Mutagenesis vol. 19 no. 6 © UK Environmental Mutagen Society 2004; all rights reserved.
DNA damaging effects of sulfur dioxide derivatives in cells from various organs of mice
Institute of Environmental Medicine and Toxicology, Research Center of Environmental Science and Engineering, Shanxi University, Taiyuan 030006, China
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Abstract |
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The DNA-damaging effects of sulfur dioxide (SO2) derivatives (a mixture of sodium sulfite and sodium bisulfite, 3:1 M/M) in the cells of various organs (brain, lung, heart, liver, stomach, spleen, thymus, mone marrow and kidney) of male mice were studied using the single cell gel electrophoresis technique (SCGE). Three groups of six mice each received an i.p. dose of SO2 derivatives (125, 250 or 500 mg/kg body wt) daily for 7 days. A control group of six mice received 200 µl of normal saline i.p. daily for 7 days. Our results show that SO2 derivatives caused significant increases in olive tail moment (OTM) in cells from all organs tested in a dose-dependent manner. These results show that SO2 derivatives can cause DNA damage to multiple organs of mice and that SO2 derivatives are systemic DNA-damaging agents, not only to the respiratory system. It is suggested that SO2 derivative exposure has a potential risk to DNA in multiple organs of mammals and might be related to carcinogenesis or other diseases related to DNA damage. Further work is required to understand the toxicological role of SO2 and its derivatives on multiple or even all organs in humans and animals. Recent research results have shown that SO2 and its derivatives can also induce an increase in the frequencies of chromosomal aberrations, sister chromatid exchanges and micronuclei in mammalian cells and cause oxidative damage in multiple organs of male and female mice. Taken together, these results suggest that SO2 and its derivatives are systemic toxic agents. However, further studies need to be performed before a definitive conclusion can be drawn.
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Introduction |
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Sulfur dioxide (SO2) is a common air pollutant. Inhaled SO2 is hydrated to produce sulfurous acid in the respiratory tract, which subsequently dissociates to form its derivatives, bisulfite and sulfite (1:3 M/M in neutral fluid). The derivatives can be absorbed into the blood or other body fluids (Shapiro, 1977
). In addition, bisulfite/sulfite enters the body in foods, beverages and drugs because sulfiting agents (sulfur dioxide, metabisulfite, bisulfite and sulfite) are widely used as preservatives. Endogenous bisulfite/sulfite is generated during the normal processing of sulfur-containing amino acids and can be formed by the metabolism of sulfur-containing drugs, including N-acetylcysteine.
Recently, several studies have shown that the SO2 derivatives (bisulfite and sulfite) may induce chromosomal aberrations (CA), sister chromatid exchanges (SCE), and micronuclei (MN) in cultured human blood lymphocytes in vitro (Meng and Zhang, 1992) and MN in mouse bone marrow cells in vivo (Meng et al., 2002a,b; Meng and Zhang, 2002). It has been reported that the frequencies of CA, SCE and MN in peripheral blood lymphocytes of workers chronically exposed to SO2 in factories were higher than in unexposed controls (Beckman and Nordenson, 1986; Meng and Zhang, 1990a,b; Yadav and Kaushik, 1996). These results suggest that SO2 and its derivatives are clastogenic and genotoxic agents (Meng and Zhang, 1992). So far, no in vivo mouse studies using the single cell gel electrophoresis assay (SCGE) reporting DNA-damaging effects of SO2 derivatives have been published. In the present study damage to DNA in cells from various organs of mice treated with SO2 derivatives at different doses was investigated with the alkaline SCGE assay.
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Materials and methods |
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Animals
Male Kunming albino mice supplied by the Division of Laboratory Animals, Shanxi Institute of Chinese Medicine, Taiyuan, 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 per day). The animals were fed standard rodent pellet diet (purchased from Shanxi Institute of Chinese Medicine, Taiyuan, China) and water ad libitum. Animals were randomly numbered and then assigned to different treatment groups.
Chemicals and treatment of SO2 derivatives
Sodium bisulfite, sodium sulfite, low melting point agarose (LMA) and normal melting point agarose (NMA) were purchased from Sigma Chemical Co. (St Louis, MO).
Three groups of six male mice each received an i.p. dose of a mixture of sodium sulfite and sodium bisulfite (3:1 M/M) (125, 250 or 500 mg/kg body wt) dissolved in 200 µl of normal saline (0.9% sodium chloride) daily for 7 days; one negative control group of six male mice each received 200 µl of saline i.p. daily for 7 days. Based on a pilot study with the mixture of sodium sulfite and sodium bisulfite (3:1 M/M) at an i.p. dose of 1000 mg/kg body wt, which resulted in lethality to 50% of mice (data not shown), a maximum dose of 500 mg/kg (0.5 LD50) and a minimum dose of 125 mg/kg (0.125 LD50) were selected.
Single cell suspension preparation
The mice were killed by cervical dislocation 24 h after the final injection. Brain, lung, heart, liver, stomach, spleen, thymus and kidney were removed immediately and washed in ice-cold phosphate-buffered saline (PBS) to remove superficial blood. The organs were minced and washed again in ice-cold PBS to remove red cells. These minced organs were then forced through a wire mesh screen in order to obtain single cell suspensions. For all samples cell viability was >95% (determined with a Malassez hemocytometer using the Trypan blue exclusion technique).
The bone marrow was removed by flushing each femur with 0.1 ml of fetal bovine serum (FBS). The cells from each femur were then pooled and centrifuged at 2000 r.p.m. for 5 min. After most of the supernatant was discarded, the cell pellet was resuspended in the remaining FBS. For all samples cell viability was >99%.
The single cell suspension was then mixed with 0.6% LMA at 37°C.
Slide preparation
The SCGE technique described by Singh et al. (1988) was followed with some minor modifications. The experimental steps were as follows. An aliquot of 100 µl of 0.6% NMA solution was added to a fully frosted microscope slide, immediately covered with a clean coverslip and allowed to solidify. Then the coverslip was carefully replaced with one covered with 75 µl of prewarmed (37°C) cell mixture/LMA layer. After the agarose layer had solidified, the coverslip was removed and a top layer of 85 µl of 0.6% LMA was added. When the top agarose layer had solidified, the coverslip was removed and the slide immersed in a jar containing cold lysing solution (2.5 mM NaCl, 100 mM EDTA, 1% sodium sarcosinate and 10 m M Tris, pH 10.0, to which 1% Triton X-100 and 10% DMSO were freshly added). The slides were left at 4°C for at least 1 h.
Electrophoresis and staining
The slides were removed from the lysing solution and placed in a horizontal electrophoresis box which was filled with freshly made electrophoreses buffer (300 mM NaOH, 1 mM EDTA, pH 13.0) to a level 0.2–0.3 cm above the slides. The cells were immersed in the alkali for 20 min to allow DNA unwinding and expression of single-strand breaks and alkali-labile sites. Electrophoresis was performed in the same buffer at room temperature for 20 min by applying an electric current of 300 mA at a voltage of 1 V/cm. All of these steps were conducted under yellow light to prevent the occurrence of additional DNA damage. After electrophoresis the slides were placed horizontally and Tris buffer (0.4 M Tris, pH 7.5) was gently added to neutralize the excess alkali. The slides were allowed to sit for 5 min and this was repeated three times. Subsequently the slides were air dried and then stored at 4°C until analysis for DNA migration.
Just prior to analysis 100 µl of ethidium bromide (20 µg/ml) was added to each slide and covered with a coverslip. After 20 min the coverslips were removed and the slides rinsed in distilled water to remove excess ethidium bromide. The slides were again covered with coverslips.
Image analysis
Slides were examined at 400x magnification using a fluorescence microscope (Leica Microscopy, Germany) connected through a digital camera (Leica DC100) to a Comet Image Processing and Analysis System, version 4 (Kinetic Imaging Ltd, Liverpool, UK). Images of 25 randomly selected cells were analyzed from each slide investigated (two slides per mouse). For each group (six male mice), 300 cells were scored. Olive tail moment (OTM), defined as the product of the distance between the barycenters of the head and tail, by the percentage of DNA in the tail of the comet was used to evaluate the extent of DNA damage in individual cells (Olive et al., 1993; Poul et al., 2004).
Statistical analysis
All values are expressed as the mean ± SD from each group of six mice. The mean OTM value of 50 cells from each organ was calculated for each individual animal (Hartmann et al., 2004). The differences between the averages of six treated mice and the untreated control mice were compared using the Dunnett test after one-way ANOVA. A level of P < 0.05 was considered statistically significant. The linear correlation test was used to define the dose–response relationship.
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Results |
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The mean body weight gain was not different in mice exposed to SO2 derivatives with respect to their corresponding control groups during the experimental period, although the final weights of both control and the SO2 derivative-exposed groups were significantly increased relative to the start values (data not shown).
Tables I and II show the OTM values in the cells from all organs tested (brain, lung, heart, liver, stomach, spleen, thymus, bone marrow and kidney) of male mice treated with SO2 derivatives (sodium sulfite and sodium bisulfite, 3:1 M/M) at various doses (125, 250 or 500 mg/kg body wt) and a control group. SO2 derivatives at all doses tested significantly increased the OTM of DNA in cells from all organs tested. Tables I and II also show that DNA damage (OTM) in these cells increased with the dose of SO2 derivatives (for brain, lung, heart, liver, stomach, spleen, thymus, bone marrow and kidney from male mice, r = 0.96, 0.96, 0.95, 0.99, 0.98, 0.99, 0.99, 0.97 and 0.99, respectively).
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Table III shows the percentage of cells with comet tails from all organs tested (brain, lung, heart, liver, stomach, spleen, thymus, bone marrow and kidney) of male mice exposed to SO2 derivatives at the doses tested and a control group. SO2 derivatives at all doses tested caused significant increases in the percentage of cells with comet tails in the cells from all organs tested in a dose-dependent manner (r = 0.95–0.98).
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Discussion |
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SO2 is a well-known environmental pollutant released into the atmosphere from the combustion of fossil fuel (Rall, 1974
). Inhaled SO2 can easily be hydrated to form its derivatives bisulfite and sulfite (1:3 M/M in neutral fluid) (Shapiro, 1977). As food additives, sulfiting agents were first used in 1664 and approved in the USA as long ago as the 1800s. With such long experience in their use, it is easy to understand why these substances have been regarded as safe. They are currently used for a variety of preservative properties, including controlling microbial growth, preventing browning and spoilage and bleaching some foods. It is estimated that up to 500 000 (<0.05% of the population) sulfite-sensitive individuals live in the USA. Recently it was reported that SO2 and its derivatives can increase CA, SCE and MN in different kinds of mammalian cells in vivo and in vitro (Beckman and Nordenson, 1986; Meng and Zhang, 1990a,b, 1992, 2002; Yadav and Kaushik, 1996; Meng et al., 2002a). In the present study we evaluated sodium bisulfite and sodium sulfite for their ability to induce DNA damage in various organs of mice using the SCGE technique. The SCGE test or comet assay is a straightforward visual method for the detection of DNA damage in interphase cells. This technique is especially sensitive in detecting DNA single-strand breaks, alkaline-labile damage and excision repair sites in individual cells. Compared with other classical methods of detection of DNA damage, SCGE has the advantage of showing DNA damage in individual cells. It is a direct, sensitive, simple, rapid and powerful technique used in toxicological studies. It is generally assumed that SCGE performed under alkaline conditions primarily detects single-strand breaks and alkali-labile sites in DNA (Singh et al., 1988). In order to conduct SCGE for each organ, a protocol of cell recovery was first established. All cell suspensions from the different organs had good and acceptable viability (>95%) and low levels of DNA damage were observed in the control groups during the comet assay: all the controls showed low values for OTMs (<0.40).
Analysis of OTMs in Tables I and II showed a strong positive, dose-dependent response in cells from all organs tested. The results imply that SO2 and its derivatives are systemic DNA-damaging agents. SO2 derivatives can cross the alveolar–capillary barrier, reach the circulating blood and all organs and then damage DNA in the cells in various organs (Meng and Zhang, 2002; Meng, 2003).
The ratio of cells with comet tails reached >50% even after low SO2 derivative exposure (125 mg/kg body wt), except for thymus and bone marrow (Table III). This implies that DNA in these cells is very sensitive to the toxicological effects of SO2 and its derivatives.
The alkaline comet assay shows that SO2 derivatives might be strong DNA-breaking agents at the various doses tested. The genotoxic activity of SO2 and its derivatives appears to be more evident in the comet assay than their mutagenic activity in the PCR-based deletion screening test for gpt gene mutation in CHO-AS52 cells (Meng and Zhang, 1999). The apparent lack of sensitivity of the gpt gene mutation test to sodium sulfite and sodium bisulfite might be attributed to the generation of a particular type of damage and a high capacity of cells to repair or selectively eliminate heavily damaged cells.
The mechanism of DNA damage induced by SO2 and its derivatives is not clear and further studies are needed. The genetic effects of the SO2 derivatives sulfite and bisulfite have been examined in bacterial and mammalian cells. It has been suggested that DNA damage induced by SO2 and its derivatives might involve the following factors. 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). Transitional mutations (G:C 
A:T) induced by bisulfite were reported in Escherichia coli and bacteriophage T4 (Mukai et al., 1970; Summers and Drake, 1971). The one-electron oxidation of bisulfite produces the sulfur trioxide radical anion, which reacts rapidly with molecular oxygen in cells to form a highly biologically reactive peroxyl radical, a reactive oxygen species (ROS) (Shi and Mao, 1994; Kilic, 2003). ROS can react with DNA to produce damage. If the damage is not correctly repaired by base excision repair, it can lead to the formation of DNA single-strand breaks and/or induction of mutations. Furthermore, a lot of free radicals are produced during sulfite and bisulfite oxidation, such as 
, 
and 
(Singh and Pathak, 1990; Shi and Mao, 1994). These free radicals can damage DNA and induce mutation (Singh and Pathak, 1990; Shi and Mao, 1994; Meng and Zhang, 1999). It has been shown that SO2 inhalation results in a significant increase in lipid peroxidation levels in multiple organs from mice of both sexes (Meng, 2003).
The present results demonstrate that SCGE under alkaline conditions can efficiently be applied to DNA damage studies in different types of cell populations, such as cells from various organs after treatment with SO2 derivatives. SCGE can be selected as an assay to provide a biomarker to evaluate the impact of exposure to SO2 and its derivatives. In this study a linear correlation was found between DNA damage caused by SO2 derivatives in cells from various mouse organs.
It has been reported that SO2 increases the frequencies of CA, SCE and MN in different types of cells. These results confirm that SO2 is a clastogenic and genotoxic agent in cells from different compartments (Meng and Zhang, 1992). In a recent work it was demonstrated that SO2 can cause oxidative stress and changes in the activities of antioxidative enzymes in cells from multiple organs of mice of both sexes. It is suggested that SO2 is a systemic oxidative DNA-damaging agent (Meng, 2003). In the present study SO2 derivatives caused DNA damage in all organs tested from male mice. Combined analysis of the experimental results mentioned above leads to the conclusion that SO2 and its derivatives are systemic toxic agents.
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Acknowledgments |
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This research program was supported by grant 30230310 from the National Natural Science Foundation of China and by a grant (20031092) from the Natural Science Foundation of Shanxi Province.
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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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References |
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Received on April 1, 2004; revised on September 9, 2004; accepted on September 13, 2004.
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