| | Toluene inhalation induced 8-hydroxy-2′-deoxyguanosine formation as the peroxidative degeneration in rat organsReceived 30 September 2002; received in revised form 12 December 2002; accepted 15 January 2003. Abstract The effect of toluene inhalation on oxidative damage in rat organs was examined. Male Wistar rats was inhaled toluene (1500 ppm for 4 h a day) for 7 days. Quantitatively and immunohistochemically, oxidative DNA damage, lipid peroxide (LPO) and superoxide dismutase (SOD) were examined. As a marker of the oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine (8-OH-dG) immunoreactivity increased in the lung, liver and kidney. The amount of 8-OH-dG also increased in liver and kidney significantly. In the testis, the amount of 8-H-dG did not increase, however 8-OH-dG immunoreactivity enhanced in the spermatogonia. SOD immunoreactivity increased in the lung, liver and kidney. However, 4-hydroxy-nonenal immunoreactivity and the amount of LPO did not change in each organ. Thus, oxidative damage by toluene is mainly DNA damage, especially, the oxidative DNA damage observed in the lung, liver and kidney for the increase of the immunoreactivity and amount of 8-OH-dG.
1. Introduction  Toluene is the most commonly abused reagent in the young generation. The toluene, which is the chief ingredient of the organic solvent such as paint thinner, is a well known neurotoxic material, and it is suggested to influence behavior [1]. Recently, many influences of toluene on neurons were reported [2], [3], [4], [5]. We previously examined a disorder of the central nervous system by toluene inhalation using molecular biological and immunohistochemical techniques [6], [7], [8]. However, there have been few studies about the oxidative DNA damage the toluene inhalation in various organs including neurons [9], [10], [11], [12], [13], [14]. In the present study, we elucidated the peroxidative damage in various organs due to toluene inhalation, examining immunoreactivity of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), formation of oxidative DNA damage, 4-hydroxy-nonenal (4-HNE), formation of lipid peroxidation, and superoxide dismutase (SOD), enzymes of the activated oxygen elimination, and quantitatively analyzing amount of 8-OH-dG and lipid peroxide (LPO). 2. Materials and methods 2.1. Animal treatment Male Wistar rats weighing 180–200 g were obtained from Japan SLC (Shizuoka, Japan) and were bred on a 12 h light–12 h dark schedule with food and water available ad libitum. Rats were exposed to toluene (1500 ppm for 4 h per day) for 7 days. Control rats in separate chambers were exposed to air concurrently with the toluene groups. 2.2. Immunohistochemical staining For immunohistochemical analysis, animals were perfused through the ascending aorta with 0.01 M phosphate-buffered saline (PBS, pH 7.2) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia (sodium pentobarbital, 100 mg/kg, i.p.) 24 h after the last inhalation. The cerebrum, cerebellum, lung, heart, liver, kidney, testis and epididymis were removed and postfixed in the same fixative overnight at 4°C. Each organ was dissected and cut in 2 mm slices. Tissue was then embedded in paraffin, and 5 μm-thick sections were cut. After deparaffinization, the sections were treated with 0.3% H2O2 in methanol for 30 min for inactivation of endogenous peroxidase, 250 μg/ml RNase for 1 h at 37°C and then incubated in blocking solution for 10 min. Sections were incubated with diluted anti-8-OH-dG (1–10 μg/ml, QED Bioscience, USA or 1–10 μg/ml, JICA, Japan), 4-HNE (1:500, Alpha Diagnostic, USA) and SOD (1:200, OXIS Health Products, USA). The immunostaining was carried out using an HISTOFINE SAB-PO (Multi) kit (Nichirei, Japan) following the manufacturer's instructions (incubating with a mixture of biotinylated goat anti-mouse IgG and biotinylated goat anti-rabbit IgG antibodies for 10 min, followed by incubation with horseradish peroxidase-labeled streptavidin for 5 min). The immunostaining was visualized by incubating with 0.02% 3,3′-diaminobenzidine and 0.03% hydrogen peroxide in PBS. Controls for the specificity of the immunohistochemistry involved omission of the primary antibody. 2.3. Enzyme-linked immunosorbent assay (ELISA) of 8-OH-dG For quantitative analysis of 8-OH-dG, animals were asphyxiated with CO2 24 h after the final inhalation. Blocks of the hippocampus, cerebellum, lung, heart, liver, kidney, testis and epididymis were frozen with liquid nitrogen and preserved at −80°C until use. The DNA was extracted with a DNA Extractor WB kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer's instructions. Briefly, 0.5 g of each tissue were homogenized in a Potter-type homogenizer and then centrifuged at 10,000×g for 20 s at 4°C to separate the nuclear fraction. Two hundred micrograms of DNA from each tissue was hydrolyzed. The 8-OH-dG levels were determined using 8-OH-dG-ELISA kits (8-OH-dG Check, JICA, Japan) according to the manufacturer's instructions. In brief, 96-well flat-bottomed ELISA plates were applied with 50 μl hydrolyzed DNA and 8-OH-dG standard, ranging from 0.125 to 10 ng/ml, and then applied with anti-8-OH-dG monoclonal antibody. Plates were incubated overnight at 4°C. After plates were rinsed three times with PBS with 0.05% Tween 20, 100 μl enzyme linked second antibody was added at room temperature with shaking for 1 h. After plates were rinsed three times as described above, 3,3′,5,5′-tetramethylbenzidine was added each well, and they were incubated at room temperature with shaking for 15 min. The enzyme reaction was stopped by adding 100 μl 1 M phosphoric acid per well, and the absorbance at 450 nm was recorded on a microplate reader (MTP-F2, Corona, Japan). The 8-OH-dG levels were determined by interpolation with standard curves assayed on individual plates. A two-way analysis of variance (ANOVA), non-parametric technique, was used on ranked data to test for overall effects of treatment. Treatment was considered significant if P<0.01. 2.4. Quantitative analysis of LPO The LPO levels were determined using a spectrum brightness method. Briefly, 0.5 g of each tissue preserved at −80°C was homogenized in a Potter-type homogenizer. Fifty microliters of LPO standard, 1, 1, 3, 3-tetraethoxypropane ranging from 0.0 to 0.22 mg/ml, and 50 μl homogenate of each tissue were applied with 0.2 ml of 8.1% sodium dodecyl sulfate and then incubated for 45 min at room temperature. Thereafter, they were applied with 0.75 ml of 20% acetic acid (pH 3.5), 0.75 ml of 0.8% 2-thiobarbituric acid and 0.25 ml of distilled water and then incubated for 1 h at 95°C water bath. After they were cooled down to room temperature, a 2.5 ml mixture liquid of n-butyl alcohol and pyridine (15:1) was added with violent shaking for 10 min. Then they were centrifuged at 3000 rpm for 15 min at room temperature to separate the top layer, and the absorbance at 532 nm was recorded on a spectrophotometer. LPO levels were determined by interpolation with standard curves. A two-way ANOVA, non-parametric technique, was used on ranked data to test for the overall effects of treatment. Treatment was considered significant if P<0.01. 3. Results 3.1. Immunohistochemical staining of 8-OH-dG Results of immunohistochemical findings of 8-OH-dG are summarized in Table 1. In the brain, immunoreactivity of 8-OH-dG was observed only in the granule cells of the cerebellum of toluene treated rats. However, purkinje cells in the cerebellum, and pyramidal cells and granule cells in the hippocampus were not stained in controls or treated rats. In the lung, immunoreactivity was enhanced in nuclei of blood vessels, bronchiole endothelium cells and interstitial cells by toluene exposure. Hepatic cell nuclei showed immunopositivity in toluene inhalation (Fig. 1). In the kidney, enhancement of immunoreactivity was observed in glomerulus nuclei in toluene treated rats (Fig. 2). In the testis, immunoreactivity was enhanced in spermatogonia of tubli seminiferi, but the epididymis was not enhanced by toluene exposure. 3.3. Immunohistochemical staining of 4-HNE No organ showed any change in the 4-HNE immunopositivity after toluene inhalation. 3.5. Immunohistochemical staining of SOD Results of immunohistochemical findings of SOD are summarized in Table 4. Immunoreactivity of SOD was observed only in the capillary endothelium cells of the hippocampus, in alveolar epithelial cells, in hepatic cells (Fig. 4), in myocardial cells and in the capillary endothelium cells of the kidney glomerulus (Fig. 5) of toluene treated rats. Testis and epididymis showed immunopositivity in toluene inhalation rats, but there were no significant differences from control rats. 4. Discussion With regard to the mechanism of cell and tissue injury by organic solvents, immediate peroxidative injury to the lipid, especially the cell membrane was reported [15], [16]. In reactive oxygen species that cause peroxidative damage, there are superoxide, hydrogen peroxide, hydroxy radical, and singlet oxygen. The generated superoxide is immediately reduced to hydrogen peroxide by SOD. Superoxide or hydrogen peroxide accumulates in the organism when the enzyme balance of these active oxygen deletion systems breaks [17], [18]. For the peroxidative damage, there is a different mechanism between the formation of lipid peroxide and 8-OH-dG. Hydrogen peroxide has a good permeability of the membrane, and is reduced with a metallic ion, and generates a hydroxy radical. This hydroxy radical causes hydration of deoxiguanosine in the eighth place. This 8-OH-dG is taken into the DNA chain by DNA polymerase as the results of the oxidation damage [19]. Hydroxy radical peroxidizes the lipid and the lipid, generated lipid peroxide, and the peroxidation of the cell membrane, and changes the penetration of the membrane. In this study, we examined whether the peroxidative injury was the mechanism of the cell and tissue injury by toluene. Then, 4-HNE, as the lipid peroxidation product, and 8-OH-dG, the DNA injury product, were examined immunohistochemically. In addition, we performed a quantitative analysis of 8-OH-dG and LPO. Enhancement of 8-OH-dG immunoreactivity and an increase in the formation of 8-OH-dG were observed in the lung, the liver, and the kidney in toluene treated rats. That is, it was suggested that oxidative injury of DNA had been generated in the lung, the liver, and the kidney. In the lung, liver, and kidney, the possibility that the cell function disorder, because of cell death, and the mutation caused by DNA reproduction obstruction occurred was suggested. The mechanism of this DNA damage is suggested as follows. In the lung, liver, and kidney, the toluene taken into the cell generated a superoxide. From the superoxide, SOD immediately caused hydrogen peroxide that becomes a hydroxy radical. It appeared that 8-OH-dG was generated for oxidative injury of DNA by the hydroxy radical. The immunoreactivity of 4-HNE, one index of the lipid peroxidation was not changed in all internal organs after toluene inhalation. Therefore, the lipid peroxide synthesis in these internal organs was not clear. That is, in the lung, liver and kidney, a difference between the lipid peroxide formation and 8-OH-dG formation was observed. It is not clear whether these differences depended on the distribution of SOD or increase of SOD synthesis in the internal organs. Therefore, SOD was examined immunohistochemically. The immunoreactivity of SOD increased in alveolar epithelial cells, hepatic cells, glomerulus, and myocardial cells. When oxidative damage of DNA increased, it appeared that the active oxygen deletion system, which reduced that damage, was activated by an increase in the immunoreactivity of SOD in the lung, liver, and kidney in these organs. In the immunohistochemical studies of 8-OH-dG and 4-HNE in the hippocampus and cerebellum and the quantitative analysis of 8-OH-dG by the ELISA method, the change was hardly seen except for the increase in immunoreactivity of 8-OH-dG in the granular layer of the cerebellum after toluene exposure. That is, peroxidation damage could not be observed clearly in the brain. In general, it was suggested that a comparatively strong oxidation stress has been received in the brain and the nerve cells because the basal metabolic rate is active. In the brain, it is suggested that a tenfold or greater amount of active oxygen is always produced compared with the other organs. From the present results, in the brain it was suggested that a reduction in the amount of active oxygen by the active oxygen deletion system such as SOD, glutathione peroxidase, and the catalase, worked strongly. Under the influence of toluene to the brain, the functional change might be major oxidative damage. In conclusion, according to the references [20], [21], we exposed the toluene (1500 ppm for 4 h per day) for 7 days to rat in this study as a toluene-inhalation model, and examined the peroxidation damage in various internal organs after toluene inhalation. As a result, oxidative injury of DNA became clear only in the lung, the liver, and the kidney, but lipid peroxide synthesis was not clear in the organs examined. It was suggested that the oxidative injury was strongly induced in those organs that were concerned with absorption, decomposition, the metabolism, and the excretion of the toluene directly. References [1].
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