Diversity Assessment of Toxic Cyanobacterial Blooms during Oxidation.

Fresh-water sources of drinking water are experiencing toxic cyanobacterial blooms more frequently. Chemical oxidation is a common approach to treat cyanobacteria and their toxins. This study systematically investigates the bacterial/cyanobacterial community following chemical oxidation (Cl2, KMnO4, O3, H2O2) using high throughput sequencing. Raw water results from high throughput sequencing show that Proteobacteria, Actinobacteria, Cyanobacteria and Bacteroidetes were the most abundant phyla. Dolichospermum, Synechococcus, Microcystis and Nostoc were the most dominant genera. In terms of species, Dolichospermum sp.90 and Microcystis aeruginosa were the most abundant species at the beginning and end of the sampling, respectively. A comparison between the results of high throughput sequencing and taxonomic cell counts highlighted the robustness of high throughput sequencing to thoroughly reveal a wide diversity of bacterial and cyanobacterial communities. Principal component analysis of the oxidation samples results showed a progressive shift in the composition of bacterial/cyanobacterial communities following soft-chlorination with increasing common exposure units (CTs) (0-3.8 mg·min/L). Close cyanobacterial community composition (Dolichospermum dominant genus) was observed following low chlorine and mid-KMnO4 (287.7 mg·min/L) exposure. Our results showed that some toxin producing species may persist after oxidation whether they were dominant species or not. Relative persistence of Dolichospermum sp.90 was observed following soft-chlorination (0.2-0.6 mg/L) and permanganate (5 mg/L) oxidation with increasing oxidant exposure. Pre-oxidation using H2O2 (10 mg/L and one day contact time) caused a clear decrease in the relative abundance of all the taxa and some species including the toxin producing taxa. These observations suggest selectivity of H2O2 to provide an efficient barrier against toxin producing cyanobacteria entering a water treatment plant.

A Medium-Throughput System for In Vitro Oxidative Stress Assessment in IPEC-J2 Cells.

The feed industry continuously seeks new molecules with antioxidant capacity since oxidative stress plays a key role in intestinal health. To improve screening of new antioxidants, this study aims to set up an assay to assess oxidative stress in the porcine small intestinal epithelial cell line IPEC-J2 using plate-reader-based analysis of fluorescence. Two oxidants, H2O2 and menadione, were tested at 1, 2 and 3 mM and 100, 200 and 300 µM, respectively. Trolox (2 mM) was used as the reference antioxidant and the probe CM-H2DCFDA was used to indicate intracellular oxidative stress. Cell culture, reactive oxygen species (ROS) production and assessment conditions were optimized to detect a significant ROS accumulation that could be counteracted by pre-incubation with trolox. Menadione (200 µM) reproducibly increased ROS levels, H2O2 failed to do so. Trolox significantly decreased intracellular ROS levels in menadione (200 µM)-exposed cells in a consistent way. The system was further used to screen different concentrations of the commercially available antioxidant ELIFE®. Concentrations between 100 and 200 ppm protected best against intracellular ROS accumulation. In conclusion, the combination of CM-H2DCFDA fluorescence analysis by a plate-reader, trolox as a reference antioxidant and 200 µM of menadione as a stressor agent, provides a replicable and reliable medium-throughput setup for the evaluation of intracellular oxidative stress in IPEC-J2 cells.

Biomarkers of oxidative damage in bacteria for the assessment of sanitation efficacy in lettuce wash water.

In the fresh produce industry, validation of sanitation efficacy is critical to prevent cross-contamination of produce. The current validation approaches are either based on time-consuming plate counting assays or indirect measurements of chemical properties of wash water. In the study, the focus was to identify biomarkers that can provide direct assessment of oxidative damage in bacteria upon exposure to sanitizers in the presence of fresh produce and correlation of these oxidative biomarkers with logarithmic inactivation of bacteria. Two endogenous bacterial biomarkers, protein carbonylation and thiol oxidation, were evaluated for assessing oxidative damage in Escherichia coli O157:H7 and Listeria innocua during sanitation of pre-cut lettuce leaves with NaOCl or H2O2. Results show that NaOCl treatment was more effective than H2O2 for oxidation of both the intracellular thiols and protein carbonylation in the selected strains. Statistical analysis of the measurements illustrates that oxidation of the intracellular thiol induced by NaOCl or H2O2 was correlated with logarithmic reduction of E. coli O157:H7 and L. innocua. In contrast, changes in the protein carbonylation content were not correlated with reduction in bacterial cell viability. In summary, these results provide a novel approach to validate sanitation efficacy for the fresh produce industry.

Toxicity assessment of hydrogen peroxide on Toll-like receptor system, apoptosis, and mitochondrial respiration in piglets and IPEC-J2 cells.

In this study, expressions of toll-like receptors (TLRs) and apoptosis-related genes in piglets and mitochondrial respiration in intestinal porcine epithelial cells were investigated after hydrogen peroxide (H2O2) exposure. The in vivo results showed that H2O2 influenced intestinal expressions of TLRs and apoptosis related genes. H2O2 treatment (5% and 10%) downregulated uncoupling protein 2 (UCP2) expression in the duodenum (P < 0.05), while low dosage of H2O2 significantly increased UCP2 expression in the jejunum (P < 0.05). In IPEC-J2 cells, H2O2 inhibited cell proliferation (P < 0.05) and caused mitochondrial dysfunction via reducing maximal respiration, spare respiratory, non-mitochondrial respiratory, and ATP production (P < 0.05). However, 50 uM H2O2 significantly enhanced mitochondrial proton leak (P < 0.05). In conclusion, H2O2 affected intestinal TLRs system, apoptosis related genes, and mitochondrial dysfunction in vivo and in vitro models. Meanwhile, low dosage of H2O2 might exhibit a feedback regulatory mechanism against oxidative injury via increasing UCP2 expression and mitochondrial proton leak.

Assessment of hepatoprotective and nephroprotective potential of withaferin A on bromobenzene-induced injury in Swiss albino mice: possible involvement of mitochondrial dysfunction and inflammation.

Bromobenzene is a well-known environmental toxin which causes liver and kidney damage through CYP450-mediated bio-activation to generate reactive metabolites and, consequently, oxidative stress. The present study aimed to evaluate the possible protective role of withaferin A against bromobenzene-induced liver and kidney damage in mice. Withaferin A (10 mg/kg) was administered orally to the mice for 8 days before intragastric intubation of bromobenzene (10 mmol/kg). As results of this experiment, the levels of liver and kidney functional markers, lipid peroxidation, and cytokines (TNF-α and IL-1ß) presented an increase and there was a decrease in anti-oxidant activity in the bromobenzene-treated group of mice. Pre-treatment with withaferin A not only significantly decreased the levels of liver and kidney functional markers and cytokines but also reduced oxidative stress, as evidenced by improved anti-oxidant status. In addition, the mitochondrial dysfunction shown through the decrease in the activities of mitochondrial enzymes and imbalance in the Bax/Bcl-2 expression in the livers and kidneys of bromobenzene-treated mice was effectively prevented by pre-administration of withaferin A. These results validated our conviction that bromobenzene caused liver and kidney damage via mitochondrial pathway and withaferin A provided significant protection against it. Thus, withaferin A may have possible usage in clinical liver and kidney diseases in which oxidative stress and mitochondrial dysfunction may be existent.

Visual and digital comparative tooth colour assessment methods and atomic force microscopy surface roughness.

This study compared digital and visual colour tooth colour assessment methods in a sample of 99 teeth consisting of incisors, canines and pre-molars. The teeth were equally divided between Control, Ozicure Oxygen Activator bleach and Opalescence Quick bleach and subjected to three treatments. Colour readings were recorded at nine intervals by two assessment methods, VITA Easyshade and VITAPAN 3D MASTER TOOTH GUIDE, giving a total of 1782 colour readings. Descriptive and statistical analysis was undertaken using a GLM test for Analysis of Variance for a Fractional Design set at a significance of P < 0.05. Atomic force micros copy was used to examine treated ename surfaces and establish surface roughness. Visual tooth colour assessment showed significance for the independent variables of treatment, number of treatments, tooth type and the combination tooth type and treatment. Digital colour assessment indicated treatment and tooth type to be of significance in tooth colour change. Poor agreement was found between visual and digital colour assessment methods for Control and Ozicure Oxygen Activator treatments. Surface roughness values increased two-fold for Opalescence Quick specimens over the two other treatments, implying that increased light scattering improved digital colour reading. Both digital and visual colour matching methods should be used in tooth bleaching studies to complement each other and to compensate for deficiencies.

Double-face activity of resveratrol in voluntary runners: assessment of DNA damage by comet assay.

Voluntary runners are subjected to a massive increase in reactive oxygen/nitrogen species production, which can promote different oxidative stress-related diseases such as premature aging, neurodegenerative disorders, and cancer. The aims of this work were to evaluate the following in peripheral blood cells of voluntary runners: (i) DNA status; (ii) susceptibility to the in vitro insult induced by hydrogen peroxide (H(2)O(2)) as a breaking agent; (iii) capabilities of 3,5,4'-trihydroxystilbene (RESV) in counteracting DNA damage. Twenty-five male voluntary runners were compared with 20 sedentary men, as age-matched controls, and DNA status was evaluated with different versions of comet assay: alkaline, neutral, and Fpg enzyme-modified version to measure 8-OH-deoxyguanosine (8-oxo-dG) levels. The H(2)O(2) and/or RESV treatments were performed directly on agarose-embedded cells (atypical comet assay). The results evidenced DNA damage and levels of 8-oxo-dG higher in runners than in sedentary control subjects. The runners' DNA was more prone to the in vitro-induced oxidative insult (200 µM H(2)O(2)) than that of the control group. Resveratrol (100 µM), depending on the individual basal DNA status, was able to switch from antioxidant to pro-oxidant. Our results, on the one hand, validated the proposed in vitro experimental protocol in order to measure individual DNA status. On the other hand, our data point out the importance of monitoring the athletes' redox status before subjecting them to dietary supplementation treatment.

In vitro assessment of the chemotherapeutic action of a specific hydrogen peroxide, peracetic, acetic, and peroctanoic acid-based formulation against the free-living stages of Ichthyophthirius multifiliis (Ciliophora).

Traditionally, malachite green administrated as in-bath treatment was the most effective and common strategy used in freshwater aquaculture systems to control infections of the ciliate protozoan parasite Ichthyophthirius multifiliis Fouquet, 1876. After the ban of malachite green in the USA and Europe to be used in fish for human consumption, there has been extensive research destined to find efficacious replacements. Recently, peracetic acid-based compounds have demonstrated a strong cytotoxic effect in vitro and in vivo against I. multifiliis. In the present study, we tested the efficacy of a hydrogen peroxide, peracetic, acetic and peroctanoic acid-based formulation (HPPAPA) to eliminate the free-living stages of I. multifiliis (tomonts, cysts and theronts). The results obtained showed that the administration of low doses (8, 12 or 15 mg/l) of a specific HPPAPA-based product during a short window of exposure (60 min) kills nearly all free-living stages of I. multifiliis (theronts, tomonts and cysts) within the window of treatment (∼100% mortality for all the stages; one-way ANOVA, P ≤ 0.001). Of note, even the lowest concentration of HPPAPA tested (8 mg/l) was able to disrupt normal cyst development and therefore theront release. The demonstrated in vitro efficacy of the peracetic acid-based product tested on the present study suggests its great potential to control I. multifiliis infections in commercial aquacultural systems.

Spectrophotometric assessment of the effects of 10% carbamide peroxide on enamel translucency.

Tooth shade results from the interaction between enamel color, enamel translucency and dentine color. A change in any of these parameters will change a tooths color. The objective of this study was to evaluate the changes occurring in enamel translucency during a tooth whitening process. Fourteen human tooth enamel fragments, with a mean thickness of 0.96 mm (+/- 0.3 mm), were subjected to a bleaching agent (10% carbamide peroxide) 8 hours per day for 28 days. The enamel fragment translucency was measured by a computer controlled spectrophotometer before and after the bleaching agent applications in accordance with ANSI Z80.3-1986--American National Standard for Ophthalmics--nonprescription sunglasses and fashion eyewear-requirements. The measurements were statistically compared by the Mann-Whitney non-parametric test. A decrease was observed in the translucency of all specimens and, consequently, there was a decrease in transmittance values for all samples. It was observed that the bleaching procedure significantly changes the enamel translucency, making it more opaque.

Spectrophotometric assessment of the effects of 10 percent carbamide peroxide on enamel translucency

Tooth shade results from the interaction between enamel color, enamel translucency and dentine color. A change in any of these parameters will change a tooth’s color. The objective of this study was to evaluate the changes occurring in enamel translucency during a tooth whitening process. Fourteen human tooth enamel fragments, with a mean thickness of 0.96 mm (± 0.3 mm), were subjected to a bleaching agent (10 percent carbamide peroxide) 8 hours per day for 28 days. The enamel fragment translucency was measured by a computer controlled spectrophotometer before and after the bleaching agent applications in accordance with ANSI Z80.3-1986 - American National Standard for Ophthalmics - nonprescription sunglasses and fashion eyewear-requirements. The measurements were statistically compared by the Mann-Whitney non-parametric test. A decrease was observed in the translucency of all specimens and, consequently, there was a decrease in transmittance values for all samples. It was observed that the bleaching procedure significantly changes the enamel translucency, making it more opaque.

An assessment of the role of reactive oxygen species and redox signaling in norepinephrine-induced apoptosis and hypertrophy of H9c2 cardiac myoblasts.

Cardiac myocytes, upon exposure to increasing doses of norepinephrine (NE), transit from hypertrophic to apoptotic phenotype. Since reactive oxygen species (ROS) generation is attributed to both phenomena, the authors tested whether an elevation in intracellular ROS level causes such transition. H9c2 cardiac myoblasts upon treatment with hypertrophic and apoptotic doses of NE (2 and 100 microM, respectively) transiently induced intracellular ROS at a comparable level, while 200 microM H(2)O(2), another proapoptotic agonist, showed robust and sustained ROS generation. Upon analysis of a number of redox-responsive transcription factors as the downstream targets of ROS signaling, the authors observed that NE (2 and 100 microM) and H(2)O(2) (200 microM) were ineffective in inducing NF-kappaB while both the agonists upregulated AP-1 and Nrf-2. However, the extents of induction of AP-1 and Nrf-2 were not in direct correlation with the respective ROS levels. Also, AP-1 activities induced by two doses of NE were intrinsically different, since at 2 microM, it primarily induced FosB, and at 100 microM it activated Fra-1. Differential induction of FosB and Fra-1 was also reiterated in adult rat myocardium injected with increasing doses of NE. Therefore, NE induces hypertrophy and apoptosis in cardiac myocytes by distinct redox-signaling rather than a general surge of ROS.

A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations.

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA-binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining beta-cell viability. For these reasons we have developed novel methods for the specific assessment of beta-cell content and viability in human islets based on cellular composition analysis through laser scanning cytometry (LSC) coupled with identification of beta-cell-specific apoptosis at the mitochondrial level. Our novel analytical methods hold promise to prospectively analyze clinical islet transplantation preparations and predict functional performance, as suggested by the observed correlation with in vivo analysis of islet potency in immunodeficient rodents.

Assessment of antioxidative activity of lipid- and water-soluble vitamins in human whole blood. Comparative analysis between a biological test and chemical methods.

Assessment of the antioxidant activity of vitamins and other compounds is of interest in the understanding of their in vivo effects. In this study, we have investigated the activity of several lipid and water-soluble vitamins in human whole blood. Measurements were carried out using a biological test that enables the evaluation of both red blood cells and plasma resistance against free radical activity induced by 2,2'-azobis (2-amidinopropane)hydrochloride (AAPH). Antioxidant activity of vitamins has been determined by using the biological test versus chemical methods (chemiluminescence, DMPD radical). We have observed strong anti-oxidant potentials for vitamins B6 and B9 with biological tests, but not with chemical methods. At 10 microM, the vitamin B9 efficiency in inhibiting radical-induced red blood cell hemolysis was almost three times higher than vitamin C efficiency and two times higher than alpha-tocopherol efficiency. Antioxidant activity was not observed for vitamins B1 or B2, nor for retinol. The weak activity of beta-carotene still remains to be investigated particularly in relation to oxygen pressure. Our study demonstrated that the biological test is more useful than the chemical methods employed in this instance, for the evaluation of antioxidant capacity of lipophilic and putatively biologically active compounds.

Effect of internal bleaching agents on dentinal permeability of non-vital teeth: quantitative assessment.

The aim of this in vitro study was to assess quantitatively dentin permeability of pulpless teeth after intracoronal bleaching therapy with three different agents. Twenty-four maxillary central incisors were randomly assigned to four groups according to the bleaching agent used: I--non-bleached control; II--37% carbamide peroxide; III--sodium perborate/20% hydrogen peroxide paste; IV--27% carbamide peroxide. After standard access and root-canal preparation the access opening, biomechanical preparation and root-canal filling, a cervical glass ionomer plug was prepared and intracoronal bleaching procedures were carried out in a standardized fashion. The access cavities were opened and the teeth were externally sealed and immersed in a 10% copper sulfate aqueous solution for 30 min, in vacuum for the first 5 min. Then, samples were removed, dried with absorbing paper and immersed in a 1% rubianic acid alcohol solution, for the same above-mentioned period in solution and in vacuum. Copper ion penetration was indicated by the rubianic acid staining. Mean values and SD for the experimental groups were: I--7.88% (+/-1.33), II--16.94% (+/-5.72); III--11.45% (+/-3.90) and IV--8.98% (+/-4.19). Data were submitted to one-way anova. The results showed that the 37% carbamide peroxide provided the highest increase in dentin permeability, followed by sodium perborate with 20% hydrogen peroxide. The 27% carbamide peroxide provided the lowest results and showed statistical similarity to the control group. On basis of these findings, it may be concluded that, among the tested intracoronal bleaching agents, 37% carbamide peroxide presented an optimized overall performance in increasing dentinal permeability.

Assessment of the effect of Fucus vesiculosus extract on the labeling of blood constituents with technetium-99m and the histological modifications on the shape of the red blood cells.

Natural products are widely used as food or food additives or medicines for humans. We are trying to develop a model to assess the possible toxic properties of natural products, such as Fucus vesiculosus, utilized in popular medicine. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used in various procedures in nuclear medicine. This labeling procedure depends on a reducing agent, and stannous chloride is used. There is evidence that this labeling may be altered by drugs. We have investigated the possibility that F. vesiculosus extract is capable of altering the labeling of blood elements with 99mTc. Blood was incubated with F. vesiculosus extract and stannous chloride solution and Tc-99m added. Blood was centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P or BC were also precipitated, centrifuged and insoluble (IF) and soluble (SF) were separated. The percentages of radioativity (%ATI) in BC, IF-P and IF-BC were calculated. Histological preparations of the RBC treated with F. vesiculosus revealed that this extract is capable of promoting important modifications on the shape of the RBC. The%ATI decreased on BC from 93.6+/-2.3 to 29.0+/-2.7, on IF-P from 77.6+/-1.2 to 7.5+/-1.0 and on IF-BC from 80.0+/-3.4 to 12.6+/-4.8. Once the RBC labeling procedure with 99mTc depends on the presence of stannous (+2) ions, the substances present in the F. vesiculosus extract should increase the valence of these ions to stannic (+4). This would decrease the%ATI on blood elements and indicate the presence of oxidant agents in the F. vesiculosus extract.

Assessment of structural features of the pseudomonas siderophore pyochelin required for its ability to promote oxidant-mediated endothelial cell injury.

We previously showed that iron chelated to the Pseudomonas aeruginosa siderophore pyochelin enhances oxidant-mediated injury to pulmonary artery endothelial cells by catalyzing hydroxyl radical (HO(*)) formation. Therefore, we examined pyochelin structural/chemical features that may be important in this process. Five pyochelin analogues were examined for (i) capacity to accentuate oxidant-mediated endothelial cell injury, (ii) HO(*) catalytic ability, (iii) iron transfer to endothelial cells, and (iv) hydrophobicity. All compounds catalyzed similar HO(*) production, but only the hydrophobic ones containing a thiazolidine ring enhanced cell injury. Transfer of iron to endothelial cells did not correlate with cytotoxicity. Finally, binding of Fe(3+) by pyochelin led to Fe(2+) formation, perhaps explaining how Fe(3+)-pyochelin augments H(2)O(2)-mediated cell injury via HO(*) formation. The ability to bind iron in a catalytic form and the molecule's thiazolidine ring, which increases its hydrophobicity, are key to pyochelin's cytotoxicity. Reduction of Fe(3+) to Fe(2+) may also be important.

In-vivo and in-vitro assessment of the free-radical-scavenger activity of Ginkgo flavone glycosides at high concentration.

Free radicals are involved in numerous skin diseases, especially inflammatory reactions and photosenescence. To identify possible free-radical scavenging by an original terpene-free Ginkgo biloba extract containing 33% Ginkgo flavone glycosides, mostly quercetin and kaempferol derivatives, we studied its activity by means of in-vitro and in-vivo experiments, using superoxide dismutase (SOD) as a positive control. By means of an in-vitro electron-spin resonance (ESR) assay we compared the activity of the Ginkgo extract with that of its two aglycones, quercetin and kaempferol. Quercetin and Ginkgo extract had significant antioxidant properties without pro-oxidant effect. In contrast, kaempferol, above an optimum antioxidant concentration, behaved as a pro-oxidant. The in-vivo experiments were conducted on an anti-inflammatory model. The cutaneous blood flux which reflects the skin inflammatory level was recorded by means of a laser Doppler perfusion imager. The data confirmed the free-radical-scavenging property of both Ginkgo extract and SOD. The Ginkgo extract significantly inhibited (37%) cutaneous blood flux to the same extent as SOD. These data confirmed the antioxidant property of Ginkgo extract. A complementary spin-trapping technique would enable identification of the free radicals involved. This Ginkgo extract should be useful for protection of the skin against free radicals.

Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage.

Liquid packaging boards and blanks were examined for microbial contaminants. A total of 218 strains were identified and representatives of the most frequent species were characterized for their potential for food spoilage. Contaminants found were aerobic spore-forming bacteria, mostly Bacillus megaterium, B. licheniformis, B. cereus group, B. pumilus, Paenibacillus macerans, P. polymyxa, P. pabuli and B. flexus. Production of amylolytic, proteolytic, lipolytic and phospholipolytic enzymes was common. Approximately 50% of the B. cereus group strains were positive in the diarrhoeal enterotoxin immunoassay test or in the enterotoxin reversed passive latex agglutination test. Strains capable of growth at 6 degrees C were found among B. cereus group, P. pabuli, P. validus, B. megaterium and P. polymyxa. All b. licheniformis strains grew at 55 degrees C. The spores of B. licheniformis were most resistant to hydrogen peroxide. The B. cereus group strains were recognizable by fatty acid components not present in any of the other paperboard strains, 11-methyldodecanoic acid (13:0 iso) and trans-9-hexadecenoic acid (16:1 omega 7 trans), each contributing 7% or more to the total cellular fatty acids.

Risk assessment of oxidant gases and particulate air pollutants: uncertainties and research needs.

The assessment of risks to human health associated with exposure to oxidant air pollutants has not received adequate attention despite the recognized public health threat posed by the ubiquitous presence of these compounds in the environment. In this article, research needs and uncertainties at each of the steps in the risk assessment of oxidant air pollutants are identified: hazard identification, dose-response assessment, exposure assessment, and risk characterization. Many of these limitations and uncertainties arise at the interface between the laboratory and the regulatory arenas. Therefore, as a case study, relevant methodologic problems associated with the application of experimental findings to the risk assessment of respirable dusts are also discussed. These issues include the extrapolation of animal data to the human case and extrapolation from high-dose to environmentally relevant, low-level exposures.