Chemical-induced Oxidative Stress Research Articles

Investigations of biological activities of aqueous extracts of Prosopis africana in rats with chemical-induced oxidative stress

Investigations of biological activities of aqueous extracts of Prosopis africana in rats with chemical-induced oxidative stress

Aqueous Extract of Cocoa Phenolic Compounds Protects Differentiated Neuroblastoma SH-SY5Y Cells from Oxidative Stress

Cocoa is a rich source of polyphenols, especially flavanols and procyanidin oligomers, with antioxidant properties, providing protection against oxidation and nitration. Cocoa phenolic compounds are usually extracted with methanol/ethanol solvents in order to obtain most of their bioactive compounds; however, aqueous extraction seems more representative of the physiological conditions. In this study, an aqueous extract of cocoa powder has been prepared and chemically characterized, and its potential protective effect against chemically-induced oxidative stress has been tested in differentiated human neuroblastoma SH-SY5Y cells. Neuronal-like cultured cells were pretreated with realistic concentrations of cocoa extract and its major monomeric flavanol component, epicatechin, and then submitted to oxidative stress induced by a potent pro-oxidant. After one hour, production of reactive oxygen species was evaluated by two different methods, flow cytometry and in situ fluorescence by a microplate reader. Simultaneously, reduced glutathione and antioxidant defense enzymes glutathione peroxidase and glutathione reductase were determined and the results used for a comparative analysis of both ROS (reactive oxygen species) methods and to test the chemo-protective effect of the bioactive products on neuronal-like cells. The results of this approach, never tested before, validate both analysis of ROS and indicate that concentrations of an aqueous extract of cocoa phenolics and epicatechin within a physiological range confer a significant protection against oxidative insult to neuronal-like cells in culture.

Comet assay: a versatile but complex tool in genotoxicity testing.

The comet assay is a versatile method for measuring DNA strand breaks in individual cells. It can also be applied to cells isolated from treated animals. In this review, we highlight advantages and limitations of this in vivo comet assay in a regulatory context. Modified versions of the standard protocol detect oxidized DNA bases and may be used to reveal sites of DNA base loss, DNA interstrand crosslinks, and the extent of DNA damage induced indirectly by reactive oxygen species elicited by chemical-induced oxidative stress. The assay is, however, at best semi-quantitative, and we discuss possible approaches to improving DNA damage quantitation and highlight the necessity of optimizing protocol standardization to enhance the comparability of results between laboratories. As a genotoxicity test in vivo, the in vivo comet assay has the advantage over the better established micronucleus erythrocyte test that it can be applied to any organ, including those that are specific targets of chemical carcinogens or those that are the first sites of contact of ingested or inhaled mutagens. We illustrate this by examples of its use in risk assessment for the food contaminants ochratoxin and furan. We suggest that improved quantitation is required to reveal the full potential of the comet assay and enhance its role in the battery of in vivo approaches to characterize the mechanisms of toxicity and carcinogenicity of chemicals and to aid the determination of safe human exposure limits.

Mining a human transcriptome database for chemical modulators of NRF2.

Nuclear factor erythroid-2 related factor 2 (NRF2) encoded by the NFE2L2 gene is a transcription factor critical for protecting cells from chemically-induced oxidative stress. We developed computational procedures to identify chemical modulators of NRF2 in a large database of human microarray data. A gene expression biomarker was built from statistically-filtered gene lists derived from microarray experiments in primary human hepatocytes and cancer cell lines exposed to NRF2-activating chemicals (oltipraz, sulforaphane, CDDO-Im) or in which the NRF2 suppressor Keap1 was knocked down by siRNA. Directionally consistent biomarker genes were further filtered for those dependent on NRF2 using a microarray dataset from cells after NFE2L2 siRNA knockdown. The resulting 143-gene biomarker was evaluated as a predictive tool using the correlation-based Running Fisher algorithm. Using 59 gene expression comparisons from chemically-treated cells with known NRF2 activating potential, the biomarker gave a balanced accuracy of 93%. The biomarker was comprised of many well-known NRF2 target genes (AKR1B10, AKR1C1, NQO1, TXNRD1, SRXN1, GCLC, GCLM), 69% of which were found to be bound directly by NRF2 using ChIP-Seq. NRF2 activity was assessed across ~9840 microarray comparisons from ~1460 studies examining the effects of ~2260 chemicals in human cell lines. A total of 260 and 43 chemicals were found to activate or suppress NRF2, respectively, most of which have not been previously reported to modulate NRF2 activity. Using a NRF2-responsive reporter gene in HepG2 cells, we confirmed the activity of a set of chemicals predicted using the biomarker. The biomarker will be useful for future gene expression screening studies of environmentally-relevant chemicals.

Functional Role of p53 in the Regulation of Chemical-Induced Oxidative Stress.

The nuclear transcription factor p53, discovered in 1979, has a broad range of biological functions, primarily the regulation of apoptosis, the cell cycle, and DNA repair. In addition to these canonical functions, a growing body of evidence suggests that p53 plays an important role in regulating intracellular redox homeostasis through transcriptional and nontranscriptional mechanisms. Oxidative stress induction and p53 activation are common responses to chemical exposure and are suggested to play critical roles in chemical-induced toxicity. The activation of p53 can exert either prooxidant or antioxidant activity, depending on the context. In this review, we discuss the functional role of p53 in regulating chemical-induced oxidative stress, summarize the potential signaling pathways involved in p53's regulation of chemically mediated oxidative stress, and propose issues that should be addressed in future studies to improve understanding of the relationship between p53 and chemical-induced oxidative stress.

The discovery of antioxidants in marine microorganisms and their protective effects on the hepatic cells from chemical-induced oxidative stress

The marine environment is emerging as a biodiversity resource for the discovery of natural molecules or chemical scaffolds with pharmaceutical activity. Marine microbes have a tremendous ability to sense and respond to their surroundings to survive in a stressful environment by producing different molecules. As oxidative stress is directly or indirectly involved in various pathological conditions in humans, we believe that marine-derived antioxidant molecules will have a great prospect as a novel antioxidant molecule. We, in this work, explored the marine microbial resources from the Gulf of Mannar, Bay of Bengal, India. High-throughput screening of antioxidant molecule producing marine microbes has revealed that extract from Kocuria marina CDMP 10, can effectively reduce the DPPH free radical. Methanolic crude extract obtained by the freeze-thawing was fractionated and purified by using activity guided purification with the help of reverse phase HPLC and analysed through UPLC-MS. Chemical analysis, as well as MS-spectra, indicated that marine bacteria K. marina CDMP 10 derived antioxidant fraction contains the short peptides. The antioxidant activity of the three highly hydrophobic peptides, (Ser–Ser–Gln, Phe–Glu, Asp–Ile and Leu–Glu) was confirmed by in vitro as well as a cell-based assay. These small peptide molecules are noncytotoxic and can prevent the human cells from chemical-induced oxidative stress. Ser–Ser–Gln peptide demonstrated a potent free radical scavenging activity in Hepatocellular carcinoma cell lines. This study suggests that these short peptides from K. marina CDMP 10 may serve as a potential pharmaceutical candidate with antioxidant activity.

CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish.

The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(-)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.

Hepatoprotective effect of Solanum surattense leaf extract against chemical- induced oxidative and apoptotic injury in rats

BackgroundOf over 35 Saudi plants traditionally used to treat liver disorders, majority still lack scientific validations. We therefore, evaluated the anti-oxidative, anti-apoptotic and hepatoprotective potential of Solanum surattense leaves total ethanol-extract (SSEE).MethodsThe cytoprotective (4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide/ MTT assay) and anti-apoptotic (caspase-3/7) potential of SSEE (25–200 μg/mL) were assessed in cultured HepG2 cells against dichlorofluorescein (DCFH)-induced toxicity. The hepatoprotective salutation of SSEE (100 and 200 mg/kg.bw/day) in carbon tetrachloride (CCl4)-intoxicated rats was evaluated by serum biochemistry and histopathology. The anti-oxidative activity of SSEE (31.25–500 μg/mL) was tested by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and linoleic acid bleaching assays. Also, SSEE was subjected to qualitative phytochemical analysis, and standardized by validated high-performance liquid chromatography (HPTLC).ResultsSSEE at doses 50, 100 and 200 μg/mL showed HepG2 cell proliferative and protective potential by about 61.0, 67.2 and 95%, respectively through inhibition of caspase-3/7 against DCFH-toxicity. In CCl4-injured rats, SSEE (200 mg/kg) significantly (P < 0.001) normalized serum transaminases, alkaline phosphatase, bilirubin, cholesterol, triglycerides, and total protein, including tissue malondialdehyde and nonprotein sulfhydryls levels, supported by the liver histopathology. SSEE further showed strong in vitro anti-oxidative and anti-lipid peroxidative activities, evidenced by the presence of alkaloids, flavonoids, tannins, sterols and saponins. Identification of β-sitosterol (3.46 μg/mg) strongly supported the anti-oxidative and hepatoprotective salutation of SSEE.ConclusionOur findings suggest the therapeutic potential of S. surattense against chemical-induced oxidative stress and liver damage. However, isolation of the active principles and elucidation of mechanism of action remain to be addressed.

Sufficiency of hypoxia-inducible 2-oxoglutarate dioxygenases to block chemical oxidative stress-induced differentiation of human embryonic stem cells

Hypoxia benefits undifferentiated pluripotent stem cell renewal, and 2-oxoglutarate (2OG) dioxygenases have been implicated in pluripotent stem cell induction and renewal. We show in human embryonic stem cells (hESC) that an ambient oxygen-induced oxidative stress response elicited by culture in a hypoxic atmosphere (0.5% O2) correlates with the expression of 2OG dioxygenases, which oxidise DNA (TET1, 2, 3) and histone H3 (KDM4C), the former reflected by elevation in genomic 5-hydroxymethylcytosine (5hmC). siRNA-mediated targeting of KDM4C and TET1–3 induces hESC differentiation. Under ambient atmospheric oxygen (21% O2), exposure to a low inhibitory concentration of sodium arsenite (NaAsO2, IC10), as a model of chemically-induced oxidative stress, suppresses antioxidant gene expression, reduces mitochondrial membrane potential and induces hESC differentiation. Co-administration of the antioxidant N-acetyl-L-cysteine promoted anti-oxidant, pluripotency and 2OG dioxygenase gene expression, elevated genomic hydroxymethylation and blocked induction of differentiation. Transient ectopic expression of KDM4C or TET1 in ambient atmospheric oxygen achieved the same. Our study substantiates a role for 2OG-dependent dioxygenases in hypoxia's promotion of undifferentiated hESC self-renewal.

Protective effect of Atriplex suberecta extract against oxidative and apoptotic hepatotoxicity.

Atriplex suberecta I. Verd is a known phytomedicinal species of Atriplex; however, studies into its bioactivity remain inconclusive. The in vitro and in vivo antioxidative and hepatoprotective potential of A. suberecta ethanol-extract (ASEE) was assessed in the present study. 1,1-diphenyl-2-picrylhydrazyl radical scavenging and β-carotene bleaching assays revealed that ASEE possesses free radical scavenging and anti-lipid peroxidative activities. These results were supported by the in vitro protection of HepG2 hepatoblastoma cells via abating 2,7-dichlorofluorescein-activated oxidative and apoptotic molecules (caspase-3/-7). In carbon tetrachloride-treated rats, the oral administration of ASEE significantly normalized serum biomarkers of liver function (serum glutamate oxaloacetate, serum pyruvate transaminase, alkaline phosphatase, γ-glutamyl transferase and bilirubin) and the lipid profile (total cholesterol, high-density lipoprotein, low-density lipoprotein, triglycerides and malondialdehyde), including tissue non-protein sulfhydryl and total protein levels. These results were also supported by liver histopathology, which demonstrated that the therapeutic effect of ASEE was comparable to silymarin. Furthermore, phytochemical analysis of ASEE revealed the presence of flavonoids, alkaloids, tannins and saponins. Rutin, an antioxidant flavonoid, was identified using the validated high-performance thin-layer chromatography method. In conclusion, this is the first report on the therapeutic potential of A. suberecta against chemical-induced oxidative stress and liver damage.

Modulatory properties of Telfairia occidentalis leaf extract on pancytopenia, electrolyte imbalance and renal oxidative damage in rats

Telfairia occidentalis is a medicinal plant acclaimed to possess many therapeutic potential. The study was carried out to investigate the possible modulatory effects of aqueous leaf extract of T. occidentalis on chemical-induced oxidative stress, renal dysfunction and pancytopenia in rats. Twenty-four (24) adult male rats were divided into 4 groups of 6 rats each. Group I received distilled water, group II, III and IV were administered 5 mg/kg body weight of cadmium chloride to induce oxidative stress. Group III and IV were treated with 200 and 400 mg/kg body weight respectively of aqueous leaf extract of T. occidentalis for 14 days while rats in group II were left untreated. Results obtained showed that administration of cadmium caused significant suppression of haematopoiesis, depleted endogenous antioxidants and increased lipid peroxidation in the kidney as well as alteration of serum electrolytes. Treatment with graded doses of T. occidentalis leaf extract significantly reversed the cadmium induced-biochemical insults as the extract restored the normal renal integrity and boosted renal antioxidant status in rats. The leaf extract also caused significant boost in haematopoiesis in the rats. This result confirmed that aqueous extract of T. occidentalis effectively maintain electrolyte balance, modulates pancytopenia and oxidative renal damage in rats suggesting its protective potentials on anaemia and renal disorders.

A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae.

Chemical-induced oxidative stress and the biochemical pathways that protect against oxidative damage are of particular interest in the field of toxicology. To rapidly identify oxidative stress-responsive gene expression changes in zebrafish, we developed a targeted panel of antioxidant genes using the Affymetrix QuantiGene Plex (QGP) platform. The genes contained in our panel include eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1a, gstp1, gclc, nqo1, prdx1, gpx1a, sod1, sod2), a stress response gene (hsp70), an inducible DNA damage repair gene (gadd45bb), and three reference genes (actb1, gapdh, hprt1). We tested this platform on larval zebrafish exposed to tert-butyl hydroperoxide (tBHP) and cadmium (Cd), two model oxidative stressors with different modes of action, and compared our results with those obtained using the more common quantitative PCR (qPCR) method. Both methods showed that exposure to tBHP and Cd induced expression of prdx1, gstp1, and hmox1a (2- to 12-fold increase via QGP), indicative of an activated Nrf2 response in larval zebrafish. Both compounds also elicited a general stress response as reflected by elevation of hsp70 and gadd45bb, with Cd being the more potent inducer. Transient changes were observed in sod2 and gpx1a expression, whereas nqo1, an Nrf2-responsive gene in mammalian cells, was minimally affected by either tBHP or Cd chemical exposures. Developmental expression analysis of the target genes by QGP revealed marked upregulation of sod2 between 0-96hpf, and to a lesser extent, of sod1 and gstp1. Once optimized, QGP analysis of these experiments was accomplished more rapidly, using far less tissue, and at lower total costs than qPCR analysis. In summary, the QGP platform as applied to higher-throughput zebrafish studies provides a reasonable cost-effective alternative to qPCR or more comprehensive transcriptomics approaches to rapidly assess the potential for chemicals to elicit oxidative stress as a mechanism of chemical toxicity.

Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology, including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity.One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore, in the current study, we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration.Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM), as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes, NQO1 and SRXN1. Interestingly, exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover, rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH+) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation.

Phycoerythrin averts intracellular ROS generation and physiological functional decline in eukaryotes under oxidative stress.

In vitro antioxidant virtue and life-prolonging effect of phycoerythrin (PE; a pigment protein isolated from Phormidium sp. A09DM) have been revealed in our previous reports (Sonani et al. in Age 36:9717, 2014a; Sonani et al. in Process Biochem 49:1757-1766, 2014b). It has been hypothesized that the PE expands life span of Caenorhabditis elegans (bears large resemblance with human aging pathways) due to its antioxidant virtue. This hypothesis is tested in present study by checking the effect of PE on intracellular reactive oxygen species (ROS) generation and associated physiological deformities using mouse and human skin fibroblasts, C. elegans, and Drosophila melanogaster Oregon R + and by divulging PE's structural attributes responsible for its antioxidant asset. PE treatment displayed noteworthy decrease of 67, 48, and 77% in ROS level in mouse fibroblast (3T3-L1), human fibroblast, and C. elegans N2, respectively, arisen under chemical-induced oxidative stress. PE treatment delayed the development of paraquat-induced Alzheimer phenotype by 14.5% in C. elegans CL4176. Furthermore, PE improved the locomotion of D. melanogaster Oregon R + under oxidative stress with simultaneous up-regulation in super-oxide dismutase and catalase activities. The existence of 52 Glu + Asp + His + Thr residues (having metal ion sequestration capacity), 5 phycoerythrobilin chromophores (potential electron exchangers) in PE's primary structure, and significant hydrophobic patches on the surface of its α- and β-subunits are supposed to collectively contribute in the antioxidant virtues of PE. Altogether, results support the hypothesis that it is the PE's antioxidant asset, which is responsible for its life-prolonging effect and thus could be exploited in the therapeutics of ROS-associated abnormalities including aging and neurodegeneration in eukaryotes.

Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5′-Luc constructs – down to −160bp from the TSS – showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from −160 to −74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene – a well-known p53 activator – increased the expression of the p53 responsive positive control and the CYP2A6-5′-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5′-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6.

OP25 - The pivotal role of proteasome enhancement in ageing, longevity and proteostasis across species

Ageing is a natural biological process, determined by both genetic and environmental/stochastic factors. It is characterized by a gradual decline of physiological function and the eventual failure of organismal homeostasis. The phenomenon is represented in vitro by replicative senescence. The proteasome is one of the major cellular proteolytic systems. It is responsible for the degradation of normal proteins as well as of damaged proteins (abnormal, misfolded and otherwise modified proteins) that tend to accumulate during ageing. Impaired proteasome function has been tightly correlated with ageing both in vivo and in vitro. We have shown that proteasome is down-regulated during replicative senescence of human fibroblasts. Its partial inhibition triggers a premature senescence phenotype that is p53-dependent. By contrast, its genetic activation through overexpression of proteasome subunits confers lifespan extension and maintenance of youthful morphological features for longer. We have also identified natural compounds that activate the transcription factor Nrf2, a key molecule involved in cellular protection against chemically-induced oxidative stress thus promoting proteasome activation. Proteasome induction results in lifespan extension and delayed establishment of senescence morphology, consistent with the effects of proteasomal genetic activation. More recently, we have revealed the results of proteasome activation on the lifespan of the Caenorhabditis elegans model. Our initial results confirm the beneficial effects of the proteasome genetic activation on the lifespan of the nematode. We also provide evidence that proteasome activation might be a potential strategy to minimize protein homeostasis deficiencies underlying aggregation-related diseases such as Alzheimer’s or Huntington’s. We are currently screening for compounds with proteasome activating properties that could be used in preventive or therapeutic approaches against Alzheimer’s disease. In conclusion, our results show that there is a dynamic interconnection between the proteasome, the proteostatic mechanisms and the progression of ageing and age-related diseases.

Morin hydrate attenuates the acrylamide-induced imbalance in antioxidant enzymes in a murine model.

Liver diseases are among the most serious health issues nowadays. Hepatocellular carcinoma, one of the most lethal types of cancer worldwide, can be caused by chemically-induced oxidative stress. In the present study, we aimed to evaluate the protective effects of morin hydrate (MH) against acrylamide (AA)-induced hepatotoxicity in male ICR mice. The mice were randomly allocated into 4 groups [the control, the group subcutaneously injected with AA alone (50 mg/kg body weight), the group subcutaneously injected with AA (50 mg/kg body weight) and MH (5 mg/kg body weight) and the group subcutaneously injected with AA (50 mg/kg body weight) and MH (15 mg/kg body weight) for 5 consecutive days]. Histopathological evaluations were performed and the levels of serum hepatic enzymes were analyzed to determine initial liver injury, and the mice in the AA-treated groups were compared with the mice receiving no treatment and with the mice administered MH in combination with AA. Furthermore, oxidative stress, hepatic inflammation and the levels of DNA damage-related markers were evaluated to determine the extent of liver damage induced by AA within a short-term period. The subcutaneous administration of AA induced severe hepatic injury, and combined treatment with AA and MH resulted in a significant improvement in all evaluated parameters. This recovery was most obvious in the group receiving AA and 15 mg/kg body weight dose of MH. The findings of our study demonstrated that MH protected mice from severe hepatic injury induced by AA. Moreover, MH is a natural polyphenolic compound, and thus it has potential for use in the treatment of severe liver diseases, in place of many synthetic drugs.

Phytonutrient Effects of Date Pit Extract against Azoxymethane--Induced Oxidative Stress in the Rat Colon.

Plants and their by-products offer a diverse mixture of chemical constituents like natural antioxidants. Date- pits are rich in phenolic compounds that have antioxidant potential. The main objective of this study was to investigate the protective effect of a date-pit extract (DPE) against AOM-induced colonic carcinogenicity and oxidative stress. Thirty-two weanling male Sprauge-Dawley rats were randomly divided into four groups (eight rats in each group). All rats were fed basic diet and water ad libitum, and randomly distributed per treatment groups as follows: negative controls injected with normal saline once a week for two weeks, a cancer group injected intra-peritoneally with azoxymethane (15mg/kg body weight) for two consecutive weeks, and DPE treated groups receiving the extract via the oral route (1.5ml/day) for the entire experiment in the presence or absence of AOM injection. Results showed that DPE contained phytonutrients that were capable of inhibiting chemically-induced oxidative stress in the rat colonic cells. In those animals that consumed DPE, a protective effect was observed against AOM-induced oxidative stress in rat colonic cells as evident by a significant decrease in MDA and oxidized DCF formation in AOM injected and DPE fed groups. It is concluded that DPE has potential antioxidant and anticarcinogenic properties.

Overexpression of a monomeric form of the bovine odorant-binding protein protects Escherichia coli from chemical-induced oxidative stress

Mammalian odorant-binding proteins (OBPs) are soluble lipocalins produced in the nasal mucosa and in other epithelial tissues of several animal species, where they are supposed to serve as scavengers for small structurally unrelated hydrophobic molecules. These would include odorants and toxic aldehydes like 4-hydroxy-2-nonenal (HNE), which are end products of lipid peroxidation; therefore OBP might physiologically contribute to preserve the integrity of epithelial tissues under oxidative stress conditions by removing toxic compounds from the environment and, eventually, driving them to the appropriate degradative pathways. With the aim of developing a biological model based on a living organism for the investigation of the antioxidant properties of OBP, here we asked whether the overexpression of the protein could confer protection from chemical-induced oxidative stress in Escherichia coli. To this aim, bacteria were made to overexpress either GCC-bOBP, a redesigned monomeric mutant of bovine OBP, or its amino-terminal 6-histidine-tagged version 6H-GCC-bOBP. After inducing overexpression for 4 h, bacterial cells were diluted in fresh culture media, and their growth curves were followed in the presence of hydrogen peroxide (H2O2) and tert-Butyl hydroperoxide (tBuOOH), two reactive oxygen species whose toxicity is mainly due to lipid peroxidation, and menadione, a redox-cycling drug producing the superoxide ion. GCC-bOBP and 6H-GCC-bOBP were found to protect bacterial cells from the insulting agents H2O2 and tBuOOH but not from menadione. The obtained data led us to hypothesize that the presence of overexpressed OBP may contribute to protect bacterial cells against oxidative stress probably by sequestering toxic compounds locally produced during the first replication cycles by lipid peroxidation, before bacteria activate their appropriate enzyme-based antioxidative mechanisms.

RNA interference of MRCK‐1 in Caenorhabditis elegans to explore oxidative stress responses (802.26)

The Caenorhabditis elegans myotonic dystrophy kinase‐related, Cdc42 binding kinase (MRCK‐1) is activated by the Rho GTPase CDC‐42 (cell division control protein 42), which contains a conserved redox‐sensitive sequence motif on the phosphoryl binding loop. If reactive oxygen species (ROS) can activate CDC‐42, MRCK‐1 may be required to elicit a downstream response to oxidative stress. To test this, mrck‐1 expression was targeted using two RNA interference (RNAi) constructs in transgenic lines of C. elegans that produce green fluorescent protein (GFP) driven by redox‐sensitive promoters. After heat‐shock and chemically‐induced oxidative stress, GFP expression levels were quantified using epifluorescent microscopy. Preliminary data from heat‐shock protein reporter strain CL2070 (hsp‐16.2:GFP) indicate no significant GFP expression changes in the RNAi‐treated worms before or after heat stress, but further studies with different oxidative stressors and reporter strains are underway. If reduction of mrck‐1 is found to affect oxidative stress responses, a new mechanism for prevention of degenerative diseases and aging could be suggested.Grant Funding Source: Supported by the Henson Undergraduate Research fund.