LACK OF THE ANTIMUTAGENIC EFFECT OF ASCORBIC ACID ON THE GENOTOXICITY OF ALBENDAZOLE IN MOUSE BONE MARROW CELLS

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1 Bull Vet Inst Pulawy 53, , 2009 LACK OF THE ANTIMUTAGENIC EFFECT OF ASCORBIC ACID ON THE GENOTOXICITY OF ALBENDAZOLE IN MOUSE BONE MARROW CELLS FULYA USTUN ALKAN AND SULEYMAN SENER Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Istanbul University, Avcilar, 34320, Istanbul, Turkey Received for publication April 21, 2009 Abstract The present study was designed to evaluate the genotoxicity of albendazole (ABZ) in mice using the micronucleus test. Mice were treated by gavage with 500, 1,000, and 1,500 mg of ABZ kg ¹ b.w., which corresponds to 1/6, 1/3, and 1/2 of the oral LD 50 of ABZ. In the second part of the study, the possible protective role of vitamin C (vit. C) was investigated against the genotoxic effect of ABZ. The mice received 200 mg of vit C kg -1 b.w. simutaneously with ABZ. Bone marrow samples were taken 48 h after the treatment. ABZ induced a statistically significant increase (P<0.001) in the percentage of micronucleaoted polychromatic erythrocytes (MNPCE) in the 1,000 and 1,500 mg of ABZ kg -1 b.w. treatment groups when compared with the negative control. On the other hand, the percentage of induced MNPCE was reduced at various levels in all ABZ treated groups simultaneously treated with an oral administration of vit. C. There were no statistically significant differences in the MNPCE frequency of these groups. The results of the study indicated that ABZ is a potential genotoxic agent, and that no protective effects of vit. C were observed against the genotoxicity of ABZ. Key words: mice, albendazole, vitamin C, micronucleus, genotoxicity. Albendazole (ABZ) is a benzimidazole carbamate (methyl 5-propylthio-1H-benzimidazole-2-yl carbamate), frequently used as an anthelmintic in veterinary and human medicine due to its broad antiparasitic activity and efficacy (5, 6). ABZ, as well as benzimidazoles, exerts anthelmintic activity by binding with cytoskeletal protein tubulin, thereby inhibiting the polimerisation of tubulins into microtubules. It has been suggested that the antimitotic activity of benzimidazoles is related to the inhibition of microtubule formation, which results in the loss of absorptive functions within the parasites and the disruption of the mitotic spindle and cell division (23, 24, 36). ABZ has been approved for human and veterinary use for over 20 years, and its teratogenicity and other damaging effects such as leukopoenia, alopecia, and gastric irritation in laboratory and farm animals are well known (15, 22, 30). Although several studies have been performed regarding the genotoxicity of the anthelmintics, there are few studies on the potential genotoxicity of ALB (31, 32). The results of the in vitro and in vivo studies regarding the genotoxic effects of ABZ are controversial. In the 2004 EMEA report, ABZ was found to give negative results in bacterial mutation tests, in an in vitro metaphase analysis of Chinese hamster ovary cells, and in an in vitro cell transformation assay. In the same report, it was mentioned that ABZ, which had been isolated from a formulated product gave a positive result in cytogenetic testing, such as in vivo mouse bone marrow micronucleus assay. The results of the mutagenicity tests on ABZ and albendazole sulphoxide (ABZSO) indicated that ABZ was an in vivo somatic cell mutagen, probably acting as an aneuploidogen, and it was reported that these results were consistent with the substances all being in vivo aneugens (6). In vivo and in vitro cytogenetic studies in humans showed that ABZ treatment increases the frequency of micronucleus (MN) (30, 31). Furthermore, non-disjunction of chromosomes in humans was at concentrations similar to those observed in plasma from patients treated with ABZ (32). The results of all these studies indicated the potential aneugenic effect of ABZ. In recent years, there is an increasing awareness that certain naturally occurring substances in plants and other sources have protective effects against environmental mutagens and carcinogens (39). On this basis, several studies with various chemotherapeutics have been performed on the antimutagenic and anticarcinogenic effects of vitamins on different organisms (2, 4, 12, 37, 38). Vit. C is a well-known potential antioxidant, and has been reported to reduce mutations in several ways, especially by scavenging reactive oxygen species (ROS) or electrophiles (3, 9,

2 494 16). Although an antigenotoxic effect and antioxidant protection are assumed for ascorbic acid against the oxidative damage of DNA mediated by ROS that can be generated during normal metabolism, as well as through exposure to chemotherapeutics and carcinogens, ascorbic acid sometimes seems to have co-genotoxic activity instead of the normal antigenotoxic action (13, 27, 33). Vit. C showed in vivo and in vitro antimutagenic effects against the mutagenicity of chemotherapeutic agents, such as cyclophosphamide (CYP), ethyl methane sulphonate, cisplatin, and mitomycin C (10, 11, 16, 26, 39). Despite these effects; however, it has also been shown to enhance the cytogenetic damage caused by thiotepa or l-ethionine, and the cytotoxicity of mitomycin C in human lymphocytes in vitro (19, 21). Experimental in vivo and in vitro studies have yielded conflicting results, suggesting that vit. C can be either genotoxic or antigenotoxic as a repair stimulant, depending on the organisms and species studied. Considering the lack of published data on the genotoxicity of ABZ relating to in vivo genotoxicity in particular, the present study was designed to evaluate the genotoxicity of short term ABZ exposure at high concentrations and to clarify whether this drug can cause chromosomal damages in the bone marrow polychromatic erythrocytes of treated mice. In the second part of the study, the role of vit. C in the form of L-ascorbic acid was investigated in order to determine if vit. C enhances or ameliorates the genotoxic effect induced by ABZ. Material and Methods Chemicals. Albendazole 98% (methyl 5- propylthio-2-benzimidazole-carbamate) and cyclophosphamide monohydrate 97% were obtained from Acros Organics (Belgium). Ascorbic acid and foetal calf serum were purchased from Amresco and Biological Industries (Israel), respectively. Giemsa and May-Grünwald stains were purchased from Merck (Germany). Animals. CD-1 strain male mice, aged 6-8 weeks and weighing g, obtained from Pendik Veterinary Control and Research Institute (Turkey), were used. The animals were housed in polypropylene cages and acclimatised for two weeks in the animal house of the department, maintained at 25 ±2ºC and humidity 50% ±5% with a 12 h light/dark period. Feed and water were provided ad libitum. The experimental protocol was approved by the Istanbul University Veterinary Faculty Ethic Committee. Dose selection. It was reported that the ABZ oral lethal dose (LD 50 ) was >3,000 mg/kg for mice (6). Therefore, the highest dose was determined as 1,500 mg/kg b.w., 50% of the LD 50. ABZ was dissolved in dimethylsulphoxide (DMSO). CYP known to cause micronucleus was used for the positive control group due to its known clastogenic and mutagenic activity (18, 34). According to many authors, who have studied the antimutagenic effects of ascorbic acid in vivo, the treatment protocols that gave the best results in terms of the reduction of chromosome damage were those in which vit. C was administrated in simultaneous treatment with the clastogenic agent. Therefore, the dose of vit. C was selected on the basis of the literature data, which was previously reported to give the highest protective effects (2, 17). CYP and vit. C were dissolved in physiological saline and distilled water, respectively. Experimental design. A total of 72 male mice were used in different control and treatment groups of the micronucleus test (eight mice in each group). The experimental treatment groups were (1) untreated control DMSO; (2) CYP 100 mg/kg b.w.; (3) ABZ 500 mg/kg b.w.; (4) ABZ 1,000 mg/kg b.w.; (5) ABZ 1,500 mg/kg b.w.; (6) ABZ 500 mg/kg b.w. + vit. C 200 mg/kg b.w.; (7) ABZ 1,000 mg/kg b.w. + vit. C 200 mg/kg b.w.; (8) ABZ 1,500 mg/kg b.w. + vit. C 200 mg/kg b.w.; (9) CYP 100 mg/kg b.w. + vit. C 200 mg/kg b.w. All chemicals were administrated orally. In each case, the animals were killed 48 h after the treatment. Micronucleous test. The genotoxic effects were evaluated in the mouse bone marrow by the micronucleous test, according to Schmid s method and OECD guidelines (29, 35). All slides were coded and examined under an Olympus CAMEDIA C7070 light microscope at 1,000x magnification. MN criteria as described by Schmid was also used. In addition, to determine the cytotoxic effects of ABZ, the percentage of PCEs was calculated on the basis of the ratio of PCEs to normochromatic erythocytes (NCEs) by counting 1,000 erythrocytes (35). Statistical analysis. The statistical analysis was performed using the SPSS 11.5 statistical program. Statistical comparisons among several groups were made by a non-parametric analysis of variance (Kruskal- Wallis test) and between two groups using the Mann- Whitney U test. In both cases a difference was considered as statistically significant when P<0.05. Results No clinical signs of toxicity were seen in any of the ABZ and simultaneously vit. C treated groups. The results from the MN analysis are shown in Table 1. Animals treated with ABZ alone at the highest doses (1,000 and 1,500 mg/kg b.w.) induced statistically significant increase (P<0.001) in the incidence of MNPCE when compared with the negative control group DMSO, although there was no significant difference between these ABZ treated groups. Mice treated with the ABZ and vit. C combination simultaneously at a dose level of 200 mg/kg b.w. showed a slight decrease in the number of MNPCE without any statistical significance with respect to the ABZ treated groups. The micronucleous assay revealed a significant increase (P<0.001) in the frequency of MNPCE cells following CYP administration as compared to the negative control. The data confirmed the sensitivity of the experimental protocol followed in the detection of genotoxic effects.

3 495 Table 1 Effect of ABZ and vit. C on the frequency of MNPCE in mouse bone marrow cells Chemicals Dose (mg/kg) MNPCE/2000PCE (mean ±SE) PCE/NCE (%) (mean ±SE) Control (DMSO) ±0.46 e 0.87 ±0.01 a CYP ±1.55 a 0.55 ±0.01 f ABZ ±0.46 e 0.83 ±0.01 a.b ABZ 1, ±0.68 c,d 0.73 ±0.01 d,e ABZ 1, ±0.89 c 0.71 ±0.01 e ABZ + vit. C ±0.53 e 0.79 ±0.02 b,c ABZ + vit. C 1, ±0.85 d 0.77 ±0.01 c,d ABZ + vit. C 1, ±1.52 c,d 0.73 ±0.01 d,e CYP + vit. C ±1.53 b 0.68 ±0.02 e MNPCE - micronucleated polychromatic erythrocytes, NCE - normochromatic erythrocytes. Results show means ±SE of between eight mice in each group. a,b,c,d,e: in each column, groups with different letter superscripts indicate a significant difference in MNPCE number (P<0.05). Furthermore, CYP plus vit. C showed a significant reduction (P<0.001) in MN frequency, and it was determined that the ascorbic acid showed an antimutagenic effect against CYP mutagenicity. Regarding the cytotoxicity, the treatment with ABZ at 1,000 and 1,500 mg/kg b.w. doses induced a significant decrease (P<0.001) in the number of PCE/NCE when compared with the negative control group. The animals treated with ABZ (1,000 and 1,500 mg/kg b.w.) and simultaneously with vit. C showed a slight increase in PCE/NCE frequency, but without any statistical significance with respect to the ABZ treated groups. On the other hand, CYP plus vit. C showed a significant increase (P<0.001) in the PCE/NCE frequency when compared with the CYP treated group. Discussion The benzimidazoles are potent anti-parasitic agents, and their mode of action is thought to result from their inhibition of microtubule functions (25). The anthelmintic activity of benzimidazole 2-carbamates has been related to their selective antimitotic activity, due to the preferential binding of these agents to helmintic tubulin over mammalian tubulin (22). The ability of benzimidazoles to act as mitotic spindle poisons leads to the potential risk of aneuploidy induction in exposed cells. ABZ inhibits tubulin polimerisation, thus resulting in improper microtubule formation, the disturbance of the mitotic apparatus (spindle, kinetochores), and leads to MN formation, either by chromosome fragments or by whole chromosomes (31, 32). The micronucleous test detects the genomic alterations arising from chromosomal damage and/or damage to the mitotic apparatus caused by clastogenic or aneugenic agents, respectively (1, 14). MN in young erythrocytes arises primarily from chromosomal fragments or lagging chromosomes that are not incorporated into daughter nucleus at the time of cell division in the erythropoietic blast cells, and changes in the incidence of MNPCEs are considered to reflect chromosomal damage (1, 7, 14). From the results of the presented study, it is evident that the ABZ treatment increased the frequency of MN where there was a positive correlation between the increased drug concentration and the induction of MN in the bone marrow cells of mice. While the lowest dose of ABZ (500 mg/kg bw) was unable to induce MN significantly, the two highest concentrations (1,000 and 1,500 mg/kg b.w.) significantly increased the number of MN. Furthermore, the frequency of MNPCE induced at the highest dose of ABZ was approximately three times higher than control value. The numeric increase in MNPCE between the two highest doses of ABZ was probably due to the absorption of the drug. It was reported that ABZ is poorly absorbed and rapidly metabolised, and the deleterious effects of ABZ in exposed individuals may depend on the concentrations reached during the treatment period and on the tubulin binding capacity of the drug (22, 23). Therefore, in the period of 48 h, the highest dose (1,500 mg/kg b.w.) of ABZ probably can not be absorbed well and could not show a statistically significant higher MN frequency than the lower dose (1,000 mg/kg b.w.) in a dosedependent manner. MN formation may be due to the fact that this drug is a benzimidazole derivative, which acts as an inhibitor for the tubulin polymerisation that is in charge of vital processes, including the motility and nutrient uptake in the parasite and chromosomal segregation at mitosis in exposed cells (20). The effects of the drug ABZ reported in the study concurred with the results of previous reports about the aneugenic and mutagenic effects of ABZ (30-32). Ramírez et al. (31, 32) showed that ABZ induced MN and produced non-disjunction events in human lymphocytes treated in vitro, and suggested that the mechanism is capable of inducing aneuploidy and the final consequence of MN formation, based on the

4 496 chromosome non-disjunction during cell replication as a result of disruption of the mitotic spindle. Recently, Oztas et al. (30) have investigated the genotoxic effect of ABZ in pediatric patients with hepatic hydatid disease. The authors demonstrated that ABZ treatment increased the frequency of sister chromatid exchange and MN, and they concluded that ABZ might be genotoxic to human lymphocytes in vivo. Therefore, we suggested that the increase in the frequency of PCEs containing MN in the bone marrows of ABZ treated mice could occur due to the changes at the stage of cell proliferation and differentiation, and that ABZ may potentially be a genotoxic agent. It was reported that vit. C acted as an antimutagenic agent in bone marrow cells when administered with clastogenic agents (39). Vit.C decreased the frequency of micronucleoated cells induced by the potent chemotherapeutic agents such as cisplatin, cyclophosphamide, mitomycin-c, and bleomycin in bone marrows of mice (11, 26). It was suggested that vit. C may reduce the mutation induction by chemicals in different ways: by scavenging harmful species, free radicals, or electrophiles, which damage DNA and other cell targets; inhibiting the promutagen bioactivation by blocking the oxidation processes, or by reacting with the electrophilic metabolite(s) of a promutagen (16, 28). In the present study it is worth mentioning that vit. C decreased the MN frequency induced by CYP. CYP is an alkylating agent and it reacts with the electron rich areas of the susceptible molecules, such as nucleic acids and proteins after biochemical activation (18). The protective effect elicited by vit. C against CYP may be due to the antioxidant property of the vitamin, which is an important free radical scavenger and offers protection to DNA from oxidative free radical damage (26). However, the protective effect of vit. C against ABZ genotoxicity was not seen. The failure of the antigenotoxic effect of ascorbic acid against chromosomal damage caused by ABZ may be explained due to the fact that the dose of vit. C used in this study was not sufficient enough to reduce the MN frequency, and the other most relevant factor is the mechanism of the micronucleus formation. The mode of action of ABZ gives rise to ideas about chromosomal damage, and we suggested that MN formation can result from the nonoxidative mechanisms, and that ABZ may cause chromosomes with damaged centromeres and chromosomes that fail to correctly attach to the spindle, or that the DNA damage can be based on the chromosome non-disjunction as a result of improper microtubule formation and was not seen disturbance of the mitotic spindle due to the inhibition of tubulin polimerisation by the interaction of the carbamate group of ABZ with microtubular proteins (31, 32). Therefore, vit. C has not shown a protective effect against this type of MN formation, due to its anti-mutagenic action not having an effect on aneugenic agents. However, we cannot clearly identify the MN formation mechanism that MN can be formed either by clastogenic or aneugenic effect. Further experiments should be performed to identify the presence of centromeres in MN by using centromere specific DNA probes and thereby differentiate between MN of clastogenic and aneugenic origin (7, 8). The results of the study indicated that ABZ induced MN either by chromosome fragments or by whole chromosomes at statistically significant levels in the treated cells compared with the negative control. According to the mode of action of ABZ and the previous genotoxicity studies of ABZ, the formation of MN may be possibly based on aneuploidy induction, thus producing MN (30, 31). In conclusion, ABZ is a frequently used chemotherapeutic agent for the treatment of several helminthiases in veterinary and human medicine, and our observations have demonstrated that ABZ has a genotoxic effect on mice erythrocytes, and that the administrated dose of vit.c has not shown a protective effect against this genotoxic activity under the present experimental conditions. 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