Autophagy dysregulation in trichloroethene-mediated inflammation and autoimmune response.

Trichloroethene (TCE), an organic solvent extensively used for degreasing metals, can cause inflammatory autoimmune disorders [i.e., systemic lupus erythematosus (SLE) and autoimmune hepatitis] from both environmental and occupational exposure. Autophagy has emerged as a pivotal pathogenic factor in various autoimmune diseases. However, role of autophagy dysregulation in TCE-mediated autoimmunity is largely unknown. Here, we investigate whether autophagy dysregulation contributes to pathogenesis of TCE-mediated autoimmune responses. Using our established mouse model, we observed TCE-treated mice had elevated MDA-protein adducts, microtubule-associated protein light chain 3 conversion (LC3-II/LC3-I), beclin-1, phosphorylation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) phosphorylation in the livers of MRL+ /+ mice. Suppression of oxidative stress with antioxidant N-acetylcysteine (NAC) effectively blocked TCE-mediated induction of autophagy markers. On the other hand, pharmacological autophagy induction with rapamycin significantly reduced TCE-mediated hepatic inflammation (NLRP3, ASC, Caspase1 and IL1-ß mRNA levels), systemic cytokines (IL-12 and IL-17) and autoimmune responses (ANA and anti-dsDNA levels). Taken together, these results suggest that autophagy plays a protective role against TCE-mediated hepatic inflammation and autoimmunity in MRL+ /+ mice. These novel findings on the regulation of autophagy could help in designing therapeutic strategies for chemical exposure-mediated autoimmune responses.

Differential Expression of miRNAs in Trichloroethene-Mediated Inflammatory/Autoimmune Response and Its Modulation by Sulforaphane: Delineating the Role of miRNA-21 and miRNA-690.

Trichloroethene (TCE), an occupational and ubiquitous environmental contaminant, is associated with the induction of autoimmune diseases (ADs). Although oxidative stress plays a major role in TCE-mediated autoimmunity, the underlying molecular mechanisms still need to be delineated. Altered non-coding RNAs, including the expression of microRNAs (miRNAs), can influence target genes, especially related to apoptosis and inflammation, and contribute to ADs. Therefore, the objective of this study was to delineate the contribution of miRNAs in TCE-mediated inflammatory and autoimmune response. To achieve this, we treated female MRL+/+ mice with TCE (10 mmol/kg in corn oil, i.p., every fourth day) with/without antioxidant sulforaphane (SFN; 8 mg/kg in corn oil, i.p., every other day) for 6 weeks. With the use of miRNA microarray, 293 miRNAs were analyzed, which included 35 miRNAs that were relevant to inflammation and ADs. Among those 35 miRNAs, 8 were modulated by TCE and/or TCE+SFN exposure. TCE treatment led to increased expression of 3 miRNAs and also decreased expression of 3 miRNAs. Interestingly, among the 35 differentially expressed miRNAs, antioxidant SFN modulated the expression of 6 miRNAs. Based on the microarray findings, we subsequently focused on two miRNAs (miRNA-21 and miRNA-690), which are known to be involved in inflammation and autoimmune response. The increases in miRNA-21 and miR-690 (observed using miRNA microarray) were further validated by RT-PCR, and the TCE-mediated increases in miR-21 and miR-690 were ameliorated by SFN treatment. Modulating miR-21 and miR-690 by respective inhibitors or mimics suppressed the expression of NF-κB (p65) and IL-12 in RAW 264.7 cells. Our findings suggest a contributory role of miR-21 and miR-690 in TCE-mediated and its metabolite dichloroacetyl chloride (DCAC)-mediated inflammation and autoimmune response and support that antioxidant SFN could be a potential therapeutic candidate for inflammatory responses and ADs.

Gut microbiome-host interactions in driving environmental pollutant trichloroethene-mediated autoimmunity.

Trichloroethene (TCE), a widely used industrial solvent, is associated with the development of autoimmune diseases (ADs), including systemic lupus erythematosus and autoimmune hepatitis. Increasing evidence support a linkage between altered gut microbiome composition and the onset of ADs. However, it is not clear how gut microbiome contributes to TCE-mediated autoimmunity, and initial triggers for microbiome-host interactions leading to systemic autoimmune responses remain unknown. To achieve this, female MRL+/+ mice were treated with 0.5 mg/ml TCE for 52 weeks and fecal samples were subjected to 16S rRNA sequencing to determine the microbiome composition. TCE exposure resulted in distinct bacterial community revealed by ß-diversity analysis. Notably, we observed reduction in Lactobacillaceae, Rikenellaceae and Bifidobacteriaceae families, and enrichment of Akkermansiaceae and Lachnospiraceae families after TCE exposure. We also observed significantly increased colonic oxidative stress and inflammatory markers (CD14 and IL-1ß), and decreased tight junction proteins (ZO-2, occludin and claudin-3). These changes were associated with increases in serum antinuclear and anti-smooth muscle antibodies and cytokines (IL-6 and IL-12), together with increased PD1 + CD4+ T cells in TCE-exposed spleen and liver tissues. Importantly, fecal microbiota transplantation (FMT) using feces from TCE-treated mice to antibiotics-treated mice induced increased anti-dsDNA antibodies and hepatic CD4+ T cell infiltration in the recipient mice. Our studies thus delineate how imbalance in gut microbiome and mucosal redox status together with gut inflammatory response and permeability changes could be the key factors in contributing to TCE-mediated ADs. Furthermore, FMT studies provide a solid support to a causal role of microbiome in TCE-mediated autoimmunity.

Redox-sensitive Nrf2 and MAPK signaling pathways contribute to trichloroethene-mediated autoimmune disease progression.

Trichloroethene (TCE) exposure is associated with the induction of autoimmune diseases (ADs). Although oxidative stress plays a major role in TCE-mediated autoimmunity, the underlying molecular mechanisms still need to be delineated. Dysregulation of redox-sensitive nuclear factor (erythroid-derived 2)-like2 (Nrf2), resulting in uncontrolled antioxidant and cytoprotective genes, and pro-inflammatory MAPK signaling pathways could be critical in TCE-mediated disease progression. This study was, therefore, focused on establishing status and contribution of Nrf2 and MAPK signaling in TCE-mediated inflammatory and autoimmune responses, especially during disease progression. To achieve these objectives, time-response studies were conducted by treating female MRL+/+ mice with TCE (0.5 mg/mL, a dose relevant to human exposure) for 24, 36 and 52 wks. TCE exposure led to reduction in Nrf2 expression, but increased phos-NF-κB (p65) and iNOS along with increased phosphorylation of MAPKs (p38, ERK and JNK) and downstream pro-inflammatory cytokines IL-12, TNF-α and RANTES in the livers in a time-dependent manner. These changes were also associated with time-dependent increases in liver protein carbonyls and induction of serum anti-dsDNA antibodies (marker of systemic lupus erythematosus disease), further supporting the role of oxidative stress and Nrf2/MAPK signaling in TCE-mediated autoimmune response progression. The mechanistic role of MAPK in TCE-mediated autoimmunity was further established by treating MRL+/+ mice with sulforaphane (SFN; 8 mg/kg, i.p., every other day) along with TCE (10 mmol/kg, i.p., every 4th day) for 6 wks using an established protocol, and by in vitro treatment of T cells with dichloroacetyl chloride (a TCE metabolite) with/without p38 MAPK inhibitor. SFN treatment attenuated the TCE-mediated phosphorylation of p38 MAPK. More importantly, treatment with SFN or p38 inhibitor led to suppression of downstream pro-inflammatory cytokines IL-12 and TNF-α. These findings thus support the contribution of Nrf2 and MAPK signaling pathways and help in delineating novel potential therapeutic targets against TCE-mediated autoimmunity.

Aberrant Gut Microbiome Contributes to Intestinal Oxidative Stress, Barrier Dysfunction, Inflammation and Systemic Autoimmune Responses in MRL/lpr Mice.

Microbiome composition and function have been implicated as contributing factors in the pathogenesis of autoimmune diseases (ADs), including systemic lupus erythematosus (SLE), rheumatoid arthritis and autoimmune hepatitis (AIH). Furthermore, dysbiosis of gut microbiome is associated with impaired barrier function and mucosal immune dysregulation. However, mechanisms by which gut microbiome contributes to the ADs and whether antioxidant treatment can restore gut homeostasis and ameliorate the disease outcome are not known. This study was, therefore, focused on examining the involvement of gut microbiome and host responses in the pathogenesis of SLE using unique female mouse models (C57BL/6, MRL+/+ and MRL/lpr) of 6 and 18 weeks with varying degrees of disease progression. Fecal microbiome diversity and composition, gut oxidative stress (OS), barrier function and inflammation, as well as systemic autoimmunity were determined. Interestingly, each mouse strain had distinct bacterial community as revealed by ß-diversity. A lower Firmicutes/Bacteroidetes ratio in 6-week-old MRL/lpr mice was observed, evidenced by decrease in Peptostreptococcaceae under Firmicutes phylum along with enrichment of Rikenellaceae under Bacteroidetes phylum. Additionally, we observed increases in colonic OS [4-hydroxynonenal (HNE)-adducts and HNE-specific immune complexes], permeability changes (lower tight junction protein ZO-2; increased fecal albumin and IgA levels) and inflammatory responses (increased phos-NF-κB, IL-6 and IgG levels) in 18-week-old MRL/lpr mice. These changes were associated with markedly elevated AD markers (antinuclear and anti-smooth muscle antibodies) along with hepatic portal inflammation and severe glomerulonephritis. Notably, antioxidant N-acetylcysteine treatment influenced the microbial composition (decreased Rikenellaceae; increased Akkeransiaceae, Erysipelotrichaceae and Muribaculaceae) and attenuated the systemic autoimmunity in MRL/lpr mice. Our data thus show that gut microbiome dysbiosis is associated with increased colonic OS, barrier dysfunction, inflammatory responses and systemic autoimmunity markers. These findings apart from delineating a role for gut microbiome dysbiosis, also support the contribution of gut OS, permeability changes and inflammatory responses in the pathogenesis of ADs.

Interplay and roles of oxidative stress, toll-like receptor 4 and Nrf2 in trichloroethene-mediated autoimmunity.

Previous studies in MRL+/+ mice suggest involvement of oxidative stress (OS) in trichloroethene (TCE)-mediated autoimmunity. However, molecular mechanisms underlying the autoimmunity remain to be fully elucidated. Even though toll-like receptors (TLRs) and Nuclear factor (erythroid-derived 2)-like2 (Nrf2) pathways are implicated in autoimmune diseases (ADs), interplay of OS, TLR and Nrf2 in TCE-mediated autoimmune response remains unexplored. This study was, therefore, undertaken to clearly establish a link among OS, TLR4 and Nrf2 pathways in TCE-induced autoimmunity. Groups of female MRL+/+ mice were treated with TCE, sulforaphane (SFN, an antioxidant) or TCE + SFN (TCE, 10 mmol/kg, i.p., every 4th day; SFN, 8 mg/kg, i.p., every other day) for 6 weeks. TCE exposure led to greater formation of serum 4-hydroxynonenal (HNE)-protein adducts, HNE-specific circulating immune complexes (CICs) and protein carbonyls which were associated with significant increases in serum antinuclear antibodies (ANAs). Moreover, incubation of splenocytes from TCE-treated mice with HNE-modified proteins resulted in enhanced splenocyte proliferation and cytokine release evidenced by increased expression of cyclin D3, Cyclin-dependent kinase 6 (CDK6) and phospho-pRb as well as increased release of IL-6, TNF-α and INF-γ. More importantly, TCE exposure resulted in increased expression of TLR4, MyD88, IRAK4, NF-kB and reduced expression of Nrf2 and HO-1 in the spleen. Remarkably, SFN supplementation not only attenuated TCE-induced OS, upregulation in TLR4 and NF-kB signaling and downregulation of Nrf2, but also ANA levels. These results, in addition to providing further support to a role of OS, also suggest that an interplay among OS, TLR4 and Nrf2 pathways contributes to TCE-mediated autoimmune response. Attenuation of TCE-mediated autoimmunity by SFN provides an avenue for preventive and/or therapeutic strategies for ADs involving OS.

Enhancing the Nrf2 Antioxidant Signaling Provides Protection Against Trichloroethene-mediated Inflammation and Autoimmune Response.

Trichloroethene (trichloroethylene, TCE) and one of its reactive metabolites dichloroacetyl chloride (DCAC) are associated with the induction of autoimmunity in MRL+/+ mice. Although oxidative stress plays a major role in TCE-/DCAC-mediated autoimmunity, the underlying molecular mechanisms still need to be delineated. Nuclear factor (erythroid-derived 2)-like2 (Nrf2) is an oxidative stress-responsive transcription factor that binds to antioxidant responsive element (ARE) and provides protection by regulating cytoprotective and antioxidant gene expression. However, the potential of Nrf2 in the regulation of TCE-/DCAC-mediated autoimmunity is not known. This study thus focused on establishing the role of Nrf2 and consequent inflammatory responses in TCE-/DCAC-mediated autoimmunity. To achieve this, we pretreated Kupffer cells (KCs) or T cells with/without tert-butylhydroquinone (tBHQ) followed by treatment with DCAC. In both KCs and T cells, DCAC treatment significantly downregulated Nrf2 and HO-1 expression along with induction of Keap-1 and caspase-3, NF-κB (p65), TNF-α, and iNOS, whereas pretreatment of these cells with tBHQ attenuated these responses. The in vitro findings were further verified in vivo by treating female MRL+/+ mice with TCE along with/without sulforaphane. TCE exposure in mice also led to reduction in Nrf2 and HO-1 but increased phospho-NF-κB (p-p65) and iNOS along with increased anti-dsDNA antibodies. Interestingly, sulforaphane treatment led to amelioration of TCE-mediated effects, resulting in Nrf2 activation and reduction in inflammatory and autoimmune responses. Our results show that TCE/DCAC mediates an impairment in Nrf2 regulation. Attenuation of TCE-mediated autoimmunity via activation of Nrf2 supports that antioxidants sulforaphane/tBHQ could be potential therapeutic agents for autoimmune diseases.

Redox regulation of hepatic NLRP3 inflammasome activation and immune dysregulation in trichloroethene-mediated autoimmunity.

Trichloroethene (TCE) exposure is associated with the development of various autoimmune diseases (ADs), including autoimmune hepatitis (AIH) and systemic lupus erythematosus (SLE), potentially through the generation of excessive reactive oxygen and nitrogen species (RONS; oxidative stress). However, the mechanisms by which oxidative stress contributes to these TCE-mediated ADs are not fully understood, and are the focus of current investigation. Female MRL+/+ mice were treated with TCE along with or without antioxidant N-acetylcysteine (NAC) for 6 weeks (TCE, 10 mmol/kg, i. p., every 4th day; NAC, 250 mg/kg/day via drinking water). TCE-treated mice had elevated antinuclear antibodies (ANA) and 4-hydroxynonenal (HNE)-specific circulating immune complexes, suggesting the association of TCE-induced oxidative stress with autoimmune response. In addition, TCE exposure led to prominent lobular inflammation with sinusoid dilation, increased sinusoidal cellularity and increased staining for proliferating cell nuclear antigen (PCNA), confirming inflammatory and hepatocellular cell proliferation. Importantly, TCE exposure resulted in the activation of hepatic inflammasome (NLRP3 and caspase-1) and up-regulation of pro-inflammatory cytokine IL-1ß, and these changes were attenuated by NAC supplementation. TCE treatment also led to dysregulation of hepatic immune response as evident from markedly increased hepatic lymphocyte infiltration (especially B cells) and imbalance between Tregs (decreased) and Th17 cells (increased). Interestingly, TCE-mediated dysregulation of various hepatic and splenic immune cells was also effectively attenuated by NAC. Taken together, our findings provide evidence for TCE-mediated inflammasome activation, infiltration of various immune cells, and skewed balance of Treg and Th17 cells in the liver. The attenuation of TCE-mediated hepatic inflammasome activation and immune responses by NAC further supports a critical role of oxidative stress in TCE-mediated inflammation and autoimmunity. These novel findings could help in designing therapeutic strategies for such ADs.

Cytochrome P450 2E1-deficient MRL+/+ mice are less susceptible to trichloroethene-mediated autoimmunity: Involvement of oxidative stress-responsive signaling pathways.

Reactive trichloroethene (TCE) metabolites and oxidative stress are involved in TCE-mediated autoimmunity, as evident from our earlier studies in MRL+/+ mice. However, molecular mechanisms underlying the autoimmunity remain largely unknown. Cytochrome P450 2E1 (CYP2E1), the major enzyme responsible for TCE metabolism, could contribute to TCE-induced toxic response through free radical generation. The current study was, therefore, aimed to further evaluate the significance of TCE metabolism leading to oxidative stress and autoimmune response by using MRL+/+ mice that lack CYP2E1. The Cyp2e1-null MRL+/+ mice were generated by backcrossing Cyp2e1-null mice (B6N; 129S4-Cyp2e1) to MRL +/+ mice. Female MRL+/+ and Cyp2e1-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4th day) for 6 weeks; their respective controls received corn oil only. TCE treatment in MRL+/+ mice induced oxidative stress, evident from significantly increased serum malondiadelhyde (MDA)-protein adducts, their antibodies and reduced liver GSH levels. TCE treatment also modulated Nrf2 pathway with decreased Nrf2 and HO-1, and elevated NF-κB (p65) expression in the liver. TCE exposure also led to increases in serum antinuclear antibodies (ANA) and anti-double stranded DNA antibodies (anti-dsDNA). Although TCE treatment in Cyp2e1-null MRL+/+ mice also led to increases in serum MDA-protein adducts and their antibodies, changes in liver GSH, Nrf2, HO-1 and NF-κB along with increases in serum ANA, anti-dsDNA, the alterations in the oxidative stress and autoimmunity markers in these mice were less pronounced compared to those in MRL+/+ mice. These findings support the contribution of CYP2E1-mediated TCE metabolism in autoimmune response and an important role of Nrf2 pathway in TCE-mediated autoimmunity.

Contribution of poly(ADP-ribose)polymerase-1 activation and apoptosis in trichloroethene-mediated autoimmunity.

Trichloroethene (TCE), a common environmental toxicant and widely used industrial solvent, has been implicated in the development of various autoimmune diseases (ADs). Although oxidative stress has been involved in TCE-mediated autoimmunity, the molecular mechanisms remain to be fully elucidated. These studies were, therefore, aimed to further explore the contribution of oxidative stress to TCE-mediated autoimmune response by specifically assessing the role of oxidative DNA damage, its repair enzyme poly(ADP-ribose)polymerase-1 (PARP-1) and apoptosis. To achieve this, groups of female MRL +/+ mice were treated with TCE, TCE plus N-acetylcysteine (NAC) or NAC alone (TCE, 10 mmol/kg, i.p., every 4th day; NAC, 250 mg/kg/day in drinking water) for 6 weeks. TCE treatment led to significantly higher levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the livers compared to controls, suggesting increased oxidative DNA damage. TCE-induced DNA damage was associated with significant activation of PARP-1 and increases in caspase-3, cleaved caspase-8 and -9, and alterations in Bcl-2 and Bax in the livers. Moreover, the TCE-mediated alterations corresponded with remarkable increases in the serum anti-ssDNA antibodies. Interestingly, NAC supplementation not only attenuated elevated 8-OHdG, PARP-1, caspase-3, cleaved caspase-9, and Bax, but also the TCE-mediated autoimmune response supported by significantly reduced serum anti-ssDNA antibodies. These results suggest that TCE-induced activation of PARP-1 followed by increased apoptosis presents a novel mechanism in TCE-associated autoimmune response and could potentially lead to development of targeted preventive and/or therapeutic strategies.

Environmental Agents, Oxidative Stress and Autoimmunity.

Oxidative stress (OS) plays an important role in the pathogenesis of a variety of autoimmune diseases (ADs) and many environmental agents participate in this process. Environmental agents, including trichloroethylene (TCE), silica, pristane, mercury, and smoke, are known to induce an autoimmune response, potentially through OS-mediated mechanisms. Here, we focus on unraveling the targets and signaling pathways that have been mechanistically linked with OS, as a result of exposure to these and numerous other environmental agents, and their impact on the immune system in triggering ADs. Antioxidants and molecular targets impeding autoimmunity by targeting specific signaling pathways are also reviewed. The review not only provides an overview of the current knowledge and evidence showing strong associations between environmental exposures, OS, and ADs, but also plausible mechanisms by which OS causes autoimmunity/ADs. We also discuss areas that require additional approaches, such as unraveling specific events/mechanisms leading to such devastating diseases and measures to prevent or attenuate such diseases.

Trichloroethene metabolite dichloroacetyl chloride induces apoptosis and compromises phagocytosis in Kupffer Cells: Activation of inflammasome and MAPKs.

Exposure to trichloroethene (TCE), an occupational and ubiquitous environmental contaminant, is associated with the development of several autoimmune diseases, including autoimmune hepatitis (AIH). However, mechanisms contributing to TCE-mediated AIH are not known. Earlier, we have shown that dichloroacetyl chloride (DCAC), one of the reactive metabolites of TCE with strong acylating capability, can elicit an autoimmune response at much lower dose than TCE in female MRL+/+ mice. Furthermore, Kupffer cells (KCs), the liver resident macrophages, are crucial for hepatic homeostasis, but can also participate in the immunopathogenesis of AIH. However, contribution of KCs in TCE-mediated AIH and the underlying mechanisms are not understood. We hypothesized that increased apoptosis and delayed clearance of apoptotic bodies, due to compromised KC function, will result in the breakdown of self-tolerance, autoimmunity, and ultimately AIH. Therefore, using an in vitro model of immortalized mouse KCs, we investigated the contribution of DCAC in TCE-mediated AIH. KCs were treated with different concentrations of DCAC and apoptosis was measured by Annexin V and PI staining. Also, the impact of DCAC on phagocytic potential of KCs was evaluated. Furthermore, markers of inflammasome (NLRP3 and caspase1) were analyzed by real-time PCR and Western blot analysis. DCAC treatment resulted in significantly increased early and late-stage apoptosis, accompanied with inflammasome activation (NLRP3 increases). DCAC treatment resulted in decreased phagocytic function of KCs in a dose-dependent manner, with reduced MFG-E8 levels (phagocytotic function). Furthermore, DCAC exposure led to induction of phos-ERK and phos-AKT signaling. These findings suggest that DCAC induces apoptosis and inflammasome activation, while compromising the phagocytic function of KCs. Our data support that increased apoptosis and impaired KC function by DCAC could be contributory to TCE-mediated AIH.

Autoimmune potential of perchloroethylene: Role of lipid-derived aldehydes.

Tetrachloroethene (perchloroethylene, PCE), an ubiquitous environmental contaminant, has been implicated in inducing autoimmunity/autoimmune diseases (ADs), including systemic lupus erythematosus (SLE) and scleroderma in humans. However, experimental evidence suggesting the potential of PCE in mediating autoimmunity is lacking. This study was, therefore, undertaken to explore PCE's potential in inducing/exacerbating an autoimmune response. Six-week old female MRL+/+ mice, in groups of 6 each, were treated with PCE (0.5mg/ml) via drinking water for 12, 18 and 24weeks and markers of autoimmunity and oxidative stress were evaluated. PCE exposure led to significant increases in serum anti-nuclear antibodies (ANA), anti-dsDNA and anti-scleroderma-70 (anti-Scl-70) antibodies at 18weeks and, to a greater extent at 24weeks, suggesting that PCE exposure exacerbated autoimmunity in our animal model. The increases in autoantibodies were associated with time-dependent increases in malondialdehyde (MDA)-protein adducts and their antibodies, as well as significantly decreased levels of antioxidants GSH and SOD. The splenocytes isolated from mice treated with PCE for 18 and 24weeks showed greater Th17 cell proliferation and increased release of IL-17 in culture supernatants following stimulation with MDA-mouse serum albumin adducts, suggesting that MDA-modified proteins may act as an immunologic trigger by activating Th17 cells and contribute to PCE-mediated autoimmunity. Our studies thus provide an experimental evidence that PCE induces/exacerbates an autoimmune response and lipid-derived aldehydes (such as MDA) contribute to this response.

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

Significance of Lipid-Derived Reactive Aldehyde-Specific Immune Complexes in Systemic Lupus Erythematosus.

Even though systemic lupus erythematosus (SLE) is associated with high morbidity and mortality rates among young and middle-aged women, the molecular mechanisms of disease pathogenesis are not fully understood. Previous studies from our laboratory suggested an association between oxidative stress and SLE disease activity (SLEDAI). To further assess the role of reactive oxygen species (ROS) in SLE, we examined the contribution of lipid-derived reactive aldehydes (LDRAs)-specific immune complexes in SLE. Sera from 60 SLE patients with varying SLEDAI and 32 age- and gender- matched healthy controls were analyzed for oxidative stress and related markers. Patients were divided into two groups based on their SLEDAI scores (<6 and ≥ 6). Both SLEDAI groups showed higher serum 4-hydroxynonenal (HNE)-/malondialdehyde (MDA)-protein adducts and their specific immune complexes (HNE-/MDA-specific ICs) together with IL-17 than the controls, but the levels were significantly greater in the high SLEDAI (≥ 6) group. Moreover, the serum levels of anti-oxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase (CAT) were significantly reduced in both patient groups compared to controls. Remarkably, for the first time, our data show that increased HNE-/MDA-specific ICs are positively associated with SLEDAI and elevated circulating immune complexes (CICs), suggesting a possible causal relationship among oxidative stress, LDRA-specific ICs and the development of SLE. Our findings, apart from providing firm support to an association between oxidative stress and SLE, also suggest that these oxidative stress markers, especially the HNE-/MDA-specific ICs, may be useful in evaluating the prognosis of SLE as well as in elucidating the mechanisms of disease pathogenesis.

iNOS null MRL+/+ mice show attenuation of trichloroethene-mediated autoimmunity: contribution of reactive nitrogen species and lipid-derived reactive aldehydes.

Earlier studies from our laboratory in MRL+/+ mice suggest that free radicals, especially overproduction of reactive nitrogen species (RNS) and lipid-derived reactive aldehydes (LDRAs), are associated with trichloroethene (TCE)-mediated autoimmune response. The current study was undertaken to further assess the contribution of RNS and LDRAs in TCE-mediated autoimmunity by using iNOS-null MRL+/+ mice. iNOS-null MRL+/+ mice were obtained by backcrossing iNOS-null mice (B6.129P2-Nos2(tm1Lau)/J) to MRL +/+ mice. Female MRL+/+ and iNOS-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4(th) day) for 6 weeks; their respective controls received corn oil only. TCE exposure led to significantly increased iNOS mRNA in livers, iNOS protein in livers and sera, increased nitrotyrosine (NT) formation in both livers and sera, induction of MDA-/HNE-protein adducts in livers and their respective antibodies in sera along with significant increases in serum antinuclear antibodies (ANA) and anti-dsDNA in MRL+/+ mice. Even though in iNOS-null MRL+/+ mice, the iNOS and NT levels were negligible in both TCE-treated and untreated groups, TCE treatment still led to significant increases in MDA-/HNE-protein adducts and their respective antibodies along with increases in serum ANA and anti-dsDNA compared to controls. Most remarkably, the increases in serum ANA and anti-dsDNA induced by TCE in the iNOS-null MRL+/+ mice were significantly less pronounced compared to that in MRL+/+ mice. Our results provide further evidence that both RNS and LDRAs contribute to TCE-induced autoimmunity in MRL+/+ mice, and iNOS deficiency attenuates this autoimmune response.

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen.

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a tumorigenic response on chronic aniline exposure.

Disruption of cytochrome P4501A2 in mice leads to increased susceptibility to hyperoxic lung injury.

Hyperoxia contributes to acute lung injury in diseases such as acute respiratory distress syndrome. Cytochrome P450 (CYP) 1A enzymes have been implicated in hyperoxic lung injury, but the mechanistic role of CYP1A2 in pulmonary injury is not known. We hypothesized that mice lacking the gene Cyp1a2 (which is predominantly expressed in the liver) will be more sensitive to lung injury and inflammation mediated by hyperoxia and that CYP1A2 will play a protective role by attenuating lipid peroxidation and oxidative stress in the lung. Eight- to ten-week-old WT (C57BL/6) or Cyp1a2(-/-) mice were exposed to hyperoxia (>95% O2) or maintained in room air for 24-72 h. Lung injury was assessed by determining the ratio of lung weight/body weight (LW/BW) and by histology. Extent of inflammation was determined by measuring the number of neutrophils in the lung as well as cytokine expression. The Cyp1a2(-/-) mice under hyperoxic conditions showed increased LW/BW ratios, lung injury, neutrophil infiltration, and IL-6 and TNF-α levels and augmented lipid peroxidation, as evidenced by increased formation of malondialdehyde- and 4-hydroxynonenal-protein adducts and pulmonary isofurans compared to WT mice. In vitro experiments showed that the F2-isoprostane PGF2-α is metabolized by CYP1A2 to a dinor metabolite, providing evidence for a catalytic role for CYP1A2 in the metabolism of F2-isoprostanes. In summary, our results support the hypothesis that hepatic CYP1A2 plays a critical role in the attenuation of hyperoxic lung injury by decreasing lipid peroxidation and oxidative stress in vivo.

Nitrosative stress and nitrated proteins in trichloroethene-mediated autoimmunity.

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.

Mice deficient in the gene for cytochrome P450 (CYP)1A1 are more susceptible than wild-type to hyperoxic lung injury: evidence for protective role of CYP1A1 against oxidative stress.

Hyperoxia contributes to acute lung injury in diseases such as acute respiratory distress syndrome in adults and bronchopulmonary dysplasia in premature infants. Cytochrome P450 (CYP)1A1 has been shown to modulate hyperoxic lung injury. The mechanistic role(s) of CYP1A1 in hyperoxic lung injury in vivo is not known. In this investigation, we hypothesized that Cyp1a1(-/-) mice would be more susceptible to hyperoxic lung injury than wild-type (WT) mice, and that the protective role of CYP1A1 is in part due to CYP1A1-mediated decrease in the levels of reactive oxygen species-mediated lipid hydroperoxides, e.g., F2-isoprostanes/isofurans, leading to attenuation of oxidative damage. Eight- to ten-week-old male WT (C57BL/6J) or Cyp1a1(-/-) mice were exposed to hyperoxia (>95% O2) or room air for 24-72 h. The Cyp1a1(-/-) mice were more susceptible to oxygen-mediated lung damage and inflammation than WT mice, as evidenced by increased lung weight/body weight ratio, lung injury, neutrophil infiltration, and augmented expression of IL-6. Hyperoxia for 24-48 h induced CYP1A expression at the mRNA, protein, and enzyme levels in liver and lung of WT mice. Pulmonary F2-isoprostane and isofuran levels were elevated in WT mice after hyperoxia for 24 h. On the other hand, Cyp1a1(-/-) mice showed higher levels after 48-72 h of hyperoxia exposure compared to WT mice. Our results support the hypothesis that CYP1A1 protects against hyperoxic lung injury by decreasing oxidative stress. Future research could lead to the development of novel strategies for prevention and/or treatment of acute lung injury.