N-Acetylcysteine protects against trichloroethene-mediated autoimmunity by attenuating oxidative stress

Abstract

Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, is known to induce autoimmunity both in humans and animal models. However, mechanisms underlying TCE-mediated autoimmunity remain largely unknown. Previous studies from our laboratory in MRL+/+ mice suggest that oxidative stress may contribute to TCE-induced autoimmune response. The current study was undertaken to further assess the role of oxidative stress in TCE-induced autoimmunity by supplementing with an antioxidant N-acetylcysteine (NAC). Groups of female MRL+/+ mice were given TCE, NAC or TCE+NAC for 6 weeks (TCE, 10mmol/kg, i.p., every 4th day; NAC, 250mg/kg/day through drinking water). TCE exposure led to significant increases in serum levels of anti-nuclear, anti-dsDNA and anti-Sm antibodies. TCE exposure also led to significant induction of anti-malondiadelhyde (MDA)- and anti-hydroxynonenal (HNE)-protein adduct antibodies which were associated with increased ANA in the sera along with increased MDA-/HNE-protein adducts in the livers and kidneys, and increases in protein oxidation (carbonylation) in the sera, livers and kidneys, suggesting an overall increase in oxidative stress. Moreover, TCE exposure also resulted in increased release of IL-17 from splenocytes and increases in IL-17 mRNA expression. Remarkably, NAC supplementation attenuated not only the TCE-induced oxidative stress, IL-17 release and mRNA expression, but also the markers of autoimmunity, as evident from decreased levels of ANA, anti-dsDNA and anti-Sm antibodies in the sera. These results provide further support to a role of oxidative stress in TCE-induced autoimmune response. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for preventive and/or therapeutic strategies.

Keywords: Autoimmune diseases; Carbonylation; N-Acetylcysteine; Oxidative stress; Trichloroethene.

Fig. 1

Anti-MDA-protein adduct antibodies (A) and anti-HNE-protein adduct antibodies in the sera of MRL+/+ mice treated with TCE or TCE + NAC for 6 weeks. The values are means ± SD. *p < 0.05 vs. controls; ^#p < 0.05 vs. TCE-treated mice.

Fig. 2

MDA-protein adducts (A) and HNE-protein adducts (B) in the livers and kidneys of MRL+/+ mice treated with TCE or TCE + NAC for 6 weeks. The values are means ± SD. *p < 0.05 vs. controls; ^#p < 0.05 vs. TCE-treated mice.

Fig. 3

Protein carbonyl content in the sera (A), livers (B) or kidneys (C) of MRL+/+ mice treated with TCE or TCE + NAC for 6 weeks. The values are means ± SD. *p < 0.05 vs. controls; ^#p < 0.05 vs. TCE-treated mice.

Fig. 4

The ANA (A), anti-dsDNA antibodies (B) and anti-SM antibodies in the sera of MRL+/+ mice treated with TCE or TCE + NAC for 6 weeks. The values are means ± SD. *p < 0.05 vs. controls; ^#p < 0.05 vs. TCE-treated mice.

Fig. 5

Correlation of (A) serum anti-MDA-protein adduct antibodies, and (B) serum anti-HNE-protein adduct antibodies with serum ANA. Spearman’s rank correlation was used to calculate correlation coefficient.

Fig. 6

Release of IL-17 into splenocyte cultures of MRL+/+ mice treated with TCE or TCE + NAC for 6 weeks. Splenocytes were incubated with medium alone, ConA (5 μg/ml) or anti-mouse CD3/CD28 antibodies (2.5 μg/ml/1 μg/ml) for 72 h, and the release of IL-17 into cultures was measured by specific ELISAs. The values are means ± SD of 6 animals in each group. US: unstimulated (medium alone) cells. *p < 0.05 vs. controls; ^#p < 0.05 vs. TCE-treated mice.

Fig. 7

Real-time PCR analysis of IL-17 mRNA expression in the spleens of MRL+/+ mice treated with TCE or TCE + NAC for 6 weeks. The fold changes of IL-17 mRNA (2^−ΔΔCT) expression in spleens are shown. Values are means ± SD. *p < 0.05 vs. controls; ^#p < 0.05 vs. TCE-treated mice.

Fig. 8

The plausible mechanisms of TCE-induced autoimmune response and its attenuation by NAC supplementation.