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Nitrosative stress and nitrated proteins in trichloroethene-mediated autoimmunity.

Wang G, Wang J, Luo X, Ansari GA, Khan MF - PLoS ONE (2014)

Bottom Line: TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers.Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB.These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, has been linked to a variety of autoimmune diseases (ADs) including SLE, scleroderma and hepatitis. Mechanisms involved in the pathogenesis of ADs are largely unknown. Earlier studies from our laboratory in MRL+/+ mice suggested the contribution of oxidative/nitrosative stress in TCE-induced autoimmunity, and N-acetylcysteine (NAC) supplementation provided protection by attenuating oxidative stress. This study was undertaken to further evaluate the contribution of nitrosative stress in TCE-mediated autoimmunity and to identify proteins susceptible to nitrosative stress. Groups of female MRL +/+ mice were given TCE, NAC or TCE + NAC for 6 weeks (TCE, 10 mmol/kg, i.p., every 4th day; NAC, ∼ 250 mg/kg/day via drinking water). TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers. Proteomic analysis identified 14 additional nitrated proteins in the livers of TCE-treated mice. Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB. Remarkably, NAC supplementation not only ameliorated TCE-induced nitrosative stress as evident from decreased iNOS, NT, nitrated proteins, NF-κB p65 activation and increased GSH levels, but also the markers of autoimmunity, as evident from decreased levels of autoantibodies in the sera. These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for designing therapeutic strategies.

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2D gel profile of proteins in livers of control (A, B), TCE-treated (C, D) or TCE+NAC-treated (E, F) MRL+/+ mice.The protein spots for nitrated proteins, identified by western blot (B, D and F), also matched to the 2D gel protein profile in (A, C and F - CBB G250 stained). The numbers used for the spots are the same as in the Tables.
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pone-0098660-g005: 2D gel profile of proteins in livers of control (A, B), TCE-treated (C, D) or TCE+NAC-treated (E, F) MRL+/+ mice.The protein spots for nitrated proteins, identified by western blot (B, D and F), also matched to the 2D gel protein profile in (A, C and F - CBB G250 stained). The numbers used for the spots are the same as in the Tables.

Mentions: Since our ELISA results showed increased NT in the livers following TCE exposure, it was of interest to identify the nitrated proteins. To achieve that, 2D gels for each sample were run in duplicate, one for CBB G250 stain and the other one for Western blot analysis. The 2D gel protein profile of a representative control sample is shown in Fig. 5A, and Fig. 5B is the corresponding Western blot map of nitrated proteins of the same sample. The nitrated proteins shown in black circles (Fig. 5B) were matched to the 2D gel protein profiles in Fig. 5A. Similarly, the 2D gel protein profiles of liver extract from a TCE-treated or a TCE+NAC-treated mouse is shown in Fig. 5C and Fig. 5E, whereas Fig. 5D and Fig. 5F show the corresponding Western blot map of nitrated protein spots of the same samples. The black circled spots (Fig. 5D and Fig. 5F) were also matched to the 2D gel protein profiles in Fig. 5C and Fig. 5E. As shown in the figures, the nitrated proteins spots were found in the pI range of pH 4.9 to 8.8 and molecular weights of 19.1 to 128.3 kDa. The 2D gel protein profiles of randomly chosen three controls, three TCE-treated and three TCE+NAC-treated mice matched well when analyzed by using SameSpots program (data not shown). Among the three controls, we identified 21 nitrated protein spots which were also present in TCE-treated protein extracts. The protein extracts from TCE-treated mice showed remarkably greater protein nitration, which was evident from increased number of spots for nitrated proteins. We identified a total of 39 nitrated protein spots among the three TCE-treated samples, out of which 18 were found only in the TCE-treated protein extracts (Fig. 5D). Interestingly, we only identified a total of 24 nitrated protein spots among the three TCE+NAC-treated samples, which were also observed in the TCE-treated protein extracts (Fig. 5F). These nitrated protein spots (same protein spots present in at least two samples) were picked up and subjected to MALDI TOF/TOF MS/MS analyses.


Nitrosative stress and nitrated proteins in trichloroethene-mediated autoimmunity.

Wang G, Wang J, Luo X, Ansari GA, Khan MF - PLoS ONE (2014)

2D gel profile of proteins in livers of control (A, B), TCE-treated (C, D) or TCE+NAC-treated (E, F) MRL+/+ mice.The protein spots for nitrated proteins, identified by western blot (B, D and F), also matched to the 2D gel protein profile in (A, C and F - CBB G250 stained). The numbers used for the spots are the same as in the Tables.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043737&req=5

pone-0098660-g005: 2D gel profile of proteins in livers of control (A, B), TCE-treated (C, D) or TCE+NAC-treated (E, F) MRL+/+ mice.The protein spots for nitrated proteins, identified by western blot (B, D and F), also matched to the 2D gel protein profile in (A, C and F - CBB G250 stained). The numbers used for the spots are the same as in the Tables.
Mentions: Since our ELISA results showed increased NT in the livers following TCE exposure, it was of interest to identify the nitrated proteins. To achieve that, 2D gels for each sample were run in duplicate, one for CBB G250 stain and the other one for Western blot analysis. The 2D gel protein profile of a representative control sample is shown in Fig. 5A, and Fig. 5B is the corresponding Western blot map of nitrated proteins of the same sample. The nitrated proteins shown in black circles (Fig. 5B) were matched to the 2D gel protein profiles in Fig. 5A. Similarly, the 2D gel protein profiles of liver extract from a TCE-treated or a TCE+NAC-treated mouse is shown in Fig. 5C and Fig. 5E, whereas Fig. 5D and Fig. 5F show the corresponding Western blot map of nitrated protein spots of the same samples. The black circled spots (Fig. 5D and Fig. 5F) were also matched to the 2D gel protein profiles in Fig. 5C and Fig. 5E. As shown in the figures, the nitrated proteins spots were found in the pI range of pH 4.9 to 8.8 and molecular weights of 19.1 to 128.3 kDa. The 2D gel protein profiles of randomly chosen three controls, three TCE-treated and three TCE+NAC-treated mice matched well when analyzed by using SameSpots program (data not shown). Among the three controls, we identified 21 nitrated protein spots which were also present in TCE-treated protein extracts. The protein extracts from TCE-treated mice showed remarkably greater protein nitration, which was evident from increased number of spots for nitrated proteins. We identified a total of 39 nitrated protein spots among the three TCE-treated samples, out of which 18 were found only in the TCE-treated protein extracts (Fig. 5D). Interestingly, we only identified a total of 24 nitrated protein spots among the three TCE+NAC-treated samples, which were also observed in the TCE-treated protein extracts (Fig. 5F). These nitrated protein spots (same protein spots present in at least two samples) were picked up and subjected to MALDI TOF/TOF MS/MS analyses.

Bottom Line: TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers.Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB.These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, has been linked to a variety of autoimmune diseases (ADs) including SLE, scleroderma and hepatitis. Mechanisms involved in the pathogenesis of ADs are largely unknown. Earlier studies from our laboratory in MRL+/+ mice suggested the contribution of oxidative/nitrosative stress in TCE-induced autoimmunity, and N-acetylcysteine (NAC) supplementation provided protection by attenuating oxidative stress. This study was undertaken to further evaluate the contribution of nitrosative stress in TCE-mediated autoimmunity and to identify proteins susceptible to nitrosative stress. Groups of female MRL +/+ mice were given TCE, NAC or TCE + NAC for 6 weeks (TCE, 10 mmol/kg, i.p., every 4th day; NAC, ∼ 250 mg/kg/day via drinking water). TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers. Proteomic analysis identified 14 additional nitrated proteins in the livers of TCE-treated mice. Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-κB. Remarkably, NAC supplementation not only ameliorated TCE-induced nitrosative stress as evident from decreased iNOS, NT, nitrated proteins, NF-κB p65 activation and increased GSH levels, but also the markers of autoimmunity, as evident from decreased levels of autoantibodies in the sera. These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for designing therapeutic strategies.

Show MeSH
Related in: MedlinePlus