Safrun Mahmood

Effect of Vitamin C Supplementation on Oxidative DNA Damage in an Experimental Model of Lead-Induced Hypertension

Aims: Chronic exposure to lead results in sustained hypertension in humans and experimental animals. We investigated the possible role of reactive oxygen species (ROS) and their impact on DNA damage in lead-induced hypertension. Further the effect of short-term supplementation of vitamin C is also demonstrated. Methods: Male Wistar rats were treated with either lead acetate (100 ppm) alone or lead acetate plus vitamin C (20 mg/rat/day). The control rats were fed regular rat chow. Blood pressure, antioxidants, total antioxidant status as measured by ferric-reducing antioxidant power, nitric oxide (NO) metabolites, malondialdehyde (MDA) and 8-hydroxy 2-deoxyguanosine were determined after 0, 1, 2 and 3 months. Results: The lead-exposed group showed a significant rise in blood pressure, lipid peroxidation (MDA) and a substantial oxidative damage to the DNA. A significant fall in NO metabolites, total antioxidant levels and ferric-reducing antioxidant power was also observed in this group. Concomitant administration of vitamin C ameliorated hypertension, normalized NO levels and abrogated lipid peroxidation. Also, it completely prevented oxidative damage to the DNA. Conclusions: These findings point to enhanced ROS-mediated inactivation and sequestration of NO which can potentially contribute to hypertension, lipid peroxidation, reduced antioxidant status and oxidative DNA damage. The beneficial effects of vitamin C on these parameters support the role of increased ROS activity in the pathogenesis of these abnormalities in this model.

Extracellular Matrix Remodeling in Takayasu's Arteritis: Role of Matrix Metalloproteinases and Adventitial Inflammation

Takayasus arteritis (TA) is an inflammatory fibrosing arteritis affecting predominately the aorta and its main branches. Pathogenesis of this disease remains enigmatic. Despite the numerous studies, the role of adventitia in vascular lesion formation in the setting of TA has been ignored. Virtually nothing is known about the mechanism regulating inflammation in the adventitia in the setting of TA. The present study included subjects with Takayasus arteritis and normal healthy control subjects. Isolated T cells from peripheral blood mononuclear cells (PBMCs) using nylon wool and HUT-78 (human cutaneous T lymphoma cell line) were stimulated with PHA for 24 h. Stimulated cell were fixed with paraformaldehyde and fractionated into membrane, cytosolic and nuclear fractions. These cellular fractions were co-cultured with human fibrosarcoma cell line (HT-1080) and transcriptional expression of matrix metalloproteinases (MMP-1, 3, 9 and TIMP-1) was determined using semiquantitative RT-PCR. Stimulation of MMPs-TIMP synthesis by HT-1080 cells was mimicked by a membranous fraction derived from activated T-cell isolated from TA subjects and activated HUT-78 cells, whereas cytosolic and nuclear fractions were ineffective. In conclusion, for the first time we provide evidence for the presence of a cell surface-specific antigenic moiety on T-cells of TA subjects, which is responsible for activation of fibroblasts (cells predominantly present in adventitia) to enhance MMP production and, therefore, may lead to extracellular matrix degradation.

Changes in the chemical composition of surfactant-like particles secreted by rat small intestine in response to different dietary fats.

Consumption of dietary oil, viz., corn, fish, coconut, or olive, induced the secretion of surfactant-like particles (SLP) in rat intestine. These lipoprotein particles differ in (i) levels of alkaline phosphatase activity, (ii) lipid composition, and (iii) FA composition in response to feeding of different oils. The secreted particles had similar buoyancy (1.07–1.08 g/mL) and cholesterol/phospholipid molar ratios (0.61–0.72) except that feeding coconut oil to rats produced SLP with a low (0.18) cholesterol/phospholipid molar ratio compared to control animals. It is concluded from these observations that feeding different oils induces the secretion of lipoprotein particles in rat intestine with different chemical compositions.

Functional Role of Sialic Acid in IgG Binding to Microvillus Membranes in Neonatal Rat Intestine

A close parallelism exists between sialylation and endocytotic activity of the small intestine during postnatal development in rats. Thus, the binding of 125I-labelled IgG to microvillus membranes and its relationship to membrane sialic acid has been studied in suckling rat intestine, during (a) postnatal development; (b) cortisone-induced precocious maturation, and (c) after desialylation of brush borders by neuraminidase treatment. Neuraminidase-treated membranes exhibited low (42%, p < 0.001) IgG binding. The observed decrease in IgG binding to desialylated membranes was associated with a decrease in the value of affinity constant, (–Ka = 0.4 × 106 M–1 in control and 0.23 × 106 M–1 in desialylated membranes). The number of IgG-binding sites (2.3 nmol/mg protein) was unchanged under these conditions. A similar decrease (50%) in IgG binding to brush borders was also observed in cortisone-injected pups. This was associated with reduced sialic acid (37%) content of the membranes compared to the controls. The value of –Ka was reduced from 0.4 × 106 M–1 in the control to 0.3 × 106 M–1 in the hormone-injected pups. The number of binding sites (n) was decreased from 2.2 to 1.4 nmol/mg protein under these conditions. Low concentrations of calcium (0.1–1.6 mM) in the incubation medium enhanced IgG binding (p < 0.001) to brush borders in pups but there was no change in binding of IgG to the membranes at 2 mM Ca2+ concentration compared to controls. Addition of Zn2+ or Mg2+ did not affect IgG binding under these conditions. These findings suggest a functional role of Ca2+ and sialic acid residues of the membrane glycans in IgG-receptor interactions in suckling rat intestine.

Doxycycline Promotes Carcinogenesis & Metastasis via Chronic Inflammatory Pathway: An In Vivo Approach

Background Doxycycline (DOX) exhibits anti-inflammatory, anti-tumor, and pro-apoptotic activity and is being tested in clinical trials as a chemotherapeutic agent for several cancers, including colon cancer. Materials & Methods In the current study, the chemotherapeutic activity of doxycycline was tested in a rat model of colon carcinogenesis, induced by colon specific cancer promoter, 1,2, dimethylhydrazine (DMH) as well as study the effect of DOX-alone on a separate group of rats. Results Doxycycline administration in DMH-treated rats (DMH-DOX) unexpectedly increased tumor multiplicity, stimulated progression of colonic tumor growth from adenomas to carcinomas and revealed metastasis in small intestine as determined by macroscopic and histopathological analysis. DOX-alone treatment showed markedly enhanced chronic inflammation and reactive hyperplasia, which was dependent upon the dose of doxycycline administered. Moreover, immunohistochemical analysis revealed evidence of inflammation and anti-apoptotic action of DOX by deregulation of various biomarkers. Conclusion These results suggest that doxycycline caused chronic inflammation in colon, small intestine injury, enhanced the efficacy of DMH in tumor progression and provided a mechanistic link between doxycycline-induced chronic inflammation and tumorigenesis. Ongoing studies thus may need to focus on the molecular mechanisms of doxycycline action, which lead to its inflammatory and tumorigenic effects.

Receptor for advanced glycation end products (RAGE), inflammatory ligand EN-RAGE and soluble RAGE (sRAGE) in subjects with Takayasu's arteritis

Takayasus arteritis (TA) is a rare primary and granulomatous large vessel vasculitis of unknown origin that predominantly affects the aorta and itsmajor branches. It has been shown toaffect individuals of anyage, gender and a wide variety of ethnic and racial population worldwide. The pathophysiology of TA is complex and multi-factorial and the exact pathogenesis remains to be elucidated; however, participation of autoimmunity, inflammation and oxidative stress has potentially been implicated [1–3]. Receptor for advanced glycation endproducts (RAGE) is amemberof the immunoglobulin superfamily and is expressed on the surface of various cells e.g. endothelium, mononuclear phagocytes, lymphocytes and smooth muscle cells. Extracellular newly identified RAGE-binding protein (EN-RAGE or S100A12), an inflammatory ligand of RAGE, is a member of the S100 protein family. Since its discovery twodecades back [4], the inflammatory role of RAGE and EN-RAGE interaction is nowwell established in various chronic inflammatory disorders. RAGE has a circulating truncated variant isoform and soluble RAGE (sRAGE), corresponds to its extracellular domain only. By competing with cell-surface RAGE for ligand binding, sRAGE contributes to the removal/neutralization of circulating ligands, thus, functioning as a decoy [5–7]. Previously, we demonstrated low sRAGE levels showing inverse correlation with MMPs in a group of forty TA subjects as compared to controls [8]. Keeping in mind the importance of RAGE biology in various diseases, we thought it worth to determine the expression of RAGE and its inflammatory ligand EN-RAGE at transcriptional level in peripheral blood mononuclear cells (PBMCs) fromTA subjects. For this, 24 subjectswith TA and 24 normal healthy controls were recruited as described previously [3,8,9]. The Institutional Ethics Committee approved the present study. Venous blood was collected from overnight fasted individuals in the morning from antecubital vein and processed as described previously [5,8]. Semi-quantitative RT-PCR was performed for determining the transcriptional expression of RAGE andEN-RAGE in PBMCs [5]. Circulating levels of sRAGE were determined in all the study subjects using commercially available enzyme-linked immunoassay as per manufacturers instructions (DRG00, RD p=0.003, Fig. 1E). However, no significant correlation of sRAGE levels was observed with either RAGE or EN-RAGE mRNA in TA subjects. This is the first report in literature which demonstrates that mRNA levels of RAGE and its ligand EN-RAGE are up-regulated in subjects with TA. This study also demonstrates a positive correlation of RAGE and ENRAGE, in subjectswith TA for thefirst time. Our data is supported by other reports, which demonstrate that RAGE expression increases stably in certain pathological processes, and is associated with ligand-rich microenvironments [6,7]. In a previous study, we demonstrated that RAGE and EN-RAGE genes are augmented in non-diabetic CAD subjects and showed their positive correlation with the severity of disease [5]. Ligation of this receptor with its ligands activates the downstream signaling pathways such as NF-κB [10]. In addition, RAGE and EN-RAGE interactions could further induce the expression of adhesion molecules, migration and proliferation of leukocytes and augment cytokine release from the lymphocytes. Excessive expression of these genes at sites of inflammation may feasibly form a positive feedback loop that may further aggravate underlying inflammation. In corroboration to this, other reports in literature and our work have clearly demonstrated a potential participation of various cytokines, chemokines and MMPs in the pathophysiology of TA [3,8,9,11]. Significantly increased RAGE–EN-RAGE expression in TA further confirms a prevalent oxidative stress and inflammatory milieu in such a situation. Presence of various functional splice variants of RAGE is another important characteristic of this particular receptor. The production of endogenous secretory RAGE by alternative splicing may influence the actions of ligands on the full form of RAGE, located on the plasma membrane [12]. Finding increased RAGE mRNA levels in TA subjects, one

Chemopreventive role of olive oil in colon carcinogenesis by targeting noncoding RNAs and methylation machinery: Chemopreventive role of olive oil in colon carcinogenesis

Epigenetic therapy induced by dietary components has become a strong interest in the field of cancer prevention. Olive oil, a potent dietary chemopreventive agent, control colon cancer, however, its role in epigenetic therapy remains unclear. Thus, we aimed to investigate the effect of olive oil in a preclinical model of colon cancer by targeting genetic and epigenetic mechanisms. DMH was used to induce colon cancer in rats; while olive oil was given to separate group of rats along with DMH treatment. Tumor burden and incidence in DMH and DMH + olive oil‐treated rats was observed by macroscopic examination and histoarchitectural studies. Potent anti‐inflammatory, anti‐angiogenic and pro‐apoptotic activity of olive oil was explored by gene expression and immunohistochemical studies. The effect of olive oil on epigenetic alterations was examined by detecting promoter methylation with MS‐HRM and dysregulation of miRNA by TaqMan MicroRNA Assay. We observed that olive oil administration lowered tumor incidence and inhibited the development of tumors in DMH‐treated rats. Olive oil markedly decreased the expression of inflammatory and angiogenic markers and restored the expression of pro‐apoptotic markers in DMH‐treated rats. Furthermore, the inverse relationship between gene expression and DNA methylation, deviant miRNA pattern and miRNA silencing mediated by aberrant DNA methylation was also seen in DMH‐treated rats, which was potentially reversible upon olive oil treatment. Our study concludes that olive oil may play a role in the epigenetic therapy by altering NF‐κB and apoptotic pathways via targeting noncoding RNAs and methylation machinery that affecting epigenome to prevent colon carcinogenesis.