M. Firoze Khan

Markers of Oxidative and Nitrosative Stress in Systemic Lupus Erythematosus: Correlation with Disease Activity

OBJECTIVE Free radical-mediated reactions have been implicated as contributors in a number of autoimmune diseases, including systemic lupus erythematosus (SLE). However, the potential for oxidative/nitrosative stress to elicit an autoimmune response or to contribute to disease pathogenesis, and thus be useful when determining a prognosis, remains largely unexplored in humans. This study was undertaken to investigate the status and contribution of oxidative/nitrosative stress in patients with SLE. METHODS Sera from 72 SLE patients with varying levels of disease activity according to the SLE Disease Activity Index (SLEDAI) and 36 age- and sex-matched healthy controls were evaluated for serum levels of oxidative/nitrosative stress markers, including antibodies to malondialdehyde (anti-MDA) protein adducts and to 4-hydroxynonenal (anti-HNE) protein adducts, MDA/HNE protein adducts, superoxide dismutase (SOD), nitrotyrosine (NT), and inducible nitric oxide synthase (iNOS). RESULTS Serum analysis showed significantly higher levels of both anti-MDA/anti-HNE protein adduct antibodies and MDA/HNE protein adducts in SLE patients compared with healthy controls. Interestingly, not only was there an increased number of subjects positive for anti-MDA or anti-HNE antibodies, but also the levels of both of these antibodies were statistically significantly higher among SLE patients whose SLEDAI scores were > or = 6 as compared with SLE patients with lower SLEDAI scores (SLEDAI score <6). In addition, a significant correlation was observed between the levels of anti-MDA or anti-HNE antibodies and the SLEDAI score (r = 0.734 and r = 0.647, respectively), suggesting a possible causal relationship between these antibodies and SLE. Furthermore, sera from SLE patients had lower levels of SOD and higher levels of iNOS and NT compared with healthy control sera. CONCLUSION These findings support an association between oxidative/nitrosative stress and SLE. The stronger response observed in serum samples from patients with higher SLEDAI scores suggests that markers of oxidative/nitrosative stress may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.

Protein adducts of malondialdehyde and 4-hydroxynonenal in livers of iron loaded rats: quantitation and localization

Pathophysiological mechanisms for hepatocellular injury, fibrosis and/or cirrhosis in hepatic iron overload are poorly understood. An increase in intracellular transit pool of iron can catalyze peroxidation of lipids to produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Covalent binding of such lipid aldehydes with proteins may cause impairment in cellular function and integrity. This investigation was focused on quantitative determination of MDA and HNE-protein adducts, and to establish a correlation between iron deposition and formation and localization of MDA and HNE-protein adducts, using immunohistochemistry. To achieve iron overload, male SD rats were fed a 2.5% carbonyl iron-supplemented diet for six weeks, while control animals received standard diet. Total iron as well as low molecular weight chelatable iron (LMWC-Fe) in the hepatic tissue of rats fed the iron supplemented diet increased significantly ( approximately 14- and approximately 15-fold, respectively). Quantitative ELISA for MDA-and HNE-protein adducts showed remarkable increases of 186 and 149%, respectively, in the liver homogenates of rats fed the iron-supplemented diet. Sections of liver stained for iron showed striking iron deposits in periportal (zone 1) hepatocytes, which was less dramatic in midzonal (zone 2) cells. Livers from iron-loaded rats showed strong, diffuse staining for both MDA and HNE adducts, which was highly pronounced in centrilobular (zone 3) hepatocytes, but was also evident in midzonal cells (zone 2). The demonstration of greater formation of both MDA and HNE-protein adducts provides evidence of iron-catalyzed lipid peroxidation in vivo. Although in this model of iron overload there was no evidence of tissue injury, our results provide an account of some of the initiating factors or early molecular events in hepatocellular damage that may lead to the pathological manifestations seen in chronic iron overload.

LIPID PEROXIDATION-DERIVED ALDEHYDE-PROTEIN ADDUCTS CONTRIBUTE TO TRICHLOROETHENE-MEDIATED AUTOIMMUNITY VIA ACTIVATION OF CD4+ T CELLS

Lipid peroxidation is implicated in the pathogenesis of various autoimmune diseases. Lipid peroxidation-derived aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are highly reactive and bind to proteins, but their role in eliciting an autoimmune response and their contribution to disease pathogenesis remain unclear. To investigate the role of lipid peroxidation in the induction and/or exacerbation of autoimmune response, 6-week-old autoimmune-prone female MRL+/+ mice were treated for 4 weeks with trichloroethene (TCE; 10 mmol/kg, ip, once a week), an environmental contaminant known to induce lipid peroxidation. Sera from TCE-treated mice showed significant levels of antibodies against MDA-and HNE-adducted proteins along with antinuclear antibodies. This suggested that TCE exposure not only caused increased lipid peroxidation, but also accelerated autoimmune responses. Furthermore, stimulation of cultured splenic lymphocytes from both control and TCE-treated mice with MDA-adducted mouse serum albumin (MDA-MSA) or HNE-MSA for 72 h showed significant proliferation of CD4+ T cells in TCE-treated mice as analyzed by flow cytometry. Also, splenic lymphocytes from TCE-treated mice released more IL-2 and IFN-gamma into cultures when stimulated with MDA-MSA or HNE-MSA, suggesting a Th1 cell activation. Thus, our data suggest a role for lipid peroxidation-derived aldehydes in TCE-mediated autoimmune responses and involvement of Th1 cell activation.

Acute hematopoietic toxicity of aniline in rats

In the present study, acute hematopoietic toxicity of aniline as a function of time was investigated in rats. The animals were given a single oral dose of aniline hydrochloride (2 mmol/kg) and euthanized at zero (control), 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h following the treatment. The blood methemoglobin level increased dramatically and attained a peak level of 37% (31 fold greater than the controls) at 0.5 h. Thereafter, the increases were less pronounced and the level declined with time. Spleen weight to body weight ratio remained unchanged up to 24 h, but increased approximately 25% at 48 h. Lipid peroxidation (MDA content) in the spleen increased by 39% at 24 h and remained steady even at 48 h. MDA-protein adducts, as quantitated by a competitive ELISA, showed 94, 126 and 265% increases in the spleen homogenates at 12, 24 and 48 h, respectively, following the treatment. However, no changes were observed in the splenic protein oxidation. Morphological examination showed congestion of splenic blood vessels and marked expansion of red pulp at 24 and 48 h. These studies suggest that aniline related changes in the blood are reflected very early as evident from increases in the methemoglobin content, whereas changes in the spleen appear later and are characterized by splenic weight changes, increased lipid peroxidation, MDA-protein adduct formation and morphological changes after a single high dose exposure. The increased lipid peroxidation in the spleen also suggests that free radical-mediated reactions could be the potential mechanism of splenic toxicity of aniline and lipid peroxidation precedes protein oxidation.

Fatty acid conjugates of xenobiotics

In this review, we discuss the formation and toxicity of fatty acid conjugates of xenobiotics. Conjugates formed in vivo and in vitro and those detected as contaminants are reviewed. Due to the lipophilic nature of these conjugates, they may accumulate in various body organs and cause toxic manifestations. In vivo formation of these fatty acid conjugates appears to be catalyzed by enzyme(s). Fatty acid ethyl esters are the most widely studied esters and have been implicated in the onset or pathogenesis of myocardial and pancreatic diseases in alcoholics. In experimental animals, studies on 2-chloroethyl linoleate, palmitoylpentachlorophenol and oleoyl and linoleoyl anilides clearly indicate that lipid conjugates of xenobiotics are involved in target organ toxicity. These findings warrant further detailed studies to isolate and identify other fatty acid conjugates and to evaluate their toxicity. Thorough toxicokinetic and metabolic studies are also needed to identify putative toxic agents. Identifying these agents could help in understanding the mechanism of pathogenesis associated with lipid conjugation. Finally fatty acid conjugates of drugs (prodrugs), have been shown to have increased half-lives and long-lasting dose-response. Thus these conjugates may be useful for enhancing the therapeutic potential of drugs and should be explored further.

Chronic exposure to trichloroethene causes early onset of SLE-like disease in female MRL +/+ mice

Trichloroethene (TCE) exacerbates the development of autoimmune responses in autoimmune-prone MRL +/+ mice. Although TCE-mediated autoimmune responses are associated with an increase in serum immunoglobulins and autoantibodies, the underlying mechanism of autoimmunity is not known. To determine the progression of TCE-mediated immunotoxicity, female MRL +/+ mice were chronically exposed to TCE through the drinking water (0.5 mg/ml of TCE) for various periods of time. Serum concentrations of antinuclear antibodies increased after 36 and 48 weeks of TCE exposure. Histopathological analyses showed lymphocyte infiltration in the livers of MRL +/+ mice exposed to TCE for 36 or 48 weeks. Lymphocyte infiltration was also apparent in the pancreas, lungs, and kidneys of mice exposed to TCE for 48 weeks. Immunoglobulin deposits in kidney glomeruli were found after 48 weeks of exposure to TCE. Our results suggest that chronic exposure to TCE promotes inflammation in the liver, pancreas, lungs, and kidneys, which may lead to SLE-like disease in MRL +/+ mice.

Activation of oxidative stress-responsive signaling pathways in early splenotoxic response of aniline

Aniline exposure causes toxicity to the spleen, which leads to a variety of sarcomas, and fibrosis appears to be an important preneoplastic lesion. However, early molecular mechanisms in aniline-induced toxicity to the spleen are not known. Previously, we have shown that aniline exposure results in iron overload and induction of oxidative stress in the spleen, which can cause transcriptional upregulation of fibrogenic/inflammatory cytokines via activation of oxidative stress (OS)-responsive signaling pathways. To test this mechanism, male SD rats were treated with aniline (1mmol/kg/day via gavage) for 7 days, an experimental condition that precedes the appearance of fibrosis. Significant increases in both NF-kappaB and AP-1 binding activity was observed in the nuclear extracts of splenocytes from aniline-treated rats as determined by ELISAs, and supported by Western blot data showing increases in p-IkappaBalpha, p-p65 and p-c-Jun. To understand the upstream signaling events which could account for the activation of NF-kappaB and AP-1, phosphorylation patterns of IkappaB kinases (IKKalpha and IKKbeta) and mitogen-activated protein kinases (MAPKs) were pursued. Our data showed remarkable increases in both p-IKKalpha and p-IKKbeta in the splenocytes from aniline-treated rats, suggesting their role in the phosphorylation of both IkappaBalpha and p65 subunits. Furthermore, aniline exposure led to activation of all three classes of MAPKs, as evident from increased phosphorylation of extracellular-signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK1/2) and p38 MAPKs, which could potentially contribute to the observed activation of both AP-1 and NF-kappaB. Activation of upstream signaling molecules was also associated with simultaneous increases in gene transcription of cytokines IL-1, IL-6 and TNF-alpha. The observed sequence of events following aniline exposure could initiate a fibrogenic and/or tumorigenic response in the spleen.

Malondialdehyde-protein adducts in the spleens of aniline-treated rats: Immunochemical detection and localization

Previously it was reported that aniline exposure in rats induces increased lipid peroxidation and formation of malondialdehyde (MDA)-protein adducts in the spleen. In order to further elucidate the role of MDA-protein adducts in the splenic toxicity of aniline, studies were conducted to detect and localize these adducts in the spleen. Rabbit polyclonal antisera to MDA-keyhole limpet hemocyanin were employed for immunohistochemical localization and Western blot analyses of MDA-protein adducts in the spleens of aniline-treated (65 mg/kg/d aniline in the drinking water for 30 d) and control rats. For immunohistochemical localization of MDA-protein adducts in the spleen, a new approach using alkaline phosphatase-fast red (red color) to demonstrate bound primary antibodies was adopted, providing a sharper and increased contrast compared to horseradish peroxidase-diaminobenzidine (brown color) methodology. This new approach allowed us to differentiate the changes in aniline-treated spleens, which had extensive brownish deposits of iron proteins. Spleens from aniline-treated rats showed intense staining for these adducts in the red pulp areas (where iron was also localized), especially within the sinusoidal macrophages. Spleens from control rats showed only mild staining for adducts and only traces of iron. Western blot analyses of splenic microsomal proteins from aniline-treated and control rats showed the presence of 13 different MDA-modified proteins. However, 26-, 32-, and 44-kD proteins were more prominent in the aniline-treated rats. The colocalization of MDA-protein adducts with iron in the red pulp of the spleen suggests that iron-catalyzed lipid peroxidation leading to formation of MDA-protein adducts could be a potential mechanism for splenic toxicity of aniline.

Cellular and biochemical indices of bronchoalveolar lavage for detection of lung injury following insult by airborne toxicants

Cellular and biochemical profiles of bronchoalveolar lavage (BAL) material after inhalation or intratracheal exposure to various airborne toxicants clearly reflect that BAL has the potential of being a useful tool for the rapid screening of lung injury. The cellular and biochemical responses not only predict inflammation, extent of tissue damage and toxic nature of the substances, but could also help in understanding the molecular mechanisms of pathogenicity. Depending upon the changes of BAL in animals acutely exposed to a pulmonary toxicant, future in-depth studies along with complete histopathological evaluations could be made. Also, the assessment of macromolecules of pharmacological importance in the lavage, especially the secretory products of alveolar macrophages and other lung cell types, could be very useful in predicting the toxic potential of various airborne substances and could also serve as important indicators of developing chronic lung diseases and, therefore, necessitate further studies.

IRON EXACERBATES ANILINE-ASSOCIATED SPLENIC TOXICITY

Our earlier studies have shown that aniline exposure in rats causes time- and dose-dependent accumulation of iron in the spleen, which may exacerbate aniline splenotoxicity by catalyzing free-radical reactions. The present studies were conducted to test whether aniline-induced splenic toxicity could be potentiated by iron overload. For 30 d male Sprague-Dawley rats received the following treatments: 0.5 mmol/kg/d aniline hydrochloride (AH) by gavage (AH group); 3% carbonyl iron-supplemented diet (IR group); 0.5 mmol/kg/d AH by gavage and iron-supplemented diet (AH + IR group); or no treatments (controls). Treatment-related significant increases in total iron, low molecular weight chelatable iron, lipid peroxidation, and protein oxidation were observed in the spleens of all the groups compared to control. However, these changes were much greater in the combined AH + IR group. The aniline-induced morphological changes in the spleen were consistent with our earlier observations, but were more pronounced in the AH + IR group. The increased toxicity, as evident from greater oxidative stress and morphological changes in the AH + IR group, suggests that iron potentiates the splenic toxicity of aniline.