Assessment of the antioxidant capacity of natural fruit extracts by inhibition of probe decay and plasma lipid peroxidation.

The oxidation of lipids, proteins, and DNA induced by reactive oxygen species has been implicated in the development of various diseases, and the role of antioxidants has received much attention. Free-radical scavenging antioxidants play an important role in the defense network in vivo, and assessment of the capacity of antioxidants has been the subject of extensive studies and controversy, but there is no universal method by which antioxidant capacity can be measured accurately. In the present study, the assessment of the antioxidant capacity of natural fruit extracts was examined for radical scavenging and inhibition of lipid peroxidation. It was found that the capacity of fruit extracts for scavenging of both hydrophilic and lipophilic free radicals and for antioxidation can be assessed from the effect on the probe decay and the inhibition of plasma lipid peroxidation respectively. The importance of these two factors in the assessment of antioxidant capacity is discussed.

Induction of adaptive response and enhancement of PC12 cell tolerance by lipopolysaccharide primarily through the upregulation of glutathione S-transferase A3 via Nrf2 activation.

Increasing evidence indicates that reactive oxygen species and other physiologically existing oxidative stimuli upregulate the antioxidant system, thereby triggering the adaptive response. In this study, we focused on adaptive cytoprotection induced by lipopolysaccharide (LPS), which induces oxidative stress and inflammatory cytokines, in PC12 cells, a model of the neuronal cell. After treating PC12 cells with LPS at sublethal concentrations, we found that they developed resistance to subsequent oxidative stress induced by 13S-hydroperoxy-9Z,11E-octadecadienoic acid and 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium. To determine the underlying molecular mechanisms responsible for an adaptive response induced by LPS, we studied the changes in the antioxidant system. LPS treatment resulted in an increase in the gene expression of glutathione S-transferase A3 (GST-A3) by up to 60-fold as well as in GST enzyme activity. A GST inhibitor and GST A3 small interfering RNA effectively attenuated the adaptive response. The nuclear factor erythroid 2 p45-related factor 2 (Nrf2) was transcriptionally activated by LPS. Nrf2 small interfering RNA effectively attenuated the increase in GST A3 mRNA level as well as the adaptive response induced by LPS. In addition, peripheral injection of LPS at sublethal concentrations increased GST enzyme activity in mouse brain. These findings, taken together, indicate that stimulation with LPS at sublethal concentrations induces an adaptive response and enhances PC12 cell tolerance, primarily through the induction of GST A3 via the transcriptional activation of the Nrf2 signaling pathway.

Antioxidant capacity of BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, and uric acid as evaluated by ORAC method and inhibition of lipid peroxidation.

The role of radical-scavenging antioxidant against oxidative stress has received much attention. The antioxidant capacity has been assessed by various methods. Above all, oxygen radical absorbance capacity (ORAC) has been frequently employed [Prior et.al., J. Agric. Food Chem.2005, 53, 4290]. In the present study, the antioxidant capacity of 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653) and uric acid was assessed by ORAC method using pyranine as a reference probe and compared with that against lipid peroxidation of human plasma. It was found that BO-653 was assessed to be much less potent than uric acid by ORAC method, whereas BO-653 exerted much higher antioxidant activity than uric acid against plasma lipid peroxidation. The reason for such discrepancy is discussed. The results suggest that ORAC method is suitable for the assessment of free radical scavenging capacity, but not for the assessment of antioxidant capacity against lipid peroxidation in plasma.

Characterization of novel furan compounds on the basis of their radical scavenging activity and cytoprotective effects against glutamate- and lipopolysaccharide-induced insults.

There is increasing evidence indicating that free radicals and oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) are related to the onset and development of neurodegenerative diseases. In order to prevent and/or delay these diseases, the use of radical-scavenging antioxidants and inhibitors against oxygenases has received much attention. In the present study, we examined the radical-scavenging activity and cytoprotective effects of some novel furan compounds with potent inhibitory activity against oxygenases such as COX-1, COX-2, and 5-LOX. The radical-scavenging activity was assessed by studying the bleaching of beta-carotene by free radicals generated from an azo initiator. In this assay system, the rate constants for scavenging peroxyl radicals by furan S and furan L was estimated to be 2 x 10(4) and 3 x 10(4) M(-1) s(-1), respectively. We also investigated the cytoprotective effects of these compounds against cell death induced by several toxins. We found that the furan compounds exhibited cytoprotective effects against PC12 cell death induced by linoleic acid hydroperoxide, primary neuronal cell death induced by glutamate, and cell death induced by lipopolysaccharide. These results suggest the beneficial effects of the furan compounds against disorders related to glutamate and lipopolysaccharide.

Simple assessment of radical scavenging capacity of beverages.

The radical-scavenging antioxidants play an important role against oxidative stress in the defense system in vivo. The beneficial effects of antioxidants contained in foods and beverages have been well-accepted, and their antioxidant capacity has been assessed by various methods. In the present study, a simple method is proposed in which the total radical scavenging capacity is assessed from the bleaching of pyranine and pyrogallol red induced by free radicals generated from azo initiator. The total content of antioxidants contained in red wine, green tea, and cassis drink and their reactivities toward peroxyl radicals were measured from the lag phase and rate of bleaching using pyranine and pyrogallol red as a probe, respectively. It was found that this method to follow the bleaching of two probes by visible light spectrophotometer is convenient and applicable for assessment of total radical scavenging capacity of both content and activity of the antioxidants contained in beverages.

Effect of mercury(II) on Nrf2, thioredoxin reductase-1 and thioredoxin-1 in human monocytes.

OBJECTIVES: Human blood levels of mercury are commonly 10nM, but may transiently reach 50-75nM after dental amalgam placement or removal. Controversy persists about the use of mercury because the effects of these 'trace' levels of mercury are not clear. Concentrations of mercury > or =5000nM unequivocally alter redox balance in blood cells including monocytes. In the current study, we tested a hypothesis that concentrations of mercury <100nM altered levels and activities of key proteins that maintain monocytic redox balance. METHODS: Human THP1 monocytes were exposed to 10-75nM of Hg(II) for 6-72h, with or without activation by lipopolysaccharide (LPS). The redox management proteins Nrf2 and thioredoxin-1 (Trx1) were separated by electrophoresis, then quantified by immunoblotting. The activity of the seleno-enzyme thioredoxin reductase (TrxR1), important in maintaining Trx1 redox balance, was measured by cell-free and cell-dependent assays. RESULTS: Concentrations of Hg(II) between 10-75nM increased Nrf2 levels (3.5-4.5 fold) and decreased Trx1 levels (2-3 fold), but these changes persisted <24h. Hg(II) potently inhibited (at concentrations of 5-50nM) TrxR1 activity in both cell-free and intracellular assays. Furthermore, Hg(II) transiently amplified LPS-induced Nrf2 levels by 2-3 fold and limited LPS-induced decreases in Trx1. All effects of Hg(II) were mitigated by pre-adding N-acetyl-cysteine (NAC) or sodium selenide (Na2SeO3), supplements of cellular thiols and selenols, respectively. SIGNIFICANCE: Our results suggest that nanomolar concentrations of Hg(II) transiently alter cellular redox balance in monocytes that trigger changes in Nrf2 and Trx1 levels. These changes indicate that monocytes have a capacity to adapt to trace concentrations of Hg(II) that are introduced into the bloodstream after dental amalgam procedures or fish consumption. The ability of monocytes to adapt suggests that low levels of mercury exposure from dental amalgam may not overtly compromise monocyte function.

Assessment of antioxidative activity of extract from fermented grain food mixture using chemical and cellular systems.

Fermented food is a rich source of antioxidants and micronutrients with the potential to prevent various human diseases. The increasing evidence indicates that in addition to its direct action, radical-scavenging antioxidants may modulate the cellular antioxidant system such as glutathione. In the present study, we investigated the antioxidant activity of Antioxidant Biofactor (AOB) extracts, a mixture of commercially available fermented grain food by using chemical and cellular experimental systems. In the former system, the total radical scavenging capacity was assessed from the bleaching of pyranine and pyrogallol red that is induced by free radicals generated from an azo initiator. In this assay system, the radical scavenging capacity per gram of AOB was estimated to be 95 micromol. On the other hand, the cytoprotective effect of AOB was also investigated on the basis of PC12 cell death induced by 6-hydroxydopamine. In this cellular system, AOB extract exhibited a cytoprotective effect only when the cells were pretreated with AOB. This pretreatment resulted in a significant increase in the levels of cellular glutathione as well as regulator of glutathione synthesis, such as the cystine/glutamate exchange transport system (xCT). This evidence suggests that AOB possesses both direct and indirect antioxidant activities to cope with oxidative insults.

Sublethal concentrations of diverse gold compounds inhibit mammalian cytosolic thioredoxin reductase (TrxR1).

UNLABELLED: Thioredoxin reductase (TrxR) reduces thioredoxin (Trx), thereby contributing to cellular redox balance, facilitating the synthesis of deoxy-ribose sugars for DNA synthesis, and regulating redox-sensitive gene expression. Auranofin is a gold compound that potently inhibits TrxR. This inhibition is one suspected mechanism of auranofin's therapeutic benefit in the treatment of rheumatoid arthritis. The use of other gold compounds to treat cancer or inflammatory disease may rely on their ability to inhibit TrxR. In the current study, we tested the hypothesis that a variety of gold compounds may inhibit TrxR. METHODS: We exposed rat-TrxR1 to auranofin, gold sodium thiomalate, sodium aurothiosulfate, triphenyl phosphine gold chloride, or gold acetate, and measured TrxR activity ex vivo. We then compared TrxR1 inhibitory levels of gold compounds to those that inhibited mitochondrial activity of THP1 monocytes and OSC2 epithelial cells, estimated by succinate dehydrogenase activity. RESULTS: All gold compounds inhibited TrxR1 at concentrations ranging from 5 to 4000 nM (50% inhibitory concentration). The oxidation state of gold did not correlate with inhibitory potency, but ligand configuration was important. Au(I)-phosphine compounds (triphenyl phosphine gold chloride and auranofin) were the most potent inhibitors of TrxR. All TrxR1 inhibitory concentrations were sublethal to mitochondrial activity in both THP1 and OSC2 cells. CONCLUSIONS: Diverse types of gold compounds may be effective inhibitors of TrxR1 at concentrations that do not suppress cellular mitochondrial function. Inhibition may be optimized to some degree by altering the ligand configuration of the compounds. These results support future study of a variety of Au compounds for therapeutic development as inhibitors of TrxR1.

Gold-induced reactive oxygen species (ROS) do not mediate suppression of monocytic mitochondrial or secretory function.

UNLABELLED: The toxicity of anti-rheumatic gold compounds has limited their use and development, yet both the toxicological and therapeutic actions of these compounds remain unclear. In the current study, we tested the hypothesis that intracellular reactive oxygen species (ROS) induced by Au(I) or Au(III) compounds mediate their ability to suppress mitochondrial activity. METHODS: Human THP1 monocytes were exposed to HAuCl(4) x 3H(2)O (Au(III)), or the anti-rheumatic compounds auranofin (AF) or gold sodium thiomalate (GSTM) for 6-72 h, after which mitochondrial activity (succinate dehydrogenase) was measured. To assess the role of cellular redox status as a mediator of mitochondrial suppression, monocytes were pre-treated with a pro-oxidant (t-butyl hydroquinone, t-BHQ) or antioxidant (N-acetyl cysteine, NAC ). ROS levels were measured 0-24h post-gold addition to determine their role as mediators of mitochondrial activity suppression. RESULTS: AF was the most potent inhibitor of mitochondrial activity, followed by Au(III) and GSTM. Only Au(III) induced intracellular ROS; no ROS formation was observed in response to AF or GSTM exposure. Although anti- and pro-oxidants had some effects on mitochondrial suppression of Au compounds, collectively the data do not support redox effects or ROS formation as major mediators of Au-compound mitochondrial suppression. CONCLUSIONS: Our results do not indicate that ROS and redox effects play major roles in mediating the cytotoxicity of AF, GSTM or Au(III).

Extracellular environment as one mediator of blue light-induced mitochondrial suppression.

OBJECTIVES: The current study tested the hypothesis that the extracellular environment mediates mitochondrial suppression of oral epithelial cells and fibroblasts by blue light. METHODS: We exposed Balb fibroblasts (Balb), normal human epidermal keratinocytes (NHEK), and oral squamous carcinoma cells (OSC2) to blue light (30-120J/cm2) in different cell-culture media and in phosphate buffered saline (PBS). Mitochondrial activity (MTT method) was used to assess cellular response 72 h post-light exposure. Cell-culture media were replaced or supplemented before or after light exposure to assess the variables of exposure time and medium degradation as mediators of blue light-induced effects. RESULTS: Mitochondrial activity of NHEK was not suppressed by exposure to blue light regardless of extracellular conditions. The mitochondrial activity of OSC2 and Balb cells was suppressed most when cells were exposed to light in cell-culture medium (versus PBS). Blue light suppressed mitochondrial activity more when irradiated medium remained in contact with the cells at least 1h, indicating a time-dependence of the medium effects. Neither a replacement nor a supplementation of medium components reduced blue light-induced mitochondrial suppression. SIGNIFICANCE: Our results suggest that tissue environments influence cellular responses to blue light and that these environments should be considered when assessing any biological effects of blue light during the photopolymerization of restorative resins.

Change of color and translucency by light curing in resin composites.

OBJECTIVE: This study evaluated color and translucency changes caused by light curing resin composite materials. METHODS: The CIELAB parameters (L*, a* and b*) of disks of A2 and opaque A2 shades of Charisma (Heraeus-Kulzer), Solare (GC) and Filtek Supreme (3M) were evaluated on the backings of black, white and the material itself both before and after light curing to evaluate color and translucency changes (by means of calculating deltaE* and the translucency parameter, respectively). RESULTS: Solare and Filtek Supreme showed significantly smaller color changes during light curing than Charisma; however, the value of deltaE* of all the products/shades was still in the clinically unacceptable range. Regarding translucency changes during light curing, the A2 and opaque A2 shades of Charisma showed a statistically significant increase, although no difference was observed in the other products. CONCLUSIONS: Solare and Filtek Supreme tended to show less changes in translucency and color during light curing compared to Charisma. Nevertheless, the changes in color during light curing were still in the range of unacceptable color change. Therefore, direct shade matching of these materials for a precise shade match should be performed by using the cured material.

Staining of hybrid composites with coffee, oolong tea, or red wine.

This study examined the surface staining mechanism of a photopolymerized composite by coffee, oolong tea, and red wine. Dental composite was subjected to an experimental 24-hour staining cycle: 17-hour immersion in artificial saliva solution containing 0.3% mucin followed by 7-hour immersion in coffee, tea, or wine. After one, two, and four weeks, digital images of the composite surface were analyzed in grayscale mode with an imaging analyzer. Specimens polished but not immersed were used as a baseline measurement for color change. Additionally, the effects of mechanical brushing and chlorhexidine on drink-induced staining were examined. Wine caused the most severe staining, followed by tea and coffee. After four weeks of immersion, brushing reduced surface staining by wine. On the contrary, chlorhexidine increased the staining effect of tea and coffee (p<0.05) when compared to the control specimens. In conclusion, we showed that common drinks stained the dental composite, but each by a specific mechanism that depended on external conditions such as the presence of chlorhexidine.

Decreased zinc availability affects glutathione metabolism in neuronal cells and in the developing brain.

A deficit in zinc (Zn) availability can increase cell oxidant production, affect the antioxidant defense system, and trigger oxidant-sensitive signals in neuronal cells. This work tested the hypothesis that a decreased Zn availability can affect glutathione (GSH) metabolism in the developing rat brain and in neuronal cells in culture, as well as the capacity of human neuroblastoma IMR-32 cells to upregulate GSH when challenged with dopamine (DA). GSH levels were low in the brain of gestation day 19 (GD19) fetuses from dams fed marginal Zn diets throughout gestation and in Zn-deficient IMR-32 cells. γ-Glutamylcysteine synthetase (GCL), the first enzyme in the GSH synthetic pathway, was altered by Zn deficiency (ZD). The protein and mRNA levels of the GCL modifier (GCLM) and catalytic (GCLC) subunits were lower in the Zn-deficient GD19 fetal brain and in IMR-32 cells compared with controls. The nuclear translocation of transcription factor nuclear factor (erythroid-derived 2)-like 2, which controls GCL transcription, was impaired by ZD. Posttranslationally, the caspase-3-dependent GCLC cleavage was high in Zn-deficient IMR-32 cells. Cells challenged with DA showed an increase in GCLM and GCLC protein and mRNA levels and a consequent increase in GSH concentration. Although Zn-deficient cells partially upregulated GCL subunits after exposure to DA, GSH content remained low. In summary, results show that a low Zn availability affects the GSH synthetic pathway in neuronal cells and fetal brain both at transcriptional and posttranslational levels. This can in part underlie the GSH depletion associated with ZD and the high sensitivity of Zn-deficient neurons to pro-oxidative stressors.

Action of 6-amino-3-pyridinols as novel antioxidants against free radicals and oxidative stress in solution, plasma, and cultured cells.

Free radical-mediated lipid peroxidation has been implicated in the pathogenesis of various diseases. Lipid peroxidation products are cytotoxic and they modify proteins and DNA bases, leading eventually to degenerative disorders. Various synthetic antioxidants have been developed and assessed for their capacity to inhibit lipid peroxidation and oxidative stress induced by free radicals. In this study, the capacity of novel 6-amino-2,4,5-trimethyl-3-pyridinols for scavenging peroxyl radicals, inhibiting plasma lipid peroxidation in vitro, and preventing cytotoxicity induced by glutamate, 6-hydroxydopamine, 1-methyl-4-phenylpyridium (MPP(+) ), and hydroperoxyoctadecadienoic acid was assessed. It was found that they exerted higher reactivity toward peroxyl radicals and more potent activity for inhibiting the above oxidative stress than alpha-tocopherol, the most potent natural antioxidant, except against the cytotoxicity induced by MPP(+). These results suggest that the novel 6-amino-3-pyridinols may be potent antioxidants against oxidative stress.

Assessment of radical scavenging capacity and lipid peroxidation inhibiting capacity of antioxidant.

The role of radical scavenging antioxidants against oxidative stress has received much attention, and the antioxidant capacity has been assessed by various methods. Among them, a method that measures the effect of antioxidant on decay of the probe is one of the most widely used methods. The present study was performed to compare the two methods to assess the antioxidant capacity, one to follow the decay of the probe and the other to measure lipid peroxidation products in human plasma. It was shown that the method following probe decay was suitable for assessment of radical scavenging capacity of antioxidant, but not for the capacity to inhibit lipid peroxidation in plasma. This is true whether a hydrophilic or lipophilic probe is used. Such different results arise from the fact that the efficacy of inhibition of lipid peroxidation by antioxidants depends on the fate of antioxidant-derived radical and interaction between antioxidants as well as the capacity of free radical scavenging. Thus, the capacity of antioxidants for inhibition of lipid peroxidation should be assessed from the effect on the extent of oxidation, not from the effect on probe decay.

Interactions between stainless steel, shear stress, and monocytes.

Angioplasty with stent placement is commonly used to treat coronary atherosclerosis. However, 20-40% of stainless steel stents restenose within 6 months via a prolonged inflammatory response mediated by monocytic infiltration and cytokine secretion. In the current study, we tested a hypothesis that blood flow and monocytes interact to alter stent corrosion. We assessed the effects of THP1 monocytes on the corrosion rate of 316L stainless steel (316LSS) under shear stress (0.5-50 dyn/cm(2)). In addition, THP1 cytokine secretion was determined using cytokine arrays and ELISA analyses. Data were compared using ANOVA and Tukey post hoc analysis (alpha = 0.05). Monocytes significantly lowered 316LSS corrosion rates without limiting current density. However, shear stress alone did not alter the corrosion rate of 316LSS. THP1 cells adhered to the 316LSS surface at all flow rates. Exposure to the 316LSS/corrosion test under high fluid flow rates increased (>twofold) the secretion of 7 of the 42 cytokines tested (angeogenin, GRO, I309, interleukin 8, interleukin 6, interleukin 1beta, and macrophage chemoattractant protein-1). Each of these cytokines play a role in wound healing, macrophage differentiation, and cell proliferation, all hallmarks of in-stent restenosis. Furthermore, only IL8 levels were significantly higher than any of the system controls during the 316LSS/corrosion test conditions. The IL8 levels from the 316LSS/corrosion tests were not significantly different from the +LPS control. Together, these data suggest that monocytic cells maybe activated by exposure to 316LSS stents and could contribute to in-stent restenosis and altered corrosion of the stent.

Colour and translucency of opaque-shades and body-shades of resin composites.

The purpose of this study was to evaluate the optical properties--not only the translucency but also the colours--of opaque-shade resin composites. The CIELAB parameters (L*, a* and b*) of disks of A2 and opaque A2 (OA2) shades of Charisma (Heraeus-Kulzer), Solare (GC) and Filtek Supreme (3M) were evaluated on backings of black, white and the material itself to calculate the translucency parameter (TP) and the colour differences (delta E*) between A2 and OA2. A two-way analysis of variance (anova) for the TP indicated a less statistically significant TP value in the OA2 shade than the A2 shade for all products. As for the products, Charisma showed a statistically greater TP value than the other two products. Regarding the delta E* between A2 and OA2, all the products revealed clinically perceptible colour differences (delta E* > 3.3). Hence, we must take the colour differences of opaque-shade resin composites into consideration, as well as the translucency of the materials, for a clinically acceptable colour match of the restoration.