Evaluation of the in vitro and in vivo Genotoxicity of Almond Skins

Biomed Environ Sci, 2011; 24(4): 415 421 415 Original Article Evaluation of the in vitro and in vivo Genotoxicity of Almond Skins ZHANG XiaoPeng, XIANG Qian, CUI WenMing, JIA XuDong, and LI Ning # Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100021, China Abstract Objective It aims to study potential genotoxicity of almond skins. Methods A bacterial reverse mutation assay was performed on S. typhimurium strains TA97, TA98, TA100, TA102, and TA1535 in the absence or presence of S 9 mixture at a dose range of 312.5 to 5 000 µg/plate. A micronucleus test and a mammalian bone marrow chromosome aberration tests were performed in Swiss Albino (CD 1) mice at doses of 625, 1 250, and 2 500 mg/kg bw used. Results Almond skins exerted no mutagenic activity in various bacterial strains of Salmonella typhimurium in either the absence or the presence of metabolic activation at all doses tested. Various doses of almond skins did not affect the proportions of immature to total erythrocytes, the number of micronuclei in the immature erythrocytes, or the number of structural and numerical chromosomal aberrations of Swiss albino mice. Conclusion Almond skins are not genotoxic under the conditions of the in vitro bacterial reverse mutation assay and two in vivo tests micronucleus test and mammalian bone marrow chromosome aberration test, which supports the safety of almond skins for dietary consumption. Key words: Almond skins; Genotoxicity; Mutagenicity; Micronuclei; Chromosome aberration Biomed Environ Sci, 2011; 24(4): 415 421 doi: 10.3967/0895 3988.2011.04.013 ISSN: 0895 3988 www.besjournal.com(full text) CN: 11 2816/Q Copyright 2011 by China CDC INTRODUCTION Previous studies have demonstrated that increased consumption of almonds and other nuts can decrease the occurrence of heart disease, prevent oxidative stress and DNA damage caused by smoking, and reduce the risk of colon cancer [1 6]. Besides dietary fibre, protein, monounsaturated fatty acid, vitamins and minerals, almonds contain a wide variety of phenolic acids and flavonoids, which are generally classified as phytochemicals conducive to promoting health and reducing the risk of chronic diseases [7 8]. Almond skins, the seed coat of almonds, account for 47% 72% of total phenolic acids and flavonoids of a whole almond [9]. A study found that these flavonoids were bioavailable, and increased the resistance of low density lipoprotein (LDL) to oxidation and decreased oxidative damage to DNA [10]. Considering these potential health benefits, almond skins can be used as a component of dietary supplements. To this end, the safety of almond skins should be warranted. Toxicity tests conducted in our laboratory indicate that almond skins are of low oral toxicity, with an oral medial lethal dose (LD 50 )>10 g/kg body weight (oral toxicity study in rats) and a no observed adverse effect level (NOAEL) of 10% (w/w) (90 day feeding test in rats) [11], but their potential to cause genotoxicity has not previously been investigated. In the present study, the in vitro mutagenic potential of almond skins was evaluated by a bacterial reverse mutation test, while the ability of almond skins to induce cytogenetic damage and chromosome aberration was assessed by a mouse bone marrow erythrocyte micronucleus test and a mammalian bone marrow chromosome aberration # Correspondence should be addressed to LI Ning. Tel: 86 10 67779118. Fax: 86 10 67776535. E mail: lining65@yahoo.com Biographical note of the first author: ZHANG XiaoPeng, female, born in 1977, assistant professor, majoring in food toxicology. Received: October 12, 2010; Accepted: November 19, 2010

416 Biomed Environ Sci, 2010; 24(4): 415 421 test separately. Materials MATERIALS AND METHODS Almond skins, provided by the Almond Board of California (California, USA), were turned into brown powder by milling the seed coat of almonds. It was kept at 4 C and freshly prepared by suspending it in water at the start of each test. In vitro test Bacterial Reverse Mutation Test The histidine requiring Salmonella typhimurium (S. typhimurium) strains TA97, TA98, TA100, TA102, and TA1535 (WOOJUNG BSC Inc., Korea) were cultured in a nutrient broth at 37 C by shaking. In a cytopreliminary toxicity study, all strains were tested by using three plates per dose (ranging from 312.5 to 5 000 µg/plate) of almond skins to determine an appropriate range of concentrations for the mutagenicity study. The preliminary study was conducted with and without metabolic activation. Cultures were assessed for a reduced rate of spontaneously occurring colonies and a visible thinning of the bacterial lawn. Based on the results of this experiment (discussed in Section 3.1), a range of concentrations from 312.5 to 5 000 µg/plate was selected for the mutagenicity study. The bacterial reverse mutation test (preincubation plate incorporation assay) was performed with or without metabolic activation (S 9 obtained from Aroclor 1254 induced rat livers) [12 14]. The nutrient broth culture of the bacterial strain (ph 7.4, containing per L of medium: 10 g peptone, 5 g beef extract, 5 g NaCl, 2.6 g K 2 HPO 4 3H 2 0, 1 L distilled water) cultured overnight (0.1 ml), 0.1 ml test substance dissolved in water, 0.5 ml S 9 mix or PBS buffer (ph 7.4) were mixed and then preincubated at 30 37 C for 20 30 min before they were added to 2 ml of molten top agar (containing 0.6% agar, 0.5% NaCl, and 10 ml of 0.5 mmol/l histidine biotin solution for the S. typhimurium strains) maintained at approximately 45 C. The ingredients were mixed and poured onto a minimal agar Petri culture dish (1.5% agar, 40% glucose, phosphate buffer). Following incubation at approximately 37 C for 48 to 72 h, the number of revertants was counted. The test sample was assayed in a triplicate at 5 concentrations (312.5, 625, 1 250, 2 500, and 5 000 µg/plate), and two independent experiments were performed for each bacterial strain. Negative (water only) and positive control experiments were also conducted. The positive control mutagens are listed in Table 1. Table 1. Positive Control Substances Used in the Bacterial Reverse Mutation Test Strain ( ) S 9 mix Concentration ( mutagen) (+) S 9 mix Concentration (mutagen) TA97 9 Aminoacridin 50 2 AA 2 TA98 AF2 0.1 B a P 5 TA100 AF2 0.01 B a P 5 TA102 Mitomycin C 0.5 2 AA 5 TA1535 AF2 0.5 2 AA 5 The test substance was considered to be mutagenic if either: 1) a two fold or greater increase in the mean number of revertants per plate was observed compared to the mean number of revertants per plate in the appropriate negative control in at least one of the tester strains, in the absence of cytotoxicity; or 2) a dose related increase in the mean number of revertants per plate, compared to the mean number in the appropriate negative control, was observed in at least 2 3 concentrations of the test substance and in at least one bacterial strain, in the absence of cytotoxicity [14 15]. In vivo Tests Animals The dose range finding study and two in vivo studies were carried out in Swiss Albino (CD 1) mice, which were obtained from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China), of 7 9 weeks of age, subjected to a general physical examination upon receipt, and acclimatized for 7 d. Animals were housed in cages (5 animals per sex per cage) and provided with food (from Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) and water (tap water) ad libitum. The housing facility was designed to maintain appropriate environmental conditions (23±2 C, 12 h light/dark cycle, 30% 70% relative humidity, and a ventilation frequency of 10 15 times/hour). All procedures were conducted in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animal, and regulations of the Office of Laboratory Animal Welfare. Dose Range finding Study In order to assess the toxicity of almond skins in mice and select dose levels for the micronucleus test and the mammalian bone marrow chromosome aberration test, a dose

Biomed Environ Sci, 2011; 24(4): 415 421 417 range finding study was conducted in 5 males and 5 females. Animals were offered with 2 500 mg Almond skins/kg body weight (bw) suspended in water by oral gavage, and were observed for 14 days. Micronucleus test Mice were randomly allocated into three treated groups and two control groups, and each group consisted of 10 animals (5/sex/group). Almond skins were administered twice at an interval of 24 h by gavage to animals at the doses of 625, 1 250, and 2 500 mg/kg bw each (based on the results of the dose range finding study). Water and cyclophosphamide (40 mg/kg bw, ip) were administered as negative and positive materials. Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity. Mice were sacrificed by carbon dioxide asphyxiation 24 or 48 h after the last treatment administered (all doses) [16 17]. For the cyclophosphamide treated group, mice were sacrificed at the 24 h time point only. Both femora were removed and the bones were cleaned of muscle tissue. The proximal ends of the femora were opened and the bone marrow was flushed into a 5 ml centrifuge tube containing 3 ml fetal bovine serum. The mixture was centrifuged for 5 min at 1 000 rpm and the resulting supernatant was discarded. After mixing, one drop of the resuspended cells was smeared onto a clean slide and air dried. Slides were briefly flamed, then fixed by immersion in 95% methanol for 10 min and stained in ordinary staining jars with Giesma Working Solution for 15 minutes. Stained slides were treated with xylene, cover slipped, and analysed microscopically. All slides were coded to ensure that that the evaluation was blinded. The proportion of immature erythrocytes (polychromatic, PCE) to total erythrocytes [PCE + mature erythrocytes (normochromatic, NCE)] was determined for each animal by analyzing 200 erythrocytes. For a valid test, the proportion of immature to total (PCE+NCE) erythrocytes was required not to be less than 20% of the negative control value. To determine the presence of micronuclei, 2 000 PCEs were analyzed per animal. Both biological and statistical significances were considered for evaluation of a positive response. The test substance was considered positive for a mutagenic response if there was a statistically significant dose related increase in the number of micronucleated PCEs for at least one of the time points, compared to the concurrent negative control group. Mammalian bone marrow chromosome aberration test Almond skins were administered once by oral gavage to animals at the same doses as that in the micronucleus test. Water and cyclophosphamide (40 mg/kg bw) were administered as the negative and positive materials. Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity. Animals were killed by carbon dioxide asphyxiation 24 or 48 h after treatment [18]. For the cyclophosphamide treated group, mice were sacrificed at the 16 h time point only. Bone marrow cell division was arrested at metaphase by intraperitoneal injection of the spindle poison, colchicines, at 4 mg/kg bw (5 ml/kg dose volume) three hours before sacrificing. Both femora were dissected out and cleaned of adherent tissue. By using syringe and needle, the proximal ends of the femora were opened and the marrow cells were flushed out with Hank s balance salts solution into clean centrifuge tubes. The recovered cells were harvested by centrifugation for 5 min at 1 000 rpm. Chromosome preparation involved hypotonic treatment of the cells, fixation and staining. The harvested cells were treated in a low osmotic solution (0.075 mol/l KCl solution) for 30 min. They were then fixed in a solution conforming methanol: glacial acetic acid (3:1) 3 times. After that, slides were prepared by dropping the harvested cultures on clean sides. The slides were stained with Giemsa s method and examined under the microscope with high multiplication. For scoring the chromosome aberrations, 100 metaphases cells from each animal were examined. Well spread metaphase cells with 40±2 chromosomes were analyzed for different chromosome aberrations. The metaphases were examined for the following aberrations: break, deletion, translocation, fragment, ring formation, dicentromere, triploid, polyploid etc. Statistical Analysis For the micronucleus test and the mammalian bone marrow chromosome aberration test, homogenous data were analyzed by using a One Way Analysis of Variance (ANOVA), and the significance of inter group differences was analyzed by using Duncan s test. Heterogeneous data were analyzed with the Kruskal Wallis test, and the significance of inter group differences between the control and treated groups was assessed by using

418 Biomed Environ Sci, 2010; 24(4): 415 421 Dunn s test. All statistical tests were performed at the P<0.05 level of significance. RESULTS Bacterial Reverse Mutation Test The preliminary cytotoxicity and range finding study proved that the almond skins were not toxic to the bacterial strains at doses of 312.5 to 5 000 μg/plate. Complete bacterial lawn and normal number of colonies were observed at all five doses. A range of 5 concentrations from 312.5 to 5 000 µg/plate were tested. In the mutagenicity study, almond skins did not increase the mean number of revertants per plate of any S. typhimurium strain with or without metabolic activation in comparison with the spontaneous reversion rate in the negative control (Table 2). The mean number of revertant colonies of the negative control was within the historical range. The positive control mutagens induced increases in revertant colonies (Table 2), confirming the validity of the assay. No reduction in the background bacterial lawn was observed at all doses in all strains. In vivo Tests In the dose range finding study, neither mortalities nor clinical signs of toxicity were observed in animals given 2 500 mg almond skins/kg body weight. According to the Limit Test of FDA guideline for a micronucleus test [17] and OECD guideline for a mammalian bone marrow chromosome aberration test [18], 2 500 mg/kg bw was selected as high dose for the following two in vivo tests. Micronucleus Test In the micronucleus test, no mortalities were recorded, and gross necropsy of the animals revealed no macroscopic findings. The criterion for a valid test was met. The proportion of immature to total erythrocytes was not affected by almond skins administration (Table 3). No statistically significant increase in the number of micronucleated PCEs was observed in any of the almond skins treated groups compared to the negative control group at either time point (Table 3). The positive control substance (cyclophosphamide) induced a marked and statistically significant increase in the number of PCEs with micronuclei, but did not affect the proportion of immature to total erythrocytes. Mammalian Bone Marrow Chromosome Aberration Test In the mammalian bone marrow chromosome aberration test, no mortalities were recorded, and gross necropsy of the animals revealed no macroscopic findings. None of the rates of structural and numerical chromosomal aberrations in all doses of almond skins treatments showed a statistically significant difference when compared with the negative control (Table 4). They were therefore considered to be of no biological relevance. The positive control substance (cyclophosphamide) induced a marked and statistically significant increase of structural and numerical chromosomal aberrations, thus indicating the high sensitivity of the test system. DISCUSSION Almond skins are seed coats of almond kernel. Almond skins contribute to 4% of almond weight but account for 47% 72% of total phenolic acids and flavonoids of a whole almond, but are always discarded. If almonds skins can be used as a component of dietary supplements, they are a good source of total phenolic acids and flavonoids. So the safety of almond skins must be clarified. In this study, the potential genotoxic effects of almond skins were assessed in vitro and in vivo. In the mutagenicity study, at the doses of 312.5 to 5 000 μg/plate, the almond skins did not cause a significant increase in the number of revertants colonies with S. typhimurium TA97, TA98, TA100, TA102, and TA1535 in either the absence or the presence of S 9 Mix. No dose dependent effect was obtained. In the micronucleus test and the chromosome aberration test, at the doses of 625, 1 250, and 2 500 mg/kg bw, almond skins did not affect the proportions of PCEs to total erythrocytes and the number of micronucleated PCEs, or induce structural and numerical chromosomal aberrations in Swiss albino mice. All assays were conducted in replicate by using multiple doses determined in preliminary dose range finding experiments. No evidence of cytotoxicity or mutagenicity was observed at any concentration tested. The bacterial reverse mutation tests were conducted with or without S 9 activation to determine whether almond skins could be converted to mutagenic metabolites

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420 Biomed Environ Sci, 2010; 24(4): 415 421 Table 3. Micronucleus Test Results Gender Almond Skins Dose Level (mg/kg bw) No. of PCE PCE/(PCE +NCE)% 24 h 48 h No. of MNPCE MNPCE/ PCE(%) No. of PCE PCE/ (PCE+ NCE) (%) No. of MNPCE MNPCE/ PCE(%) Solvent Control 109.8±2.4 54.9 3.0±1.4 1.5 109.0±9.1 54.5 3.6±2.1 1.8 625 109.8±8.9 54.9 3.8±1.9 1.9 107.8±4.4 53.9 4.6±2.7 2.3 Female 1250 109.6±8.0 54.8 4.2±2.5 2.1 109.4±7.1 54.7 5.4±2.1 2.7 2500 107.2±7.3 53.6 3.4±1.5 1.7 110.2±3.0 55.1 4.6±2.1 2.3 Positive Control 94.2±5.1 47.1 40.0±7.2 * 20.0 * Male Solvent Control 109.0±3.4 54.5 3.6±1.7 1.8 109.0±3.2 54.5 4.8±2.3 2.4 625 110.6±4.5 55.3 3.4±1.7 1.7 109.6±8.9 54.8 5.2±1.8 2.6 1250 111.2±7.7 55.6 4.8±2.2 2.4 109.6±3.8 54.8 3.8±1.6 1.9 2500 109.8±6.1 54.9 4.8±2.4 2.4 109.0±6.8 54.5 5.4±2.1 2.7 Positive Control 93.4±3.8 46.7 34.8±9.4 * 17.4 * Note. Abbreviations: bw, Body Weight; MNPCE, Micronucleated PolychromaticErythrocyte; NCE, Normochromatic Erythrocyte; No., Number; PCE, Polychromatic Erythrocyte; SD, Standard Deviation. Positive Control=Cyclophosphamide (40 mg/kg Body Weight). * Significantly Different Compared to Negative Control. Table 4. Number of Chromosome Aberration in the in vivo Mammalian Bone Marrow Chromosome Aberration Test Treatments Gender (M/F) Killing Time Dose (Hour p. a.) (mg/kg) Cells Scored Number and Type of Aberration b d t r f Other Rate of Aberration (%) P Value * Solvent Control (Water) Almond Skins Positive Control M F M F 24 500 6 0 0 3 6 0 3.00±1.00 48 500 8 0 0 2 3 0 2.60±0.55 24 500 3 0 0 2 4 0 1.80±0.84 48 500 8 0 0 3 2 0 2.60±0.89 24 625 500 11 0 0 3 4 0 3.60±1.14 24 1250 500 7 0 0 4 2 0 2.60±0.55 24 2500 500 10 0 0 3 5 0 3.60±0.55 48 2500 500 12 0 0 1 3 0 3.20±0.84 24 625 500 7 0 0 4 2 0 2.60±0.55 24 1250 500 11 0 0 3 2 0 3.20±0.45 24 2500 500 8 0 0 2 6 0 3.20±1.30 48 2500 500 8 0 0 3 5 1 3.40±0.55 M 24 40 500 43 0 0 14 21 8 17.20±3.11 * F 24 40 500 22 0 0 14 40 15 18.20±1.92 * <0.001 Note. Abbreviations: b (Break), d (Deletion), t (Translocation), r (Ring); f (Fragment), other (Dicentric, Triradial, Quadriradial etc). Hours p.a: Hours after Administration. * P<0.01 (Compared to Solvent Control Group). >0.05 following bioactivation by microsomal enzymes. Appropriate positive and negative controls were included in all three genotoxicity tests and produced the expected results, demonstrating the validity of the assays. In summary, almond skins showed no mutagenic activity in the bacterial reverse mutation test and did not induce micronuclei in

Biomed Environ Sci, 2011; 24(4): 415 421 421 the immature erythrocytes or increase the rates of structural and numerical chromosomal aberrations of Swiss albino mice. These data indicate that almond skins do not have genotoxic potential, which supports the safety of almond skins for dietary consumption. ACKNOWLEDGEMENTS We are grateful to Almond Board of California for providing almond skins. We thank Dr. ZHANG ZhenTao, Purdue University, and Jack Wood, National Center for AIDS/STD Control and Prevention, China CDC, for their assistance in the preparation of this manuscript. REFERENCES 1. Jia XD, Li N, Zhang WZ, et al. A pilot study on the effects of almond consumption on DNA damage and oxidative stress in smokers. Nutrion And Cancer, 2006; 54(2), 179 83. 2. Li N, Jia XD, Chen CY, et al. Almond consumption reduces oxidative DNA damage and lipid peroxidation in male smokers. J Nutr, 2007; 137, 2717 22. 3. Davis PA, Iwahashi CK. Whole almonds and almond fractions reduce aberrant crypt foci in a rat model of colon carcinogenesis. Cancer Letters, 2001; 165, 27 33. 4. Fraser GE. Nut consumption, lipids, and risk of a coronary event. Clin Cardiol, 1999; 22 (Suppl. III), III11 III15. 5. Hu FB, Stampfer MJ, Manson JE, et al. Frequent nut consumption and risk of coronary heart disease in women: prospective cohort study. Br Med J, 1998; 317, 1341 5. 6. Jenkins DA, Kendall CW, Augustine M. Dose response of almonds on coronary heart disease risk factors: blood lipids, oxidized low density lipoproteins, lipoprotein(a), homocysteine, and pulmonary nitric oxide a randomized, controlled, crossover trial. Circulation, 2002; 106, 1327 32. 7. Hu FB. Plant based foods and prevention of cardiovascular disease: an overview. Am J Clin Nutr, 2003; 78, 544S 5451S. 8. Hu FB, Willett WC. Optimal diets for prevention of coronary heart disease. JAMA, 2002; 288, 2569 78. 9. Milbury PE, Chen CY, Dolnikowski GG, et al. Determination of flavonoids and phenolics and their distribution in almonds. J Agric Food Chem, 2006; 54, 5027 33. 10.Chen CY, Milbury PE, Lapsley K, et al. Flavonoids from almond skins are bioavailable and act synergistically with Vitamins C and E to enhance hamster and human LDL resistance to oxidation. J Nutr, 2005; 135, 1366 73. 11.Song Y, Wang W, Cui WM, et al. A subchronic oral toxicity study of almond skins in rats. Food Chem Toxicol, 2009; doi:10.1016/j.fct.2009.10.025 12.Mortelmans K, Zeiger E. The ames salmonella/microsome mutagenicity assay. Muta Re, 2000; 455, 29 60. 13.Ames BN, McCann J, Yamasaki E. Methods for detecting carcinogens and mutagens with the Salmonella/mammalianmicrosome mutagenicity test. Mutat Res, 1975; 31, 347 64. 14.Maron D, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res, 1983; 113, 173 215. 15.U.S.FDA. Bacterial reverse mutation test. 2000a; In: Toxicological principles for the safety assessment of food ingredients. http://www.cfsan.fda.gov/~redbook/redivc1a.html 16.Odagiri Y, Uchida H, Shibazaki S. Interindividual variation in cytogenetic response to x ray and colchicine measured with the cytokinesis block micronucleus assay. Mutat Res, 1997; 381, 1 13. 17.U.S.FDA. Mammalian Erythrocyte Micronucleus Test. 2000b; In: Toxicological principles for the safety assessment of food ingredients. 18.OECD. Mammalian Bone Marrow Chromosome Aberration Test. 1997; In: OECD Guideline for the Testing of Chemicals, Section 475, adopted 21st July, 1997.