added to a 4.5 mL medium containing 78% RPMI 1640 (Sigma–Aldrich Co., USA), 20% inactivated fetal bovine serum (Gibco-Invitrogen, Denmark), antibiotics (penicillin and streptomycin) and stimulated with 2% phytohemagglutinin (PHA; Gibco-Invitrogen, Denmark) and incubated for 48 h at 37 ◦ C. The concentrations of ASA (25, 50 or 100 ␮g/mL culture medium) tested in combination with doxorubicin were established on the basis of the results of the mitotic index (MI) and chromosome aberrations analysis in the preliminary experiments (Table 1). Doxorubicin was used to induce chromosomal aberrations. It was diluted in distilled water, and added to the culture medium at the concentration of 0.2 ␮g/mL defined by preliminary experiments (0.05–40.0 ␮g/mL of doxorubicin) and according to literature data [11]. Human peripheral blood lymphocytes were incubated for 24 h before ASA and/or doxorubicin were simultaneously added to the culture medium, and left until harvest. An untreated control culture and a 0.4% DMSO treated culture were established as well. Cultures were harvested 24 h after the treatments. Colchicine was added to the cultures at a final concentration of 0.4 ␮g/mL of the culture medium 1 h prior to harvesting. Lymphocytes were stained with Giemsa (Sigma–Aldrich Co., USA). The end points analyzed were the mitotic index, total number of chromosome aberrations, and percentage of aberrant metaphases. The mitotic index was determined by scoring the number of metaphases in 1000 cells per culture for a total of 6000 cells per treatment and controls. A total of 600 well-spread metaphases containing 46 ± 1 chromosomes were scored in each treatment (100 metaphases/donor) in a coded test. The classification of aberrations was as described in the ISCN [12].

The results were tabulated and experimental values were expressed with ± standard deviations (S.D.). One-way ANOVA was carried out and the Student’s t-test was used to detect significant differences amongst different treatment groups. The level of significance set was α = 0.01. Gaps were recorded but not included in the total number of chromosome aberrations or in the number of aberrant metaphases.

3. Results The results obtained in this study showing the effects of ASA and doxorubicin on the mitotic index, total number of chromosomal aberrations and percentage of aberrant metaphases are presented in Tables 1 and 2. Results of the preliminary assay with different concentrations of ASA (1.0–150 ␮g/mL) and the respective controls are shown in Table 1. Apparently, treatment with ASA alone was neither cytotoxic nor clastogenic. In the treatments with the lowest concentrations (1.0, 5.0, 10.0, and 50.0 ␮g/mL) the cells showed mitotic index values similar to the untreated control. However, in concentrations above 50.0 ␮g/mL, there was a considerable decrease in the mitotic index when compared with untreated control, but not statistically significant (p > 0.01). No clastogenic effects were observed in the tested range of ASA concentrations. The ASA concentration of 150.0 ␮g/mL showed a tendency to increase chromosomal aberrations and was therefore excluded in the subsequent experiments. No significant variations were observed in the mitotic index or in the total number of chromosomal aberrations in the cultures treated with

Table 2 Mitotic index, total of chromosomal aberrations and percentage of aberrant metaphases in human lymphocytes treated with different concentrations of acetylsalisylic acid (ASA) and doxorubicin (DXR) and respective controls Treatments (␮g/mL)

MI (%)

Untreated control DMSO 0.4% ASA: 25.0 ASA: 50.0 ASA: 100.0 ASA: 0 ASA: 25.0 ASA: 50.0 ASA: 100.0

DXR: 0 DXR: 0 DXR: 0 DXR: 0.2 DXR: 0.2 DXR: 0.2 DXR: 0.2

Ctg

Ctb

Chb

Ex

5.3 4.7

0 2

5.3 4.9 4.6 2.7a 2.9 2.8a 2.8a

3 3 7 21 5 20 7

Total of chromosome aberrations

1 1

0 2

0 0

1 3

1 1 2 55 44 40 74

1 1 2 126 62 79 80

1 0 0 86 47 38 58

3 2 4 267 153 157 212

Percentage of aberrant metaphases ± S.D. 0.16 ± 0.4 0.50 ± 0.8 0.50 0.33 0.33 22.33 13.66 15.83 16.83

± ± ± ± ± ± ±

0.8 0.4 0.4 6.9a 7.3a,b 7.3a,b 6.6a,b

Six hundred cells were analyzed per treatment and 6000 cells were analyzed for mitotic index (MI). Ctg, chromatid gaps; Ctb, chromatid breaks; Chb, chromosomal breaks; Ex, exchange figures. a Significantly different from the untreated control (p < 0.01). b Significantly different from the DXR treatment (p < 0.01).

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0.4% DMSO, when compared with untreated controls (Tables 1 and 2). In the cells treated with the antitumor drug alone, a statistically significant reduction of the mitotic index was observed compared with the untreated control (2.7% versus 5.3%) (Table 2). All cultures treated with doxorubicin present a significant reduction in the mitotic index (p < 0.01), except for the cells treated with doxorubicin combined with 25.0 ␮g/mL of ASA (p > 0.01). The treatments with ASA were not effective in inhibiting the doxorubicin cytotoxicity in the cultured human lymphocytes. Analysis of chromosomal aberrations and aberrant metaphases showed significant differences between the doxorubicin-exposed cells and untreated controls (p < 0.01). Chromosomal breaks and exchange figures were the predominant types of aberrations found, followed by chromatid breaks. Chromosomal gaps were not observed. In fact, doxorubicin was clastogenic and an effective inducer of aberrant metaphases with chromosomal damage (22.33%) when compared with untreated controls and DMSO (0.16 and 0.50%, respectively). In lymphocyte cultures treated with combinations of ASA and doxorubicin, a significant decrease in the total number of chromosome aberrations was observed compared with doxorubicin alone (p < 0.01). This protective effect of ASA on doxorubicin-induced chromosomal damage was obtained for all combinations, and it was most evident when the lowest concentration of ASA (25.0 ␮g/mL) was simultaneously added with the antitumor drug (Table 2). Combinations with different concentrations of ASA, 25.0, 50.0, and 100 ␮g/mL, resulted in a reduction of about 38.8, 29.1, and 24.6%, respectively, in doxorubicin-induced clastogenicity when compared with lymphocytes exposed to the antitumor drug. 4. Discussion The cytogenetic characterization and classification of the different types of chromosomal aberrations have an important role in human genetics and many cancers are associated with specific types of aberrations. The identification of the main chromosomal damage and studies of the mechanisms of chromosome-related human pathologies are of wide usefulness in the research of carcinogenesis and mutagenesis [13]. In this study we evaluated the possible clastogenicity and cytotoxicity of different concentrations of ASA in cultured human lymphocytes in vitro and the anticlastogenic effect of this NSAID on doxorubicin-induced chromosomal aberrations. In the cultures, the whole peripheral blood was used, because of the various xenobiotic-metabolizing enzymes

present in erythrocytes that might represent an advantage over the isolated lymphocytes. The basal frequency of chromosomal aberrations evaluated in the cultured blood lymphocytes obtained in the present study was in line with the results obtained by Karahalil et al. [14] in 18 healthy non-smokers. According to Zeiger [15] before a substance is claimed to have antimutagenic effects, it should also be evaluated for mutagenicity and rigorously tested with the appropriate protocols. In the preliminary experiments, we tested seven concentrations of ASA (1.0–150.0 ␮g/mL) and the initial results in 100 metaphases analyzed per treatment showed that ASA was neither cytotoxic nor clastogenic, when compared with untreated control and DMSO. Although the highest concentrations of ASA showed a slight decrease in mitotic index in comparison with the untreated control, it was not statistically significant. The information in literature about the mutagenicity of ASA is contradictory. In short-term cytogenetic tests in normal human lymphocyte cultures treated with 1.0–300.0 ␮g/mL of ASA, no significant increase of chromosomal aberrations was observed at any of the concentrations tested [16]. This is not in agreement with Jarvik and Fleiss [17] who reported that ASA at 1.0 ␮g/mL induced 1.87% metaphases with chromosomal damage against 0.67% found in the untreated lymphocyte cultures. Our data on the non-clastogenic activity of ASA are in agreement with investigations that reported the genotoxicity of analgesic compounds assessed by an in vitro micronucleus assay, indicating that ASA failed to induce micronuclei in the normal rat-kidney cell line NRK-49F and ASA did not show any appreciable increases in the revertant colony in the Ames test assay with and without metabolic activation [18,19]. Further studies on the clastogenicity of ASA in vitro showed that the induction of chromosomal aberrations in mammalian V79 cells in vitro were negative [20]. Other published mutagenic data indicated that there might be a health risk. ASA administered both intraperitoneally and orally showed weak genotoxicity when measured by sister chromatid exchange and chromosomal aberrations at the highest dose tested in bone marrow cells of mice [21]. This drug also demonstrated cytotoxic and genotoxic effects for plant (Allium cepa), invertebrate (Hydra attenuate), and vertebrate (Carassius auratus gibelio) organisms, and there was a multiple increase in the cells with micronuclei and double nuclei in onion [22]. This apparent controversy could be related to the test system used, given that there are differences between the in vitro and in vivo protocols. Since ASA was not clastogenic in the present study,

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to further investigate the possible protective effects of ASA on doxorubicin-induced chromosomal damage in cultured lymphocytes, three concentrations (25, 50, and 100 ␮g/mL) were selected for the anticlastogenic experiments. Genotoxic compounds cause DNA damage by a variety of mechanisms. Indirect-acting genotoxic agents were found to modify the function of cellular proteins, leading to accumulation of endogenous DNA damage. Doxorubicin is a genotoxic agent that inhibits the activity of the enzyme topoisomerase II, resulting in the accumulation of DNA strand breaks that, if not repaired by the cell, can cause mutations [23]. Doxorubicin is an anthracycline antibiotic and is one of the most active anticancer agents used in clinical oncology, especially in the treatment of acute leukemia and lymphomas, but also in some solid tumors, such as breast, ovarian and endometrial cancers [24]. However, the clinical effectiveness of DXR treatment is diminished due to the dose-limiting side effect, cardiotoxicity, caused by the generation of oxygen radical species. According to Weijl et al. [25], despite the fact that chemotherapy-induced generation of free radicals is well demonstrated, antitumor drug-induced cell cytotoxicity, in general, does not seem to depend on free radical generation. The clinical success of doxorubicin for treating cancer is probably due to DNA intercalation and topoisomerase inhibition. The patterns of DNA damage in anthracycline-treated cancer cells seem to support the concept that direct oxidative lesions only occur if cancer cells are exposed to elevated concentrations of anthracyclines. Clinically relevant concentrations caused the formation of protein-associated DNA single- and double-strand breaks, which might result from anthracycline inhibition of topoisomerase II by forming an anthracycline–DNA topoisomerase II complex [26,27]. Several data supported the rationalization that the generation of hydroxyl radicals by anthracyclines is not the pathway that contributes to its effects on cancer cells [28]. Concurrent therapies under study are combinations of doxorubicin and antioxidant nutrients or pharmacological drugs that aim to maximize the antitumor effects of doxorubicin while attenuating the generation of free radicals and the potential cardiotoxicity [29]. Given these characteristics and the fact that doxorubicin is a good inducer of chromosomal damage, a number of studies have been using doxorubicin as a positive control in anticlastogenic and antimutagenic tests [11,30,31]. Doxorubicin was selected in this study because it is an effective clastogenic and potent carcinogenic agent, and DXR has clastogenic effects that do not require enzymatic activation.

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Based on the preliminary cytotoxicity observed in the reduction of the mitotic index values and the increase in the chromosomal aberrations frequency, a single concentration of doxorubicin (0.2 ␮g/mL) was then chosen to be tested with different concentrations of ASA. Doxorubicin at a concentration above 0.3 ␮g/mL inhibited the mitotic index in more than 70% when compared with the untreated control. Concentrations below 0.1 ␮g/mL did not induce a chromosomal aberration rate statistically different from the untreated control. The results have shown that doxorubicin produces an increase in chromosomal aberrations in human lymphocytes in vitro and a decrease in the mitotic index, characterizing both clastogenicity and cytotoxicity. A similar increase in chromosomal damage in cultured lymphocytes was observed in previous studies [11,32]. The aberrant metaphases were 22.33% after treatment with doxorubicin alone as compared with 0.16% for untreated control. The chromosomal aberrations most frequently found were chromosomal breaks and chromatid breaks. This is in line with the kind of chromosomal damage found after treatment with this antineoplastic agent, which is described in literature [33]. The data showed that all cultures treated with doxorubicin presented a statistically significant reduction in the mitotic index, despite the presence of ASA in the culture medium. In the cells simultaneously treated with ASA and doxorubicin, a significant inhibition in the total number of chromosomal aberrations and aberrant metaphases were found when compared with the frequencies in doxorubicin cultures. The lowest concentration of ASA presented the most effective reduction of doxorubicin-induced aberrant metaphases with chromosomal damage. The possible explanation, given the results obtained in this study, is that ASA, acting as an antioxidant, prevented the damages that would be induced by reactive oxygen species released by the antineoplastic doxorubicin. The exact mechanisms of anticlastogenicity of ASA are not well understood. Several dietary and pharmacological antioxidants have shown protective effects against the biological damage induced by reactive oxygen species generated by doxorubicin [30,33]. Niikawa et al. [8] suggested the possible bioantimutagenic effects of ASA on the mitomycin C-induced polychromatic erythrocytes and peripheral reticulocytes with micronuclei in mice, and the need for more intensive research to determine whether the radical scavenging activity of ASA is associated with its mechanism of suppressing chromosomal aberrations. By inhibiting cyclooxygenase activity, ASA would suppress the attending generation of superoxide anions and reduce the synergism with oxy-

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gen radicals produced by redox cycling of doxorubicin [34]. Studies in a cultured cardiomyocyte model revealed that 5-ASA tested in doxorubicin-treated cardiomyocytes suppressed the oxidative stress induced by the antitumor drug, possibly preventing the cardiotoxicity resulting from doxorubicin use [35]. ASA addition also resulted in a marked inhibition of oxidative strand breaks in øX-174 plasmid DNA induced by H2 O2 /Cu(II) or hydroquinone/Cu(II). Moreover, ASA was found to be much more potent than the hydroxyl radical scavenger mannitol in protecting against reactive oxygen speciesmediated DNA strand breaks [7]. The identification of compounds that counteract the free radicals produced by doxorubicin could be beneficial to balance the potential long-term harmful effects of anthracycline chemotherapy. Finally, the results of this study show that more investigations to elucidate the mechanisms underlying the anticlastogenic effect of ASA are well needed. Acknowledgements The authors are grateful to Vera L´ucia Cardoso de Azevedo, Ricardo Manoel da Cruz, and L´ızia M.F. Reis e Campos (UFTM) and Joana D’Arc C. Darin (FCFRP) for the technical assistance. One of the authors (Rafaela de Barros e Lima Bueno) was sponsored by PIBIC/CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico). References [1] Y. Surh, Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances, Mutat. Res. 428 (1999) 305–327. [2] L.M.G. Antunes, L.M. Pascoal, M.D.P. Bianchi, F.L. Dias, Evaluation of the clastogenicity and anticlastogenicity of the carotenoid bixin in human lymphocyte cultures, Mutat. Res. 585 (2005) 113–119. [3] L.G. Hare, J.V. Woodside, I.S. Young, Dietary salicylates, J. Clin. Pathol. 56 (2003) 649–650. [4] R.M. Peek Jr., Prevention of colorectal cancer through the use of COX-2 selective inhibitors, Cancer Chemother. Pharmacol. 54 (2004) S50–S56. [5] J.E. Drew, J.R. Arthut, A.J. Farquharson, W.R. Russell, P.C. Morrice, G.G. Duthie, Salicylic acid modulates oxidative stress and glutathione peroxidase activity in the rat colon, Biochem. Pharmacol. 70 (2005) 888–893. [6] J.A. Baron, R.S. Sandler, Non-steroidal anti-inflammatory drugs and cancer prevention, Annu. Rev. Med. 51 (2000) 511–523. [7] C.S. Hsu, Y. Li, Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention, Biochem. Biophys. Res. Comm. 293 (2002) 705–709.

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