ToxSci Advance Access originally published online on July 14, 2008
Toxicological Sciences 2008 105(2):331-341; doi:10.1093/toxsci/kfn139
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Subacute Oral Exposure to Dibromoacetic Acid Induced Immunotoxicity and Apoptosis in the Spleen and Thymus of the mice
* Department of Toxicological Science, College of Public Health, Harbin Medical University, Harbin, HeiLongjiang Province, Peoples' Republic of China, 150081
Department of Immunological Science, College of Preclinical Medicine, Harbin Medical University, Harbin, HeiLongjiang Province, Peoples’ Republic of China, 150081
1 To whom correspondence should be addressed at Department of Toxicological Science, College of Public Health, Harbin Medical University, Harbin, HeiLongJiang Province, Peoples' Republic of China, 150081. Fax: (86) 451-87502829. E-mail: libaix{at}ems.hrbmu.edu.cn.
Received March 16, 2008; accepted June 30, 2008
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ABSTRACT |
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Dibromoacetic acid (DBA) is a haloacetic acid that is present in drinking water as a by-product of chlorinated disinfection. To evaluate its potential adverse health effects, the immunotoxicological effects of DBA on the thymus and spleen of BALB/c mice were investigated. Groups of mice (10 mice per group) were administered DBA at doses of 0, 5, 20, and 50 mg/kg body weight daily for 28 days via oral gavage. The mice orally administered DBA exhibited obvious immunotoxicity, as indicated by changes in the thymus and spleen. DBA induced a dose-dependent decrease and increase in thymus weight and spleen weight, respectively. The histological changes were cortical atrophy of the thymus, white pulp shrinkage of the spleen, and apoptosis of many splenic and thymic lymphocytes; these observations were confirmed by morphometric analysis of the electron microscope scans. Lymphocytes proliferation analysis indicated that the proliferative function of the splenic and thymic lymphocytes was altered after DBA exposure. Cell death via apoptosis was analyzed with an annexin-V/propidium iodide assay by flow cytometry, and we observed that the percentage of apoptosis increased in a dose-dependent manner after DBA treatment. In addition, DBA treatment altered the expression of a few apoptosis-related genes such as Fas, TRAF2, bcl-2, and bax in a dose-dependent manner. Western blot analysis revealed increased expression of the Fas and FasL proteins. In conclusion, DBA induces obvious immunotoxicity in the thymus and spleen, and immune-cell apoptosis mediated by the Fas/FasL pathway may be the potential mechanism underlying this immunotoxicity.
Key Words: dibromoacetic acid; immunotoxicity; apoptosis; mice.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Obvious public health benefits are derived by chlorinated disinfection of drinking water. However, the process of disinfection results in the formation of many disinfection by-products (DBPs), among which trihalomethanes and haloacetic acids (HAAs) are the two most prevalent classes (Arora et al., 1997
; Boorman, 1999). HAAs include monochloroacetic acid, dichloroacetic acid, trichloroacetic acid (TCA), monobromoacetic acid, and dibromoacetic acid (DBA). Under the Disinfection By-product Rule of U.S. Environmental Protection Agency (U.S. EPA, 2003), the sum of the concentrations of 5 HAAs is limited to 60 µg/l (60 ppb). However, the level of HAAs in many drinking water sources in the United States exceeds the annual maximum contaminant level by almost 50%, and the concentration of DBA is reported to be as high as 22 µg/l (Weinberg et al., 2002). Furthermore, when there is a significant quantity of bromide in chlorinated drinking water, DBA can become the predominant HAA (Richardson et al., 2003).
The widespread potential for human exposure to HAAs via the consumption of finished drinking water contaminated with HAAs has raised questions about the health hazards of these compounds. Hence, many investigations are being performed to investigate the potential adverse effects of HAAs. Animal studies on HAAs have shown that these compounds are carcinogenic, mutagenic, and teratogenic and cause reproductive and developmental toxicity (Daniel et al., 1992; DeAngelo et al., 1996; Herren-Freund et al., 1987; Nieuwenhuijsen et al., 2000). DBA was selected for this study because toxicology data on this compound are limited as compared with that for other HAAs. Currently, there is considerable research on the reproductive toxicity and neurotoxicity of DBA in animals (Bodensteiner et al., 2004; Holmes et al., 2001; Klinefelter et al., 2004; Moser et al., 2004). DBA's long-term carcinogenicity is being evaluated by the U.S. National Toxicology Program (NTP). It has been shown that mice exposed to DBA exhibit a significant increase in hepatocellular adenomas, carcinomas, and hepatoblastomas in a dose-dependent manner (Melnick et al., 2007).
The immune system is one of the main adaptation mechanisms via which the body defends itself against harmful agents and pathogens. Maladaptive immunological alterations may lead to a broad range of disorders, including inflammatory and infectious diseases, autoimmune diseases, and tumorigenesis. With regard to immunosurveillance against tumors, immunosuppression might play a role in DBA-induced carcinogenesis. The potential adverse effects of drinking water contaminants on the immune system are a concern for both the EPA and the National Institute of Environmental Health Sciences (NIEHS). Several DBPs have been identified, and their potential effects on the immune system will be evaluated in a joint project between the EPA and the NIEHS. To establish the potential effects of DBA on the immune system, some primary studies with female B6C3F1 mice have been conducted by the NTP (NTP, 2007). In their experiment, the animals consumed drinking water containing DBA at doses of 125–2000 mg/l. Their results revealed that the consumption of DBA at some concentrations causes marked toxicological and immunomodulatory effects. Animals exposed to high doses of DBA exhibit a significant decrease in thymus weight and an increase in spleen weight. They also show a dose-dependent decrease in the antibody-forming cell response to sheep erythrocytes and an alteration in hematological parameters.
To investigate the functional changes in the immune system following exposure to DBA in greater detail, in this study that followed good laboratory practice, BALB/c mice were orally administered repeat doses of DBA for 28 days. The DBA doses administered in our study were 5, 20, and 50 mg/kg, which is similar to the high doses used in the NTP study. The thymus and spleen weights, histological changes, and the in vitro proliferative activity of lymphocytes were evaluated. In addition, we aimed to investigate whether oral exposure to DBA triggered the apoptosis of thymocytes and splenocytes. Apoptosis plays an important role in development and homeostasis in vertebrates (Steller, 1995), and it is also a vital regulatory process of the immune system. Many lymphocytes undergo apoptosis at the termination of the acute phase of an immune response. Further, apoptosis is involved in the deletion of the majority of T cells in the thymus (Palmer, 2003). This active regulatory mechanism could be a potential target for immunotoxicants that are known to destroy splenic and thymic tissues. In the present study, we investigated whether oral exposure to DBA triggered the apoptosis of thymocytes and splenocytes and if it altered the expression of apoptosis-regulating molecules such as Fas/FasL, bcl-2, bax, and TRAF-2 in mRNA or protein level (Steller, 1995).
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MATERIALS AND METHODS |
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Chemicals and Reagents
Dibromoacetic acid (purity 99%), concanavalin A (Con A, Type IV), lipopolysaccharide (LPS) and methyl thiazolyl tetrazolium (MTT) were obtained from Sigma-Aldrich (St Louis, MO). Annexin-V/propidium iodide (PI) kit was purchased from Pharmingen (Becton Dickinson Company, NJ, USA).
DBA Preparation
DBA solution of different concentrations was prepared by dissolving DBA in deionized water and adjusting the pH to approximately 6.5 with 1N NaOH. The DBA concentration is 500, 2000, and 5000 mg/l, respectively. All the solutions were kept in the refrigerator at 4°C and were made up fresh every week.
Animals and Treatment
Male and female BALB/c mice (4–6 weeks old) were obtained from Chinese Academy of Science Laboratory Animal Center. The animals received humane care in individual cages according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by National Institutes of Health. After acclimatization for 1 week, the mice were housed in standard polyethylene cages, in groups of five per cage, with wood shavings as bedding and given purified water and rodent chow ad libitum. Animal rooms were maintained at a 12-h light-dark cycle at 20 ± 3°C and 50% ± 15% relative humidity.
Experimental Design
Animals were randomly grouped by weight and divided into four groups (n = minimum of 5/dose group/sex). Mice were treated daily with 0 (deionized water; vehicle control) or 5, 20, and 50 mg/kg body weight of DBA solution by intragastric administration of 0.1 ml/10 g body weight for 28 consecutive days.
Body Weight and Immune Organ Weight
Body weight was measured and recorded every 7 days. The weights of thymus and spleen were measured when the mice were sacrificed, and the relative weights of spleen and thymus of each mouse were calculated by the formula of organ weight (mg)/body weight (g).
Thymocytes and Splenocytes Preparation
Spleen and thymus cells were harvested as described (Burchiel et al., 2004). Thymus and spleen were removed aseptically at the time of sacrifice and single-cell suspensions were prepared by forcing thymus and spleen through 400-µm stainless steel mesh. Lymphocytes were washed with Hank's balanced salt solution (HBSS) and then erythrocytes were lysed with buffered solution (pH 7.2) of 0.15M NH4Cl, 1mM KHCO3, and 0.1mM EDTA. After washing with HBSS, the cells were resuspended in RPMI1640 medium, and viable lymphocytes counts were obtained by trypan blue (Sigma-Aldrich) exclusion method using a hemacytometer. Finally, cells were adjusted into different concentrations in RPMI1640 medium supplemented with 10% fetal calf serum.
Lymphocytes Proliferation Assay In Vitro
Con A and LPS were used to evaluate T and B cell proliferation, respectively, by MTT reduction method (Mosmann, 1983; Visconti et al., 1991). Splenocytes and thymocytes were harvested from individual mice, prepared into a single-cell suspension as described above, and adjusted to 5 x 106 cells/ml in complete RPMI1640 medium. The splenocytes were exposed to mitogens in 96-well culture microtiter plates in triplicates containing 1 µg/ml of LPS or 5 µg/ml of Con A, and thymocytes were only exposed to ConA. Complete RPMI1640 medium lacking LPS or Con A was used as the control. Plates were incubated at 37°C in a humidified, 5% CO2 incubator for 48 h. Ten microliters of MTT [3-(4,5-dimethylthiazolyl-2)2,5-diphenyltetrazolium bromide] solution(5 mg/ml) was added to each well, allowing the plate to incubate for additional 4 h. At the end of the incubation, 100 µl of acidic isopropanol (0.04M HCl in absolute isopropanol) was added to each well to dissolve the converted dye formazan. The absorbance data (optical density) were measured by a microtiter plate reader at 570 nm.
Histopathologic Analysis of Thymuses and Spleens
Light microscopy.
At the time of sacrifice, the thymuses and spleens were collected, trimmed of fat, and weighed before stored in 10% buffered formalin. After fixation, one middle cross section from the spleen and one lobe of the thymus were embedded in paraffin, and five 5–6 micron sections were prepared and stained with hematoxylin and eosin for histopathological evaluation by light microscope.
Electron microscopy.
To confirm the cells apoptosis existing in spleen and thymus, we deployed the electron microscopy scan in the splenic and thymic tissues. For electron microscopy, thymus and spleen were cut into 1-mm3 cubes and fixed in 2.5% freshly depolymerized paraformaldehyde with 0.5% glutaldehyde in 0.1 mol/l of sodium cacodylate buffer, pH 7.4, for 2 h. After rinsing in sodium cacodylate buffer, the samples were partially dehydrated with ethanol and embedded in LR White resin. Ultra-thin sections were cut and collected on gold grids. Labeled ultra-thin sections were observed with a transmission electron microscope.
Flow Cytometric Analysis of Phosphatidyl Serine Externalization in Apoptosis Lymphocytes
To determine the extent of early apoptosis and necrosis in thymus and spleen, cell death was determined by staining cells with annexin-V and PI (Bertho et al., 2000). Positioning of quadrants on annexin-V/PI dot plots was performed and living cells (annexin-V–/PI–), early apoptotic/primary apoptotic cells (annexin-V+/PI–), late apoptotic/secondary apoptotic cells (annexin-V+/PI+), and necrotic cells (annexin-V–/PI+) were distinguished (Vermes et al., 1995). Therefore, the total apoptotic proportion included the percentage of cells with fluorescence annexin-V+/PI– and annexin-V+/PI+. Thymus and spleen lymphocytes were collected like above when the mice were sacrificed, adjusted to 2 x 106/ml and washed with cold phosphate-buffered saline (PBS) three times, centrifuged and suspended in a final volume of 100-µl binding solution, incubated with 5 µl annexin-V–fluorescein isothiocyanate (FITC) and 5 µl PI for 15 min in dark at room temperature, and 400 µl binding buffer (1x) was added to each sample. Samples were collected using the flow cytometer (Becton Dickinson, Conc), and data were analyzed and quantitated using Cell Quest software. At least three independent experiments were performed.
Reverse Transcription–PCR Analysis of Gene Expression Related with Apoptosis
To identify the apoptosis caused by DBA in mRNA level, the expression of several genes related with apoptosis was analyzed by reverse transcription (RT)–PCR. RNA was isolated from the tissues homogenates of spleen and thymus with Trizol reagent as described by the manufacture. First-strand cDNA was prepared with the reverse transcription system (Promega, Madison, WI). PCR using Taq polymerase (Takara bio-group, Japan) was performed using the oligonucleotide primers for mice. Primer was synthesized by Invitrogen Company, and the sequences and annealing temperatures are listed in Table 1 (Fisher et al., 2004). PCRs used the parameters as: 4 min at 94°C, 30 s at 94°C, 30 s at the appropriate annealing temperature, and 60 s at 72°C, cycled 30x, with a final incubation at 72°C 5 min, after PCR, all the products were resolved using 1.5% agarose gels.
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The Expression of Fas/FasL Protein by Immunobloting Analysis
For preparation of tissue lysates, thymuses and spleens were excised and homogenized in cold PBS. Thymus or spleen of 100 mg was lysed with 500 µl of lysis buffer (50mM Tris, pH 7.4; 1% Igepal; 150mM sodium chloride; 1mM EDTA) with protease inhibitors (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstain, 1mM phenylmethylsulfonyl fluoride, 1mM sodium orthovanadate, 1mM sodium fluoride). Lysates were clarified by centrifugation at 12,000 g for 5 min, and their protein content was determined by the Bradford protein assay. The lysates were then mixed with 4x sodium dodecyl sulfate gel-loading buffer to give 40–60 µg protein per lane. After boiling for 5 min, proteins were resolved in a 10% polyacrylamide gel and transferred to nitrocellulose membrane (pore size, 0.22 µm) by electroblotting. The membrane was blocked for 1 h with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature and subsequently incubated with the primary antibodies of Fas and FasL (purchased from Santa Cruz, CA) for 2 h at 37°C or overnight at 4°C on a shaker, then incubated with horseradish peroxidase–conjugated secondary antibodies and processed with the ECL system according to the manufacturer's protocol (Amersham Biosciences, NJ, USA). Protein visualization of β-actin expression was used as a loading control. The density of the bands was quantified by densitometry. β-Actin was used to normalize the samples. Western data were expressed as fold-change of the band density of the DBA sample relative to the vehicle control sample.
Statistical Analysis
All the data presented in this paper were analyzed using SPSS13.0. The statistical differences were determined by one-way ANOVA followed by a Bonferroni multiple-comparison tests. A p value of <0.05 was considered significant.
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RESULTS |
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Changes in Body Weight after DBA Exposure
All animals survived until the end of the study. There were no overt changes in appearance and/or behavior following DBA exposure. No significant differences in body weight were observed (p > 0.05, Table 2).
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Toxicity in Lymphatic Organs
The thymus is the central immune organ, and it is here that T cells complete their development and maturation. The spleen is a peripheral immune organ that contains T cells, B cells, and other immune cells. Therefore, thymus or spleen weights that exceed the normal reference ranges are an important indicator of potential immunotoxicity. In the present study, the mean thymus weight and its relative weight were significantly reduced in male and female mice after dosing with 20 and 50 mg/kg DBA, and the mean spleen weight and spleen relative weight were increased in the mice of both sexes in the group of 20 and the 50 mg/kg (p < 0.05). The absolute number of cells harvested per spleen and thymus was significantly decreased in the group of 20 and 50 mg/kg (Table 3).
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Lymphoproliferation Assay
During mitogenesis, the mitogens Con A and LPS activate T cells and B cells, respectively. Changes in the proliferation of T and B cells following DBA exposure were measured via a MTT assay. In BALB/c mice, exposure to 20 and 50 mg/kg DBA causes an apparent inhibition in B-cell and thymic T-cell proliferations. However, in the present experiment, the splenic T-cell proliferation in 20 and 50 mg/kg dose groups was slightly increased as compared to that in 0 mg/kg dose group (Fig. 1).
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Histopathological Analysis of the Thymus and Spleen
Light Microscopy
After the mice were sacrificed (five from each group), their thymuses and spleens were used for histological and pathological examinations. The examinations revealed atrophy of the thymic cortex, diminution in size of the white pulp, and shrinkage of the splenic germinal centers (Fig. 2). The changes in the 20- and 50-mg/kg dose groups were more severe.
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Electron Microscopy
Microphotographs of the thymus and spleen of the animals of each group treated with different doses of DBA were obtained. The images showed widespread karyopyknosis, cytochondriome degeneration, and also apoptosis of the lymphocytes in these organs; these changes were particularly notable in the thymus and spleen of the 20- and 50-mg/kg dose groups (Fig. 3).
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The histopathological features observed by light and electron microscopy indicated that oral exposure to DBA may lead to morphological changes in the immune organs and that a number of immune cells in these organs had undergone apoptosis in male and female mice.
Apoptosis and Cell Death
After the oral administration of DBA for 28 days, the effects of DBA on cell death in the thymus and spleen were examined by flow cytometry analysis. After the mice were sacrificed, the thymocytes and splenocytes were stained with annexin-V and PI. DBA exposure increases the apoptosis of thymocytes and splenocytes in mice; this was indicated by the increased numbers of annexin V+ and annexin-V+/PI+cells in the treated groups as compared to that in the control group. Detailed data are presented in Table 4 and Figure 4 (p < 0.05 or p < 0.01).
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Effect of DBA on the mRNA Expression of Some Apoptosis-Related Genes in Mice
The histopathological and electron microscopic examinations revealed that many lymphocytes undergone apoptosis. Therefore, the mRNA expressions of some apoptosis-related genes were examined by RT-PCR in the thymus and spleen of the mice orally administered different DBA doses (Fig. 5). The ratio of the optical density of the RT-PCR products of the genes to the RT-PCR product of β-actin (a housekeeping gene) is presented in Figure 5B. The mRNA expression of Fas was nearly increased by 2–2.5 fold in the thymus and fivefold that in the spleen of the mice of the groups treated with different DBA doses (p < 0.05). The expression of the TRAF2 gene increased by twofold in the thymus and by nearly fivefold in the spleen of the mice of the groups treated with different DBA doses. While the mRNA expression of bax in the thymus and spleen was slightly upregulated and that of bcl-2 was slightly decreased in the thymus. Although the change in the expression level was not statistically significant in the thymus, the expression of the bcl-2 gene in the spleen decreased 1.5-fold (p < 0.05). Thus, DBA caused an obvious increase in the mRNA expression of Fas and TRAF2 in the thymus and spleen and a decrease in the mRNA expression of bcl-2 in the spleen of BALB/c mice.
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DBA Induced Expression of the Fas and FasL Proteins
Expression of the Fas and FasL proteins in the thymus and spleen was examined by semi-quantitative western blotting. Blotting with anti-Fas and anti-FasL antibodies revealed bands at 45 and at 37 kDa corresponding to the Fas and FasL, respectively. Bands density was normalized per β-actin content. It revealed that DBA induced an increase in the expression of these proteins (shown in Fig. 6). The expression of Fas was increased by 2.4-fold in the thymus and nearly twofold in the spleen and the expression of FasL was increased by 2.3-fold in the thymus and spleen.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DBA is a haloacetic acid that is a TCA analog. However, there is limited information available about this compound. In recent years, many toxic endpoints such as spermatotoxicity, hepatotoxicity, neuromuscular toxicity, carcinogenicity, and reproductive and developmental toxicity endpoints have been identified in animals (Bodensteiner et al., 2004
; Christian et al., 2002; Holmes et al., 2001; Klinefelter et al., 2004; Linder et al., 1994; Moser et al., 2004).
Many chemicals can induce immunotoxicity via multiple mechanisms; however, these mechanisms are not clearly understood. A number of chemically induced immunotoxic effects are characterized as immunosuppressive effects (Tryphonas et al., 1991; Wood et al., 1992), which manifest as marked decreases in immune function. In animals, these toxic effects may manifest as thymus atrophy, splenomegaly, inhibition of immune-cell function, or even apoptosis of lymphocytes.
The main aim of this study was to examine the immunotoxicity and immunocytes apoptosis that occurred in BALB/c mice treated with DBA by oral gavage for 28 days. We administered different doses of DBA, ranging from 5 to 50 mg/kg. This range is partly similar to that used in the NTP study (NTP, 2007) that demonstrated the obvious toxicity of DBA to the immune system. However, the actual dose to which each mouse in our experiment was exposed is lower than that in the NTP study. In the NTP experiment, the mice were exposed to DBA by allowing them free access to DBA-contaminated drinking water, and in this study, the daily water intake of each mouse was approximately 6 ml. In contrast, in our study, the mice were administered DBA by oral gavage, and each mouse ingested approximately 0.2 ml DBA solution. After DBA treatment, no obvious physical changes such as morbidity, mortality, or alterations in body weight were observed in any treatment group. However, we found that the spleen weight of the mice was significantly increased, while thymus weight was decreased in the 20- and 50-mg/kg dose groups. The diminished thymic weight in animals could be associated with the decrease in thymic cell counts; a similar reduction in splenic cells was also observed, in spite of spleen enlargement. The splenomegaly may be associated with the expansion of red pulp (erythrocytes) area.
Similar to many chemicals, such as 3,4-dichloropropionanilide, TCDD, ethanol, and cadmium (Blaylock et al., 1992; Cuff et al., 1996; Han et al., 1993; Pathak and Khandelwal, 2007), DBA caused splenomegaly and thymus atrophy. In the NTP study, thymus or spleen weights exceeding the normal reference ranges were considered important indicators of potential immunotoxicity (Luster et al., 1992). The most plausible explanations for these are inhibition of lymphocytes proliferation and the induction of lymphocytes apoptosis in thymus or spleen (Kamath et al., 1997; Vandebriel et al., 1999). The ability of DBA to inhibit cell proliferation was confirmed by the results of the MTT reduction assay, in which mitogens were omitted from the incubation mixture. The mitogen response is initiated by the binding of mitogen to mitogen receptors, and a decreased mitogen response may be due to a decrease in the expression of mitogen receptors on lymphocytes (Chakrabarti et al., 1994). We observed significant alteration of T- and B-cell proliferation in the thymus or spleen in vitro with the stimulation of the mitogens Con A and LPS. T cells complete their maturation in the thymus, and the spleen contains mature B cells (60%), T cells, and other cells. We can conclude from our results that the proliferative activity of immature T cells in the thymus and that of mature B lymphocytes were inhibited after the mice were exposed to DBA. However, the proliferation of T cells in the spleen was increased especially in the dose group of 20 mg/kg. The mechanism is probably due to that the T cells were induced apoptosis through death receptor pathway in the spleen. It is proved that some molecules like Fas-associated death domain (FADD/Mort-1) and caspase 8 can not only induce apoptosis by the death receptors pathway in T lymphocytes but also have distinct, essential role in promoting T-cell proliferation. Furthermore, these molecules are required for proliferation of mature T cells, but not developing pro–T-cells. The mechanism of the signaling components promoting T-cell proliferation remains unclear. It is possible that FADD and caspase 8 act immediately downstream of the TCR/CD3 complex (Newton et al. 1998; Salmena et al. 2003; Zhang et al., 1998).
Examination of histopathological features revealed some morphological changes in the thymus and spleen of the DBA-exposed mice. In the thymus, the cortical layer became thin, while in the spleen, the white pulp diminished and the germinal centers shrunk, and the red pulp enlarged; these features were particularly notable in the 20- and 50-mg/kg dose groups. Further, the widespread cytochondriome degeneration and karyopyknosis of lymphocytes in the thymus and spleen were examined by electron microscope scanning. Since there were extensive morphological changes of apoptosis in the thymus and spleen, these changes were examined with annexin-V/PI assays in order to determine the potential of DBA to induce necrosis and apoptosis of lymphocytes in the thymus and spleen in greater detail. Flow cytometry revealed a dose-dependent increase in apoptosis and necrosis following exposure to all the DBA concentrations used in this study. It is quite plausible that DBA-induced apoptosis is partially responsible for the suppression of the cell proliferative response to mitogens. Thus, thymus atrophy and decrease in the white pulp in the spleen could be the result of apoptosis induction and the inhibition of lymphocyte proliferation.
T cells undergo development and maturation in the thymus. Developing thymocytes sequentially rearrange their TCR and β-chain genes to generate the T-cell repertoire. Such T cells undergo a meticulous selection process that eliminates autoreactive and nonfunctional cells from this repertoire, and apoptosis plays a crucial role in this selection process (Palmer, 2003). Similar to other chemicals, DBA-induced thymocytes apoptosis can lead to alterations in the T-cell repertoire, such that the immune system may tend to react strongly toward self-antigens and weakly against foreign antigens (Blaylock et al., 1992; Okasha et al., 2001). The spleen is a peripheral lymphoid organ where lymphocytes are activated on being presented with foreign antigens; these activated lymphocytes are sensitive to signals that lead to apoptosis.
Apoptosis is triggered through two pathways: the death receptor pathway and the mitochondrial pathway. The death receptor pathway involves two receptors that are members of the TNFR3 superfamily: TNFR1 and CD95 (Fas/APO-1). These receptors induce cell death on binding with their ligands (receptor-mediated cell death). The ligand for TNFR1 is TNF, which is a pleiotropic cytokine with various biological activities. In contrast, the ligand for Fas (FasL) is mainly expressed on T lymphocytes, where it mediates the normal elimination of autoreactive lymphocytes (Ju et al., 1999). In the present study, we used RT-PCR analysis to evaluate the expression of a few apoptosis-regulating genes, namely, Fas, bcl-2, bax, and TRAF2. The mRNA expression of Fas and TRAF2 in the thymus and spleen increased after in vivo exposure to different doses of DBA. DBA exposure caused moderate or nonsignificant changes in the mRNA expression levels of the anti-apoptotic gene (bcl-2) and pro-apoptotic gene (bax) that participate in the mitochondrial pathway of apoptosis. TRAF2 promotes lymphocytes differentiation and can mediate the activation of the noncanonical nuclear factor-kappa B (NF-
B) pathway. TRAF2 overexpression can inhibit CD95 and TNF-RI–mediated apoptosis and cause a switch to NF-B/AP-1 activation (Bertho et al., 2000). The upregulation of Fas and FasL may have serious consequences for T-cell differentiation in the thymus. It has been demonstrated that Fas/FasL interactions are involved in the process of negative selection in the thymus (Castro et al., 1996). During T-cell maturation, Fas plays an important role in positive and negative selections, during which T cells that respond to self-antigens are clonally deleted, while those that respond to nonself (foreign) antigens are selected and sent to the periphery (Camacho et al., 2004; Dearstyne and Kerkvliet, 2002; Palmer, 2003). In the spleen, the expression of the Fas, bcl-2, and TRAF2 genes was increased. This is because the peripheral lymphocytes were activated following stimulation with a foreign antigen, i.e., DBA, and the death receptor pathway or the mitochondrial pathway was activated. Consequently, the fine balance between the expression of anti-apoptotic and apoptotic genes was altered, resulting in the induction of apoptosis. Furthermore, western blot analysis of Fas and FasL showed that the expressions of these proteins were increased in the thymus and spleen. The data clearly suggested that DBA might mediate cell death in the thymus and spleen through the death receptor pathway with the possible involvement of Fas/FasL interaction.
In summary, the present study clearly demonstrates that exposure to DBA leads to suppression of the immune response; this suppression is characterized by thymus atrophy and splenomegaly, reduction in the response to polyclonal mitogens, and increased apoptosis. We demonstrated that the DBA-induced suppression of the immune response is directly related to the induction of apoptosis; this is an important step toward understanding the mechanism underlying DBA-induced toxicity. Our study is primarily proved that the apoptosis induced by DBA occurring in the thymus and spleen is mediated by the Fas/FasL death receptor pathway.
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