SO2 inhalation induces protein oxidation, DNA–protein crosslinks and apoptosis in rat hippocampus
Introduction
SO2 is a common air pollutant, and human exposure to SO2 has become increasingly widespread due to the combustion of fossil fuels. The epidemiological studies have linked SO2 exposure with many respiratory responses (Schwela, 2000; Herbarth et al., 2001); however, recent researches indicate that SO2 was a oxidative agent to all organs tested from mice (brain, lung, heart, liver, stomach, spleen, kidney and testis) (Meng, 2003); SO2 also caused DNA breaks and ultrastructure changes in various cells from multiple organs (Meng et al., 2005; Meng and Liu, 2007). Especially, SO2 inhalation enhanced lipid peroxidation levels in brains of rat and guinea pig (Haider et al., 1981; Yargicoğlu et al., 1999; Kilic, 2003; Kucukatay et al., 2003, Kucukatay et al., 2007; Meng and Zhang, 2003) and was responsible for DNA breaks of brain in mice (Meng et al., 2005), and these injuries in brain were more serious than that in other organs. Also, SO2 derivatives changed the characteristics of voltage-gated sodium and potassium channels in rat hippocampal neurons (Meng and Sang, 2002; Sang and Meng, 2003). These results provide evidence for the possible neurotoxicity of SO2, but little information is available about its mechanisms.
SO2 is a gas and exists in aqueous solution at neutral pH as an equilibrium between bisulfite and sulfite ions (NaHSO3 and Na2SO3, 3:1 M/M), defined as SO2 derivatives (Shapiro, 1977; Meng et al., 2004; Du et al., 2006). Our preliminary data show that pre-treating mice with antioxidant, such as sea buckthorn seed oil, could attenuate SO2 inhalation-induced changes in the antioxidant system (Wu and Meng, 2003). It is suggested that SO2-induced toxicity may involve the formation of sulfur- and oxygen-centered free radicals, such as SO3−, SO4− and SO5−, during the process of SO2 derivative-oxidation (Shi, 1994; Shi and Mao, 1994). These free radicals can attack nucleic acids, especially some spots in purine and pyridine, result in base substitution and DNA breaks, and eventually induce mutation (Hayatsu and Miura, 1970; Hayatsu and Miller, 1972; Pagano et al., 1990; Reist et al., 1998; Meng and Zhang, 1999). Moreover, these radicals can react with proteins and lipids (Reist et al., 1998), lead to lipid peroxidation of cell membrane in tissues (Curtis et al., 1988; Meng, 2003; Meng et al., 2005), and affect protein structures and functions. Since the central neuronal system is one of the tissues highly sensitive to free radicals, it is postulated that SO2-induced neurotoxicity may be associated with its attacking lipids, proteins and nucleic acids via generated free radicals.
Previous studies show that the free radicals generated by SO2 caused membrane lipid peroxidation in brains of rat and guinea pig (Yargicoğlu et al., 1999; Kucukatay et al., 2003; Meng and Zhang, 2003). However, protein oxidation is a more sensitive index than lipid peroxidation during testing the damages of central neuronal system (Lyras et al., 1997; Samuel et al., 2005). To date, SO2-induced protein oxidative damage in central neuronal system, usually marked by the carbonyl content (Davies, 2005), has not been investigated.
It is also reported that the free radicals from SO2 metabolism induced DNA strand breaks (Meng et al., 2005), and this could be an iron-mediated process, e.g., via the Fenton reaction (Meng, 2003). Bertoncini and Meneghini (1995) and Meneghini (1997) have proposed that the Fenton reaction-mediated OH-radicals attacked DNA at the site where iron was bound, and produced DNA–protein crosslinks (DPC) (Altman et al., 1995). DPC are thought to be important genotoxic lesions induced by environmental agents and carcinogens, implicating the formation of DPC complexes, but not clarifying the type of specific protein crosslinked with DNA. These lesions, unlike the strand breaks and other DNA lesions that are readily repaired, are relatively persistent in the cells (Oleinick et al., 1987; Ramfrex et al., 2000). Due to a poor repair capacity, DNA–protein complexes are present during DNA replication, cause a loss of important genetic material, and induce cellar apoptosis (De Flora and Wetterhahn, 1989; Costa, 1991). Therefore, DPC induction has been proposed for assessing the DNA damage, but at this point no data exist concerning the influence of SO2 on its level.
The hippocampus is the old and simple section in phylogeny, and is usually used as the model for studying the anatomy and physiology in central neuronal system due to its involving the modulation of plasticity and the function of learning and memory. In addition, the hippocampus is one of the most vulnerable regions of the brain, especially to oxidative stress. In the present study, we investigated SO2 inhalation-induced protein oxidation, DPC and following injuries by examining protein carbonyl (PCO) content, DPC coefficient, caspase-3 activity in rat hippocampus, which actually containing different cell types (neuron, astrocyte and microglia); and counting the number of TUNEL positive staining neuron in hippocampal slice, to provide the evidence for the possible mechanisms of SO2 neurotoxicity.
Section snippets
Preparation of animals
Male Wistar rats, weighing 180–200 g, were used for the present experiment. The rats were housed in metallic cages under standard conditions and divided randomly into four equal groups of six animals each: one control group and three groups exposed to SO2. The experimental groups were placed in 1 m3 exposure chambers containing continuous concentrations of 14.00±1.01 (about 5 ppm), 28.00±1.77 (about 10 ppm), and 56.00±3.44 mg/m3 (about 20 ppm) SO2 for 6 h/day (8:00 a.m.–14:00 p.m.) for 7 days,
SO2 inhalation augmented PCO content
To detect SO2-induced protein oxidative damage in central neuronal system, PCO content was tested by DNPH spectrophotometry in rat hippocampus. Fig. 1A indicates that SO2 inhalation caused a significant increase of PCO level in rat hippocampus in a concentration-dependent manner. In control group, PCO background level was 0.83±0.05 nmol/mg protein; after exposure to SO2 at different concentrations, PCO levels were elevated to 0.91±0.06, 1.16±0.13 and 1.30±0.13 nmol/mg protein, respectively. For SO
Discussion
In this study, SO2 at 14–56 mg/m3 (5–20 ppm), levels some 10–40-fold greater than the typical urban concentration (0.5 ppm), was used to examine the responses of rat hippocampus. Although the experimental concentration may be viewed as beyond the normal polluting level in the human atmosphere environment, there are three important considerations. First, the animals were subjected to regular periods of extended exposure, with relief periods between protocols (i.e., 6 h/day, for 7 days, with 18 h
Conclusion
The present results indicate that SO2 inhalation significantly augmented the PCO content and DPC coefficient with concentration-dependent properties in rat hippocampus. In addition, SO2 at higher concentrations (28 and 56 mg/m3) caused the statistical increases of caspase-3 activity and number of TUNEL positive staining neuron, and induced apoptosis. It implies that causing oxidative stress and DNA injury by attacking proteins, lipids and nucleic acids via free radicals in central neuronal
Acknowledgments
This study was supported by the National Natural Science Foundation of PR China (no. 20607013), Young Science Foundation of Shanxi Province (no. 20051043), Scientific Research Foundation for the Returned Overseas Chinese Scholars of Shanxi Province, and Program for the Top Young Academic Leaders of Higher Learning Institutions of Shanxi.
The study involving experimental animals was conducted in accordance with national and institutional guidelines for the protection of animal welfare.
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