TY - JOUR
AU1 - Zhang,, Ming
AU2 - Wang,, Yanrang
AU3 - Wang,, Qian
AU4 - Yang,, Junyu
AU5 - Yang,, Deyi
AU6 - Liu,, Jing
AU7 - Li,, Jianguo
AB - Abstract Ethylbenzene is an important industrial chemical that has recently been classified as a possible human carcinogen (International Agency of Research on Cancer class 2B), but the available data do not support the genotoxic mechanism of ethylbenzene-induced tumors in kidney. We investigated the effects of ethylbenzene on renal ultrastructure and explored the nongenotoxic mechanism of mitochondria-mediated apoptosis pathway. Forty male Sprague-Dawley rats were used as a vivo model with ethylbenzene inhalation for 13 weeks, and the metabolites of ethylbenzene, mandelic acid (MA), and phenylglyoxylic acid (PGA) in urine were examined by high-performance liquid chromatography. Meanwhile, the ultrastructure of renal tubular epithelial cells was observed, and cell apoptosis was detected via terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay. Furthermore, we investigated the expression levels of messenger RNA (mRNA) and protein of bax, bcl-2, cytochrome c, caspase-9, and caspase-3 in rat kidney. With respect to levels of MA, PGA, and MA + PGA, a significant dose-dependent increase was observed in 4335 and 6500 mg/m3 ethylbenzene-treated groups against the control group. The mitochondria of renal tubular epithelial cells became a compact and vacuolar structure in 6500 mg/m3 ethylbenzene-treated group, and ethylbenzene induced a significant increase in the number of apoptotic cells as compared to the control group. In addition, enhanced mRNA and protein expression levels of all measured genes were observed in various ethylbenzene-treated groups except the decreased bcl-2 expression levels. Our results indicated that ethylbenzene may induce apoptosis of renal tubular epithelial cells via mitochondria-mediated apoptotic pathways. MA and PGA in urine might be a parameter of biological dose in vivo after ethylbenzene inhalation. ethylbenzene, renal toxicity, apoptosis, mitochondria, mandelic acid, phenylglyoxylic acid Ethylbenzene (CAS No. 100-41-4) is produced by alkylating benzene with ethylene, and mainly used as an intermediate in the manufacture of styrene. It is also present in insecticide sprays, household degreasers, antiknocks, paints, adhesives, rust preventives, and a major component of mixed xylenes used as a general solvent. Occupational exposure to ethylbenzene mostly occurs during the process of styrene production and polymerization as well as production and use of mixed xylenes (Fishbein, 1985; Wang et al., 2006). The main route of metabolism of ethylbenzene, both in rodents and in human, is by α-hydroxylation to 1-phenylethanol. The subsequent intermediates are acetophenone, ω-hydroxyacetophenone, 1-phenyl-1,2-ethanediol, mandelic acid (MA), phenylglyoxylic acid (PGA), and finally hippuric acid and discharged into urine (Engstrom, 1984). Among these metabolites, MA and PGA have been used in biological monitoring of occupational exposure to ethylbenzene by high-performance liquid chromatography (HPLC) in human (Jang et al., 2001; Knecht et al., 2000). However, no data are available for dose relationship between levels of ethylbenzene and MA in vivo in rodents and in human, as well as PGA. Ethylbenzene induces tumors in rats and mice and has been classified as a possible human carcinogen (class 2B) by the International Agency for Research on Cancer (2000). National Toxicology Program (NTP) provided clear evidence of carcinogenic activity following inhalation exposure in male F344/N rats based on increased incidences of renal tubule tumors at the top dose level of 750 ppm (NTP, 1999). The increased tumor incidence was accompanied by increased renal tubule hyperplasia and nephropathy. After ethylbenzene is readily absorbed from the lungs and distributed throughout the body, the data available from genotoxicity studies in vivo indicate that there is no evidence of genotoxic activity in assays measuring the induction of DNA breakage, DNA adducts, micronuclei, sister chromatid exchange (SCE), or unscheduled deoxyribonucleic acid synthesis associated with an inhalation exposure route (Chan et al., 1998). An epidemiological study has been revealed to assess the genotoxicity of a number of compounds (methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene) in human lymphocytes using comet assay and indicated that ethylbenzene failed to cause genotoxicity even at a very high concentration of 200μM (Chen et al., 2008). Moreover, Norppa and Vainio (1983) reported that ethylbenzene individually does not induce SCE after 48 h of treatment of human lymphocytes in a styrene-analogues evaluation study. They also suggested that ethylbenzene itself is not an effective mutagen prior to its conversion to reactive species (Norppa and Vainio, 1983). A recent review has indicated some currently available in vivo and in vitro genotoxicity data of ethylbenzene and concluded that ethylbenzene does not induce kidney, liver, or lung tumors in rat and mice through genotoxic mechanisms (Henderson et al., 2007). Taken together, the present available data from the standard assays do not support genotoxic mechanisms for ethylbenzene-induced kidney, liver, or lung tumors in rats and mice. Thus, the nongenotoxic mechanisms, such as prolonged stimulation of cell proliferation, apoptosis, and indirect cytotoxicity associated with lysosomal overload, should be mentioned. Apoptosis is a highly ordered process that contributes to the selective elimination of cells in physiologic and pathologic situations. It is thought that the mitochondria are most pertinent in cytochrome c release, caspase activation, and final apoptosis. However, it is still unknown whether ethylbenzene induces mitochondrial dysfunction and which factors are involved in the mitochondria-mediated apoptosis pathway. In the present study, we used male Sprague-Dawley rats as a vivo model to explore the effects of ethylbenzene on histopathology and ultrastructure of renal tubule; apoptosis; and expression levels of bax, bcl-2, cytochrome c, caspase-9, and caspase-3 messenger RNA (mRNA) and proteins in apoptosis pathways in order to provide some useful clues for further study of ethylbenzene-induced renal toxicology. In addition, the levels of MA and PGA in urine were examined to assess the vivo load of ethylbenzene. MATERIALS AND METHODS Chemicals. Ethylbenzene (analytical pure) was obtained from Tianjin Chemical Reagents Company. MA (purity > 99.8%) and PGA (purity > 99.9%) were purchased from Sigma-Aldrich Co. (St Louis, MO). TRIzol Reagent and SYBR Green Mix were obtained from GIBCO/Invitrogen Corp. (Carlsbad, CA). Taq DNA polymerase and deoxy-ribonucleoside triphosphate mix were purchased from Fermentas Inc. (Burlington, Ontario, Canada). Reverse transcription kit was provided by Takara (Dalian, China). The primers for all genes were synthesized and purified by Beijing AUGCT Biotech Corp. (Beijing, China). bax, cytochrome c, bcl-2, caspase-3, and glyceraldehyde-3-phosphate (GAPDH) rabbit anti-rat antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Caspase-9 rabbit anti-rat antibody was obtained from Cell Signaling Technology, Inc. (Boston, MA). The horseradish peroxidase–linked sheep anti-rabbit IgG antibody and enhanced chemiluminescence solution were purchased from Pierce (Rockford, IL). All other reagents used were analysis grade laboratory chemicals from standard commercial suppliers. Animal treatments and sample collections. Healthy Sprague-Dawley rats, weighed 60–75 g and aged 2 weeks, were purchased from the Center of Experimental Animals, Chinese Academy of Military Medical Science. They were housed in an air-conditioned environment at 24 ± 1°C with a 12-h light/dark cycle and maintained on a standard feed, with drinking water ad libitum. All experiments were carried out in accordance with the ethical guidelines of the Animal Experimentation Committee of Tianjin Centers for Disease Control and Prevention and were compliant with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After acclimatization to the housing facility and diet for 7 days, three groups of 10 males and 10 females were allocated randomly by weight and were inhaled with different doses of ethylbenzene: 433.5 mg/m3 (100 ppm), 4335 mg/m3 (1000 ppm), and 6500 mg/m3 (1500 ppm) 6 h/day for 13 weeks. The control group was exposed to fresh air. The ethylbenzene exposures were conducted in 200-l stainless steel inhalation chambers with dynamic and adjustable laminar airflow. In order to prevent any leakage of the test atmospheres, the chambers were maintained at a negative pressure of no more than 3 mm water. The chamber temperature was maintained within the range of 21°C–25°C and the relative humidity within 30–70%. Chemical vapors were generated by passing an additional airflow through the fritted disk of a heated bubbler containing ethylbenzene and air. Under these conditions, the vaporized compounds were carried out into the main air inlet pipe of the exposure chambers. The urine samples were collected per 4 weeks during the experiment period and immediately centrifuged (3000 × g for 10 min) for analysis. All the rats were killed by cervical dislocation, and the kidney tissues were quickly dissected off and rinsed in ice-cold saline. A piece of renal tissue was subjected to routine histopathological examination. A slice of tissue fixed in 2.5% glutaraldehyde was used for transmission electron microscope. Another partition of tissue was fixed in formalin and embedded in paraffin for terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay. The remaining tissue were frozen in liquid nitrogen and stored at −80°C until analysis. Experimental ethylbenzene exposure sampling and analysis. Concentrations of ethylbenzene were monitored continuously with a Shimadzu GC-14C gas chromatograph equipped with a flame ionization detector and an automatic gas-sampling valve. The exposure levels were determined once during the exposure period of 6 h by collecting atmosphere samples through glass tubes packed with activated charcoal, in the breathing zone of the test animals. Ethylbenzene was then desorbed with carbon disulfide and analyzed on the Shimadzu GC-14C gas chromatograph, using ethylbenzene internal standards. The column temperature was maintained at 70°C. The injector and the detector were maintained at 275°C and 300°C, respectively. Nitrogen (purity > 99.999%) was employed as the carrier gas at a flow rate of 1.0 ml/min. One microliter of each sample was injected, and gas chromatograph peaks were identified based on the retention time of individual authentic standards (± 0.3%). As the concentration determined by analyzer was essentially the same as the target concentration, it will be referred to the target concentration in the study (Table 1). TABLE 1 Analytical Concentrations of Ethylbenzene in Exposure Chambers Target concentration (mg/m3) Analytical concentration (mg/m3) Range (mg/m3) Low High 433.5 432.7 ± 7.2 423.1 442.6 4335 4301 ± 47 4234 4369 6500 6534 ± 54 6463 6597 Target concentration (mg/m3) Analytical concentration (mg/m3) Range (mg/m3) Low High 433.5 432.7 ± 7.2 423.1 442.6 4335 4301 ± 47 4234 4369 6500 6534 ± 54 6463 6597 Open in new tab TABLE 1 Analytical Concentrations of Ethylbenzene in Exposure Chambers Target concentration (mg/m3) Analytical concentration (mg/m3) Range (mg/m3) Low High 433.5 432.7 ± 7.2 423.1 442.6 4335 4301 ± 47 4234 4369 6500 6534 ± 54 6463 6597 Target concentration (mg/m3) Analytical concentration (mg/m3) Range (mg/m3) Low High 433.5 432.7 ± 7.2 423.1 442.6 4335 4301 ± 47 4234 4369 6500 6534 ± 54 6463 6597 Open in new tab Determination of MA and PGA in rat urine. The assay method of MA and PGA in urine was described with some modification (Wang et al., 2006) using a HPLC method. Chromatography was performed on an XDB-C18 column (150 × 4.6 mm, internal diameter 5 μm; Agilent). The mobile phase was consisted of methanol-mixed acid (1:9, vol/vol). The mixed acid comprises 0.05% of acetic acid and 0.05% of phosphoric acid as volume ratio. The flow rate was set at 1.0 ml/min. Detection was set at ultraviolet wavelength of 225 nm. The column temperature was maintained at room temperature. One-milliliter urine sample was handled with 0.1 ml of 6M hydrochloric acid. The mixed solution was then extracted with 4 ml ethyl acetate. After vortex for 3 min, sample was centrifuged at 3500 × g for 5 min. The 500 μl of organic layer was transferred to another tube and evaporated to dryness under nitrogen. The residue was reconstituted with 200 μl of the mobile phase and mixed for 1 min. An aliquot of 5 μl of the resulting solution was injected into the liquid chromatography system for analysis. In all urine samples, creatinine (Cr) was also measured using Hitachi 902 automated analyzer according to Jaffe method. The values of MA and PGA were expressed as milligrams per gram of creatinine. All samples were coded and analyzed under blind conditions. Histopathological examination. After removal, the kidney tissues were subjected to routine histopathological examination with hematoxylin and eosin staining for light microscope examination. Ultrastructural analysis. Dissected kidney samples were fixed with 2.5% glutaraldehyde for 1 h. After rinsed twice for 15 min at a time in 0.2M phosphate buffer, the samples were postfixed in 1% buffered osmium tetroxide for 1 h, then dehydrated through graded alcohols, and embedded in epoxy resin. Ultrathin sections stained with uranyl acetate and lead citrate were made prior to examination under the transmission electron microscope (Hitachi 7500, Japan) and photographed by Megaview digital photograph system. TUNEL assay. Four micrometers of tissue slices of paraffin-embedded kidney was cut and placed onto the silanized slides (Dako, Denmark) and processed according to the recommended procedure by the kit employed (Calbiochem, Merck). The nuclei labeled with dark brown color were considered apoptotic cells when visualized at magnification of ×40 using a light microscope. Apoptotic index for kidney of control and treated rat was presented as the percentage of positive cells to the totality in one field of vision. At least three fields were counted for each slide. Isolation of total RNA and quantitative real-time PCR. Total RNA was isolated from kidney tissue using the TRIzol reagent. The A260/280 ratio was found to be in the range of 1.8–2.0, and total RNA was also electrophoresed on a 1% agarose gel to visually assess RNA quality. A 4-μg aliquot of DNase I–treated total RNA from each sample was converted to complementary DNA using a reverse transcription kit following the manufacturer’s instructions. The sequences of primer pairs and the sizes of the products are shown in Table 2. Primer testing was performed with four random samples and a negative control (Rnase-free water) for each primer set. Real-time PCR was performed using ABI Prism 7900HT PCR System (ABI, CA), and the fluorescence threshold value was calculated using SDS 2.2.1 system software. PCR was performed in a two-step method described in the manufacturer’s instructions for the SYBR PrimeScript RT-PCR Kit. The method of log (−ΔCT) was used for quantification calculations. TABLE 2 Sequences of Primer Pairs and Product Sizes of Genes Gene Sequences of primer pairs Product sizes (bp) bax F: 5′-GAGCTGCAGAGGATGATTG-3′ 90 R: 5′-CCCAGTTGAAGTTGCCATC-3′ cytochrome c F: 5′-GGCAAGCATAAGACTGGACCAA-3′ 174 R: 5′-TTTCCAAATACTCCATCAGGGTATC-3′ caspase-9 F: 5′-CCCTTCCTCGCTTCATCTC-3′ 100 R: 5′-GCTTCTGGATCCTGCTTGG-3′ caspase-3 F: 5′-GGACCTGTGGACCTGAAAAA-3′ 159 R: 5′-GCATGCCATATCATCGTCAG-3′ bcl-2 F: 5′-GGGAGATCGTGATGAAGTAC-3′ 120 R: 5′-GGAAGGAGAAGATGCCAG-3′ β-actin F: 5′-GAACCCTAAGGCCAACCGTG 3-′ 105 R: 5′-AGGCATACAGGGACAACACAGC-3′ Gene Sequences of primer pairs Product sizes (bp) bax F: 5′-GAGCTGCAGAGGATGATTG-3′ 90 R: 5′-CCCAGTTGAAGTTGCCATC-3′ cytochrome c F: 5′-GGCAAGCATAAGACTGGACCAA-3′ 174 R: 5′-TTTCCAAATACTCCATCAGGGTATC-3′ caspase-9 F: 5′-CCCTTCCTCGCTTCATCTC-3′ 100 R: 5′-GCTTCTGGATCCTGCTTGG-3′ caspase-3 F: 5′-GGACCTGTGGACCTGAAAAA-3′ 159 R: 5′-GCATGCCATATCATCGTCAG-3′ bcl-2 F: 5′-GGGAGATCGTGATGAAGTAC-3′ 120 R: 5′-GGAAGGAGAAGATGCCAG-3′ β-actin F: 5′-GAACCCTAAGGCCAACCGTG 3-′ 105 R: 5′-AGGCATACAGGGACAACACAGC-3′ Open in new tab TABLE 2 Sequences of Primer Pairs and Product Sizes of Genes Gene Sequences of primer pairs Product sizes (bp) bax F: 5′-GAGCTGCAGAGGATGATTG-3′ 90 R: 5′-CCCAGTTGAAGTTGCCATC-3′ cytochrome c F: 5′-GGCAAGCATAAGACTGGACCAA-3′ 174 R: 5′-TTTCCAAATACTCCATCAGGGTATC-3′ caspase-9 F: 5′-CCCTTCCTCGCTTCATCTC-3′ 100 R: 5′-GCTTCTGGATCCTGCTTGG-3′ caspase-3 F: 5′-GGACCTGTGGACCTGAAAAA-3′ 159 R: 5′-GCATGCCATATCATCGTCAG-3′ bcl-2 F: 5′-GGGAGATCGTGATGAAGTAC-3′ 120 R: 5′-GGAAGGAGAAGATGCCAG-3′ β-actin F: 5′-GAACCCTAAGGCCAACCGTG 3-′ 105 R: 5′-AGGCATACAGGGACAACACAGC-3′ Gene Sequences of primer pairs Product sizes (bp) bax F: 5′-GAGCTGCAGAGGATGATTG-3′ 90 R: 5′-CCCAGTTGAAGTTGCCATC-3′ cytochrome c F: 5′-GGCAAGCATAAGACTGGACCAA-3′ 174 R: 5′-TTTCCAAATACTCCATCAGGGTATC-3′ caspase-9 F: 5′-CCCTTCCTCGCTTCATCTC-3′ 100 R: 5′-GCTTCTGGATCCTGCTTGG-3′ caspase-3 F: 5′-GGACCTGTGGACCTGAAAAA-3′ 159 R: 5′-GCATGCCATATCATCGTCAG-3′ bcl-2 F: 5′-GGGAGATCGTGATGAAGTAC-3′ 120 R: 5′-GGAAGGAGAAGATGCCAG-3′ β-actin F: 5′-GAACCCTAAGGCCAACCGTG 3-′ 105 R: 5′-AGGCATACAGGGACAACACAGC-3′ Open in new tab Western blot analysis. The proteins from kidney tissues were isolated and measured as described previously (Zhang et al., 2007), and densitometry analysis was performed with Gel-Pro Analyzer Software version 3.0. GAPDH was used as an endogenous control to obtain the relative quantitative levels of protein expression. Statistical analysis. Data were represented as means ± SD. Statistical significance was assessed by ANOVA with subsequent Student-Newman-Keuls test using SPSS (13.0) software. Relationships between continuous variables were assessed using Pearson correlation coefficient analyzed by bivariate correlations method. A difference of p < 0.05 was considered statistically significant. RESULTS Clinical Monitoring of Experimental Animals There were no clinical signs of dysfunction in the treated rats over the course of the experimental period, and change trends of rat body weight were shown in Figure 1. The body weight of each group was increased along with experimental process, but the significant difference did not exist between that of the control group and any ethylbenzene-treated groups (p > 0.05). FIG. 1. Open in new tabDownload slide Change trends of rat body weight in each group over the course of the experimental period. The body weight of all experimental rats was measured on Friday of a week and represented with the mean weight of each groups. FIG. 1. Open in new tabDownload slide Change trends of rat body weight in each group over the course of the experimental period. The body weight of all experimental rats was measured on Friday of a week and represented with the mean weight of each groups. Effect of Ethylbenzene on MA and PGA Levels in Rat Urine Table 3 indicated that MA and PGA levels in 4335 and 6500 mg/m3 ethylbenzene-treated groups were significantly higher than those in the control group and 433.5 mg/m3 ethylbenzene-treated group, respectively, as well as the total amount of MA and PGA (abbreviated as MA + PGA) (p < 0.05). A linear correlation analysis of exposed ethylbenzene showed significant positive associations with MA, PGA, and MA + PGA levels (r = 0.827, 0.720, and 0.802, respectively; all p < 0.05). TABLE 3 Determination of MA and PGA Levels in Rat Urine Group MA (mg/g Cr) PGA (mg/g Cr) Total amount of MA and PGA (mg/g Cr) Control ND ND ND 433.5 mg/m3 0.084 ± 0.070 0.041 ± 0.029 0.126 ± 0.096 4335 mg/m3 0.303 ± 0.148*, # 0.168 ± 0.104*, # 0.471 ± 0.250*, # 6500 mg/m3 0.404 ± 0.154*, # 0.174 ± 0.092*, # 0.578 ± 0.237*, # Group MA (mg/g Cr) PGA (mg/g Cr) Total amount of MA and PGA (mg/g Cr) Control ND ND ND 433.5 mg/m3 0.084 ± 0.070 0.041 ± 0.029 0.126 ± 0.096 4335 mg/m3 0.303 ± 0.148*, # 0.168 ± 0.104*, # 0.471 ± 0.250*, # 6500 mg/m3 0.404 ± 0.154*, # 0.174 ± 0.092*, # 0.578 ± 0.237*, # Note. Data were represented as means ± SD. Limit of detection (LOD) of MA and PGA are 0.010 and 0.006 mg/g Cr, respectively. ND, not detected (expressed with half of the values of LOD to enter the analysis). * p < 0.05 as compared with the control group. # p < 0.05 as compared with the 433.5 mg/m3 ethylbenzene-treated group. Open in new tab TABLE 3 Determination of MA and PGA Levels in Rat Urine Group MA (mg/g Cr) PGA (mg/g Cr) Total amount of MA and PGA (mg/g Cr) Control ND ND ND 433.5 mg/m3 0.084 ± 0.070 0.041 ± 0.029 0.126 ± 0.096 4335 mg/m3 0.303 ± 0.148*, # 0.168 ± 0.104*, # 0.471 ± 0.250*, # 6500 mg/m3 0.404 ± 0.154*, # 0.174 ± 0.092*, # 0.578 ± 0.237*, # Group MA (mg/g Cr) PGA (mg/g Cr) Total amount of MA and PGA (mg/g Cr) Control ND ND ND 433.5 mg/m3 0.084 ± 0.070 0.041 ± 0.029 0.126 ± 0.096 4335 mg/m3 0.303 ± 0.148*, # 0.168 ± 0.104*, # 0.471 ± 0.250*, # 6500 mg/m3 0.404 ± 0.154*, # 0.174 ± 0.092*, # 0.578 ± 0.237*, # Note. Data were represented as means ± SD. Limit of detection (LOD) of MA and PGA are 0.010 and 0.006 mg/g Cr, respectively. ND, not detected (expressed with half of the values of LOD to enter the analysis). * p < 0.05 as compared with the control group. # p < 0.05 as compared with the 433.5 mg/m3 ethylbenzene-treated group. Open in new tab Histopathological Changes in Rat Renal Tubules With respect to the control group (Fig. 2A), Figure 2B showed significant morphological abnormality in the rat kidney in 6500 mg/m3 ethylbenzene-treated group characterized by hydropic degeneration in renal tubules in the form of swelling, necrosis, and lysis in renal tubular epithelial cells and irregular tubular lumens. FIG. 2. Open in new tabDownload slide Histopathological changes of the rat renal tubules exposed to ethylbenzene. The kidney tissue slices were stained with hematoxylin and eosin and observed by inverted phase contrast microscope. Then, representative photomicrographs were taken at ×200. (A) Control group and (B) 6500 mg/m3 ethylbenzene-treated group. FIG. 2. Open in new tabDownload slide Histopathological changes of the rat renal tubules exposed to ethylbenzene. The kidney tissue slices were stained with hematoxylin and eosin and observed by inverted phase contrast microscope. Then, representative photomicrographs were taken at ×200. (A) Control group and (B) 6500 mg/m3 ethylbenzene-treated group. Ultrastructural Changes in Rat Renal Tubules As shown in Figure 3A and B, epithelial cell is typically stylolitic with rhabditiform mitochondria in control group. However, severe ultrastructural alterations in rat renal tubules were indicated in Figure 3C and D after the treatment with 6500 mg/m3 ethylbenzene. Cells were vigorously affected, with lysosome deposit and dissolved cell organelles. The nucleus of epithelial cells became shrunken, accompanied with chromatin condensation, and margination. The nuclear membrane was crinkly and lost common shape. Furthermore, the mitochondria appeared swollen with vacuolar structure and loss of cristae. FIG. 3. Open in new tabDownload slide Ultrastructural changes of the rat renal tubular cells exposed to ethylbenzene. These ultrathin sections were stained with uranyl acetate and lead citrate and observed by transmission electron microscope. Then, representative photos were taken at ×4000 to ×10,000. (A and B) Control group and (C and D) 6500 mg/m3 ethylbenzene-treated group. Mitochondria, nucleus, swollen mitochondria with vacuolar structure and loss of cristae, shrunken nucleus, and chromatin condensation. FIG. 3. Open in new tabDownload slide Ultrastructural changes of the rat renal tubular cells exposed to ethylbenzene. These ultrathin sections were stained with uranyl acetate and lead citrate and observed by transmission electron microscope. Then, representative photos were taken at ×4000 to ×10,000. (A and B) Control group and (C and D) 6500 mg/m3 ethylbenzene-treated group. Mitochondria, nucleus, swollen mitochondria with vacuolar structure and loss of cristae, shrunken nucleus, and chromatin condensation. Effect of Ethylbenzene on Apoptosis in Rat Renal Tubular Epithelial Cells As shown in Figure 4B, TUNEL-positive cells were enhanced in 6500 mg/m3 ethylbenzene-treated group, showing apoptotic morphological changes, including chromatin condensation and nuclear fragmentation. After treatment with increased doses of ethylbenzene, the percentage of apoptotic cells were 5.9, 15.6, 21.4, and 38.0%, respectively. The apoptosis rate increased in a concentration-dependent manner, with statistical significance at all concentrations (Fig. 4C). FIG. 4. Open in new tabDownload slide Effect of ethylbenzene on apoptosis in rat renal tubular epithelial cells. The silanized kidney tissue slices were determined by TUNEL staining. Apoptotic cells were calculated at magnification of ×40 using a light microscope, and representative photomicrographs were taken from an experiment performed in triplicate. (A) Control group and (B) 6500 mg/m3 ethylbenzene-treated group. The arrows point to the apoptotic cells (TUNEL-positive cells). (C) Percentage of apoptotic cells. Data were presented as means ± SD of four independent experiments. Compared to the control group, significance difference was determined at *P < 0.05. FIG. 4. Open in new tabDownload slide Effect of ethylbenzene on apoptosis in rat renal tubular epithelial cells. The silanized kidney tissue slices were determined by TUNEL staining. Apoptotic cells were calculated at magnification of ×40 using a light microscope, and representative photomicrographs were taken from an experiment performed in triplicate. (A) Control group and (B) 6500 mg/m3 ethylbenzene-treated group. The arrows point to the apoptotic cells (TUNEL-positive cells). (C) Percentage of apoptotic cells. Data were presented as means ± SD of four independent experiments. Compared to the control group, significance difference was determined at *P < 0.05. mRNA Expression Levels As shown in Figure 5, the expression levels of caspase-3 and bax mRNA in all ethylbenzene-treated groups were higher than that in the control (p < 0.05). Compared with the control group, the expression levels of cytochrome c mRNA in the 4335 and 6500 mg/m3 ethylbenzene-treated groups were markedly enhanced, while the bcl-2 mRNA were depressed (p < 0.05). The expression levels of caspase-9 mRNA in all ethylbenzene-treated groups were higher than that in the control group, but a statistically significant difference did not exist (p > 0.05). Furthermore, the expression levels of bax mRNA in 4335 and 6500 mg/m3 ethylbenzene-treated groups were markedly higher than that in 433.5 mg/m3 group (p < 0.05). The cytochrome c mRNA levels in 6500 mg/m3 ethylbenzene-treated group were markedly higher than those in 433.5 and 4335 mg/m3 groups (p < 0.05). Compared with 433.5 mg/m3 group, the bcl-2 mRNA levels in 4335 and 6500 mg/m3 groups were progressively depressed (p < 0.05). FIG. 5. Open in new tabDownload slide Expression levels of bax, cytochrome c, caspase-9, caspase-3, and bcl-2 mRNA in rat kidneys exposed to different doses of ethylbenzene by quantitative real-time PCR. PCR was performed using a two-step method on the ABI Prism 7900HT Sequence Detection System. The fluorescence threshold value was calculated using SDS 2.2.1 System Software. The log transformation of −ΔCT method was used for quantification calculations. Data were presented as means ± SD of three independent experiments performed in triplicate. Compared to the control group, significance difference was determined at *p < 0.05; compared to the 433.5 mg/m3 ethylbenzene-treated group, significance difference was determined at #p < 0.05; and compared to the 4335 mg/m3 ethylbenzene-treated group, significance difference was determined at †p < 0.05. FIG. 5. Open in new tabDownload slide Expression levels of bax, cytochrome c, caspase-9, caspase-3, and bcl-2 mRNA in rat kidneys exposed to different doses of ethylbenzene by quantitative real-time PCR. PCR was performed using a two-step method on the ABI Prism 7900HT Sequence Detection System. The fluorescence threshold value was calculated using SDS 2.2.1 System Software. The log transformation of −ΔCT method was used for quantification calculations. Data were presented as means ± SD of three independent experiments performed in triplicate. Compared to the control group, significance difference was determined at *p < 0.05; compared to the 433.5 mg/m3 ethylbenzene-treated group, significance difference was determined at #p < 0.05; and compared to the 4335 mg/m3 ethylbenzene-treated group, significance difference was determined at †p < 0.05. Protein Expression Levels Results of one representative Western blot were shown in Figure 6. Compared to the control group, bax and caspase-3 protein expression levels in 4335 and 6500 mg/m3 ethylbenzene-treated groups and cytochrome c and caspase-9 protein expression levels in all ethylbenzene-treated groups were all significantly enhanced (p < 0.05). The bcl-2 protein expression levels in all ethylbenzene-treated groups were lower than that of the control group with statistical significance (p < 0.05). Furthermore, compared to 433.5 mg/m3 ethylbenzene-treated group, caspase-9 and caspase-3 protein expression levels in 4335 and 6500 mg/m3 ethylbenzene-treated groups were significantly increased, respectively (p < 0.05). The bcl-2 protein expression levels in 6500 mg/m3 ethylbenzene-treated group were significantly lower than those of the 433.5 and 4335 mg/m3 ethylbenzene-treated groups and control group (p < 0.05). FIG. 6. Open in new tabDownload slide Expression levels of bax, cytochrome c, caspase-9, caspase-3, and bcl-2 protein in rat kidneys exposed to different doses of ethylbenzene by Western blot. Tissues were isolated and loaded into each lane for 7.5% SDS-polyacrylamide gel electrophoresis, followed by blot analysis with different polyclonal anti-rat antibodies as described in “Materials and Methods” section. Representative X-ray autoradiographs for different proteins are shown: groups in lanes 1, 2, 3, and 4 were exposed to 0, 433.5, 4335, and 6500 mg/m3 ethylbenzene, respectively. Optical densities of different proteins were performed using a Gel-Pro Analyzer version 3.0 software. Data were presented as means ± SD of three independent experiments performed in triplicate. Compared to the control group, significance difference was determined at *p < 0.05; compared to the 433.5 mg/m3 ethylbenzene-treated group, significance difference was determined at #p < 0.05; and compared to the 4335 mg/m3 ethylbenzene-treated group, significance difference was determined at †p < 0.05. FIG. 6. Open in new tabDownload slide Expression levels of bax, cytochrome c, caspase-9, caspase-3, and bcl-2 protein in rat kidneys exposed to different doses of ethylbenzene by Western blot. Tissues were isolated and loaded into each lane for 7.5% SDS-polyacrylamide gel electrophoresis, followed by blot analysis with different polyclonal anti-rat antibodies as described in “Materials and Methods” section. Representative X-ray autoradiographs for different proteins are shown: groups in lanes 1, 2, 3, and 4 were exposed to 0, 433.5, 4335, and 6500 mg/m3 ethylbenzene, respectively. Optical densities of different proteins were performed using a Gel-Pro Analyzer version 3.0 software. Data were presented as means ± SD of three independent experiments performed in triplicate. Compared to the control group, significance difference was determined at *p < 0.05; compared to the 433.5 mg/m3 ethylbenzene-treated group, significance difference was determined at #p < 0.05; and compared to the 4335 mg/m3 ethylbenzene-treated group, significance difference was determined at †p < 0.05. DISCUSSION Ethylbenzene figures among the volatile organic chemicals frequently detected in the environment. Exposure of the general population to low concentration of ethylbenzene arises from its presence in indoor and outdoor air due to several sources, such as vehicle exhausts, household consumer products containing mixed xylenes, cigarette smoke, and fossil fuels (Holzer et al., 1976) apart from the industrial emission. Inhalation was reported to be the principal route of ethylbenzene exposure for the general population (Wang et al., 2006). The same route of exposure is also relevant for workers involved in the production and use of ethylbenzene as an intermediate in rubber and chemical manufacturing industries (Fishbein, 1985). Levels of ethylbenzene in blood can reflect recent exposure. During the past two decades, National Health and Nutrition Examination Survey has reported the median or geometric mean value of ethylbenzene level in blood among serial subsamples of adults (1988–1994), nonsmokers (1999–2000), and participants aged 20 years and older (2001–2002 and 2003–2004), which was about 0.060 μg/l or slightly lower. Similar levels were also observed in sample of southwestern U.S. residents, showing about two to three times higher than levels among low-income children in a Midwestern U.S. city (Centers for Disease Control and Prevention, National Health and Nutrition Examination Surveys, 2009). PGA is the carboxylated product of MA in the metabolite route of ethylbenzene. Some research has shown that the relationship between the excretion of MA and PGA is not constant and may be influenced by numerous factors, in particular the level of ethylbenzene exposure and the sampling time (Perbellini et al., 1988). Knecht et al. (2000) viewed several good correlations between external exposure and the total amount of the metabolites MA and PGA excreted, corresponding to 90% of the absorbed ethylbenzene. The present study indicated a positive linear correlation between the total amount of these two metabolites in urine samples and external exposed ethylbenzene, which was in general in accordance with the findings of the previous study (Knecht et al., 2000). Furthermore, the individual metabolite of MA and PGA was linearly correlated with external exposure in our study, respectively. These results might provide some available data on biomonitoring of ethylbenzene in vivo. Nevertheless, MA and PGA are also metabolites of styrene and phenyl glycol; concurrent exposure to these substances with ethylbenzene can limit the specificity of the metabolite analysis (Knecht et al., 2000). A number of subchronic and chronic toxicity studies have been conducted with ethylbenzene, including 13-week and 2-year studies in mouse and rat, showing the multiorganic toxicity, such as ototoxicity and neurotoxicity (Cappaert et al., 1999, 2000, 2001, 2002; Mellert et al., 2007; Vyskocil et al., 2008). In the present study, the inhalation route of exposure was used consistent with all these performed studies. The results of clinical signs of dysfunction in the experimental animals are negative, as well as alteration of rats’ body weights. However, the rat kidney was impaired after the ethylbenzene exposure, indicating hydropic degeneration of rat renal tubular epithelial cells in pathological morphology. Furthermore, the ultrastructural results of these epithelial cells indicated the shrunken nucleus lysosome deposit as well as swollen and vacuolar mitochondria. Thus, ethylbenzene might result in mitochondrial dysfunction and the typical representations purport the occurrence of apoptosis. Apoptosis is a tightly controlled process in which cell death is executed for maintaining steady state under physiological conditions and for responding to various stimuli. In the present study, we directly observed ethylbenzene-caused apoptotic morphological changes with TUNEL assay, including chromatin condensation and nuclear fragmentation in renal tubules, demonstrating that ethylbenzene induced apoptosis in renal tubules. This finding suggests that apoptosis is involved in the toxic mechanism of ethylbenzene. It is not clear, however, which pathway participates in the course of apoptosis induced by ethylbenzene. Apoptosis induced by chemicals in vivo (Shih et al., 2003) and in vitro (Bhatt et al., 2008; Mao et al., 2007; Song et al., 2008) was found to primarily involve the mitochondria-mediated pathway. The initial investigations of bcl-2 family members have shown their important roles in regulating mitochondria-mediated apoptosis. bcl-2 family proteins consist of some antiapoptotic and proapoptotic members. bcl-2 itself functions as a repressor of apoptosis, whereas bax, another member of the family, acts as a promoter of cell death. bcl-2 has been shown to form a heterodimer with bax, and the ratio of bax/bcl-2 plays an important role in determining the release of cytochrome c from the mitochondria into the cytosol (Borner, 2003). Some previous studies have demonstrated that several poisons can influence the expression of bax and bcl-2 proteins either at the transcriptional level or at the translational level (Hossain et al., 2009; Xu et al., 2008). However, whether ethylbenzene may influence apoptosis-related gene expressions has not been reported. The present study manifested the upregulation in gene expression of bax and cytochrome c and a downregulation of bcl-2, leading to an increase in bax/bcl-2 ratio. So far, bcl-2 appears to be a critical prosurvival protein of the bcl-2 family in the Sertoli cells. Moreover, there existed a significant dose-effect relationship. Furthermore, bax is essential to mitochondrial outer membrane permeabilization during apoptosis. bax affects the permeabilization of the outer mitochondrial membrane, allowing proteins in the mitochondrial intermembrane space, such as cytochrome c, to escape into the cytosol where they can induce the caspases activation and cell death (Antignani and Youle, 2006; Green and Kroemer, 2004). In the mitochondria-mediated apoptosis pathway, toxic insults cause the disruption of the mitochondrial inner membrane releasing cytochrome c, which complexes with apoptosis initiation factor, the latter forming a hexameric complex that binds procaspase-9 and promotes its activation to caspase-9 (Chang and Yang, 2000; Earnshaw et al., 1999; Philchenkov, 2003). Closely behind, caspase-3, a common and terminal protease in various apoptosis pathways (including mitochondria-mediated and death receptor–dependent pathways), is activated and apoptosis occurs. The present study showed the mRNA expression of caspase-9 in all ethylbenzene-treated groups and caspase-3 in 4335 and 6500 mg/m3 ethylbenzene-treated groups were enhanced. This result implies that caspase-9 and caspase-3 play a pivotal role in ethylbenzene-induced apoptosis of rat renal tubule epithelial cells. Taken together, mitochondria-mediated apoptotic pathway has been definitely proven to be involved in ethylbenzene-induced apoptosis of the rat renal tubule epithelial cells since the cytochrome c released into the cytosol and caspase-9 are the major molecules associated with mitochondria-mediated apoptotic pathway. Furthermore, the majority of the current study was focused on the effects of only singular chemical, whereas organism is daily exposed to mixtures of various products in the environment. Hence, it is noted that toxicity studies focusing on mixtures to low concentration for long time should be regarded as priority in the next studies. In conclusion, ethylbenzene may exhibit renal toxicity and result in apoptosis, possibly ascribed to the activation of mitochondria-mediated pathway, which might elucidate the previous studies of nonavailable data of genotoxicity. MA and PGA in urine might be available on ethylbenzene biomonitoring in vivo. These detailed mechanisms, of course, need further investigation. This study herein has provided preliminary but important data for further study of renal toxicity resulting from ethylbenzene. FUNDING Key Projects of Tianjin Municipal Natural Science Foundation (No. 07JCZDJC08500, No. 09JCZDJC20900); Scientific Fund of Tianjin Bureau of Public Health (No. 07KZ72). References Antignani A Youle RJ How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr. Opin. Cell Biol. 2006 18 685 689 Google Scholar Crossref Search ADS PubMed WorldCat Bhatt K Feng L Pabla N Liu K Smith S Dong Z Effects of targeted Bcl-2 expression in mitochondria or endoplasmic reticulum on renal tubular cell apoptosis Am. J. Physiol. Renal Physiol. 2008 294 F499 F507 Google Scholar Crossref Search ADS PubMed WorldCat Borner C The Bcl-2 protein family: sensors and checkpoints for life-or-death decisions Mol. Immunol. 2003 39 615 647 Google Scholar Crossref Search ADS PubMed WorldCat Cappaert NL Klis SF Baretta AB Muijser H Smoorenburg GF Ethyl benzene-induced ototoxicity in rats: a dose-dependent mid-frequency hearing loss J. Assoc. Res. Otolaryngol. 2000 1 292 299 Google Scholar PubMed OpenURL Placeholder Text WorldCat Cappaert NL Klis SF Muijser H de Groot JC Kulig BM Smoorenburg GF The ototoxic effects of ethyl benzene in rats Hear. Res. 1999 137 91 102 Google Scholar Crossref Search ADS PubMed WorldCat Cappaert NL Klis SF Muijser H Kulig BM Ravensberg LC Smoorenburg GF Differential susceptibility of rats and guinea pigs to the ototoxic effects of ethyl benzene Neurotoxicol. Teratol. 2002 24 503 510 Google Scholar Crossref Search ADS PubMed WorldCat Cappaert NL Klis SF Muijser H Kulig BM Smoorenburg GF Simultaneous exposure to ethyl benzene and noise: synergistic effects on outer hair cells Hear. Res. 2001 162 67 79 Google Scholar Crossref Search ADS PubMed WorldCat Centers for Disease Control and Prevention, National Health and Nutrition Examination Surveys Ethylbenzene. In Fourth National Report on Human Exposure to Environmental Chemicals 2009 Atlanta, GA Department of Health and Human Services, Centers for Disease Control and Prevention 470 472 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Google Scholar Chan PC Hasemani JK Mahleri J Aranyi C Tumor induction in F344/N rats and B6C3F1 mice following inhalation exposure to ethylbenzene Toxicol. Lett. 1998 99 23 32 Google Scholar Crossref Search ADS PubMed WorldCat Chang HY Yang X Proteases for cell suicide: functions and regulation of caspases Microbiol. Mol. Biol. Rev. 2000 64 821 846 Google Scholar Crossref Search ADS PubMed WorldCat Chen CS Hseu YC Liang SH Kuo JY Chen SC Assessment of genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene to human lymphocytes using comet assay J. Hazard. Mater. 2008 153 351 356 Google Scholar Crossref Search ADS PubMed WorldCat Earnshaw WC Martins LM Kaufmann SH Mammalian caspases: structure, activation, substrates, and functions during apoptosis Annu. Rev. Biochem. 1999 68 383 424 Google Scholar Crossref Search ADS PubMed WorldCat Engstrom KM Metabolism of inhaled ethylbenzene in rats Scand. J. Work Environ. Health 1984 10 83 87 Google Scholar Crossref Search ADS PubMed WorldCat Fishbein L An overview of environmental and toxicological aspects of aromatic hydrocarbons. IV. Ethylbenzene Sci. Total Environ. 1985 44 269 287 Google Scholar Crossref Search ADS PubMed WorldCat Green DR Kroemer G The pathophysiology of mitochondrial cell death Science 2004 305 626 629 Google Scholar Crossref Search ADS PubMed WorldCat Henderson L Brusick D Ratpan F Veenstra G A review of the genotoxicity of ethylbenzene Mutat. Res. 2007 635 81 89 Google Scholar Crossref Search ADS PubMed WorldCat Holzer G Oro J Bertsch W Gas chromatographic-mass spectrometric evaluation of exhaled tobacco smoke J. Chromatogr. 1976 126 771 785 Google Scholar Crossref Search ADS PubMed WorldCat Hossain S Liu HN Nguyen M Shore G Almazan G Cadmium exposure induces mitochondria-dependent apoptosis in oligodendrocytes Neurotoxicology 2009 30 544 554 Google Scholar Crossref Search ADS PubMed WorldCat International Agency for Research on Cancer Ethylbenzene IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 2000 Vol. 77 France IARC Press, Lyon 227 266 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Google Scholar Jang JY Droz PO Kim S Biological monitoring of workers exposed to ethylbenzene and co-exposed to xylene Int. Arch. Occup. Environ. Health 2001 74 31 37 Google Scholar Crossref Search ADS PubMed WorldCat Knecht U Reske A Woitowitz HJ Biological monitoring of standardized exposure to ethylbenzene: evaluation of a biological tolerance (BAT) value Arch. Toxicol. 2000 73 632 640 Google Scholar Crossref Search ADS PubMed WorldCat Mao WP Ye JL Guan ZB Zhao JM Zhang C Zhang NN Jiang P Tian T Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria Toxicol. In Vitro 2007 21 343 354 Google Scholar Crossref Search ADS PubMed WorldCat Mellert W Deckardt K Kaufmann W van Ravenzwaay B Ethylbenzene: 4- and 13-week rat oral toxicity Arch. Toxicol. 2007 81 361 370 Google Scholar Crossref Search ADS PubMed WorldCat Norppa H Vainio H Induction of sister-chromatid exchanges by styrene analogues in cultured human lymphocytes Mutat. Res. 1983 116 379 387 Google Scholar Crossref Search ADS PubMed WorldCat National Toxicology Program (NTP) Toxicology and carcinogenesis studies of ethylbenzene (CAS No. 100-41-4) in F344/N Rats and B6C3F1 mice (inhalation studies) Natl. Toxicol. Program Tech. Rep. Ser. 1999 466 1 231 PubMed OpenURL Placeholder Text WorldCat Perbellini L Mozzo P Turri PV Zedde A Brugnone F Biological exposure index of styrene suggested by a physiologico-mathematical model Int. Arch. Occup. Environ. Health 1988 60 187 193 Google Scholar Crossref Search ADS PubMed WorldCat Philchenkov AA Caspases as regulators of apoptosis and other cell functions Biochemistry (Mosc) 2003 68 365 376 Google Scholar Crossref Search ADS PubMed WorldCat Shih CM Wu JS Ko WC Wang LF Wei YH Liang HF Chen YC Chen CT Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells J. Cell. Biochem. 2003 89 335 347 Google Scholar Crossref Search ADS PubMed WorldCat Song Y Liang X Hu Y Wang Y Yu H Yang K p, p'-DDE induces mitochondria-mediated apoptosis of cultured rat Sertoli cells Toxicology 2008 253 53 61 Google Scholar Crossref Search ADS PubMed WorldCat Vyskocil A Leroux T Truchon G Lemay F Gendron M Gagnon F El Majidi N Viau C Ethyl benzene should be considered ototoxic at occupationally relevant exposure concentrations Toxicol. Ind. Health 2008 24 241 246 Google Scholar Crossref Search ADS PubMed WorldCat Wang JZ Wang XJ Tang YH Shen SJ Jin YX Zeng S Simultaneous determination of mandelic acid enantiomers and phenylglyoxylic acid in urine by high-performance liquid chromatography with precolumn derivatization J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2006 840 50 55 Google Scholar Crossref Search ADS PubMed WorldCat Xu J Lian LJ Wu C Wang XF Fu WY Xu LH Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice Food Chem. Toxicol. 2008 46 1488 1494 Google Scholar Crossref Search ADS PubMed WorldCat Zhang M Wang A He W He P Xu B Xia T Chen X Yang K Effects of fluoride on the expression of NCAM, oxidative stress, and apoptosis in primary cultured hippocampal neurons Toxicology 2007 236 208 216 Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
TI - Involvement of Mitochondria-Mediated Apoptosis in Ethylbenzene-Induced Renal Toxicity in Rat
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfq046
DA - 2010-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/involvement-of-mitochondria-mediated-apoptosis-in-ethylbenzene-induced-ublzD4QCqf
SP - 295
EP - 303
VL - 115
IS - 1
DP - DeepDyve
ER -