INDUCTION OF MICRONUCLEI IN RAT BONE MARROW AFTER SUBCHRONIC INHALATION EXPOSURE TO MIXTURE OF BENZENE, CYCLOHEXANONE AND CYCLOHEXANE

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1 Bull. Vet. Inst. Pulawy 48, , 2004 INDUCTION OF MICRONUCLEI IN RAT BONE MARROW AFTER SUBCHRONIC INHALATION EXPOSURE TO MIXTURE OF BENZENE, CYCLOHEXANONE AND CYCLOHEXANE NATÁLIA KOVALKOVIČOVÁ 1, IRENA ŠUTIAKOVÁ 2, JAROSLAV LEGÁTH 1, ĽUBOMÍR LEGÁTH 3, MARCEL FALIS 1, JURAJ PISTL 1 AND GABRIEL KOVÁČ 1 1 University of Veterinary Medicine, 2 Research Institute of Veterinary Medicine, 3 University of Pavol Jozej Šafárik, Košice, Slovakia, kovalkovicova@uvm.sk Received for publication March 04, Abstract The induction of micronuclei was evaluated in 24 Wistar rats (12 male and 12 female) after subchronic inhalation exposure to the mixture of benzene, cyclohexanone and cyclohexane, in the dose of 0.72 g/m 3 ; 5 d per week; for 105 d, against control group (6 male and 6 female). In the exposed group of females the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was significantly increased compared with the control group (10.5 ± 5.419/1000 PCEs against ± 1.722/1000 PCEs; P < 0.05). The significant increase in MNPCEs was observed also in exposed group of males compared with the control ( ± 6.431/1000 PCEs against ± 1.472/1000 PCEs; P < 0.05). This showed the genotoxic effect of the mixture in the tested animals. The significant changes in the frequency of micronucleated normochromatic erythrocytes (MNNCEs) as well as the ratio of PCEs/NCEs in exposed animals were not recorded. Key words: rat, erythrocyte bone marrow test, benzene, cyclohexane, cyclohexanone, genotoxicity, micronucleus. There are about chemicals in daily use that can have adverse biological effects at very low concentrations. Although the study of single chemicals is important in obtaining fundamental information on chemical of interest, human and animal exposure to chemicals, occupational or environmental, is rarely limited to a single chemical. In daily life, both humans and animals are constantly exposed to indoor and outdoor air pollutants; many natural or synthetic agents are present in the food, beverages, medicines, and cosmetics. Thus, each of us has a background exposure to chemical mixtures. The most risky groups are men exposed also occupationally and exposure of them is a dynamic fenomenon, in that constant changes occur with respect to the number of chemicals and their concentrations in relation to time (22). Many toxic substances do not always have acute toxic effects; however, they exhibit considerable potential for chronic toxicity which is most frequently accompanied by impairment of the immune system or of genetic material, even at concentrations less than those necessary for general toxic effect. In this study we evaluated the genotoxic potential of the mixture of benzene, cyclohexanone and cyclohexane after subchronic inhalation exposure in rats by the erythrocyte bone marrow assay. Benzene, cyclohexanone and cyclohexane are important substances widely used in industry. Most of the population occupationally exposed to these agents are exposed for a long period. Material and Methods Animals. Thirty six clinically healthy male and female Wistar rats SPF, 6-week-old, were obtained from Velaz Praha, Czech Republic. There were 12 males weighing ± g and 12 females weighing ± g in the experimental group. The control group consisted of 6 males with mean b.wt ± g and 6 females with mean b.wt ± 9.81 g. Animals were housed individually in plastic cages and acclimatized for 1 week before the experiment. Animals were observed prior to the study to ensure that they were healthy. Only the animals found to be in a clinically acceptable condition were assigned to the study. During the study, food and water were offered ad libitum. Animal quarters were maintained at 22 o C (± 2 o C); 30 70% relative humidity on a 12 h light/dark cycle (24). Chemical substances in tested mixture. The tested mixture contained benzene, cyclohexanone and cyclohexane manufactured by Chemko Strážske,

2 342 Slovak Republic in the ratio 4.2 (benzene) : 8.08 (cyclohexanone) : 59.8 (cyclohexane). The ratio was calculated according to long-term measurement of the concentrations of these substances in the air of the chemical plants. The features of the substances are indicated in Table 1 as reported by Marhold (19). Table 1 The features of the chemical substances in the tested mixture Substance Molecular formula Mol. wt. Benzene C 6 H 6 78 Cyclohexanone C 6 H 10 O 98 Cyclohexane C 6 H Dose and exposure. The tested mixture was applicated by inhalation apparatus at a dose of 0.72 g/m 3 for 2 h/d, 5 d/week for 105 d. Animals were whole-body exposed. Animal sacrifice, bone marrow cell isolation, and slide preparation. Before the animals were killed, a 10-ml centrifuge tube was filled with 5 ml of foetal calf serum for each individual (PAN SYSTEMS GmbH, Biotechnologische Producte). All the rats were sacrificed 24 h after the end of exposure by vertebral cervical dislocation and both femora were removed. The bones were then freed from muscle manually by the use of gauze. The proximal and distal ends of the femur were carefully shortened with scissors until a small opening to the marrow canal became visible. With the needle about 3.0 ml serum was pulled from the tube into plastic syringe. Then the needle was inserted a few mm into the proximal part of the marrow canal and the marrow was gently flushed in the prepared test-tube. The tubes were then centrifuged at 800 rpm for 5 min. The supernatant was removed with Pasteur pipette. A small drop of the viscous sediment was put on the end of a slide and spread by pulling the material behind a cover glass held at an angle of 45 degrees. The preparations were then air dried and fixed in absolute methanol for 6 min. Cells were stained with May- Grűnwald (3 min) and Giemsa-Romanowski (15 min). Scoring and statistical analysis. Criteria for scoring follow those of Mac Gregor et al. (17) and OECD Guideline 474 (25). At high magnification one thousand polychromatic erythrocytes (PCEs) per animal from coded slides were screened for the presence of micronuclei (MNPCEs). Separately, the number of micronucleated mature erythrocytes (MNNCEs) was registered in a total of 1000 normochromatic erythrocytes (NCEs) per animal. Toxicity to bone marrow was estimated by the relationship between polychromatic (PCEs) and normochromatic erythrocytes (NCEs) frequency. The ratio of PCEs to NCEs was determined in the first 1000 erythrocytes scored per animal. The Student s test was used to compare MNPCEs and MNNCEs values and PCE to NCE ratios between treated and control animals for statistical significance. Results Micronucleus frequencies and distributions of micronuclei observed in polychromatic and normochromatic erythrocytes as well as the ratio of polychromatic to normochromatic erythrocytes of female rats are shown in Table 2. The subchronic inhalation exposure to the tested mixture resulted in significant increase in the number of micronuclei in polychromatic erythrocytes (MNPCEs) of the female rats compared with the control group (10.5 ± 5.419/1000 PCEs against ± 1.722/1000 PCEs; P < 0.05). The significant increase in MNPCEs was observed also in the exposed male group compared with the control ( ± 6.431/1000 PCEs against ± 1.472/1000 PCEs; P < 0.05). Micronucleus frequencies and distributions of micronuclei in polychromatic and normochromatic erythrocytes as well as the ratio of polychromatic to normochromatic erythrocytes of males are shown in Table 3. No significant differences in micronucleated normochromatic erythrocytes and ratio of PCEs/NCEs in exposed rats were observed. Disscussion Although the evaluation and health risk assessment of each chemical entity taken separately is generally well regulated at the national and international levels, the question of possible interactions of the chemicals in the mixture is still a matter of debate. Not only workers in industry and agriculture but also the general population may be potentially at risk of exposure to low doses of various chemicals, at the workplace or through food and drinking water (3). Exposure to chemical mixtures can produce unexpected effects additive, antagonistic, synergistic, or potentiated. The chemical mixtures that produce such effects contain at least 1 relatively lipophilic and 1 relatively hydrophilic chemical. The relative differences in lipophilia and hydrophilia are reflected by the octanol:water partition coefficients of the chemicals Kow (defined as the ratio of such quantity of chemical dissolved in the octanol phase to that dissolved in the water phase of an octanol-water mixture). The Kow data range from less than -1.0 to more than 6.0. Higher values indicate more lipophilic character, and lower values indicate more hydrophilic character. It is well established that a lipophilic chemical (higher Kow) facilitates the absorption of at least 1 hydrophil (lower Kow) in the mixture. As a result of the facilitated absorption, chemicals that alone would be innocuous are absorbed at levels that produce toxic effects (39).

3 343 Table 2 Micronucleus frequencies and distributions in bone marrow cells of female rats after subchronic inhalation exposure to mixture of benzene, cyclohexanone and cyclohexane Animal PCEs MNPCEs in PCEs NCEs MNNCEs in NCEs Ratio PCEs/NCEs C C C C C C ± ± ± E E E E E E E E E E E E ± 5.419* ± a ± a a: no significant difference; * statistical significance (P < 0.05); ** statistical significance (P < 0.01); C1 C6: control animals; E1 E12: exposed animals; SD: standard deviation; PCEs: number of polychromatic erythrocytes screened; MNPCEs: number of micronucleated polychromatic erythrocytes; NCEs: number of normochromatic erythrocytes screened; MNNCEs: number of micronucleated normochromatic erythrocytes; ratio PCEs/NCEs: ratio of polychromatic to normochromatic erythrocytes; MN: micronuclei Table 3 Micronucleus frequencies and distributions in bone marrow cells of male rats after subchronic inhalation exposure to mixture of benzene, cyclohexanone and cyclohexane Animal PCEs MNPCEs in PCEs NCEs MNNCEs in NCEs Ratio PCEs/NCEs C C C C C C ± ± ± E E E E E E E E E E E E ± 6.431* ± a ± a a: no significant difference; * statistical significance (P < 0.05); ** statistical significance (P < 0.01); C1 C6: control animals; E1 E12: exposed animals; SD: standard deviation; PCEs: number of polychromatic erythrocytes screened; MNPCEs: number of micronucleated polychromatic erythrocytes; NCEs: number of normochromatic erythrocytes screened; MNNCEs: number of micronucleated normochromatic erythrocytes; ratio PCEs/NCEs: ratio of polychromatic to normochromatic erythrocytes; MN: micronuclei

4 344 Prediction of the effects of a mixture based on the knowlewdge of each of the constituents requires detailed information on the composition of the mixture, exposure level, mechanism of action, and receptor of the individual compounds (3). We tested a combination of 3 chemicals in a 105-d inhalation study in Wistar rats at the dose of 0.72 g/m 3 administered to the animals for 2 h/d, 5 d/week. The chemicals comprised benzene (Kow value 2.13), cyclohexanone (Kow 0.81), cyclohexane (Kow 4.72) in the ratio of 4.2 : 8.08 : Literary data about possible effects of this mixture of chemical substances are not available, but may be predicted from the knowledge of each of the constituents. Rats have been chosen for the study of the genotoxic effects of the mixture because they play a dominant role as an animal model in toxicology testing and an enormous amount of data concerning pharmacokinetics, metabolism, distribution and excretion of various chemical substances were obtained during their toxicological evaluation. Benzene genotoxicity has been demonstrated in several in vivo studies which have evaluated different endpoints such as micronuclei (27), chromosomal aberrations and sister chromatid exchanges in lymphocytes of occupationally exposed workers (11, 18, 28, 34, 36, 37). The study by Sasiadek (29) has shown that benzene causes cytogenetic changes in specific chromosomes, especially of the C-group chromosomes 6 12 a X. The comet assay has been successfully used to detect the DNA damage induced in liver and blood cells of mice (35) and in lymphocytes of exposed human subjects (1). Fabiani et al. (8) evaluated the genotoxicity of benzene metabolites in peripheral blood mononuclear cells under different culture conditions. Cytogenetic damage, including structural aberrations (5, 15), micronuclei (14) and sister chromatid exchanges, have been also demonstrated in bone marrow cells, splenocytes and peripheral blood lymphocytes of mice or rats exposed to benzene intraperitoneally (6), orally (4, 5) or by inhalation (2, 7, 9, 10, 21, 31, 32, 38). The induction of micronuclei in bovine lymphocytes in vitro by exposure to benzene was studied by Piešová and Šiviková (26). Benzene metabolite hydroquinone induces the chromosomal aberrations and micronuclei in mouse bone marrow cells (20, 40). The majority of studies of genotoxic effects induced by inhalation of benzene have dealt with chromosome damage in mouse bone marrow cells after chronic or subchronic exposure. The tested concentrations ranged from 1 to 300 ppm for as long as 13 weeks (9, 10, 32, 33) and showed a dose-dependent increase in micronucleated polychromatic erythrocytes (9, 10). There is little information regarding genotoxicity of cyclohexanone. Its embryotoxicity and teratogenicity was observed in mice and rats (12, 23, 30). The mutagenic activity of cyclohexane of atmospheric air dust was studied on various strains of Drosophila melanogaster (16). Its tumourigenic potential in mouse skin was assessed by Gupta and Mehrotra (13). The findings obtained in this study of an increased frequency of micronucleated polychromatic erythrocytes from the bone marrow of rats treated subchronically with the mixture of benzene, cyclohexanone and cyclohexane by inhalation is, to our knowledge, the first report of a genotoxic effect of the mixture on this target tissue. 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