Mutagenesis, Vol. 17, No. 3, 215-217,
May 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Induction effects of sulfur dioxide inhalation on chromosomal aberrations in mouse bone marrow cells
Institute of Environmental Medicine and Toxicology, Department of Life Sciences, Shanxi University, Taiyuan 030006, People's Republic of China
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Abstract |
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To investigate the induction of chromosome aberrations (CA) in mouse bone marrow cells by sulfur dioxide (SO2) inhalation, mice were treated by SO2 exposure for 4 h/day for 7 days at various concentrations of SO2, then mitotic indices and CA in mouse bone marrow cells were analyzed. The present results show that SO2 might increase the frequencies of CA and aberrant cells in mouse bone marrow in a dose-dependent manner. The frequencies (%) of aberrant cells in mouse bone marrow induced by SO2 at concentrations of 0, 7, 14, 28 and 56 mg/m3 were 1.81, 3.00, 3.58, 4.26, 4.86, respectively. At low concentrations SO2 induced only chromatid-type CA, while at high concentrations SO2 induced both chromatid-type and chromosome-type CA. SO2 inhalation decreased the mitotic indices of bone marrow cells. The results imply that SO2 inhalation may inhibit mitoses and increase CA frequencies of bone marrow cells and that it is a clastogenetic and genotoxic agent. Long exposure to SO2 pollution at low concentrations in the environment may be a potential risk for induction of cytogenetic damage in vivo in humans.
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Introduction |
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Sulfur dioxide (SO2) is a common air pollutant (Shapiro, 1977
). Epidemiological studies have linked SO2 exposure to many respiratory diseases, including lung cancer (Andersson et al., 1998; Marsh et al., 1998). Recent studies have reported the promotive effects of SO2 exposure on rat lung tumorigenesis (Ohyama et al., 1999). Several studies have shown that the frequencies of chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) in peripheral blood lymphocytes of workers chronically exposed to SO2 in factories were higher than in controls (Schneider and Kalkins, 1970; Beckman and Nordenson, 1986; Meng and Zhang, 1990; Yadav and Kaushik, 1996). SO2 derivatives (bisulfite and sulfite) induce CA, SCE and MN in cultured human blood lymphocytes in vitro, and these increases occur in a dose-dependent manner (Meng and Zhang, 1992). However, cytogenetic damage due to SO2 inhalation in bone marrow cells of mammals has not been reported. The detection of CA in bone marrow cells as well as in peripheral blood lymphocytes is a very sensitive index of damage produced by ionizing radiation (Jenssen and Ramel, 1976; Chaubey et al., 1978; Morales-Ramirez et al., 1994; Abrasson-Zetterberg et al., 1995) and by chemical mutagens (Mavournin et al., 1990; Shelby and Witt, 1995). In the present study we have evaluated SO2 inhalation for the ability to induce CA in mouse bone marrow cells in vivo. This is the first study on the effects of SO2 inhalation in inducing significant increases in CA frequencies in mouse bone marrow cells in vivo. |
Materials and methods |
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Animals
Male and female Kunming mice were supplied by the Division of Laboratory Animals, China Institute for Radiation Protection (Taiyuan, China) and were ~6 weeks old. The mice weighed between 20 and 25 g on the day of the experiment. Animals were housed in the departmental animal facility for 4 days after receipt prior to each experiment. The animal room was maintained at 22 ± 2°C with 50% humidity and time-controlled lighting (12 h light/day). The animals were fed standard rodent pellet diet (China Institute for Radiation Protection) and water ad libitum.
SO2inhalation treatments
Animals were randomly numbered and then assigned to different treatment groups. They were divided into five equal groups: one control group and four groups exposed to SO2 at 7, 14, 28 and 56 mg/m3). These groups were subdivided into two subgroups of eight animals each: one male and one female.
Various concentrations of SO2 were administered to the animals in the SO2 groups in exposure chambers for 4 h (8:00–12:00 a.m.)/day for 7 days. The control group was exposed to filtered air in another identical chamber for the same period of time. Animals were placed in a 1 m3 exposure chamber. The gas was delivered to animals via a tube positioned at the upper level of the chamber and distributed homogeneously via a fan in the chamber. The SO2 was diluted with fresh air at the intake port of the chamber to yield the desired SO2 concentrations. SO2 within the chambers was measured every 30 min by pararosaniline hydrochloride spectrophotometry in order to monitor the SO2 concentrations (Xi et al., 1995).
At the end of the experimental period the mice were deprived of food for 24 h and then prepared for the experimental procedure.
For determination of the relationship between CA formation and relative duration of exposure to SO2, four groups of 10 male mice each were exposed to SO2 at 14 mg/m3 and then killed at 1, 3, 5 and 7 days post-treatment. One group of 10 male mice without SO2 exposure were killed as a control.
Slide preparation and CA assay
Twenty-two hours after the last SO2 inhalation treatment the animals were injected with colchicine (2 mg/kg) and 2 h later they were killed by cervical dislocation. Bone marrow chromosomes were prepared and slides were stained with Giemsa (Prestone et al., 1987). One slide was prepared from each mouse. All the slides were coded and 100 well spread metaphase cells per animal were scored blind by the same observer. Mitotic index (MI) was calculated from 1000 cells/animal and expressed as a percentage. CA were scored following the method of the WHO (1985) and Prestone et al. (1987). The aberration frequencies of chromatid and chromosome types per cell were calculated. Gaps were recorded but not included in the frequency of aberrations per cell. Statistical calculations were carried out on the percentages of aberrant cells.
Statistical analysis
The statistical significance of the differences between the SO2-treated and control groups was determined. Student's t-test was used for MI and frequencies of aberrant cells, the 
2 test for CA frequencies.
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Results |
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CA frequency and SO2 inhalation
In the current study SO2 was tested for potential clastogenicity in both male and female mice using the mouse bone marrow CA test. Our results showed that the responses of male and female mice to SO2 inhalation were very similar; no sex difference was observed (data not shown). The results of chromosome analysis in bone marrow cells from male and female mice are together summarized in Table I
. The frequencies of total CAs and total aberrant cells increased with SO2 dosage. Two types of structural CAs, i.e. chromatid breaks and isochromatid breaks, were observed. Dicentrics, rings, translocations and other types were not found in our experiments, so breaks were the predominant types of SO2 inhalation-induced aberrations (Table I). The frequencies of chromosome-type aberrations (isochromatid breaks) increased only at high concentrations (56 and 84 mg/m3) of SO2, and not at low concentrations (<28 mg/m3). This implies that SO2 inhalation at low concentrations induced mainly chromatid-type aberrations and at relatively high concentrations (56 and 84 mg/m3) induced both chromatid- and chromosome-type aberrations in bone marrow cells of mice. These results imply that SO2 is a clastogenic and genotoxic agent.
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Table I
also shows that SO2 inhalation decreased the MI of mouse bone marrow cells in a dose-dependent manner. This implies that SO2 is a mitotic inhibitor. However, a decrease in MI could also result from a delay in cell cycling due to cell toxicity, death or delay for DNA repair. Further study is needed to understand the relationship between SO2 and mitosis of mouse bone marrow cells.
Time–response relationship
Table II shows that the CA frequencies induced by SO2 inhalation increased with the duration of SO2 exposure. The highest and most significant increase in CA frequency was in the group exposed to SO2 for 7 days, the longest duration in this study. This implies that the CA frequency increases in a duration-dependent manner. The time–response relationships of CA formation and SO2 exposure in male and female mice were very similar.
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Discussion |
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Human exposure to the environmental pollutant SO2 has become increasingly widespread due to the combustion of fossil fuels (Shapiro, 1977
). Consequently, it has become important to study the genetic effects of SO2 in mammals and humans. CA analysis of mouse bone marrow cells is one of the most useful methods to assess such effects. However, no study on SO2 inhalation-induced CA and the dose–effect relationship has previously been carried out in vivo. Although our data are still insufficient, this is the first experimental evidence that SO2 inhalation induces CA in mouse bone marrow cells in a dose-dependent manner in vivo. These results have confirmed our previous observation that SO2 is a clastogenic and genotoxic agent (Meng and Zhang, 1990, 1992).
Inhaled SO2 can be easily hydrated to yield sulfurous acid in the respiratory tract, which subsequently dissociates to form sulfite/bisulfate and hydrogen ions (Gunison and Benton, 1971). The genetic effects of bisulfite have been examined in bacterial and mammalian cells. At high concentrations and at pH values between 5 and 6 bisulfite modifies DNA in vitro and deaminates cytosine to uracil (Fishbein, 1976; Shapiro, 1977). Recently we reported that bisulfite increases the frequencies of mutants in CHO-AS52 cells and causes a deletion mutation of the xathine-guanine phosphoribosyltransferase locus (gpt) (Meng and Zhang, 1999). Several possibilities still remain to explain the mechanism of SO2-induced CA formation. It is possible that bisulfite induces chromosomal damage via DNA modification and deamination of cytosine to uracil, an enzyme-catalyzed process that has been proposed for mammalian cells. A second possible mechanism for SO2-induced chromosomal damage may involve formation of sulphur- and oxygen-centered free radicals, such as SO-3, SO-4 and SO-5 (Reist et al., 1998), either via autoxidation of bisulfite or by enzyme-catalyzed oxidation (Meng and Zhang, 1992). The one-electron oxidation of bisulfite produces the sulfur trioxide radical anion, which reacts rapidly with molecular oxygen to form a peroxyl radical. Free radicals generated by SO2 can damage nucleic acids and induce mutations (Reist et al., 1998). Moreover, these radicals can react with proteins and cause sulfitolysis in peptide structures and this may alter membrane properties (Reist et al., 1998). Also, these radicals can react with lipids and lead to lipid peroxidation in several tissues (Curtis et al., 1988). While constantly being subjected to oxidative stress, aerobic organisms are protected against oxidative damage by a variety of antioxidant systems under normal conditions. Oxidative damage, however, may occur when the antioxidant potential is decreased and/or when the oxidative stress increases. In conclusion, further work on the formation of free radicals by SO2 and the effects of radicals on various kinds of biological molecules is required to understand the mechanism of SO2 toxicity.
It is known that bone marrow cells cycle about once every 11–13 h and so during the SO2 exposure periods of 1, 3, 5 and 7 days in this study the cells would pass through 2, 6, 10 and 14 cell cycles on average, respectively. Thus, in theory CAs induced by SO2 in any cycle other than the last cycle will be lost at cell division. However, in this study the frequencies of CA induced by SO2 increased with the relative duration of SO2 exposure. We suggest that bone marrow cells passing through the last cycle in mice exposed to SO2 for 1, 3, 5 and 7 days were actually exposed to SO2 for 2, 6, 10 and 14 generations, respectively. The biological macromolecular damage induced by SO2 might accumulate with an increase in the generations exposed to SO2 and this might be the reason why CAs induced by SO2 in bone marrow cells increased with exposure duration, although some aberrations were lost at cell division. On the other hand, cells with seriously damaged genetic material may not enter the next cycle (generation) or even die, however, some macromolecular damage that does not influence cell division could accumulate with SO2 exposure generation by generation to so serious a degree that it could indirectly cause damage to the genetic material and lead to an increase in CA frequency in bone marrow cells exposed to SO2. So, the longer the duration of SO2 exposure, the higher the CA frequency, as shown in Table II.
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Acknowledgments |
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This study was supported by a grant (no. 30070647) from the National Natural Science Foundation of China and by a grant from the National Natural Science Foundation of Shanxi Province, China.
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Notes |
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1 To whom correspondence should be addressed. Tel: +86 351 7011895; Fax: +86 351 7011895; Email: zqmeng{at}sxu.edu.cn
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Received on September 21, 2001; accepted on January 8, 2002.
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