Transcriptome analysis provides new insights into the tolerance and aerobic reduction of Shewanella decolorationis Ni1-3 to bromate.

As a possible human carcinogen, bromate is easily formed in drinking water and wastewater treatments using advanced oxidation technology. Microbial reduction is a promising method to remove bromate, but little is known about aerobic bromate reduction as well as the molecular mechanism of tolerance and reduction to bromate in bacteria. Herein, bromate reduction by isolate under aerobic conditions was reported for the first time. Shewanella decolorationis Ni1-3, isolated from an activated sludge recently, was identified to reduce bromate to bromide under both aerobic and anaerobic conditions. RNA-Seq together with differential gene expression analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify that bromate triggered the expression of genes for oxidative stress protection (e.g., ohr, msrQ, dsbC, gpo, gorA, and gst), DNA damage repair (e.g., dprA, parA, and recJ), and sulfur metabolism (e.g., cysH, cysK, and cysP). However, the genes for lactate utilization (e.g., lldF and dld), nitrate reduction (e.g., napA and narG), and dissimilatory metal reduction (e.g., mtrC and omcA) were down-regulated in the presence of bromate. The results contribute to revealing the molecular mechanism of resistance and reduction in S. decolorationis Ni1-3 to bromate under aerobic conditions and clarifying the biogeochemical cycle of bromine. KEY POINTS: • Aerobic bromate reduction by pure culture was observed for the first time • Strain Ni1-3 effectively reduced bromate under both aerobic and anaerobic conditions • ROS and SOS response genes were strongly induced in the presence of bromate.

Potassium bromate (KBrO3) modulates oxidative stress and inflammatory biomarkers in sodium hydroxide (NaOH) - induced Crohn's colitis in Wistar rats.

Potassium bromate (KBrO3) present in consumed ozonised water was recently documented to exacerbate experimental gastric ulcer. Information, however, is vague as regards its effects in the colon where water reabsorption occurs. In this study, we observed the possible effects of KBrO3 on oxidative stress and inflammatory biomarkers in sodium hydroxide (NaOH) - induced Crohn's colitis (CC). Wistar rats (180-200 g) were divided into six groups (n = 10): (i) control; (ii) untreated CC (induced by 1.4% NaOH; intra-rectal administration); and (iii-vi) CC treated with vitamin E, KBrO3, vitamin E+KBrO3, and sulphazalazine, respectively, for 7 days. Body weight and stool score were monitored daily. By day 3 and 7, excised colon was evaluated for ulcer scores and biochemical and histological analysis. Blood samples collected on days 3 and 7 were assayed for haematological indices using standard methods. Data were subjected to analysis of variance (ANOVA) and p ≤ 0.05 considered significant. Platelet/lymphocyte ratio, colonic ulcer score, malondialdehyde, and mast cells were significantly decreased while colonic sulfhydryl, and Ca2+- and Na+/K+-ATPase activities were increased following KBrO3 treatment compared with untreated CC. These findings suggest that KBrO3 may mitigate against NaOH-induced CC via inhibiting mast cell population and oxidative and inflammatory content but stimulating colonic sulfhydryl and Ca2+- and Na+/K+-ATPase activities.

Oxidative-stress-driven mutagenesis in the small intestine of the gpt delta mouse induced by oral administration of potassium bromate.

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.

Propolis prevents inhibition of apoptosis by potassium bromate in CCD 841 human colon cell.

Previously, we demonstrated that biotransformation of propolis by some special strains of Lactobacillus plantarum might decrease the allergenic molecules in propolis. In this study, we aimed to investigate the effect of biotransformation of propolis on its antioxidant effect and its protective effect against potassium bromate-induced cancer in human colon cell line. Propolis samples were treated with different solutions (ethanol, polyethylene glycol, and water), and ultrasonication was applied at 40 Hz (5, 10, and 15 minutes) in order to facilitate solvation of solid samples. Fermentations were performed by L. plantarum strains (ISLG-2, ATCC-8014, and Visbyvac). The phenolic content of propolis was determined with liquid chromatography-mass spectrometry/mass spectrometry (LCMS/MS). The antioxidant activity (antioxidant enzymes, lipid peroxidation) and apoptosis markers (caspase 3,8,9, cytochrome-c, tumour necrosis factor-related apoptosis-inducing ligand-R1 and R2 [TRAIL], and apoptosis protease activating factor-1 [APAF-1] levels) were determined in CCD 841-human colon cell line after induction of oxidative stress by potassium bromate. All propolis samples in different solvents induced apoptosis and 4 biotransformed (by L. plantarum ISL-2 strain and L. plantarum ATCC 8014 strain) propolis samples with low allergenic molecules demonstrated similar inductions of apoptosis in CCD841 cell line. In conclusion, reduction of allergenic molecules in propolis via biotransformation did not change the antioxidant and protective effects of propolis, and it is suggested as a potential therapeutic molecule in prevention of colon cancer caused by oxidative stress for all patients. SIGNIFICANCE OF THE STUDY: This study is the first investigation that shows protective effect of propolis against potassium bromate toxicity by means of decreasing lipid peroxidation and reversing the main molecule levels in intrinsic and extrinsic pathway of apoptosis. Biotransformed propolis samples by L. plantarum ISL-2 and ATCC 8014 strain with low allergen molecule content has also the same effect in potassium bromate toxicity in CCD841 colon cell. Our data contributed that propolis as a natural compound might be a good candidate due to its minimal toxicity and lack of any adverse effects to prevent carcinogenic effect of potassium bromate.

A general dose-response relationship for chronic chemical and other health stressors and mixtures based on an emergent illness severity model.

Current efforts to assess human health response to chemicals based on high-throughput in vitro assay data on intra-cellular changes have been hindered for some illnesses by lack of information on higher-level extracellular, inter-organ, and organism-level interactions. However, a dose-response function (DRF), informed by various levels of information including apical health response, can represent a template for convergent top-down, bottom-up analysis. In this paper, a general DRF for chronic chemical and other health stressors and mixtures is derived based on a general first-order model previously derived and demonstrated for illness progression. The derivation accounts for essential autocorrelation among initiating event magnitudes along a toxicological mode of action, typical of complex processes in general, and reveals the inverse relationship between the minimum illness-inducing dose, and the illness severity per unit dose (both variable across a population). The resulting emergent DRF is theoretically scale-inclusive and amenable to low-dose extrapolation. The two-parameter single-toxicant version can be monotonic or sigmoidal, and is demonstrated preferable to traditional models (multistage, lognormal, generalized linear) for the published cancer and non-cancer datasets analyzed: chloroform (induced liver necrosis in female mice); bromate (induced dysplastic focia in male inbred rats); and 2-acetylaminofluorene (induced liver neoplasms and bladder carcinomas in 20,328 female mice). Common- and dissimilar-mode mixture models are demonstrated versus orthogonal data on toluene/benzene mixtures (mortality in Japanese medaka, Oryzias latipes, following embryonic exposure). Findings support previous empirical demonstration, and also reveal how a chemical with a typical monotonically-increasing DRF can display a J-shaped DRF when a second, antagonistic common-mode chemical is present. Overall, the general DRF derived here based on an autocorrelated first-order model appears to provide both a strong theoretical/biological basis for, as well as an accurate statistical description of, a diverse, albeit small, sample of observed dose-response data. The further generalizability of this conclusion can be tested in future analyses comparing with traditional modeling approaches across a broader range of datasets.

Modulation of hypersensitivity to oxidative DNA damage in ATM defective cells induced by potassium bromate by inhibition of the Poly (ADP-ribose) polymerase (PARP).

The ataxia telangiectasia mutated (ATM) protein is a pivotal multifunctional protein kinase predominantly involved in DNA damage response, as well as in maintaining overall functional integrity of the cells. Apart from playing its major role in regulating the cellular response to DNA damage, ATM, when mutated, can additionally determine oxidative stress, metabolic syndrome, mitochondrial dysfunction and neurodegeneration. In the present paper we aim to investigate the levels of oxidative stress potentially induced by the oxidizing rodent renal carcinogen KBrO3 in ATM-defective lymphoblastoid cell lines (LCLs) established from four classical AT patients (with different ATM mutations), one AT variant with reduced hypersensitivity to X rays, obligate AT heterozygotes and wild type intrafamilial control. A possible modulatory involvement of PARP in potentially induced oxidative stress is also evaluated following its inhibition with 3-aminobenzamide (3-AB). Treatments with KBrO3 clearly showed a marked hypersensitivity of the ATM-defective LCLs, including the AT variant. A marked and statistically significant reduction of KBrO3-induced chromosomal damage following inhibition of PARP by 3-AB, was observed in all AT LCLs, but not in those from the AT variant, AT heterozygotes and wild type intrafamilial control. This result is suggestive of a modulatory involvement of PARP in the hypersensitivity of ATM-defective cells to DNA oxidative damage.

Identification of the Multifaceted Chemopreventive Activity of Curcumin Against the Carcinogenic Potential of the Food Additive, KBrO3.

BACKGROUND: Potassium bromate (KBrO3), a food additive, has been used in many bakery products as an oxidizing agent. It has been shown to induce renal cancer in many in-vitro and in-vivo experimental models. OBJECTIVES: This study evaluated the carcinogenic potential of potassium bromate (KBrO3) and the chemopreventive mechanisms of the anti-oxidant and anti-inflammatory phytochemical, curcumin against KBrO3-induced carcinogenicity. METHOD: Lactate dehydrogenase (LDH) cytotoxicity assay and morphological characteristics were used to assess curcumin's cytoprotective potential against KBrO3 toxicity. To assess the chemopreventive potential of curcumin against KBrO3-induced oxidative insult, intracellular H2O2 and the nuclear concentration of the DNA adduct 8- OHdG were measured. PCR array, qRT-PCR, and western blot analysis were used to identify dysregulated genes by KBrO3 exposure. Furthermore, immunofluorescence was used to evaluate the ciliary loss and the disturbance of cellular tight junction induced by KBrO3. RESULTS: Oxidative stress assays showed that KBrO3 increased the levels of intracellular H2O2 and the DNA adduct 8-OHdG. Combination of curcumin with KBrO3 efficiently reduced the level of H2O2 and 8-OHdG while upregulating the expression of catalase. PCR array, qRT-PCR, and western blot analysis revealed that KBrO3 dysregulated multiple genes involved in inflammation, proliferation, and apoptosis, namely CTGF, IL-1, and TRAF3. Moreover, qRT-PCR and immunofluorescence studies showed that KBrO3 negatively affected the tight junctional protein (ZO-1) and induced a degeneration of primary ciliary proteins. The negative impact of KBrO3 on cilia was markedly repressed by curcumin. CONCLUSION: Curcumin could potentially be used as a protective agent against carcinogenicity of KBrO3.

Effect of Potassium Bromate on the Liver of Adult Male Albino Rat and A Possible Protective Role of Vitamin C: Histological, Immunohistochemical, and Biochemical Study.

Potassium bromate (KBrO3 ) is a food additive which is used primarily as a maturing agent for flour. It is proved as a toxic agent with significant reduction in the activities of antioxidant capacity. The therapeutic efficacy of vitamin C as antioxidant may provide a possible solution to KBrO3 mediated oxidative damage. Twenty four adult male albino rats were used to evaluate the protective role of vitamin C against KBrO3 induced hepatotoxicity and divided into four groups; Group 1 (control), Group 2: received 30 mg/Kg/day vitamin C orally for 4 weeks, Group 3: received 20 mg/Kg/dose KBrO3 orally twice weekly for 4 weeks and Group 4: received both KBrO3 and vitamin C. Liver specimens were processed for histological study by light and electron microscopes and stained immunohistochemically to detect glial fibriller acidic protein (GFAP). Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated as well as the levels of malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) activities in all dissected tissues were determined. KBrO3 induced histological alterations in the form of degeneration, cellular infiltration and significant increase in collagen deposition in portal tracts with a significant increase in immunoexpression of GFAP. Significant rise in serum levels of AST, ALT, and MDA in liver tissues were recorded. However, levels of GSH and SOD were significantly decreased. Most of these changes were improved by vitamin C treatment. In conclusion, vitamin C ameliorates the histological and biochemical alterations of the liver induced by KBrO3 . Anat Rec, 299:1256-1269, 2016. © 2016 Wiley Periodicals, Inc.

Nigella sativa fixed and essential oil modulates glutathione redox enzymes in potassium bromate induced oxidative stress.

BACKGROUND: Nigella sativa is an important component of several traditional herbal preparations in various countries. It finds its applications in improving overall health and boosting immunity. The current study evaluated the role of fixed and essential oil of Nigella sativa against potassium bromate induced oxidative stress with special reference to modulation of glutathione redox enzymes and myeloperoxidase. METHODS: Animals; 30 rats (Sprague Dawley) were divided in three groups and oxidative stress was induced using mild dose of potassium bromate. The groups were on their respective diets (iso-caloric diets for a period of 56 days) i.e. control and two experimental diets containing N. sativa fixed (4%) and essential (0.3%) oils. The activities of enzymes involved in glutathione redox system and myeloperoxidase (MPO) were analyzed. RESULTS: The experimental diets modulated the activities of enzymes i.e. glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) positively. Indices of antioxidant status like tocopherols and glutathione were in linear relationship with that of GPx, GR and GST (P<0.01). MPO activities were in negative correlation with GST (P<0.01) but positive correlation with some other parameters. CONCLUSIONS: Our results indicated that both Nigella sativa fixed and essential oil are effective in improving the antioxidant indices against potassium bromate induced oxidative stress.

Chemoprotective effect of taurine on potassium bromate-induced DNA damage, DNA-protein cross-linking and oxidative stress in rat intestine.

Potassium bromate (KBrO3) is widely used as a food additive and is a major water disinfection by-product. It induces multiple organ toxicity in humans and experimental animals and is a probable human carcinogen. The present study reports the protective effect of dietary antioxidant taurine on KBrO3-induced damage to the rat intestine. Animals were randomly divided into four groups: control, KBrO3 alone, taurine alone and taurine+ KBrO3. Administration of KBrO3 alone led to decrease in the activities of intestinal brush border membrane enzymes while those of antioxidant defence and carbohydrate metabolism were also severely altered. There was increase in DNA damage and DNA-protein cross-linking. Treatment with taurine, prior to administration of KBrO3, resulted in significant attenuation in all these parameters but the administration of taurine alone had no effect. Histological studies supported these biochemical results showing extensive intestinal damage in KBrO3-treated animals and greatly reduced tissue injury in the taurine+ KBrO3 group. These results show that taurine ameliorates bromate induced tissue toxicity and oxidative damage by improving the antioxidant defence, tissue integrity and energy metabolism. Taurine can, therefore, be potentially used as a therapeutic/protective agent against toxicity of KBrO3 and related compounds.

Bisphenol a promotes cell survival following oxidative DNA damage in mouse fibroblasts.

Bisphenol A (BPA) is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER) is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3) or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway.

Comparative reactivity of myeloperoxidase-derived oxidants with mammalian cells.

Myeloperoxidase is an important heme enzyme released by activated leukocytes that catalyzes the reaction of hydrogen peroxide with halide and pseudo-halide ions to form various hypohalous acids. Hypohalous acids are chemical oxidants that have potent antibacterial, antiviral, and antifungal properties and, as such, play key roles in the human immune system. However, increasing evidence supports an alternative role for myeloperoxidase-derived oxidants in the development of disease. Excessive production of hypohalous acids, particularly during chronic inflammation, leads to the initiation and accumulation of cellular damage that has been implicated in many human pathologies including atherosclerosis, neurodegenerative disease, lung disease, arthritis, inflammatory cancers, and kidney disease. This has sparked a significant interest in developing a greater understanding of the mechanisms involved in myeloperoxidase-derived oxidant-induced mammalian cell damage. This article reviews recent developments in our understanding of the cellular reactivity of hypochlorous acid, hypobromous acid, and hypothiocyanous acid, the major oxidants produced by myeloperoxidase under physiological conditions.

Genetic instability in lymphoblastoid cell lines expressing biallelic and monoallelic variants in the human MUTYH gene.

The MUTYH DNA glycosylase counteracts mutagenesis by removing adenine misincorporated opposite DNA 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated polyposis (MAP). The impact on genetic instability of the p.Tyr179Cys and p.Arg245His MUTYH variants was evaluated in lymphoblastoid cell lines (LCLs) derived from MAP patients and their relatives in comparison to wild-type LCLs. No difference in MUTYH expression was identified between wild type and LCLs with the p.Tyr179Cys, while the p.Arg245His mutation was associated with an unstable MUTYH protein. LCLs homozygous for the p.Tyr179Cys or the p.Arg245His variant contained increased DNA 8-oxodG levels and exhibited a mutator phenotype at the PIG-A gene. The extent of the increased spontaneous mutation frequency was 3-fold (range 1.6- to 4.6-fold) in four independent LCLs carrying the p.Tyr179Cys variant, while a larger increase (6-fold) was observed in two p.Arg245His LCLs. A similar hypermutability and S-phase delay following treatment with KBrO3 was observed in LCLs homozygous for either variant. When genetic instability was investigated in monoallelic p.Arg245His carriers, mutant frequencies showed an increase which is intermediate between wild-type and homozygous cells, whereas the mutator effect in heterozygous p.Tyr179Cys LCLs was similar to that in homozygotes. These findings indicate that the type of MUTYH mutation can affect the extent of genome instability associated with MUTYH inactivation. In addition, the mild spontaneous mutator phenotype observed in monoallelic carriers highlights the biological importance of this gene in the protection of the genome against endogenous DNA damage.

Protective effects of Sonchus asper (L.) against KBrO3-induced oxidative stress in rat testis.

Sonchus asper is used traditionally in the treatment of kidney inflammation, hormonal imbalance and impotency. Sonchus asper methanolic extract (SAME) was investigated for its possible preventive effect against potassium bromate (KBrO3) induced oxidative damages in male rats using biochemical, molecular and histopathological markers in this study. 5 groups, each group of 6 rats were taken kept under standard conditions. Group 1 remained untreated while Group II was given 20 mg/kg KBrO3 orally (in aqueous saline) and Group III, and IV were treated with 100; 200 mg/kg b.w., of SAME after 48 h of KBrO3 treatment. KBrO3 administration in rats significantly altered (P<0.01) the serum level of reproductive hormones, activities of antioxidant enzymes and glutathione contents (GSH), which was significantly reversed P<0.001) by co-treatment of 100 mg/kg and 200 mg/kg b.w., SAME. Administration of SAME in rats also significantly P<0.001) reversed the lipid peroxidation induced by KBrO3 in rats, which could be due to the presence of some plant bioactive constituents.

Coordinated action of the Fanconi anemia and ataxia telangiectasia pathways in response to oxidative damage.

Fanconi anemia (FA) and ataxia telangiectasia (AT) share common traits such chromosomal instability and proneness to hematological cancers. Both AT and FA cell lines, and patients, are characterized by abnormally high levels of oxidative stress markers. The key FA protein FANCD2 is phosphorylated on Ser 222 by ATM after ionizing radiation (IR), thus allowing normal activation of the S-phase checkpoint, and ATM cells are known to be hypersensitive to oxidative damage. In this work we show that FANCD2 deficient cells have a defective S-phase checkpoint after Hydrogen Peroxide (H(2)O(2)) induced oxidative damage. ATM dependent phosphorylation of FANCD2 at the S222 residue is necessary for normal S-phase checkpoint activation after oxidative stress, while FANCD2 monoubiquitination at K561 is dispensable. We also show that FANCD2 is not required for base excision repair of 8-oxoG and other DNA lesions (abasic sites, uracils), while treatments that exclusively induce 8-oxoG, but not DNA double strand breaks, fail to activate FANCD2 monoubiquitination, thus indicating that the known accumulation of 8-oxoG in FA cells reflects an overproduction of ROS rather than defective processing of oxidized bases. We conclude that the handling of DNA damage after H(2)O(2)-induced oxidative stress requires the coordinated action of FANCD2 and ATM.

Taurine bromamine: a potent oxidant of tryptophan residues in albumin.

Taurine is the most abundant free amino acid in leukocytes and can react with HOBr to produce taurine bromamine (Tau-NHBr). The aim of this study was to assess the ability of Tau-NHBr to oxidize tryptophan, either free or as a residue in albumin. We have demonstrated that Tau-NHBr is a powerful oxidant for tryptophan. Importantly, in comparison to taurine chloramine, HOCl or HOBr, Tau-NHBr exhibits a degree of selectivity for tryptophan. Oxidation of albumin by Tau-NHBr resulted in emission of light, and the quantum yield was more than 10-fold more efficient than that of the other oxidants. The fluorescence band corresponding to oxidized albumin (λ(ex) 350/λ(em) 450), which is characteristic of the formation of formylkynurenine, was significantly higher in reactions using Tau-NHBr. Excitation of the fluorescent probe 8-anilino-1-naphthalenesulfonate at 295 nm was used to assess the depletion of tryptophan residues in albumin. Results from this experiment further supported a higher efficiency of oxidation of tryptophan residues by Tau-NHBr. Other parameters of protein oxidation, including cysteine depletion and formation of carbonyl groups, were not significantly different between the oxidants tested. In conclusion, these results indicate that Tau-NHBr has a higher affinity for tryptophan residues in proteins.

Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.

An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay.

MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh(-/-) mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity.

DNA topoisomerase I inhibitors ameliorate seizure-like behaviors and paralysis in a Drosophila model of epilepsy.

The Drosophila DNA topoisomerase type I mutant allele, top1JS is an effective general seizure-suppressor mutation, reverting seizure-sensitive phenotypes of several mutant strains in a genetic model of epilepsy. Seizure-suppression is caused by reduced transcription of the top1 (topoisomerase I gene) gene [Song J, Hu J, Tanouye MA. (2007) Seizure suppression by top1 mutations in Drosophila. J Neurosci 27(11):2927-2937]. Here, we examine the possibility that pharmaceutical inhibition of Top1 (topoisomerase I protein) enzymatic activity may also be effective at reducing seizure phenotypes. We investigate the effect of vertebrate Top1 inhibitor camptothecin (CPT) along with two related compounds, apigenin and kaempferol, when fed to seizure-sensitive mutant Drosophila. All three Top1 inhibitors were found to suppress phenotypes in these mutants. In particular, for drug treatments, the recovery time from seizure and paralysis is greatly reduced compared with untreated animals. Intriguingly we find that chronic drug treatments result in a small reduction in seizure sensitivity. Taken together, the results suggest that Top1 inhibitors may have the potential to be developed into effective anti-epileptic drugs, especially for brain tumor patients presenting with epilepsy.

Hypothiocyanous acid is a more potent inducer of apoptosis and protein thiol depletion in murine macrophage cells than hypochlorous acid or hypobromous acid.

Hypohalous acids are generated by activated leucocytes, via the formation of H(2)O(2) and the release of peroxidase enzymes (myeloperoxidase and eosinophil peroxidase). These species are important bactericidal agents, but HOCl (hypochlorous acid) and HOBr (hypobromous acid) have also been implicated in tissue damage in a number of inflammatory diseases. HOSCN (hypothiocyanous acid; cyanosulfenic acid) is a milder, more thiol-specific, oxidant than HOCl or HOBr and as such may be a more potent inducer of cellular dysfunction due to selective targeting of critical thiol residues on proteins. In the present study, HOCl and HOBr are shown to react rapidly with macrophage (J774A.1) cells, resulting in a greater extent of cell lysis compared with HOSCN. However, HOSCN induces apoptosis and necrosis with greater efficacy, and at lower concentrations, than HOCl or HOBr. Apoptosis occurs in conjunction with an increased release of cytochrome c into the cytosol, but no associated increase in caspase activity. Similarly, apoptosis is observed on treating the cells in the presence of a caspase inhibitor, suggesting that it is mediated by a caspase-independent pathway. HOSCN oxidized protein thiols more efficiently than either HOCl or HOBr. The greater efficacy of HOSCN in inducing apoptosis is attributed to selective damage to critical mitochondrial membrane protein thiol groups, resulting in increased permeability and subsequent leakage of cytochrome c into the cytosol. This induction of damage by HOSCN may be of critical importance in people with elevated levels of SCN(-) (thiocyanate ions) arising from cigarette smoking, and plays a role in the pathologies associated with this biological insult.