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Nitrotyrosine formation in splenic toxicity of aniline
Toxicology 194 (2003) 95–102
Nitrotyrosine formation in splenic toxicity of aniline M. Firoze Khan∗ , Xiaohong Wu, Bhupendra S. Kaphalia, Paul J. Boor, G.A.S. Ansari Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA Received 23 June 2003; received in revised form 28 July 2003; accepted 12 August 2003
Abstract Splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis and development of a variety of sarcomas in rats. However, the mechanisms of this selective splenic toxicity are not well understood. Previously we showed that aniline exposure causes oxidative damage to spleen. To further explore the oxidative mechanisms of aniline toxicity, we evaluated the contributions of nitric oxide. Nitric oxide reacts with superoxide anion to form peroxynitrite, a powerful oxidant that converts the tyrosine residues of proteins to nitrotyrosine (NT). Therefore, aim of this study was to establish the role of nitric oxide through the formation and localization of NT in the spleen of rats exposed to aniline. Male Sprague–Dawley (SD) rats were given 1 mmol/kg per day aniline hydrochloride in water by gavage for 7 days, while the controls received water only. Immunohistochemical analysis for NT showed an intense staining in the red pulp areas of spleen from aniline-treated rats, localized in macrophages and sinusoidal cells. Occasionally mild NT immunostaining was also evident in the white pulp. Western blot analyses of the post-nuclear fraction of the spleens showed major nitrated proteins with molecular weights of 49, 30 and 18 kDa. Immunohistochemical analysis of inducible nitric oxide synthase (iNOS) also showed increased expression in the red pulp of the spleens from aniline-treated rats; the cellular localization was similar to nitrated proteins. These studies suggest that oxidative stress in aniline toxicity also includes aberration in nitric oxide production leading to nitration of proteins. Functional consequences of such nitration will further elucidate the contribution of nitric oxide to the splenic toxicity of aniline. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Nitrotyrosine; iNOS; Oxidative stress; Aniline; Spleen; Immunohistochemistry
1. Introduction Exposure to aniline causes selective toxicity to the spleen leading to vascular congestion, hyperplasia, fibrosis and a variety of sarcomas in rats (Bus and Popp, 1987; Khan et al., 1995, 1997a, 1999a, 2003a,b). Mechanism(s) by which aniline induces selective tox-
∗ Corresponding author. Tel.: +1-409-772-6881; fax: +1-409-747-1763. E-mail address: [email protected] (M.F. Khan).
icity to the spleen is not well understood. In earlier studies, we have shown that aniline exposure results in iron overload (Khan et al., 1997a, 1999a,b), lipid peroxidation (Khan et al., 1997a,b, 1999a), increased formation of MDA–protein adducts (Khan et al., 1999a, 2003a) and protein oxidation (Khan et al., 1999a,b) in the spleen. These results suggest a critical role for iron-catalyzed generation of reactive oxygen species (ROS) in aniline-mediated splenic damage to spleen. Since oxidative and nitrosative stress could occur simultaneously, it was logical to study the possible involvement of nitric oxide (• NO) whose formation and
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contribution has not been studied in splenic toxicity of aniline. There is considerable evidence of overproduction of • NO during inflammation (Kooy et al., 1995; Goldstein et al., 1998) and a number of pathological conditions (Shin et al., 1998; Greenacre and Ischiropoulos, 2001; Oldreive and Rice-Evans, 2001). • NO vigorously reacts with superoxide to produce peroxynitrite (ONOO− ) which is a highly reactive nitrogen species (Nakazawa et al., 2000; Greenacre and Ischiropoulos, 2001). One of its reactions is nitration of the ortho position of tyrosine leading to nitrotyrosine (NT) formation in proteins (Greenacre and Ischiropoulos, 2001). Since NT is a stable end product of this reaction, it serves as a very useful biomarker in identifying the generation of • NO in vivo (Ter Steege et al., 1998). Macrophages and endothelial cells are the major biological sources of • NO which can regulate a variety of biological processes (Greenacre and Ischiropoulos, 2001; Bian and Murad, 2001). In the spleen, there are both fixed and mobile macrophages. The tissue-fixed macrophages line the sinusoids of the spleen and have a close relationship with the endothelial cells lining the sinusoids (Bian and Murad, 2001). Aniline treatment has been shown to result in expansion of the red pulp, increases in macrophages and sinusoidal cells (Khan et al., 1993, 1997a, 1999a), and up-regulation of fibrogenic cytokine transforming growth factor- 1 (Khan et al., 2003b). Even though oxidative stress appears to be an important mechanism in the selective damage to spleen following aniline insult, the contribution of • NO in the splenic toxicity is not established. Therefore, this study was focused on establishing the formation, localization and identification of NT residues, along with the regulation of inducible nitric oxide synthase (iNOS) in the spleen of rats treated with aniline.
2. Materials and methods 2.1. Animals and treatments Male Sprague–Dawley (SD) rats (∼225 g), obtained from Harlan Sprague–Dawley Inc. (Indianapolis, IN), were housed in wire-bottom cages over adsorbent paper with free access to tap water and Purina laboratory chow and maintained in a controlled environment ani-
mal room (temperature, 22 ◦ C; relative humidity, 50%; photoperiod, 12 h light/dark cycle) for 7 days prior to treatments. The animals were divided into two groups of five each. One group of animals received 1 mmol/kg per day aniline hydrochloride (Aldrich, Milwaukee, WI) in 0.5 ml of drinking water by gavage for 7 days, while the other group received equal volume of water only and served as controls. The choice of aniline dose was based on our earlier studies that showed oxidative stress in the spleen at this dose (Khan et al., 1997a, 2003b). The animals were euthanized 24 h following the last dose and spleens were aseptically removed immediately, blotted, and weighed. A portion of each spleen was stored at −80 ◦ C, while the remainder was fixed in 10% formalin for immunohistochemical studies described below. 2.2. Immunohistochemical localization of nitrated proteins For immunohistochemical staining, alkaline phosphatase-Fast Red staining method was used as described earlier (Khan et al., 2002, 2003a). The method was standardized for NT immunostaining in the spleen. Briefly, 4 m tissue sections were deparafinized in a 55 ◦ C oven for 1 h and passed through xylene and various concentrations of ethanol, and finally rehydrated with water. The slides were incubated with unmasking buffer (Vector, Burlingame, CA) at 95 ◦ C for 20 min for antigen retrieval and then subsequently incubated with different reagents for blocking the non-specific binding sites, which included peroxidase inhibitor (Pierce, Rockford, IL) for 10 min, levamisole (alkaline phosphatase inhibitor; 0.2%) for 10 min, 0.5% periodic acid for 5 min, avidin and biotin block solutions (Vector) for 10 min each, and normal serum (Signet, Dedham, MA). After removal of excess normal serum, the sections were incubated with rabbit anti-nitrotyrosine antibody (Chemicon) [1:1000 dilution in antibody diluent buffer (Dako, Carpinteria, CA)] for 1 h and then anti-rabbit IgG-biotin [1:1500 (Sigma, St. Louis, MO)] for 20 min at room temperature. The slides were thoroughly washed after each incubation. The Signet ultra streptavidin alkaline phosphatase labeling reagent was added to the tissue sections and incubated for 20 min at room temperature. Finally,
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Fast Red (Biopathology Inc., Okalahoma City, OK) staining was performed according to company’s manual. To establish the specificity of the staining, sets of additional sections were also stained with preimmune serum or anti-NT antibody pre-absorbed with NT-albumin. All sections were finally counterstained with hematoxylin (Gill’s formulation, Vector) and mounted for light microscopic examination. 2.3. Western blot analyses of nitrated proteins Western blot analysis for nitrated proteins was performed in the splenic post-nuclear fractions of control and aniline-treated rats. For the preparation of post-nuclear fractions, spleen homogenates (10%) were made in phosphate-buffered saline (PBS, pH 7.4) containing protease inhibitor cocktail (Sigma). The homogenate was centrifuged at 1000 rpm at 4 ◦ C for 10 min to obtain post-nuclear fraction. Protein was measured by DC-protein assay method (Bio-Rad, Hercules, CA). Western blot analysis of nitrated proteins in the spleen samples was done essentially as described by Towbin et al. (1979) with minor modifications. Briefly, before loading the protein samples onto the gel, protein preparations (200 g) were cleaned by using Pageprep according to company’s manual (Pierce). The proteins were separated electrophoretically by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel) and then transferred electrophoretically to polyvinyl difluoride (PVDF) membrane (Bio-Rad). The non-specific binding sites on the membrane was blocked with 1% casein in PBS at 4 ◦ C overnight and incubated with mouse monoclonal anti-nitrotyrosine antibody (1:70 dilution in 0.25% casein, Cayman Chemical) at room temperature for 3 h, and then incubated with anti-mouse IgG-horseradish peroxidase (1:2000 dilution in 0.25% casein, Cappel) at room temperature for 1 h. Membrane was washed extensively with Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 for about 30 min, and developed using 3,3-diaminobenzidine (DAB)/H2 O2 (Sigma) as substrate. 2.4. Immunohistochemical demonstration of iNOS induction in the spleen Immunohistochemical detection and localization of iNOS was achieved essentially using the method de-
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scribed above for nitrotyrosine. However, the primary antibody used in this case was anti-iNOS (polyclonal, 1:20 dilution, Transduction Laboratory, Lexington, KY).
3. Results 3.1. Immunohistochemical localization of nitrated proteins in the spleen For immunohistochemical localization of nitrated proteins in the spleen, we employed the alkaline phosphatase-Fast Red (red color) methodology as previously described (Khan et al., 2002, 2003a). This method provided an increased contrast in comparison to horseradish peroxidase-diaminobenzidine
Fig. 1. Immunohistochemical staining for nitrated proteins at low power (100×) shows negligible staining in either red pulp (R) or white pulp (W) areas in control spleen (A). In contrast, nitrated proteins show an evident, diffuse, marked increase of staining in red pulp (R) of spleen from aniline-treated rats (B).
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(HRP-DAB, brown color) methodology. The ultimate red color stain allowed us to differentiate the changes in aniline-treated rats, which had extensive brownish deposits of iron-containing proteins (hemosiderin), thus allowing for more definitive localization of nitrated proteins in the spleen. Low power observation of spleen showed little stain of nitrated proteins in control rats (Fig. 1A), whereas diffuse, marked positive stain was evident in expanded red pulp areas of spleen from aniline-treated rats (Fig. 1B). Control
spleen showed only focal staining at higher power confined to the red pulp (Fig. 2A). Spleens from aniline-treated rats, however, showed very intense NT immunoreactivity, especially in the macrophages and sinusoidal cells of the expanded red pulp. Staining was also evident in the peripheral white pulp areas (Fig. 2B). No NT immunoreactivity was evident in the spleen sections incubated with preimmune serum or anti-NT antibody pre-absorbed with NT-albumin (negative controls; data not shown).
Fig. 2. High power view (450×) of immunohistochemical staining for nitrated proteins (A and B) and iNOS (C and D). In all photomicrographs, white pulp areas are in lower left corner and red pulp is in upper right. Nitrated proteins show a marked increase in red pulp area of aniline-treated rats (B) compared to controls (A) as evidenced by diffuse NT immunoreactivity and intense staining evident in clusters of macrophages (B, arrow; not seen in controls). Similarly, iNOS staining is increased throughout red pulp of aniline-treated rats (D) compared to controls (C); intense iNOS immunostaining is again localized in macrophages (D, arrows).
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3.2. Immunohistochemical detection and localization of iNOS in the spleen For the induction and localization of iNOS in the spleen, the same immunohistochemical approach was used as described for NT. Spleens from control rats showed weak and sporadic immunostaining (Fig. 2C). On the other hand, spleens from aniline-treated rats showed intense iNOS immunoreactivity, mostly in the red pulp areas. Occasionally, staining was also evident in the white pulp areas (Fig. 2D). 3.3. Detection of nitrated proteins in the spleen Results of Western blot analysis of post-nuclear fractions of control and aniline-treated rats for nitrated proteins are presented in Fig. 3. Using anti-NT antibodies, three nitrated proteins of molecular weight 18, 30 and 49 kDa were identified. These protein bands were rather faint in the controls, indicating a basal formation of nitrated proteins. On the other
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hand, the nitrated protein bands were very intense in the aniline-treated rats, indicating greater formation of nitrated proteins and correlating with our immunohistochemical results.
4. Discussion The splenotoxic sequence of events, preceding the development of splenic sarcomas following aniline exposure, is sinusoidal congestion, hyperplasia and splenic fibrosis (Bus and Popp, 1987; Khan et al., 1993, 1999a). One serious consequence of such changes in the spleen could be a reduced ability of the spleen to participate in the immune response and/or in its phagocytic function of clearing damaged erythrocytes and infectious organisms from the blood. Therefore, it is important to delineate the mechanism of selective splenotoxic response of aniline. Even though, our earlier studies showed a role for generalized oxidative stress (Khan et al., 1997a,
Fig. 3. Western blot analyses of nitrated proteins (NT–protein adducts) in control and aniline-treated rats. Lane 1: marker proteins; lanes 2–4: controls; lanes 5–7: aniline-treated. NT–protein bands of 18, 30 and 49 kDa are more prominent in aniline-treated rats.
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1998, 1999a,b, 2003a), the generation and/or contribution of reactive nitrogen species (RNS) during aniline-induced toxicity are not known. Therefore, the present studies were focused on establishing the formation of • NO following aniline treatment. Aniline exposure, in this study, resulted in increased NT formation, especially in the sinusoidal cells of the splenic red pulp. Both ONOO− -dependent and -independent mechanisms may be responsible for the increased formation of NT in this macrophageendothelium-rich tissue (Fig. 4). The most probable mechanism could be the reaction of • NO with superoxide (O2 •− ) resulting in the formation of highly reactive ONOO− which nitrates the tyrosine residues (Nakazawa et al., 2000). Other possibility includes the reaction of nitric oxide with oxygen to form nitrogen dioxide that can abstract phenolic hydrogen of the tyrosine residue to form a phenoxy radical that in turn can react with a second nitrogen dioxide to form NT. Since nitrogen dioxide formation is a slow reaction (Beckman et al., 1994) and requires two molecules for each molecule of tyrosine, its contribution in the overall formation of NT could be minimal, but can not be ruled out (Beckman et al., 1992). The process of ONOO− formation can be catalyzed by iron
(Beckman et al., 1992) which is excessively deposited in the spleen of animals treated with aniline or its metabolites, phenylhydoxylamine and nitrosobenzene (Khan et al., 1997a, 1998, 1999a,b, 2000). Increased • NO production through iron-mediated up-regulation of iNOS as well as increased reactive oxygen species could enhance the formation of ONOO− (Aust et al., 1993; Goldstein et al., 1998; Zhou et al., 2000; Chen et al., 2001) leading to NT formation as observed in the present studies. Increases in NT formation have also been observed in other tissues following treatment with ethanol (Baraona et al., 2002), acetaminophen (Hinson et al., 1998; Michael et al., 1999) and silica (Porter et al., 2002), and tyrosine nitration often correlated with toxicity. Our earlier studies suggest that aniline-induced oxidative damage could be a potential mechanism in splenic toxicity (Khan et al., 1997a,b, 1999, 2003a). The present studies further support that contention and add a new dimension that reactive nitrogen species also contribute to aniline-induced oxidative stress. The biosynthesis of • NO by the enzyme NO synthase (NOS) proceeds by the hydroxylation of l-arginine to form NG-hydroxy-l-arginine followed by the conversion of NG-hydroxy-l-arginine to
Fig. 4. Plausible pathways leading to protein nitration in the spleen following aniline insult.
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l-citrulline and • NO (Perry and Marletta, 1998). Our immunohistochemical data showed increased iNOS immunostaining in the red pulp areas of the spleen as a result of aniline exposure. Interestingly, NT localization is congruent with iNOS localization, suggesting the generation of RNS in the immediate vicinity. Induction of iNOS in the spleen following aniline treatment, presumably as a result of iron overload, is also supported by other studies (Chen et al., 2001; Seril et al., 2002). Western blot analysis for nitrated proteins in the post-nuclear fraction of aniline-treated spleen revealed three proteins of 18, 30 and 49 kDa, indicating nitrative modification of proteins was selective. Efforts to characterize the structure and function of these proteins are underway. Even though these studies demonstrate increased NT formation and nitration of specific proteins in the spleen, further studies on the consequences of this nitration on the biological activity of the proteins are needed. In addition, it will be logical to explore the relationship between nitrated proteins and aberration of cell or organ function. Recent studies suggest that nitration of proteins by ONOO− results in the inactivation of ␣1-antiproteinase (Whiteman and Halliwell, 1996; Whiteman et al., 1996), and inhibition of catalase (Keng et al., 2000; Kocis et al., 2002) and superoxide dismutase (Pittman et al., 2002). A role for RNS in injury or disease process is further substantiated by studies using scavenger of ONOO− (glutathione) (Knight et al., 2002) and specific an iNOS inhibitor (aminoguanidine) (Shin et al., 1998; Du et al., 2002), resulting in the reversal of injury. In conclusion, our studies show that exposure to aniline results in marked increases in NT formation, apparently through iNOS induction. Macrophages and sinusoidal cells appear to be major participants as evident from NT and iNOS immunostaining. Nitration of 18, 30 and 49 kDa proteins suggests selective modification of these proteins by • NO. These studies indicate that aniline-induced nitrosative stress could contribute to the splenic toxicity of aniline.
Acknowledgements This publication was made possible by Grant ES06476 from National Institute of Environmental Health Sciences (NIEHS) and Grant HL65416 from
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National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS or NHLBI, NIH.
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Nitrotyrosine formation in splenic toxicity of aniline M. Firoze Khan∗ , Xiaohong Wu, Bhupendra S. Kaphalia, Paul J. Boor, G.A.S. Ansari Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA Received 23 June 2003; received in revised form 28 July 2003; accepted 12 August 2003
Abstract Splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis and development of a variety of sarcomas in rats. However, the mechanisms of this selective splenic toxicity are not well understood. Previously we showed that aniline exposure causes oxidative damage to spleen. To further explore the oxidative mechanisms of aniline toxicity, we evaluated the contributions of nitric oxide. Nitric oxide reacts with superoxide anion to form peroxynitrite, a powerful oxidant that converts the tyrosine residues of proteins to nitrotyrosine (NT). Therefore, aim of this study was to establish the role of nitric oxide through the formation and localization of NT in the spleen of rats exposed to aniline. Male Sprague–Dawley (SD) rats were given 1 mmol/kg per day aniline hydrochloride in water by gavage for 7 days, while the controls received water only. Immunohistochemical analysis for NT showed an intense staining in the red pulp areas of spleen from aniline-treated rats, localized in macrophages and sinusoidal cells. Occasionally mild NT immunostaining was also evident in the white pulp. Western blot analyses of the post-nuclear fraction of the spleens showed major nitrated proteins with molecular weights of 49, 30 and 18 kDa. Immunohistochemical analysis of inducible nitric oxide synthase (iNOS) also showed increased expression in the red pulp of the spleens from aniline-treated rats; the cellular localization was similar to nitrated proteins. These studies suggest that oxidative stress in aniline toxicity also includes aberration in nitric oxide production leading to nitration of proteins. Functional consequences of such nitration will further elucidate the contribution of nitric oxide to the splenic toxicity of aniline. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Nitrotyrosine; iNOS; Oxidative stress; Aniline; Spleen; Immunohistochemistry
1. Introduction Exposure to aniline causes selective toxicity to the spleen leading to vascular congestion, hyperplasia, fibrosis and a variety of sarcomas in rats (Bus and Popp, 1987; Khan et al., 1995, 1997a, 1999a, 2003a,b). Mechanism(s) by which aniline induces selective tox-
∗ Corresponding author. Tel.: +1-409-772-6881; fax: +1-409-747-1763. E-mail address: [email protected] (M.F. Khan).
icity to the spleen is not well understood. In earlier studies, we have shown that aniline exposure results in iron overload (Khan et al., 1997a, 1999a,b), lipid peroxidation (Khan et al., 1997a,b, 1999a), increased formation of MDA–protein adducts (Khan et al., 1999a, 2003a) and protein oxidation (Khan et al., 1999a,b) in the spleen. These results suggest a critical role for iron-catalyzed generation of reactive oxygen species (ROS) in aniline-mediated splenic damage to spleen. Since oxidative and nitrosative stress could occur simultaneously, it was logical to study the possible involvement of nitric oxide (• NO) whose formation and
0300-483X/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2003.08.008
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contribution has not been studied in splenic toxicity of aniline. There is considerable evidence of overproduction of • NO during inflammation (Kooy et al., 1995; Goldstein et al., 1998) and a number of pathological conditions (Shin et al., 1998; Greenacre and Ischiropoulos, 2001; Oldreive and Rice-Evans, 2001). • NO vigorously reacts with superoxide to produce peroxynitrite (ONOO− ) which is a highly reactive nitrogen species (Nakazawa et al., 2000; Greenacre and Ischiropoulos, 2001). One of its reactions is nitration of the ortho position of tyrosine leading to nitrotyrosine (NT) formation in proteins (Greenacre and Ischiropoulos, 2001). Since NT is a stable end product of this reaction, it serves as a very useful biomarker in identifying the generation of • NO in vivo (Ter Steege et al., 1998). Macrophages and endothelial cells are the major biological sources of • NO which can regulate a variety of biological processes (Greenacre and Ischiropoulos, 2001; Bian and Murad, 2001). In the spleen, there are both fixed and mobile macrophages. The tissue-fixed macrophages line the sinusoids of the spleen and have a close relationship with the endothelial cells lining the sinusoids (Bian and Murad, 2001). Aniline treatment has been shown to result in expansion of the red pulp, increases in macrophages and sinusoidal cells (Khan et al., 1993, 1997a, 1999a), and up-regulation of fibrogenic cytokine transforming growth factor- 1 (Khan et al., 2003b). Even though oxidative stress appears to be an important mechanism in the selective damage to spleen following aniline insult, the contribution of • NO in the splenic toxicity is not established. Therefore, this study was focused on establishing the formation, localization and identification of NT residues, along with the regulation of inducible nitric oxide synthase (iNOS) in the spleen of rats treated with aniline.
2. Materials and methods 2.1. Animals and treatments Male Sprague–Dawley (SD) rats (∼225 g), obtained from Harlan Sprague–Dawley Inc. (Indianapolis, IN), were housed in wire-bottom cages over adsorbent paper with free access to tap water and Purina laboratory chow and maintained in a controlled environment ani-
mal room (temperature, 22 ◦ C; relative humidity, 50%; photoperiod, 12 h light/dark cycle) for 7 days prior to treatments. The animals were divided into two groups of five each. One group of animals received 1 mmol/kg per day aniline hydrochloride (Aldrich, Milwaukee, WI) in 0.5 ml of drinking water by gavage for 7 days, while the other group received equal volume of water only and served as controls. The choice of aniline dose was based on our earlier studies that showed oxidative stress in the spleen at this dose (Khan et al., 1997a, 2003b). The animals were euthanized 24 h following the last dose and spleens were aseptically removed immediately, blotted, and weighed. A portion of each spleen was stored at −80 ◦ C, while the remainder was fixed in 10% formalin for immunohistochemical studies described below. 2.2. Immunohistochemical localization of nitrated proteins For immunohistochemical staining, alkaline phosphatase-Fast Red staining method was used as described earlier (Khan et al., 2002, 2003a). The method was standardized for NT immunostaining in the spleen. Briefly, 4 m tissue sections were deparafinized in a 55 ◦ C oven for 1 h and passed through xylene and various concentrations of ethanol, and finally rehydrated with water. The slides were incubated with unmasking buffer (Vector, Burlingame, CA) at 95 ◦ C for 20 min for antigen retrieval and then subsequently incubated with different reagents for blocking the non-specific binding sites, which included peroxidase inhibitor (Pierce, Rockford, IL) for 10 min, levamisole (alkaline phosphatase inhibitor; 0.2%) for 10 min, 0.5% periodic acid for 5 min, avidin and biotin block solutions (Vector) for 10 min each, and normal serum (Signet, Dedham, MA). After removal of excess normal serum, the sections were incubated with rabbit anti-nitrotyrosine antibody (Chemicon) [1:1000 dilution in antibody diluent buffer (Dako, Carpinteria, CA)] for 1 h and then anti-rabbit IgG-biotin [1:1500 (Sigma, St. Louis, MO)] for 20 min at room temperature. The slides were thoroughly washed after each incubation. The Signet ultra streptavidin alkaline phosphatase labeling reagent was added to the tissue sections and incubated for 20 min at room temperature. Finally,
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Fast Red (Biopathology Inc., Okalahoma City, OK) staining was performed according to company’s manual. To establish the specificity of the staining, sets of additional sections were also stained with preimmune serum or anti-NT antibody pre-absorbed with NT-albumin. All sections were finally counterstained with hematoxylin (Gill’s formulation, Vector) and mounted for light microscopic examination. 2.3. Western blot analyses of nitrated proteins Western blot analysis for nitrated proteins was performed in the splenic post-nuclear fractions of control and aniline-treated rats. For the preparation of post-nuclear fractions, spleen homogenates (10%) were made in phosphate-buffered saline (PBS, pH 7.4) containing protease inhibitor cocktail (Sigma). The homogenate was centrifuged at 1000 rpm at 4 ◦ C for 10 min to obtain post-nuclear fraction. Protein was measured by DC-protein assay method (Bio-Rad, Hercules, CA). Western blot analysis of nitrated proteins in the spleen samples was done essentially as described by Towbin et al. (1979) with minor modifications. Briefly, before loading the protein samples onto the gel, protein preparations (200 g) were cleaned by using Pageprep according to company’s manual (Pierce). The proteins were separated electrophoretically by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel) and then transferred electrophoretically to polyvinyl difluoride (PVDF) membrane (Bio-Rad). The non-specific binding sites on the membrane was blocked with 1% casein in PBS at 4 ◦ C overnight and incubated with mouse monoclonal anti-nitrotyrosine antibody (1:70 dilution in 0.25% casein, Cayman Chemical) at room temperature for 3 h, and then incubated with anti-mouse IgG-horseradish peroxidase (1:2000 dilution in 0.25% casein, Cappel) at room temperature for 1 h. Membrane was washed extensively with Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 for about 30 min, and developed using 3,3-diaminobenzidine (DAB)/H2 O2 (Sigma) as substrate. 2.4. Immunohistochemical demonstration of iNOS induction in the spleen Immunohistochemical detection and localization of iNOS was achieved essentially using the method de-
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scribed above for nitrotyrosine. However, the primary antibody used in this case was anti-iNOS (polyclonal, 1:20 dilution, Transduction Laboratory, Lexington, KY).
3. Results 3.1. Immunohistochemical localization of nitrated proteins in the spleen For immunohistochemical localization of nitrated proteins in the spleen, we employed the alkaline phosphatase-Fast Red (red color) methodology as previously described (Khan et al., 2002, 2003a). This method provided an increased contrast in comparison to horseradish peroxidase-diaminobenzidine
Fig. 1. Immunohistochemical staining for nitrated proteins at low power (100×) shows negligible staining in either red pulp (R) or white pulp (W) areas in control spleen (A). In contrast, nitrated proteins show an evident, diffuse, marked increase of staining in red pulp (R) of spleen from aniline-treated rats (B).
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(HRP-DAB, brown color) methodology. The ultimate red color stain allowed us to differentiate the changes in aniline-treated rats, which had extensive brownish deposits of iron-containing proteins (hemosiderin), thus allowing for more definitive localization of nitrated proteins in the spleen. Low power observation of spleen showed little stain of nitrated proteins in control rats (Fig. 1A), whereas diffuse, marked positive stain was evident in expanded red pulp areas of spleen from aniline-treated rats (Fig. 1B). Control
spleen showed only focal staining at higher power confined to the red pulp (Fig. 2A). Spleens from aniline-treated rats, however, showed very intense NT immunoreactivity, especially in the macrophages and sinusoidal cells of the expanded red pulp. Staining was also evident in the peripheral white pulp areas (Fig. 2B). No NT immunoreactivity was evident in the spleen sections incubated with preimmune serum or anti-NT antibody pre-absorbed with NT-albumin (negative controls; data not shown).
Fig. 2. High power view (450×) of immunohistochemical staining for nitrated proteins (A and B) and iNOS (C and D). In all photomicrographs, white pulp areas are in lower left corner and red pulp is in upper right. Nitrated proteins show a marked increase in red pulp area of aniline-treated rats (B) compared to controls (A) as evidenced by diffuse NT immunoreactivity and intense staining evident in clusters of macrophages (B, arrow; not seen in controls). Similarly, iNOS staining is increased throughout red pulp of aniline-treated rats (D) compared to controls (C); intense iNOS immunostaining is again localized in macrophages (D, arrows).
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3.2. Immunohistochemical detection and localization of iNOS in the spleen For the induction and localization of iNOS in the spleen, the same immunohistochemical approach was used as described for NT. Spleens from control rats showed weak and sporadic immunostaining (Fig. 2C). On the other hand, spleens from aniline-treated rats showed intense iNOS immunoreactivity, mostly in the red pulp areas. Occasionally, staining was also evident in the white pulp areas (Fig. 2D). 3.3. Detection of nitrated proteins in the spleen Results of Western blot analysis of post-nuclear fractions of control and aniline-treated rats for nitrated proteins are presented in Fig. 3. Using anti-NT antibodies, three nitrated proteins of molecular weight 18, 30 and 49 kDa were identified. These protein bands were rather faint in the controls, indicating a basal formation of nitrated proteins. On the other
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hand, the nitrated protein bands were very intense in the aniline-treated rats, indicating greater formation of nitrated proteins and correlating with our immunohistochemical results.
4. Discussion The splenotoxic sequence of events, preceding the development of splenic sarcomas following aniline exposure, is sinusoidal congestion, hyperplasia and splenic fibrosis (Bus and Popp, 1987; Khan et al., 1993, 1999a). One serious consequence of such changes in the spleen could be a reduced ability of the spleen to participate in the immune response and/or in its phagocytic function of clearing damaged erythrocytes and infectious organisms from the blood. Therefore, it is important to delineate the mechanism of selective splenotoxic response of aniline. Even though, our earlier studies showed a role for generalized oxidative stress (Khan et al., 1997a,
Fig. 3. Western blot analyses of nitrated proteins (NT–protein adducts) in control and aniline-treated rats. Lane 1: marker proteins; lanes 2–4: controls; lanes 5–7: aniline-treated. NT–protein bands of 18, 30 and 49 kDa are more prominent in aniline-treated rats.
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1998, 1999a,b, 2003a), the generation and/or contribution of reactive nitrogen species (RNS) during aniline-induced toxicity are not known. Therefore, the present studies were focused on establishing the formation of • NO following aniline treatment. Aniline exposure, in this study, resulted in increased NT formation, especially in the sinusoidal cells of the splenic red pulp. Both ONOO− -dependent and -independent mechanisms may be responsible for the increased formation of NT in this macrophageendothelium-rich tissue (Fig. 4). The most probable mechanism could be the reaction of • NO with superoxide (O2 •− ) resulting in the formation of highly reactive ONOO− which nitrates the tyrosine residues (Nakazawa et al., 2000). Other possibility includes the reaction of nitric oxide with oxygen to form nitrogen dioxide that can abstract phenolic hydrogen of the tyrosine residue to form a phenoxy radical that in turn can react with a second nitrogen dioxide to form NT. Since nitrogen dioxide formation is a slow reaction (Beckman et al., 1994) and requires two molecules for each molecule of tyrosine, its contribution in the overall formation of NT could be minimal, but can not be ruled out (Beckman et al., 1992). The process of ONOO− formation can be catalyzed by iron
(Beckman et al., 1992) which is excessively deposited in the spleen of animals treated with aniline or its metabolites, phenylhydoxylamine and nitrosobenzene (Khan et al., 1997a, 1998, 1999a,b, 2000). Increased • NO production through iron-mediated up-regulation of iNOS as well as increased reactive oxygen species could enhance the formation of ONOO− (Aust et al., 1993; Goldstein et al., 1998; Zhou et al., 2000; Chen et al., 2001) leading to NT formation as observed in the present studies. Increases in NT formation have also been observed in other tissues following treatment with ethanol (Baraona et al., 2002), acetaminophen (Hinson et al., 1998; Michael et al., 1999) and silica (Porter et al., 2002), and tyrosine nitration often correlated with toxicity. Our earlier studies suggest that aniline-induced oxidative damage could be a potential mechanism in splenic toxicity (Khan et al., 1997a,b, 1999, 2003a). The present studies further support that contention and add a new dimension that reactive nitrogen species also contribute to aniline-induced oxidative stress. The biosynthesis of • NO by the enzyme NO synthase (NOS) proceeds by the hydroxylation of l-arginine to form NG-hydroxy-l-arginine followed by the conversion of NG-hydroxy-l-arginine to
Fig. 4. Plausible pathways leading to protein nitration in the spleen following aniline insult.
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l-citrulline and • NO (Perry and Marletta, 1998). Our immunohistochemical data showed increased iNOS immunostaining in the red pulp areas of the spleen as a result of aniline exposure. Interestingly, NT localization is congruent with iNOS localization, suggesting the generation of RNS in the immediate vicinity. Induction of iNOS in the spleen following aniline treatment, presumably as a result of iron overload, is also supported by other studies (Chen et al., 2001; Seril et al., 2002). Western blot analysis for nitrated proteins in the post-nuclear fraction of aniline-treated spleen revealed three proteins of 18, 30 and 49 kDa, indicating nitrative modification of proteins was selective. Efforts to characterize the structure and function of these proteins are underway. Even though these studies demonstrate increased NT formation and nitration of specific proteins in the spleen, further studies on the consequences of this nitration on the biological activity of the proteins are needed. In addition, it will be logical to explore the relationship between nitrated proteins and aberration of cell or organ function. Recent studies suggest that nitration of proteins by ONOO− results in the inactivation of ␣1-antiproteinase (Whiteman and Halliwell, 1996; Whiteman et al., 1996), and inhibition of catalase (Keng et al., 2000; Kocis et al., 2002) and superoxide dismutase (Pittman et al., 2002). A role for RNS in injury or disease process is further substantiated by studies using scavenger of ONOO− (glutathione) (Knight et al., 2002) and specific an iNOS inhibitor (aminoguanidine) (Shin et al., 1998; Du et al., 2002), resulting in the reversal of injury. In conclusion, our studies show that exposure to aniline results in marked increases in NT formation, apparently through iNOS induction. Macrophages and sinusoidal cells appear to be major participants as evident from NT and iNOS immunostaining. Nitration of 18, 30 and 49 kDa proteins suggests selective modification of these proteins by • NO. These studies indicate that aniline-induced nitrosative stress could contribute to the splenic toxicity of aniline.
Acknowledgements This publication was made possible by Grant ES06476 from National Institute of Environmental Health Sciences (NIEHS) and Grant HL65416 from
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National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIEHS or NHLBI, NIH.
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