Fluro-Protein C-Phycocyanin Docked Silver Nanocomposite Accelerates Cell Migration through NFĸB Signaling Pathway.

Currently, there is a great demand for the development of nanomedicine aided wound tissue regeneration via silver doped nanoceuticals. Unfortunately, very little research is being carried out on antioxidants-doped silver nanometals and their interaction on the signaling axis during the bio-interface mechanism. In this study, c-phycocyanin primed silver nano hybrids (AgcPCNP) were prepared and analyzed for properties such as cytotoxicity, metal decay, nanoconjugate stability, size expansion, and antioxidant features. Fluctuations in the expression of marker genes during cell migration phenomena in in vitro wound healing scenarios were also validated. Studies revealed that physiologically relevant ionic solutions did not exhibit any adverse effects on the nanoconjugate stability. However, acidic, alkali, and ethanol solutions completely denatured the AgcPCNP conjugates. Signal transduction RT2PCR array demonstrated that genes associated with NFĸB- and PI3K-pathways were significantly (p < 0.5%) altered between AgcPCNP and AgNP groups. Specific inhibitors of NFĸB (Nfi) and PI3K (LY294002) pathways confirmed the involvement of NFĸB signaling axes. In vitro wound healing assay demonstrated that NFĸB pathway plays a prime role in the fibroblast cell migration. In conclusion, the present investigation revealed that surface functionalized AgcPCNP accelerated the fibroblast cell migration and can be further explored for wound healing biomedical applications.

Exploring the Pattern of Metabolic Alterations Causing Energy Imbalance via PPARα Dysregulation in Cardiac Muscle During Doxorubicin Treatment.

Cardiotoxicity by anthracycline antineoplastic drug doxorubicin is one of the systemic toxicity of the cardiovascular system. The mechanism responsible for doxorubicin cardiotoxicity and lipid metabolism remains elusive. The current study tested the hypotheses that the role of peroxisome proliferator-activated receptor α (PPARα) in the progress of doxorubicin-induced cardiomyopathy and its mechanism behind lipid metabolism. In the present study, male rats were subjected to intraperitoneal injection (5-week period) of doxorubicin with different dosages such as low dosage (1.5 mg/kg body weight) and high dosage (15 mg/kg body weight) to induce doxorubicin cardiomyopathy. Myocardial PPARα was impaired in both low dosage and high dosage of doxorubicin-treated rats in a dose-dependent manner. The attenuated level of PPARα impairs the expression of the genes involved in mitochondrial transporter, fatty acid transportation, lipolysis, lipid metabolism, and fatty acid oxidation. Moreover, it disturbs the reverse triacylglycerol transporter apolipoprotein B-100 (APOB) in the myocardium. Doxorubicin elevates the circulatory lipid profile and glucose. Further aggravated lipid profile in circulation impedes the metabolism of lipid in cardiac tissue, which causes a lipotoxic condition in the heart and subsequently associated disease for the period of doxorubicin treatment. Elevated lipids in the circulation translocate into the heart dysregulates lipid metabolism in the heart, which causes augmented oxidative stress and necro-apoptosis and mediates lipotoxic conditions. This finding determines the mechanistic role of doxorubicin-disturbed lipid metabolism via PPARα, which leads to cardiac dysfunction.

Retinoic acid attenuates nuclear factor kappaB mediated induction of NLRP3 inflammasome.

BACKGROUND: Acetylcholine (ACh), a neurotransmitter and a part of the cholinergic system, can modify immune responses. Expression of acetylcholine receptors (AChR) in immune cells, including macrophages, leads to modulation of their function. Inflammasomes are part of the innate immune system and have been linked to a variety of inflammatory diseases. The NLRP3/ASC/caspase-1/IL-1 axis has emerged as a critical signaling pathway in inflammation process initiation. The role of ACh in modulating inflammasomes in macrophages remains relatively under-explored. METHODS: The effect of AChR agonist carbachol on inflammasome expression was investigated using murine and human macrophages. Cell lysates were assessed by western blot for protein analysis. Immunofluorescence studies were used to study the translocation of p65. The experiments were conducted in the presence of NF-ĸB inhibitor, AChR antagonists, and retinoic acid (RA) to study the role of NF-ĸB, ACh receptors, and RA, respectively. RESULTS: We found that carbachol increased the expression of NLRP3 inflammasome (NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18). The treated cells also showed an increase in NF-ĸB activation. The effect of carbachol was diminished by NF-ĸB inhibitor and atropine, a mAChR antagonist. The addition of RA also significantly reduced the effect of carbachol on NLRP3 inflammasomes. CONCLUSIONS: Our current study suggests that carbachol induces NLRP3 inflammasome activation through mAChR and NF-ĸB, and that RA abolishes the inflammatory response. It reveals the potentials of co-administration of RA with cholinergic drugs to prevent inflammatory responses during cholinergic medications.

The emerging role of exosomes in innate immunity, diagnosis and therapy.

Exosomes, which are nano-sized transport bio-vehicles, play a pivotal role in maintaining homeostasis by exchanging genetic or metabolic information between different cells. Exosomes can also play a vital role in transferring virulent factors between the host and parasite, thereby regulating host gene expression and the immune interphase. The association of inflammation with disease development and the potential of exosomes to enhance or mitigate inflammatory pathways support the notion that exosomes have the potential to alter the course of a disease. Clinical trials exploring the role of exosomes in cancer, osteoporosis, and renal, neurological, and pulmonary disorders are currently underway. Notably, the information available on the signatory efficacy of exosomes in immune-related disorders remains elusive and sporadic. In this review, we discuss immune cell-derived exosomes and their application in immunotherapy, including those against autoimmune connective tissue diseases. Further, we have elucidated our views on the major issues in immune-related pathophysiological processes. Therefore, the information presented in this review highlights the role of exosomes as promising strategies and clinical tools for immune regulation.

Development of an in-situ forming, self-healing scaffold for dermal wound healing: in-vitro and in-vivo studies.

The importance of the extra-cellular matrix (ECM) for wound healing has been extensively researched. Understanding its importance, multiple ECM mimetic scaffolds have been developed. However, the majority of such scaffolds are prefabricated. Due to their stiffness, prefabricated scaffolds cannot come into direct contact with the basal skin cells at the wound bed, limiting their efficacy. We have developed a unique wound dressing, using chitosan (CH) and chondroitin sulfate (CS), that can form a porous scaffold (CH-CS PEC) in-situ, at the wound site, by simple mixing of the polymer solutions. As CH is positively and CS is negatively charged, mixing these two polymer solutions would lead to electrostatic cross-linking between the polymers, converting them to a porous, viscoelastic scaffold. Owing to the in-situ formation, the scaffold can come in direct contact with the cells at the wound bed, supporting their proliferation and biofunction. In the present study, we confirmed the cross-linked scaffold formation by solid-state NMR, XRD, and TGA analysis. We have demonstrated that the scaffold had a high viscoelastic property, with self-healing capability. Both keratinocyte and fibroblast cells exhibited significantly increased migration and functional markers expression when grown on this scaffold. In the rat skin-excisional wound model, treatment with the in-situ forming CH-CS PEC exhibited enhanced wound healing efficacy. Altogether, this study demonstrated that mixing CH and CS solutions lead to the spontaneous formation of a highly viscoelastic, porous scaffold, which can support epidermal and dermal cell proliferation and bio-function, with an enhanced in-vivo wound healing efficacy.

Nanoceutical Adjuvants as Wound Healing Material: Precepts and Prospects.

Dermal wound healing describes the progressive repair and recalcitrant mechanism of 12 damaged skin, and eventually, reformatting and reshaping the skin. Many probiotics, nutritional supplements, metal nanoparticles, composites, skin constructs, polymers, and so forth have been associated with the improved healing process of wounds. The exact mechanism of material-cellular interaction is a point of immense importance, particularly in pathological conditions such as diabetes. Bioengineered alternative agents will likely continue to dominate the outpatient and perioperative management of chronic, recalcitrant wounds as new products continue to cut costs and improve the wound healing process. This review article provides an update on the various remedies with confirmed wound healing activities of metal-based nanoceutical adjuvanted agents and also other nano-based counterparts from previous experiments conducted by various researchers.

Retinoic acid abrogates LPS-induced inflammatory response via negative regulation of NF-kappa B/miR-21 signaling.

CONTEXT: Macrophages are essential components of the immune system, with significant roles in inflammation modulation. They can be activated into pro-inflammatory M1 or anti-inflammatory M2 phenotypes, depending on their micro-environment. Molecular factors that modulate macrophage polarization are hot targets for therapeutic strategies to counter chronic inflammatory pathological conditions. OBJECTIVE: The current study aimed to elucidate the molecular mechanisms by which Retinoic acid (RA), a potent immunomodulator, suppresses LPS-induced inflammatory response in macrophages. MATERIALS AND METHODS: RAW 264.7 macrophages were treated with RA and/or LPS, and analyzed for inflammatory genes and miR-21 by PCR. The roles of miR-21 and NF-ĸB signaling pathway were also assessed by knock-down experiments, immunofluorescence, and ChIP assays. RESULTS: Pretreatment with RA quenched the LPS-induced inflammatory responses, including phagocytosis, ROS generation, and NO production. RA shifted the polarization away from the M1 state by negative regulation of IKKα/ß, p65, and miR-21. RA hindered the phosphorylation of IKKα/ß, translocation of p65 into the nucleus, and the subsequent upregulation of miR-21. Knock-in and knock-down experiments showed that miR-21 is central for the polarization shift toward the pro-inflammatory M1 state. CONCLUSION: miR-21 is involved in the LPS-induced pro-inflammatory profile of macrophages and that RA negatively regulates the inflammatory response by targeting NF-ĸB/miR-21 signaling. Our data exposes RA's potential as a pharmacological agent to manipulate miR-21 and counteract hyper-inflammatory response.

Chromatic intervention and biocompatibility assay for biosurfactant derived from Balanites aegyptiaca (L.) Del.

Extraction of biosurfactants from plants is advantageous than from microbes. The properties and robustness of biosurfactant derived from the mesocarp of Balanites aegyptiaca have been reported. However, the dark brown property of biosurfactant and lack of knowledge of its biocompatibility limits its scope. In the present work, the decolorization protocol for this biosurfactant was optimized using hydrogen peroxide. The hemolytic potential and biocompatibility based on cell toxicity and proliferation were also investigated. This study is the first report on the decolorization and toxicity assay of this biosurfactant. For decolorization of biosurfactant, 34 full factorial design was used, and the data were subjected to ANOVA. Results indicate that 1.5% of hydrogen peroxide can decolorize the biosurfactant most efficiently at 40 °C in 70 min at pH 7. Mitochondrial reductase (MTT) and reactive oxygen species (ROS) assays on M5S mouse skin fibroblast cells revealed that decolorized biosurfactant up to 50 µg/mL for 6 h had no significant toxic effect. Hemolysis assay showed ~ 2.5% hemolysis of human RBCs, indicating the nontoxic effect of this biosurfactant. The present work established a decolorization protocol making the biosurfactant chromatically acceptable. Biocompatibility assays confirm its safer use as observed by experiments on M5S skin fibroblast cells under in vitro conditions.

MicroRNA 21 Elicits a Pro-inflammatory Response in Macrophages, with Exosomes Functioning as Delivery Vehicles.

MicroRNAs can regulate inflammatory responses by modulating macrophage polarization. Although microRNA miR-21 is linked to crucial processes involved in inflammatory responses, its precise role in macrophage polarization is controversial. In this study, we investigated the functional relevance of endogenous miRNA-21 and the role of exosomes. RAW 264.7 macrophages were transfected with miR-21 plasmid, and the inflammatory response was evaluated by flow cytometry, phagocytosis, and real-time PCR analysis of inflammatory cytokines. To understand the signaling pathways' role, the cells were treated with inhibitors specific for PI3K or NFĸB. Exosomes from transfected cells were used to study the paracrine action of miR-21 on naive macrophages. Overexpression of miR-21 resulted in significant upregulation of pro-inflammatory cytokines, pushing the cells towards a pro-inflammatory phenotype, with partial involvement of PI3K and NFĸB signal pathways. The cells also secreted miR-21 rich exosomes, which, on delivery to naive macrophages, caused them to exhibit pro-inflammatory activity. The presence of miR-21 inhibitor quenched the inflammatory response. This study validates the pro-inflammatory property of miR-21 with a tendency to foster an inflammatory milieu. Our findings also reinforce the dual importance of exosomal miR-21 as a biomarker and therapeutic target in inflammatory conditions.

Engineering bioactive surfaces on nanoparticles and their biological interactions.

The successful integration of nanoparticles into biomedical applications requires modulation of their surface properties so that the interaction with biological systems is regulated to minimize toxicity for biological function. In the present work, we have engineered bioactive surfaces on gold (Au) and silver (Ag) nanoparticles and subsequently evaluated their interaction with mouse skin fibroblasts and macrophages. The Au and Ag nanoparticles were synthesized using tyrosine, tryptophan, isonicotinylhydrazide, epigallocatechin gallate, and curcumin as reducing and stabilizing agents. The nanoparticles thus prepared showed surface corona and exhibited free radical scavenging and enzyme activities with limited cytotoxicity and genotoxicity. We have thus developed avenues for engineering the surface of nanoparticles for biological applications.

An appraisal on molecular and biochemical signalling cascades during arsenic-induced hepatotoxicity.

Arsenic is a ubiquitous metalloid compound commonly found in the environment, and it is usually found in combination with sulphur and metals. Arsenic is considered as a therapeutic as well as poisoning agent since ancient times. It causes toxic effects on different organs, mainly the liver. In this review, we focused on the molecular mechanism of arsenic-induced hepatotoxicity. Here we envisaged the bridge between arsenic and hepatotoxicity with particular focus on the level of hepatic enzymes such as ALT, AST, and ALP. Here, we attempted to elucidate the role of arsenic in redox imbalance on increased oxidative stress (elevated level of ROS, MDA and NO) and decreased antioxidant levels such as reduced GSH, catalase, and SOD. Oxidative stress induces mitochondrial dysfunction via apoptosis (AKT-PKB, MAPK, PI3/AKT, PKCδ-JNK, AKT/ERK, p53 pathways), fibrosis (TGF-ß/Smad pathway), and necrosis and inflammation (TNF-α, NF-ĸB, IL-1, and IL-6). Along with that, arsenic activates caspases and Bax, decreases Bcl2 through mitochondrial dysfunction, and induces apoptosis regulatory mechanism. We believe the alteration of all these pathways leads to arsenic-induced hepatotoxicity.

c-Phycocyanin primed silver nano conjugates: Studies on red blood cell stress resilience mechanism.

Green synthesis of metal-encased nutraceutical nano-hybrids has been a target for research over the last few years. In the present investigation, we have reported temperature dependent facile synthesis of silver nanoparticles using FDA approved c phycocyanin (cPC). The cPC conjugated silver nanoparticles (AgcPCNPs) were characterized by TEM, Zeta Potential, UV-vis, XPS, FTIR, and CD Spectroscopy. The temperature optimization studies suggested the synthesis of stable AgcPCNPs at 40 °C while at higher temperature system shows aggregated appearance. Molecular docking studies predicted the exclusive interaction of C, D, I, and J chains of cPC with the surface of AgNPs. Moreover, AgcPCNPs significantly (p < 0.1 %) counteract the toxic nature of AgNPs on red blood cell by measuring parameters like total RBC count, % hemolysis, % hematocrit, coagulation time, pH, electrolyte concentrations and degree of blood cell lipid peroxidation by the anti-oxidation mechanism. Skin fibroblast in vitro cell migration result suggeststhat AgcPCNPs enhanced the degree of cell movement towards the wound area. Data obtained collectively demonstrate that AgcPCNPs can be a better agent in the dermal wound healing with reduced toxicity with the bi-phasic advantage of cPC as a wound healer and Ag nano-metal as an anti-bacterial agent.

An appraisal of cuticular wax of Calotropis procera (Ait.) R. Br.: Extraction, chemical composition, biosafety and application.

Plastic and polythene as hydrophobic materials become a grave concern due to their non-biodegradable nature, cumbersome recycling and waste management. Cuticular wax derived from Calotropis procera is explored as an eco-friendly and safe hydrophobic material. The effects of duration of exposure to solvent, solvent type, size and side of the leaf on cuticular wax yield have been studied. Leaf with the smallest area (10 cm2-25 cm2) was found to be the most suitable to isolate the wax. GC-MS analysis of the wax revealed that the wax consists of mainly esters, alkane and alkene. Mitochondrial reductase (MTT) and lactate dehydrogenase (LDH) assay have been carried out on M5S cell line at various concentrations and the results indicate that up to 1 µg/ml (acetone as solvent) and 3 µg/ml (chloroform as solvent) use of wax has no toxic effect. To evaluate the hydrophobic potential of the wax in developing hydrophobic paper water regains and contact angle has been measured. The gain in hydrophobicity of the paper is evident from the rise in contact angle (≥90˚) of paper coated with wax. Scanning electron micrograph and FTIR spectra generated physical and chemical evidence of coating of wax on paper.

Single step formation of biocompatible bimetallic alloy nanoparticles of gold and silver using isonicotinylhydrazide.

Manufacturing nanoparticles with controlled physicochemical properties using environment-friendly routes have potential to open new prospects for a variety of applications. Accordingly, several approaches have been established for manufacturing metal nanoparticles. Many of these approaches entail the use of hazardous chemicals and could be toxic to the environment, and cannot be used readily for biomedical applications. In the present work, we report a single step bio-friendly approach to formulate gold (Au), silver (Ag), and Au-Ag alloy nanoparticles with desired surface corona and composition using isonicotinylhydrazide (INH) as a reducing agent. INH also functioned as a stabilizing agent by enabling a surface corona around the nanoparticles. Remarkably, within a single step INH could also provide a handle in regulating the composition of Au and Ag in bimetallic systems without any additional chemical modification. The physicochemical and surface properties of the different nanoparticles thus obtained have been examined by analytical, spectroscopic and microscopic techniques. Cell cytotoxicity (release of lactate dehydrogenase), cell viability and intracellular reactive oxygen species (ROS) assays confirmed that the Au, Ag, and Au-Ag bimetallic nanoparticles prepared with INH are biocompatible. Finally, the presence of organic surface corona of INH on the nanoparticles was found to impart nanozyme activity and antimycobacterial sensitivity to the nanoparticles.

The pivotal role of microRNA-21 in osteoclastogenesis inhibition by anthracycline glycoside aloin.

Osteopenic disorders such as osteoporosis and rheumatoid arthritis are characterized by excessive bone resorption by osteoclasts relative to bone formation by osteoblasts. MicroRNAs are emerging as key players in bone remodeling, modulating the functions of both osteoblasts and osteoclasts. Among them, miR-21 is highly expressed in osteoclast precursors and is known to regulate genesis, differentiation, and apoptosis of osteoclasts. The pro-osteoclastogenic nature of miR-21 makes it a potential candidate as a therapeutic target to treat bone disorders. We had previously demonstrated that anthroglycoside aloin derived from Aloe vera was effective in promoting osteoblastogenesis and inhibiting osteoclastogenesis. The present study investigated the role of miR-21 in aloin's inhibitory effect on osteoclast differentiation. Aloin effectively suppressed receptor activator of nuclear factor kappa-B (NFĸB) ligand (RankL)-induced miR-21 expression via repression of NFĸB activation. MiR-21 suppression resulted in upregulation of osteoclast suppressor programmed cell death protein 4 (PDCD4), and downregulation of osteoclast marker cathepsin K. Knockdown or gain-of-function studies revealed that miR-21 was pivotal to aloin's inhibitory effect on osteoclastogenesis. This study also highlights the dynamic potential of aloin as a therapeutic agent to treat osteopenic disorders.

Review on molecular and biochemical insights of arsenic-mediated male reproductive toxicity.

Arsenic is a natural metalloid found in abundance, in the environment. Exposure to arsenic can cause health issues due to its carcinogenic nature. The primary source of arsenic contact is drinking water. Exposure to arsenic in drinking water can cause reproductive dysfunction in males through a reduction in testes weight, accessory sex organ weight, viability, and motility of sperm, epididymal sperm count, decreased gonadotrophins level, decreased testosterone, and steroidogenesis disruption. This review focuses on the mechanisms by which arsenic impairs the quality of semen, based on epidemiological observations in humans, and experimental studies in different biological research models. Arsenic-mediated male reproductive toxicity can be induced by various mechanisms such as inhibition of spermatogenesis, testosterone pathway hinderance, oxidative stress, inflammation, genotoxic effects, activation of heat shock proteins, and activation of a signaling pathway in testes (ERK/AKT/NF-kB signaling pathway), among others. The interplay between the principal mechanisms involved needs to be elucidated further in future since an overall examination of arsenic-mediated male reproductive toxicity is still a deficit.

Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study.

The appearance of Mycobacterium tuberculosis strains leading to drug resistance has caused new problems in TB treatment in various parts of the world and forces WHO to declare TB as a global emergency. With the increase of TB drug resistance, it is convinced that a more effective vaccine development will stop the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT83, have been examined as TB vaccine candidate. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of this research is to conduct amplification of MPT83 antigen cloning, and expression of its antigen on E. coli bacteria. From the result of the research, it is expected that raw material to produce TB vaccine as well as a high-quality antigen can be obtained. The band of DNA in PCR product is 660 bp, while the one in pGEMT-Easy-Mpt83 recombinant plasmid is 3678 bp. This is expressed in E. coli BL21 strain and produces 48 kDa protein as well as GST-MPT83 fusion protein.

Deciphering the molecular events during arsenic induced transcription signal cascade activation in cellular milieu.

Anthropogenic sources of arsenic poses and creates unintentional toxico-pathological concerns to humans in many parts of the world. The understanding of toxicity of this metalloid, which shares properties of both metal and non-metal is principally structured on speciation types and holy grail of toxicity prevention. Visible symptoms of arsenic toxicity include nausea, vomiting, diarrhea and abdominal pain. In this review, we focused on the dermal cell stress caused by trivalent arsenic trioxide and pentavalent arsanilic acid. Deciphering the molecular events involved during arsenic toxicity and signaling cascade interaction is key in arsenicosis prevention. FoxO1 and FoxO2 transcription factors, members of the Forkhead/Fox family, play important roles in this aspect. Like Foxo family proteins, ATM/CHK signaling junction also plays important role in DNA nuclear factor guided cellular development. This review will summarize and discuss current knowledge about the interplay of these pathways in arsenic induced dermal pathogenesis.

Role of arsenic exposure in adipose tissue dysfunction and its possible implication in diabetes pathophysiology.

Exposure to arsenic in drinking water can stimulate a diverse number of diseases that originate from impaired lipid metabolism in adipose and glucose metabolism, leading to insulin resistance. Arsenic inhibits differentiation of adipocyte and mediates insulin resistance with diminutive information on arsenicosis on lipid storage and lipolysis. This review focused on different mechanisms and pathways involved in adipogenesis and lipolysis in adipose tissue during arsenic-induced diabetes. Though arsenic is known to cause type2 diabetes through different mechanisms, the role of adipose tissue in causing type2 diabetes is still unclear. With the existing literature, this review exhibits the effect of arsenic on adipose tissue and its signalling events such as SIRT3- FOXO3a signalling pathway, Ras -MAP -AP-1 cascade, PI(3)-K-Akt pathway, endoplasmic reticulum stress protein, C/EBP homologous protein (CHOP10) and GPCR pathway with role of adipokines. There is a need to elucidate the different types of adipokines which are involved in arsenic-induced diabetes. The exhibited information brings to light that arsenic has negative effects on a white adipose tissue (WAT) by decreasing adipogenesis and enhancing lipolysis. Some of the epidemiological studies show that arsenic would causes obesity. Few studies indicate that arsenic might induces lipodystrophy condition. Further research is needed to evaluate the mechanistic link between arsenic and adipose tissue dysfunction which leads to insulin resistance.

Arsenic trioxide induces ROS activity and DNA damage, leading to G0/G1 extension in skin fibroblasts through the ATM-ATR-associated Chk pathway.

Arsenic (As) toxicity is a global health problem, affecting millions of people. Exposure to arsenic, mostly via drinking water, has been associated with cancer of skin, lungs, and blood, in addition to several kinds of skin lesions. The present study focused on the effect of arsenic trioxide (As2O3) on normal skin fibroblast cells. Specifically, the effect of As2O3 on ROS generation and oxidative stress was investigated. Proteins involved in the DNA damage signaling pathway and cell cycle were also studied. As2O3 induced the generation of intracellular ROS. Immunohistochemistry analysis revealed a dose-dependent increase in the number of 8-OHdG-positive cells, an indication of oxidative stress. Cell cycle analysis by flow cytometry demonstrated that As2O3 caused a significant percentage of cells to accumulate in the G0/G1 phase with a concomitant reduction in the S phase. Increases in the activated forms of DNA damage signaling proteins, ATM and ATR, and their effector molecules, Chk2 and p53, were also observed. In addition, expression of oncogene p21 was also increased. The study shows that exposure of normal skin fibroblast cells to As2O3 could lead to cell cycle arrest through ATM/ATR and DNA damage signaling pathways. In conclusion, we report here that arsenic trioxide increases cellular oxidative stress leading to shift in cell cycle and leads to DNA damage through ATM/ATR and the CHK-dependent signaling pathway.