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Environmental Research 96 (2004) 298–304 www.elsevier.com/locate/envres
Oxidation damage of sulfur dioxide on testicles of mice Ziqiang Meng and Wei Bai Institute of Environmental Medicine and Toxicology, Shanxi University, Wucheng Road 36, Taiyuan 030006, PR China Received 14 August 2003; received in revised form 20 January 2004; accepted 22 April 2004
Abstract The effects of sulfur dioxide (SO2) on levels of thiobarbituric acid reactive substances (TBARS), levels of reduced glutathione (GSH), and the activities of Cu,Zn-superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were investigated in testicles of Kunming albino male mice. SO2 at different concentrations (22, 56, and 112 mg/m3) was administered to animals of SO2 groups in different exposure chambers for 6 h/day for 7 days, while control groups were exposed to ﬁltered air under the same conditions. Our results show that SO2 caused lipid peroxidation and changes in antioxidative status in testicles of mice. Exposure to SO2 at all concentrations tested signiﬁcantly increased TBARS levels in testicles of mice. SO2 at all concentrations tested tended to decrease activities of SOD and GPx enzymes and levels of GSH relative to control animals, but only the decreases in SOD and GPx activities caused by SO2 exposures of higher concentrations were statistically signiﬁcant. SO2 at all concentrations tested tended to increase activities of CAT relative to control animals, but the increases of CAT activities caused by SO2 exposures of low concentrations (22 and 56 mg/m3) were statistically signiﬁcant. These results lead to the conclusion that SO2 exposure can cause oxidative damage to testicles of male mice, and SO2 is a toxin to the reproductive system of mammals, not only to the respiratory system. Further work is required to understand the toxicological role of SO2 in reproduction organs or even sperm from humans and animals. r 2004 Elsevier Inc. All rights reserved. Keywords: Sulfur dioxide; Lipid peroxidation; Testicle; Reproduction; Antioxidative enzyme; Glutathione
1. Introduction Exposures to SO2 at signiﬁcant concentrations produce toxic symptoms, thickening of the mucous layer of the respiratory tract, pneumonia, nasopharyngitis, fatigability, gastritis, and alterations in the sense of taste and smell (Ferris et al., 1967). Epidemiological investigations have pointed out that SO2 exposure increases morbidity and mortality, particularly among subjects with cardiopulmonary diseases (Glasser et al., 1967). Recently, Selevan et al. have reported that young men may experience alterations in sperm quality after exposure to periods of high air pollution, without changes in sperm numbers (Selevan et al., 2000). Analysis of the impact of SO2 on fecundability in the Corresponding author. Fax: +86-351-7011895.
E-mail address: [email protected]
(Z. Meng). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.04.008
ﬁrst unprotected menstrual cycle shows that the relationship between SO2 and fecundability was greater in couples living close to the highly SO2-polluted district (Dejmek et al., 2000). The timing of these effects is consistent with the period of sperm maturation. This is in agreement with recent ﬁndings in which sperm abnormalities originating during spermatid maturation are found in young men from the region with high levels of air pollutants, including SO2 (Selevan et al., 2000). Therefore, more studies are needed on the effects of SO2 and other air pollutants on reproductive organs of mammals and humans. Recently, several investigations have reported that exposure to SO2 at 10 ppm may cause increase in increased lipid peroxidation in brains and erythrocytes of rats (Etlik et al., 1995; Gu¨mu¨s-lu¨ et al., 1998; Yargicog˘lu et al., 1999; Meng and Zhang, 2001a, b). SO2 at various concentrations may cause an increase in
ARTICLE IN PRESS Z. Meng, W. Bai / Environmental Research 96 (2004) 298–304
lipid peroxidation in brains and livers of mice in a dosedependent manner (Meng and Zhang, 2003). However, oxidation damage in testicles of mice caused by SO2 at different concentrations has not been reported. In this study, the testicles of mice were assessed for oxidative stress and antioxidant status caused by SO2 inhalation at different concentrations. The present results show that SO2 could cause oxidative damage in testicles of mice and that SO2 is a toxic agent to testicles of mammals, not only to the respiratory system.
2. Materials and methods 2.1. Preparation of animals Kunming albino male mice, weighing 1972 g (5 weeks old), were used for the present experiment. The mice were divided at random into six equal groups of 10 animals each: three groups exposed to SO2 at 2272, 5673, and 11278 mg/m3 and three to their respective control groups. Selection of dosage and timing of treatment were based upon earlier reports about lipid peroxidation of brains and livers of mice (Meng and Zhang, 2003). The animals were housed in groups of 10 mice in metallic cages under standard conditions (2472 1C and 5075% humidity) with a 12 h light–dark cycle. The animals had ready access to food and water ad libitum. SO2 at different concentrations (2272, 5673, and 11278 mg/m3) was administered to animals of SO2 groups in 1-m3 exposure chambers for 6 h/day for 7 days. Control groups were exposed to ﬁltered air in another identical chamber for the same period of time. SO2 gas was delivered to animals via a tube positioned at the upper level of each chamber and distributed homogeneously via a fan in each chamber. The SO2 was diluted with fresh air at the intake port of the chamber to yield the desired SO2 concentrations. SO2 within the chamber was measured every 30 min with pararosaniline hydrochloride spectrophotometry in order to monitor the SO2 concentrations (Goyal, 2001). Daily food and water consumption of every cage and weekly weight of individual mice were recorded during the feeding period. The mean daily food and water consumption was estimated from the recorded values. At the end of the experimental period, mice were deprived of food for 18 h and then prepared for the experimental procedures. 2.2. Measurement of antioxidant systems and oxidation products Both SO2-exposed and control mice were killed by cervical dislocation after the ﬁnal SO2 exposure followed by the 18 h food deprivation. Immediately after the mice
were killed, testicles were removed and weighed out, and their size and color or texture were observed. Then, these organs were trimmed in ice-cold saline (0.85% w/v NaCl) and homogenized at 10% (w/v) in 1.17% KCl in 0.10 M phosphate buffer, pH 7.4, at 0–4 1C gently using a glass Potter-type homogenizer at 500–800 rpm in ice. The homogenates were ﬁltered through a muslin cloth and were centrifuged at 20,000g for 30 min at 4 1C to obtain supernatants of tissue homogenates. The resultant supernatants were immediately pipetted for measuring the levels of thiobarbituric acid reactive substances (TBARS) (as an indicator of lipid peroxidation) and reduced glutathione (GSH) and the enzymatic activities of Cu,Zn-superoxide dismutase (SOD), Sedependent glutathione peroxidase (GPx), and catalase (CAT), and protein contents. 2.2.1. Assay of SOD activity Cu,Zn-SOD activity was measured using the method of Sun et al. (1988). The ﬁnal volume of the reaction systems was 3.0 mL and contained 0.1 mM xanthine, 0.1 mM EDTA, 50 mg bovine serum albumin per liter, 25 mM nitroblue tetrazolium (NBT), 9.9 nM xanthine oxidase, and 40 mM Na2CO3 (pH 10.2). The production of formazan was determined at 560 nm in a spectrophotometer (Hitachi U-3010, Japan) and 25 1C. One unit of SOD is deﬁned as the amount of protein that inhibits the rate of NBT reduction by 50%. Data are expressed as U/mg of tissue protein. 2.2.2. Assay of GPx activity GPx activity was measured by the method of Wendel (1981), with certain modiﬁcations. Brieﬂy, a reaction mixture was prepared in a vial containing 1 mg of bnicotinamide adenine dinucleotide phosphate tetrasodium salt (Sigma, St. Louis, MO, USA) containing the following: 9.2 mL of 1 mM sodium azide in 50 mM phosphate buffer with 0.4 mM EDTA (pH 7.0), 2 mM GSH in 0.05 mL of cold deionized water, and 10 units of glutathione reductase in 0.1 mL of cold deionized water. The assay was performed using 3 mL of the reaction mixture with the addition of 50 or 100 mL of tissue supernatant, mixed by inversion, and equilibrated to 25 1C in a 1-cm light path cuvette. The absorbance was monitored at 340 nm until constant in a spectrophotometer (Hitachi U-3010, Japan). Hydrogen peroxide was then added to a ﬁnal concentration of 0.2 mM and immediately mixed by inversion, and the decrease in absorbance at 340 nm was recorded every 15 s for 5 min. Speciﬁc activity was computed based on the extinction coefﬁcient of 6.22 mM1 cm1 for b-nicotinamide adenine dinucleotide phosphate at 340 nm. One unit of GPx catalyzes the oxidation by H2O2 of 1 mM reduced glutathione to oxidized glutathione per minute at pH 7.0 at 25 1C.
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2.2.3. Assay of CAT activity CAT activity was assayed by the method of Claiborne (1985). Brieﬂy, the assay mixture consisted of 1.95 mL phosphate buffer (0.05 M, pH 7.0), 1.0 mL hydrogen peroxide (0.019 M), 50 or 100 mL of supernatants of tissue homogenates, in a ﬁnal volume of 3.0 mL. Changes in absorbance were recorded at 240 nm (Hitachi U-3010, Japan). CAT activity was calculated in terms of nanomoles of H2O2 consumed/min/mg of tissue protein. 2.2.4. Assay of GSH content Levels of GSH were measured by the method of Jollow et al. (1974). An aliquot of 1.0 mL supernatants (10%, w/v) was precipitated with 1.0 mL of sulfosalicylic acid (4% w/v). The samples were kept at 4 1C for at least 1 h and then subjected to centrifugation for 15 min at 4 1C. The assay mixture contained 0.1 mL ﬁltered aliquot, 2.7 mL phosphate buffer (0.1 M, pH 7.4), and 0.2 mL 5,50 -dithiobis-2-nitrobenzoic acid (DTNB, 40 mg/10 mL of phosphate buffer 0.1 M, pH 7.4) in a total volume of 3.0 mL. The yellow color developed was read immediately at 412 nm on a spectrophotometer (Hitachi U3010, Japan). The content of GSH in the tissue was determined by comparing its absorption with that of a curve made with known amounts of GSH. Data were expressed as nmol/mg of tissue protein. 2.2.5. TBARS assay TBARS levels, as an indicator of lipid peroxidation, were measured by a ﬂuorometric method described by Wasowicz et al. (1993), using 1,1,3,3,-tetramethoxypropane as a standard and the results are expressed as nmol/mg of tissue proteins. The supernatants (50 ml) were placed into a tube containing 1 mL of distilled water. After the addition of 1 mL of a solution containing 29 mM 2-thiobarbituric acid (TBA) in acetic acid (8.75 M), samples were placed in a water bath and heated for 1 h at 95–100 1C. After the samples had cooled, 25 ml of 5 M HCl was added and the reaction
mixture was extracted by agitation for 5 min with 3.5 mL of n-butanol. After centrifugation, the butanol phase was separated and the ﬂuorescence of the butanol extract was measured in a spectroﬂuorometer (Hitachi F-4500, Japan) using wavelengths of 525 nm for excitation and 547 nm for emission. 2.3. Protein assay In all measurements of antioxidant systems and oxidation products, the protein concentrations were evaluated according to Lowry et al. (1951) with bovine serum albumin as standard. 2.4. Statistical analysis All values were expressed as the mean7standard deviation. A single-tailed Student t-test was used for signiﬁcant differences between SO2-exposed groups and their corresponding control groups. A level of Po0.05 was accepted as statistically signiﬁcant.
3. Results The mean body weight gain was not different in mice exposed to SO2 with respect to their corresponding control groups during the experimental period, although the ﬁnal weights of both control and SO2-exposed groups were signiﬁcantly increased relative to the beginning values (data not shown). No deaths, morbidity, or distinctive clinical signs were observed after any treatment. Table 1 presents activities of SOD, GPx, and CAT in testicles of mice treated with SO2 exposure (at 2272, 5673, and 11278 mg/m3) and their control groups. SO2 inhalation at all concentrations tested caused a decrease in SOD activities in testicles from male mice, but at low concentration (2272 mg/m3) the decrease in SOD activities is not statistically signiﬁcant. Low SO2
Table 1 Effect of SO2 inhalation on activities of antioxidative enzymes in testicles of male mice SO2 (mg/m3)
863.8795.4 (11.80%) 1197.47220.5 (20.97%) 1703.97227.2* (30.09%)
4.0070.91 (6.98%) 3.2770.62* (22.70%) 3.2970.46** (19.95%)
1.14070.453* (+27.37%) 0.70070.164* (+37.25%) 0.82970.116 (+14.19%)
Note: Data are expressed as mean7standard deviation (n=10). The activities of SOD, GPx and CAT are expressed as U/mg of tissue proteins. The changed percentages of enzyme activities are expressed in parentheses. Signiﬁcantly different from control without SO2 exposure by t test at *Po0.05, ** Po0.01, ***Po0.001.
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Table 2 Effect of SO2 inhalation on levels of GSH and TBARS in testicles of male mice SO2 (mg/m3)
56.03715.73 (3.65%) 40.96716.34* (23.95%) 47.3675.36* (13.78%)
0.22370.028*** (+31.18%) 0.29670.075*** (+38.32%) 0.29770.068*** (+54.69%)
Note: Data are expressed as mean7standard deviation (n=10). Levels of TBARS and GSH are expressed as nM/mg of tissue proteins. The changed percentages of levels of TBARS and GSH are expressed in parentheses. Signiﬁcantly different from control without SO2 exposure by t test at * Po0.05, **Po0.01.
concentration (2272 mg/m3) caused no signiﬁcant decrease in GPx activities in testicles for male mice. Higher SO2 (5673 and 11278 mg/m3) caused a signiﬁcant decrease in GPx activities in testicles. For CAT, SO2 at low concentrations (22 and 56 mg/m3) caused a signiﬁcant increase in CAT activities in testicles from male mice. However, high SO2 concentration (112 mg/m3) caused no signiﬁcant increase in CAT activities in testicles from male mice. Table 2 summarizes the levels of GSH and TBARS in testicles in mice treated with SO2 exposure at 2272, 5673, and 11278 mg/m3 and their control groups. SO2 exposure at all concentrations tested signiﬁcantly caused the increase in TBARS levels in testicles from male mice. It is also shown that SO2 exposure at all concentrations tested caused decreases in GSH levels in testicles from male mice, but only the decreases in GSH levels caused by SO2 at higher concentrations (5673 and 11278 mg/ m3) were statistically signiﬁcant. From Tables 1 and 2 we can see that there is a high degree of variability in activities of SOD and CAT in the controls of each treatment set, because these mice in different SO2-concentration treatments were from different batches. This might be the reason there is a high variability in some data among the different control groups. However, the mice in each SO2-concentration treatment group and its control group were all from the same batch. The percentages of the increase or decrease between the SO2-treatment group and its control group (the animals were from same batch) were compared.
4. Discussion Sulfur dioxide (SO2) is one of the most important pollutants in the world, responsible for several cardiopulmonary diseases in humans. SO2 affects both children and adults, causing low work productivity with extremely high social and economical costs for communities. Recently, a relationship between increased ambient levels of air pollution and an increased risk
for low birth weight (LBW) has been reported (Maisonet et al., 2001; Bobak, 2000). Carbon monoxide, nitrogen dioxide, sulfur dioxide, and total suspended particle concentrations in the ﬁrst trimester of pregnancy are risk factors for low birth weight (Ha et al., 2001). Rogers et al. (2000) have presented results of a population-based case-control study of the association between maternal exposures to environmental sulfur dioxide and total suspended particulates (TSP) and the risk for having a very low birth weight (VLBW) baby, i.e., one weighing less than 1500 g at birth. It is suggested that there is an association between VLBW and maternal exposures to high levels of air pollution. Recently, it has been observed that birth rates in Teplice, a highly polluted district in Northern Bohemia of Czech, have been reduced during periods when sulfur dioxide levels were high. Dejmek et al. (2000) have reported that the relationship between SO2 and fecundability was greater in couples living close to the highly polluted district. The timing of these effects is consistent with the period of sperm maturation. This is in agreement with recent ﬁndings in which sperm abnormalities originating during spermatid maturation were found in young men from the Teplice region who were exposed to the increased levels of ambient SO2 (Selevan et al., 2000). Selevan et al. have reported that young men may experience alterations in sperm quality after exposure to periods of elevated air pollution, without changes in sperm numbers. In the present study, oxidation damages of SO2 inhalation on testicles of male mice were investigated in order to probe the roles of SO2 on reproduction of mammals. In this study, a range of SO2 concentrations was used to examine the response of the mouse testicles to this air pollutant. SO2 at 22 mg/m3 (7.86 ppm) represented a level some 16-fold greater than the typical urban concentration (0.5 ppm), but is known to induce asthmatic symptoms in healthy individuals (Meng, 2000). SO2 at 112 mg/m3 (40 ppm) was used to examine the effects of greater exposure while still working at a much lower concentration than the 800 ppm previously
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applied in some rat studies (Stratmann et al., 1991). Although the experimental conditions may be viewed as beyond the normal atmosphere encountered in the human environment, there are two important considerations. First, the animals were subjected to regular periods of extended exposure, with relief periods between protocols (i.e., 6 h per day, for 7 days, with 18 h between exposures). This may provide a corollary to individuals exposed to the gas in an occupational setting. Second, the mouse is an obligate nose-breather and approximately 95% of the inhaled SO2 is trapped in the nasal passages. Therefore, the actual concentrations of gas reaching the lungs may have been signiﬁcantly lower than those in the exposure chamber. Under normal conditions, cells possess enzymatic and nonenzymatic defenses to cope with free radicals, such as SOD, GPx, CAT, and GSH (Tardy et al., 1989; Shi, 1994). Oxidative damage, however, may occur when antioxidant potential is decreased and/or when oxidative stress is increased. Free-radical-induced oxidative damage has been implicated in the pathogenesis of a number of injury and diseases states (Freeman and Crapo, 1982; Halliwell, 1987; Max, 1987). Several studies have found that SO2 inhalation enhances lipid peroxidation in the brains of rats and guinea pigs and in the rat erythrocytes (Haider et al., 1981; Yargicog˘lu et al., 1999; Etlik et al., 1995; Meng and Zhang, 2001a, b). However, oxidation damage in testicles of mice caused by SO2 at different concentrations has not been reported. In the present investigation, we studied the oxidative stress and antioxidation status in testicles of mice following exposure of SO2 at 22, 56, and 112 mg/ m3 in order to understand the toxicological role of SO2 inhalation on testicles of animals. The present results show that SO2 inhalation increased levels of lipid peroxidation in testicles of mice, accompanied by signiﬁcant changes in activities of SOD and GPx and levels of GSH in the testicles. The level of TBARS has been shown to be an indicator of endogenous lipid peroxidation (Tappel and Zalkin, 1960). Our results show that exposure to SO2 at 22, 56, and 112 mg/m3 all signiﬁcantly increased levels of TBARS in the testicles from male mice (Table 2). Inhaled SO2 can easily be 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) (Shapiro, 1977). The derivatives can be absorbed into blood or other bodily ﬂuids (Meng, 2000). The mechanism of SO2 or bisulﬁte/ sulﬁte toxicity may involve oxidation damage in cells, tissues, and organs caused by sulfur- and oxygencentered free radicals formed in the process of sulﬁte oxidation (Shi, 1994; Shi and Mao, 1994). Table 1 indicates that SO2 exposure tended to decrease activities of SOD in testicles from mice. However, the decreases of SOD and GPx activities
caused by lower SO2 concentration were not signiﬁcant statistically; only higher SO2 concentrations caused signiﬁcant decreases of these enzyme activities. For CAT activity, lower SO2 concentrations (22 and 56 mg/ m3) caused signiﬁcant increases in CAT activities in testicles from mice, while the increase in CAT activities caused by high SO2 exposure (112 mg/m3) was nonsigniﬁcant. GPx and CAT have been considered the primary scavengers of hydrogen peroxide (Chance et al., 1979). The increases of CAT caused by SO2 exposure at lower concentrations might be an adaptive mechanism to the superﬂuous hydrogen peroxide produced by inhaled SO2. Unexpectedly, the changes in activities of GPx and CAT caused by SO2 at different concentrations in testicles of mice did not follow a linear dose–response curve. The changed percentages of the activities of GPx or CAT caused by SO2 at 22, 56, and 112 mg/m3 in testicles of mice were similar in some respects (Table 1). The decreases in activities of the antioxidant enzymes might predispose the testicles to increased free radical damage, because SOD can catalyze decomposition of superoxide radicals to produce hydrogen peroxide. GPx and CAT have been considered the primary scavengers of hydrogen peroxide (Chance et al., 1979). The decreases of GPx activity in response to SO2 can reduce the protection against free radicals and lipid peroxidation. GSH is the major cellular sulfydryl compound that serves as both a nucleophile and an effective reductant by interacting with numerous electrophilic and oxidizing compounds. It is a cofactor for GPx, which catalyzes the reduction of hydrogen peroxide to water, thereby limiting the formation of hydroxyl radical, the most toxic of the oxygen-based radicals. GSH has been proposed to play a role in detoxiﬁcation of SO2, and GSH in tissues may be lowered by SO2 exposure. Our results show that exposure to SO2 at different concentrations all tend to decrease levels of GSH in testicles of male mice. Unexpectedly, the changes of GSH levels caused by SO2 at different concentrations in testicles of mice did not follow a linear dose–response curve. Slott et al. (1989) reported that using intratesticular injections of a mixture of two GSH-depleting agents, diethylmaleate and buthionine sulfoximine, testicular GSH levels were decreased to 33–54% of control 2 h after injection and remained suppressed for 24 h. Their results showed that GSH-depleting agents selectively lowered GSH levels in the treated testis, with minimal adverse effects. Our results showed that SO2 at different concentrations caused decreases of testicular GSH levels by 4–24%. Signiﬁcant high levels of endogenous lipid peroxidation is a indicator of a biochemical disorder in cells, tissues, and organs, and an indicator of a toxicological role of some chemical toxins to the living organism. Lipid peroxidation is believed to be involved in several disease
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states, such as diabetes and neurodegerative diseases as well as the aging process (Baynes,1991). Adverse effects of the oxidative stress on testis and sperm production in the morphology and histology remain in need of further studies. The detailed mechanism of SO2-induced oxidation damage is not clear, but it may involve formation of sulfur- and oxygen-centered free radicals, such as SO 3, SO 4 and SO5 (Shi, 1994; Shi and Mao, 1994). The oneelectron oxidation of bisulﬁte, a known derivative of SO2, produces the sulfur trioxide radical anion, which reacts rapidly with molecular oxygen to form a peroxyl radical. The free radicals generated by SO2 can damage nucleic acids (Meng and Zhang, 1999; Hayatsu and Miller, 1973) and induce mutation (Meng and Zhang, 1999; Pagano et al., 1990; Hayatsu and Miura, 1970). Moreover, these radicals can react with proteins and lipids (Reist et al., 1998). Oxidative stress is also induced by SO2 at various concentrations in brains and livers of mice in a dosedependent manner (Meng and Zhang, 2003). It has been indicated that SO2 is a systemic oxidative damage agent. However, the effects of SO2 on testicles of mice were not speciﬁc. Differences among sensitivities of various organs of mice to SO2 need further studies. It is generally considered that SO2 and its in vivo derivatives—bisulﬁte/sulﬁte—are toxic to the respiratory system and can cause allergic reactions, the most common of which is bronchoconstriction in asthmatics (Ceballos-Picot et al., 1992). It may induce cytogenetic damage in human lymphocytes and act as a comutagen and cocarcinogen to link to lung cancer (Lester, 1995; Meng and Zhang, 1990, 1992, 1999, 2002; Meng et al., 2002a, b). In this study, the signiﬁcant changes in oxidative stress and antioxidation status in the testicles were found in SO2 groups. It is shown that effects of SO2 on living organism are many-sided. In summary, results from the present research show that (i) SO2 exposure resulted in a signiﬁcant increase in the lipid peroxidation process in the testicles of male mice, accompanied by changes in antioxidant status in these organs; (ii) effects of SO2 on living organisms are many-sided; (iii) SO2 is also a toxin to testicles, not only to the respiratory system. However, the short duration of the study is a limitation, and longer exposures to SO2 might lead to other toxicities. Further work is required to understand the toxicological role of SO2 on the reproduction health of mammals.
Acknowledgments This study was supported by Grant 30070647 from the National Natural Science Foundation of China and by a grant from the National Natural Science Foundation of Shanxi Province.
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