tured dog lymphocytes after a short-term oral CdO administration by the micronucleus (MN) test. The dogs were given 10 mg CdO/kg body weight per day for 3 ...
© Copyright 2006 by Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/(Online) 1559-0720/06/11203–0241 $30.00
Increased Micronucleus Frequency After Oral Administration of Cadmium in Dogs HAMIYET DÖNMEZ-ALTUNTAS,*,1 ZUHAL HAMURCU,1 NARIN LIMAN,2 HALIL DEMIRTAS,1 AND NALAN ˙IMAMOGLU1 1
Department of Medical Biology, Medical Faculty, and Department of Histology and Embryology, 2Veterinary Faculty, Erciyes University Kayseri, Turkey Received November 3, 2005; Accepted January 6, 2006
ABSTRACT Cadmium (Cd) is a toxic heavy metal that has been classified as a human carcinogen by the International Agency for Research on Cancer. The genotoxic effects of cadmium oxide (CdO) were investigated in cultured dog lymphocytes after a short-term oral CdO administration by the micronucleus (MN) test. The dogs were given 10 mg CdO/kg body weight per day for 3 and 28 d, respectively group I (n = 7) and group II (n = 6). Blood samples were collected at the beginning of feeding and at 4 and 29 d after Cd administration and cultured for 72 h. Whereas no significant increase in the MN frequency in group I was observed (p = 0.398), a significant MN induction with CdO was found in group II (p = 0.028) when compared with initial MN frequencies of dogs in both groups. Our results suggest that CdO might be directly and/or indirectly genotoxic after a monthly oral administration of CdO in dogs. Index Entries: Cadmium; cadmium oxide; dogs; feed; micronucleus.
INTRODUCTION Cadmium (Cd) is the one important environmental toxicant among heavy metals and a naturally occurring component of the Earth’s crust (1). Cd, in association with chlorides, hydroxyl, sulfhydryls, and thiol groups, forms soluble complexes and these complexes largely govern the biological activity of Cd (1). Nonoccupational exposure to Cd occurs mainly in * Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
241
Vol. 112, 2006
242
Dönmez-Altuntas et al.
the consumption of contaminated food or drinking water and cigaret smoking. Occupational exposures include the inhalation of cadmium oxide (CdO) fumes generated during heating or welding of Cd-containing materials or the inhalation of particles of metal, oxide, and pigment dust. Occupational exposure to Cd is associated with cancers of the lung and other sites, including the prostate in humans (1,2). In recent years, the possible mechanisms implicated in Cd carcinogenesis, including gene regulation, oxidative stress, disruption of cell adhesion, inhibition of DNA repair, and interference with apoptosis have been reviewed. However, there is no consensus on the potential mechanisms in Cd carcinogenesis. Cd is a poor mutagen and acts as an indirect carcinogen because it inhibits the repair of DNA damage caused by other agents (3–5). However, the cytogenetic results in subjects environmentally or occupationally exposed to Cd are contradictory (6–10). Fu et al. (7) found increased chromosomal aberrations and micronucleus (MN) rates in people environmentally exposed to Cd, whereas Forni (8). did not find this in Cd-exposed workers. There were no significant differences in sister chromatid exchange (SCE) rates between the Cd-polluted group and their controls (9), whereas there were a statistically significant increase in SCE/cell correlated with blood Cd concentrations in Greenlandic Eskimos (10). The people and the dogs, because of their close association and shared environment, are exposed to similar pollutants. Animals respond to many environmental toxicants in ways analogous to humans. Although there are several studies on the genotoxicity of Cd in humans and animals, to our knowledge no such report is available on MN frequency in dogs. The purpose of this study was to evaluate the genotoxic effects of CdO (water unsoluable) on cross-breed dog lymphocytes after a short-term oral administration of CdO, using the cytochalasin-B-blocked MN assay.
MATERIAL AND METHODS Animals Thirteen dogs (1–2 yr old; from the local animals care center) were divided into two groups with seven and six dogs in each (at equal male and female rates). They weighed about 9–10 kg, which was almost the same as the weight of cross-breed dogs used in the experiment. Cadmium oxide (Fluka Chemica, Buchs, Switzerland) dissolved in water was orally administrated in daily doses of 10 mg CdO/kg body weight for 3 d in group I and for 28 d in group II. Dogs were fed for experiment’s duration at a rate of 2% of body weight per day divided into two equal morning and evening feedings with the commercial food. The dogs were followed up for their general attitudes. Their pulse, breath, and body temperature were Biological Trace Element Research
Vol. 112, 2006
Effects of Cd on Micronucleus Frequency in Dogs
243
measured daily in group I and weekly in group II. The animal experiments were approved by the Erciyes University Ethics Committee of Animal Experiments. The blood samples were obtained prior to (0. d) and at 4 (in group I) and 29 (in group II) d after oral administration in daily doses of 10 mg CdO/kg body weight from each dog.
Micronucleus Assay For micronuclei (MNi) in binucleate (BN) cells, 0.3-mL heparinized blood samples were cultured for 72 h at 37°C in 5 mL of the culture medium Nutrient Mixture F-10 (Biol. Ind.) that was supplemented with 20% heat-inactivated fetal calf serum (Biological Industries), 1.8% phytohemagglutinin (Biol. Ind.), 100 U/mL penicillin, and 100 µg/mL streptomycin (Biol. Ind.). At 44 h of incubation, cytochalasin-B (Sigma Chemical Co., St. Louis, MO, USA) was added to the cultures to give a final concentration of 3 µg/mL, according to the method of Fenech and Morley (11). The cultures were stopped at 72 h, treated with hypotonic solution (0.1 M KCL) by the method of Balasem and Ali (12) for 3 min, and fixed in two changes of methanol–acetic acid (3 : 1). The fixed cells were spread onto glass slides and stained with 5% Giemsa for 7 min. All of the slides were coded and blind read. In order to determine intraindividual differences, different slides of two parallel cultures of one dog were prepared. Cells with two macronuclei surrounded by cytoplasm and a cell membrane were scored for the presence of MNi. The number of MNi in 1000 BN cells were scored and the frequency of MNi per 1000 BN cells were calculated for each dog. Published criteria for scoring MNi in BN cells and selecting BN cells were followed (13,14).
Statistical Analysis The frequencies of BN cells with MNi in dogs treated with CdO were compared by using the Wilcoxon test from nonparametric tests for each group.
RESULTS After oral administration in daily doses of 10 mg CdO/kg body weight, the means and standard deviations of BN cells with MN found in dog lymphocytes are presented in Table 1. MN frequencies of dogs prior to CdO treatment were compared with their posttreatment values by using the Wilcoxon test. In group I, MN frequency was not significantly induced in dogs after treatment, during which as they were orally administered daily doses of 10 mg CdO/kg body weight for 3 d (p = 0.398). In group II, significant differences were found in MN frequencies of dogs after treatBiological Trace Element Research
Vol. 112, 2006
244
Dönmez-Altuntas et al. Table 1 Frequency of MNi in Cultured Dog Lymphocytes Treated with CdO (Mean % ± SD)
a b
3 d, 10 mg/kg treatment. 28 d, 10 mg/kg treatment.
ment, during which they were orally administered daily doses of 10 mg CdO/kg body weight for 28 d (p = 0.028).
DISCUSSION Cadmium and Cd compounds were classified as a carcinogenic to humans and experimental animals (1). Indeed, Cd is a nongenotoxic carcinogen, nonmutagenic in bacterial tests and weakly mutagenic in mammalian cells in vitro (1). However, Cd compounds have been proven to be comutagenic in mammalian cell tests when combined with genotoxic agents, and this property has been explained by the inhibition of DNA repair processes by this metal (3,15). The mechanisms of Cd carcinogenesis remain largely unknown. However, because Cd is not strongly genotoxic, genotoxic events may apply indirectly. These events could include aberrant gene expression, enhanced cell proliferation, blocked apoptosis, and altered cell signaling (3–5). In fact, high concentrations of Cd might act by damaging DNA directly and can produce genotoxic and mutagenic events (4–6,16). Rates of MN in the Cd-exposed workers did not differ from those of the matched controls (8). In contrast with these results, Fu et al. (7) found increased MN rates in people environmentally exposed to Cd. It was shown that pet dogs can be an early warning sentinel for human exposure to environmental contaminants. The dogs exposed to environmental contaminants had significantly more MNi and a higher frequency of micronucleated binucleates than the control dogs (17). In this study, significant differences were found in MN frequencies of dogs after oral administration of CdO for 28 d in group II (p = 0.028), whereas MN frequencies were not significantly induced in dogs after oral administration for 3 d in group I (p = 0.398). Cd acts indirectly through genotoxic events such as inhibition of repair of DNA damage and depressed apoptosis (3–5). In our study the increase in MN frequency after a monthly oral administration of CdO showed it to be concurrent with indirect genotoxic events by Cd. On the other hand, we can say that acute Biological Trace Element Research
Vol. 112, 2006
Effects of Cd on Micronucleus Frequency in Dogs
245
short-term oral administration of CdO is insignificant, but for subacute short-term oral administration, Cd might directly damage genetic material because MN result’s from lesions/adducts at the level of DNA or chromosomes (14). However, further data about the direct genotoxic effects of Cd are required.
ACKNOWLEDGMENT The authors are grateful to Mustafa Akgül for the linguistic support in preparing the manuscript.
REFERENCES 1. IARC (International Agency for Research on Cancer), Beryllium, cadmium, mercury, and exposures in the glass manufacturing industry, in IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume, 58. IARC Scientific Publications, Lyon, pp 119–238 (1993). 2. C. G. Elinder, T. Kjellstorm, C. Hogstedt, K. Andersson, and G. Spong, Cancer mortality of cadmium workers, Br. J. Ind. Med. 42, 651–655 (1985). 3. M. Waisberg, P. Joseph, B. Hale, and D. Beyersmann, Molecular and cellular mechanisms of cadmium carcinogenesis, Toxicology 192, 95–117 (2003). 4. M. P. Waalkes, Cadmium carcinogenesis in review, J. Inorg. Biochem. 79, 241–244 (2000). 5. M. P. Waalkes, Cadmium carcinogenesis, Mutat. Res. 533, 107–120 (2003). 6. V. Verougstraete, D. Lison, and P. Hotz, A systematic review of cytogenetic studies conducted in human populations exposed to cadmium compounds, Mutat. Res. 511, 15–43 (2002). 7. J. Y. Fu, X. S. Huang, and X. Q. Zhu, Study on peripheral blood lymphocytes chromosome abnormality of people exposed to cadmium in environment, Biomed. Environ. Sci. 12, 15–19 (1999). 8. A. Forni, Comparison of chromosome aberrations and micronuclei in testing genotoxicity in humans, Toxicol. Lett. 72, 185–190 (1994). 9. K. Nogawa, I. Tsuritani, Y. Yamada, et al., Sister chromatid exchanges in the lymphocytes of people exposed to environmental cadmium, Toxicol. Lett. 32, 283–288 (1986). 10. H. C. Wulf, N. Kromann, N. Kousgaard, J. C. Hansen, E. Niebuhr, and K. Alboge, Sister chromatid exchange (SCE) in Greenlandic Eskimos: dose-response relationship between SCE and seal diet, smoking, and blood cadmium and mercury concentrations, Sci. Total. Environ. 48, 81–94 (1986). 11. M. Fenech and A. A. Morley, Measurement of micronuclei in lymphocytes, Mutat. Res. 147, 29–36 (1985). 12. A. N. Balasem and A. Ali, Establishment of dose-response relationships between doses of CS-317 γ-rays and frequencies of micronuclei in human peripheral blood, Mutat. Res. 259, 133–138 (1991). 13. M. Fenech, The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations, Mutat. Res. 285, 35–44 (1993). 14. M. Fenech, The in vitro micronucleus technique, Mutat. Res. 455, 81–95 (2000). 15. A. Hartwig and T. Schwerdtle, Interactions by carcinogenic metal compounds with DNA repair processes: toxicological implications, Toxicol. Lett. 127, 47–54 (2002). Biological Trace Element Research
Vol. 112, 2006
246
Dönmez-Altuntas et al.
16. R. R. Misra, G. T. Smith, and M. P. Waalkes, Evaluation of the direct genotoxic potential of cadmium in four different rodent cell lines, Toxicology, 126, 103–114 (1998). 17. L. C. Backer, C. B. Grindem, W. T. Corbett, L. Cullins, and J. L. Hunter, Pet dogs as sentinels for environmental contamination, Sci. Total. Environ. 274, 161–169 (2001).
Biological Trace Element Research
Vol. 112, 2006
