Sources and levels of copper affect liver copper profile, intestinal morphology and cecal microbiota population of broiler chickens fed wheat-soybean meal diets.

Super dosing copper (Cu) has long been used as an alternative to antibiotic growth-promoters in broiler chickens' diet to improve gut health. This study was designed to compare nutritional and growth-promoting levels of Cu hydroxychloride (CH) with CuSO4 on gut health bio-markers and liver mineral profile of broiler chickens. Ross 308 chicks (n = 864) were randomly assigned to eight treatments, as basal diet containing no supplemental Cu; the basal diet with 15 or 200 mg/kg Cu as CuSO4; or 15, 50, 100, 150 or 200 mg/kg Cu from CH. The highest liver Cu content was observed in birds fed the diets with 200 mg/kg CuSO4 (P < 0.01). Serum FITC-d concentration as the leaky gut marker, and liver malondialdehyde concentration were not affected. Copper level or source had no effect on cecal short chain fatty acid and the mRNA expression of five jejunal genes involved in gut integrity. Negative linear responses of Cu were observed on Lactobacillus (P = 0.032), Bacteroides (P = 0.033), and Enterobacteriaceae (P = 0.028) counts. The jejunal villus height increased in birds fed CH at 200 and 100 mg/kg (P < 0.05). Increasing Cu levels, linearly and quadratically (P < 0.001), increased Cu excretion.

Biosynthesis of eco-friendly and recyclable Pd/LDHs catalyst using the withered leaves extract for Suzuki coupling reaction.

The hydrotalcite-supported palladium (Pd) catalyst is prepared with a green and environmentally friendly route, introducing the extract of withered leaves as a dispersant and reducing agent (Pd/LDHs-B). Compared with the as-prepared catalyst (Pd/LDHs-P with the average diameter of 4.3 nm) using a chemical synthesis method with polyvinylpyrrolidone as a dispersant and ascorbic acid (Vc) as a reductant, the results indicate that the size of Pd nanoparticles in Pd/LDHs-B is smaller (ca. 3.6 nm). The Pd-LHDs-B (0.5 mmol‰) exhibits higher activity (98.66%) than Pd/LDHs-P (98.19%) in the Suzuki reaction of 4-bromotoluene and phenylboronic acid at 60°C for 30 min. Also, the reusability of Pd/LDHs-B is confirmed by recycling tests without a significant decrease in activity.

pH-activated heat shock protein inhibition and radical generation enhanced NIR luminescence imaging-guided photothermal tumour ablation.

Photothermal therapy had great potential in being a new approach of tumour ablation due to their high selectivity and low side effect. However, the shallow penetration depth of near-infrared (NIR) irradiation resulted in the limited curative effect. Herein, a novel nanomedicine was developed based on the indocyanine green-loaded vanadium oxide nanocomposites (VO2-ICG) for pH-activated NIR luminescence imaging-guided enhanced photothermal tumour ablation. In acidic tumour microenvironment, the VO2 NPs were decomposed and released VO2+, which could not only inhibit the function of 60 kDa heat shock protein (HSP60), but also generate hydroxyl radical (OH) by catalysing intratumoral H2O2. Furthermore, the ICG was also released in the decomposition process of VO2 NPs, allowing the pH-activated NIR luminescence imaging and photothermal therapy. The inhibition of HSP60 down-regulated the heat tolerance of cells and the generation of OH up-regulated the intracellular oxidative stress, which enhanced the photothermal therapeutic efficiency. Our work demonstrated a promised method to enhance photothermal therapeutic effect, highlighting the importance of HSP inhibition and OH generation in promoting cell apoptosis under mild hyperthermia.

Evaluation of aluminium mobilization from its soil mineral pools by simultaneous effect of Aspergillus strains' acidic and chelating exometabolites.

This contribution investigates aluminium mobilization from main aluminium pools in soils, phyllosilicates and oxyhydroxides, by acidic and chelating exometabolites of common soil fungi Aspergillus niger and A. clavatus. Their exometabolites' acidity as well as their ability to extract aluminium from solid mineral phases differed significantly during incubation. While both strains are able to mobilize aluminium from boehmite and aluminium oxide mixture to some extent, A. clavatus struggles to mobilize any aluminium from gibbsite. Furthermore, passive and active fungal uptake of aluminium enhances its mobilization from boehmite, especially in later growth phase, with strong linear correlation between aluminium bioaccumulated fraction and increasing culture medium pH. We also provide data on concentrations of oxalate, citrate and gluconate which are synthesized by A. niger and contribute to aluminium mobilization. Compared to boehmite-free treatment, fungus reduces oxalate production significantly in boehmite presence to restrict aluminium extraction efficiency. However, in presence of high phyllosilicates' dosages, aluminium is released to an extent that acetate and citrate is overproduced by fungus. Our results also highlight fungal capability to significantly enhance iron and silicon mobility as these elements are extracted from mineral lattice of phyllosilicates by fungal exometabolites alongside aluminium.

Agronomic Effectiveness of Granulated and Powdered P-Exchanged Mg-Al LDH Relative to Struvite and MAP.

Layered double hydroxides (LDHs) used to recover P from wastewater have recently been proposed as new slow-release fertilizers. Here, the use of P-exchanged Mg-Al LDHs as powdered or granulated fertilizer is explored and compared with monoammonium phosphate (MAP), a fully water-soluble fertilizer, and with struvite, a recycled phosphate fertilizer with lower solubility. First, these three fertilizers were compared in a 100-day incubation experiment using P diffusion visualization and chemical analysis to assess P release from either granules or powdered fertilizer in three different soils. By the end of the incubation, 74-90% of P remained within the LDH granule, confirming a slow release. Second, a pot experiment was performed with wheat (Triticum aestivum) in an acid and a calcareous soil. The granular treatment resulted in a considerably higher P uptake for MAP compared to LDH and struvite. For the powder treatments, the P uptake was less than for granular MAP and was largely unaffected by the chemical form. The LDHs and struvite showed a lower agronomic effectiveness than granular MAP, but the benefits of their use in P recycling, potential residual value, and environmental benefits may render these slow-release fertilizers attractive.

Lyophilized aqueous extracts of Mori Fructus and Mori Ramulus protect Mesenchymal stem cells from •OH-treated damage: bioassay and antioxidant mechanism.

BACKGROUND: Mori Fructus and Mori Ramulus are two traditional Chinese herbal medicines from mulberries. The present work explores their beneficial effects on •OH-treated mesenchymal stem cells (MSCs) and discusses possible mechanisms. METHODS: Lyophilized aqueous extracts of Mori Fructus (LAMF) and Mori Ramulus (LAMR) were prepared and analyzed using HPLC. LAMF and LAMR (along with morin) were further investigated for their effects on •OH-treated MSCs using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The direct antioxidation mechanisms were studied using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO•)-scavenging, 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS+•)-scavenging and 1,1-diphenyl-2-picryl-hydrazl (DPPH•)-scavenging, as well as Cu2+-reducing and Fe3+-reducing antioxidant power. Finally, the indirect antioxidant mechanism was investigated based on the UV-vis spectra of Fe2+-chelation. RESULTS: In each LAMF and LAMR, seven phytophenols were successfully measured by HPLC, including five flavonoids (morin, rutin, astragalin, isoquercitrin and luteolin) and two non-flavonoids (chlorogenic acid and maclurin). MTT assays revealed that LAMF, LAMR and morin could effectively increase the survival of •OH-treated MSCs at 10-100 µg/mL, and could effectively scavenge PTIO• (IC 50 6609.7 ± 756.6, 4286.9 ± 84.9 and 103.4 ± 0.9 µg/mL, respectively), DPPH• (IC 50 208.7 ± 3.0, 97.3 ± 3.1 and 8.2 ± 0.7 µg/mL, respectively) and ABTS+• (IC 50 73.5 ± 5.8, 34.4 ± 0.1 and 4.2 ± 0.2 µg/mL, respectively), and reduce Cu2+ (IC 50 212.5 ± 7.0, 123.2 ± 0.9 and 14.1 ± 0.04 µg/mL, respectively) & Fe3+ (IC 50 277.0 ± 3.1, 191.9 ± 5.2 and 5.0 ± 0.2 µg/mL, respectively). In the Fe2+-chelating assay, the five flavonoids produced much stronger shoulder-peaks than the two non-flavonoids within 420-850 nm. CONCLUSION: Mori Fructus and Mori Ramulus, can protect MSCs from •OH-induced damage. Such beneficial effects can mainly be attributed to the antioxidant action of phytophenols, which occurs via direct (ROS-scavenging) and indirect mechanism (Fe2+-chelating). The ROS-scavenging mechanism, however, include at least a H+-transfer and an electron-transfer (ET), and possibly includes a hydrogen-atom-transfer (HAT). In the Fe2+-chelating, flavonoids are more effective than non-flavonoids. This can be attributed to several adjacent planar chelating-sites between the 3-OH and 4-C = O, between the 4-C = O and 5-OH, or between the 3'-OH and 4'-OH in flavonoids. Such multiple-Fe2+-chelating reactions cause overlap in the UV-vis absorptions to deepen the complex color, enhance the peak strength, and form shoulder-peaks. By comparison, two non-flavonoids with catechol moiety produce only a weak single peak.

Effects of sub-acute methanol extract treatment of Calliandra portoricensis root bark on antioxidant defence capacity in an experimental rat model.

BACKGROUND: The attendant side effects associated with some synthetic drugs used in the management of diseases have led to the search for safer alternative therapies that are relatively cheaper with minimal side effects. METHODS: The methanol extract of Calliandra portoricensis root bark (CPRB) was orally administered at the doses of 5, 10, 20, and 25 mg/kg body weight for 14 consecutive days of 5 rats in each group. The control rats were given distilled water. RESULTS: The 95% methanol extract of CPRB significantly (p<0.05) scavenged NO• and OH• radicals compared to vitamin C. The level of lipid peroxidative products (malondialdehyde, MDA) was significantly (p<0.05) attenuated in a dose-dependent manner. Antioxidant enzymes including superoxide dismutase and catalase were significantly (p<0.05) exercabated in both liver and kidney in a dose-dependent manner. Furthermore, serum AST, alanine aminotransaminase and γ-glutamyltransferase (GGT) activity depicted non-significant (p>0.05) increase in the treated animals. The histological examination showed mild vacuolar, portal congestion and cell infiltration by mononuclear of the hepatic tissues. CONCLUSIONS: The study then concluded that a therapeutic dose of the methanol extract of CPRB triggered the antioxidant defence systems in male rats. It is, therefore, recommended that the doses should be carefully and clinically chosen because higher doses may cause some health risks.

Potent antioxidant and genoprotective effects of boeravinone G, a rotenoid isolated from Boerhaavia diffusa.

BACKGROUND AND AIMS: Free radicals are implicated in the aetiology of some gastrointestinal disorders such as gastric ulcer, colorectal cancer and inflammatory bowel disease. In the present study we investigated the antioxidant and genoprotective activity of some rotenoids (i.e. boeravinones) isolated from the roots of Boerhaavia diffusa, a plant used in the Ayurvedic medicine for the treatment of diseases affecting the gastrointestinal tract. METHODS/PRINCIPAL FINDINGS: Antioxidant activity has been evaluated using both chemical (Electron Spin Resonance spectroscopy, ESR) and Caco-2 cells-based (TBARS and ROS) assays. DNA damage was evaluated by Comet assay, while pERK(1/2) and phospho-NF-kB p65 levels were estimated by western blot. Boeravinones G, D and H significantly reduced the signal intensity of ESR induced by hydroxyl radicals, suggesting a scavenging activity. Among rotenoids tested, boeravinone G exerted the most potent effect. Boeravinone G inhibited both TBARS and ROS formation induced by Fenton's reagent, increased SOD activity and reduced H(2)O(2)-induced DNA damage. Finally, boeravinone G reduced the levels of pERK(1) and phospho-NF-kB p65 (but not of pERK(2)) increased by Fenton's reagent. CONCLUSIONS: It is concluded that boeravinone G exhibits an extraordinary potent antioxidant activity (significant effect in the nanomolar range). The MAP kinase and NF-kB pathways seem to be involved in the antioxidant effect of boeravinone G. Boeravinone G might be considered as lead compound for the development of drugs potentially useful against those pathologies whose aetiology is related to ROS-mediated injuries.

Kinetics of transesterification of palm oil and dimethyl carbonate for biodiesel production at the catalysis of heterogeneous base catalyst.

The transesterification of palm oil with dimethyl carbonate (DMC) for preparing biodiesel has been studied in solvent-free system at the catalysis of potassium hydroxide (KOH) as heterogeneous catalyst. Fatty acid methyl esters (FAMEs) were analyzed by GC with internal standard method. The effects of reaction conditions (molar ratio of DMC and palm oil, catalyst amount and time) on FAMEs yield were investigated. The highest FAMEs yield could reach 96.2% at refluxing temperature for 8h with molar ratio of DMC and oil 9:1 and 8.5% KOH (based on oil weight). Kinetics of the KOH-catalyzed transesterification of palm oil and DMC was researched over a temperature range of 65-75 degrees C. A pseudo first-order model was proposed. The activation energy (E(a)) was 79.1 kJ mo1(-1) and the pre-exponential factor (k(o)) was 1.26 x 10(9) min(-1) from Arrhenius equation. Further, a plausible reaction mechanism for the catalytic process with DMC as acyl acceptor was proposed.

Enhancement of disease resistance to Magnaporthe grisea in rice by accumulation of hydroxy linoleic acid.

Linoleic acid (18:2) and linolenic acid (18:3) are sources for various oxidized metabolites called oxylipins, some of which inhibit growth of fungal pathogens. In a previous study, we found disease resistance to rice blast fungus Magnaporthe grisea enhanced in 18:2-accumulating transgenic rice (F78Ri) in which the conversion from 18:2 to 18:3 was suppressed. Here, we demonstrate that 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs, respectively) inhibit growth of M. grisea more strongly than their 18:3-derived counterparts. Furthermore, in F78Ri plants, the endogenous levels of HPODEs and HODEs increased significantly, compared with wild-type plants. These results suggest that the increased accumulation of antifungal oxylipins, such as HPODEs and HODEs, causes the enhancement of disease resistance against M. grisea.

Superoxide, H2O2, and iron are required for TNF-alpha-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase.

Reactive oxygen species (ROS) play an important but not yet fully defined role in the expression of inflammatory genes such as monocyte chemoattractant protein (MCP)-1. We used complementary molecular and biochemical approaches to explore the roles of specific ROS and their molecular linkage to inflammatory signaling in endothelial cells. Adenovirus-mediated expression of superoxide dismutase and catalase inhibited TNF-alpha-induced MCP-1 gene expression, suggesting important roles of superoxide (O(2)(-).) and H(2)O(2) in MCP-1 gene activation. In addition, the iron chelator 1,2-dimethyl-3-hydroxypyridin-4-one and the hydroxyl radical scavengers dimethylthiourea and dimethyl sulfoxide inhibited TNF-alpha-induced MCP-1 expression, suggesting important roles of iron and hydroxyl radicals in inflammatory signal activation. In contrast, scavenging of peroxynitrite with 5,10,15,20-tetrakis-(4-sulfonatophenyl)prophyrinato iron (III) chloride had no effect on TNF-alpha-induced MCP-1 expression. Inhibition of NADPH oxidase, the major oxidase responsible for O(2)(-). generation, with diphenylene iodonium suppressed TNF-alpha-induced MCP-1 mRNA accumulation. Rac1 is an upstream signaling molecule for the activation of NADPH oxidase and O(2)(-). generation. Expression of dominant negative N17Rac1 by adenovirus suppressed TNF-alpha-induced MCP-1 mRNA levels and MCP-1 protein secretion. Expression of N17Rac1 inhibited TNF-alpha-induced MCP-1 and NF-kappaB transcriptional activity. These data suggest that ROS such as superoxide and H(2)O(2) derived from Rac1-activated NADPH oxidase mediate TNF-alpha-induced MCP-1 expression in endothelial cells.

Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol.

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O(2)*-) and hydrogen peroxide (H(2)O(2)). The electron transfer chain of mitochondria is a well documented source of H(2)O(2); however, the release of O(2)*- from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O(2)*-, elucidating the mechanisms underlying the release of O(2)*- from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O(2)*-. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochrome c proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O(2)*- release from mitochondria were estimated at approximately 0.04 nmol/min/mg protein, a value increased approximately 8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-based p-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O(2)*- production from mitochondria by approximately 55%, thus suggesting that a large portion of O(2)*- exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O(2)*- in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.

Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0.

During maturation of oocytes, Cl(-) conductance (G(Cl)) oscillates and intracellular pH (pH(i)) increases. Elevating pH(i) permits the protein synthesis essential to maturation. To examine whether changes in G(Cl) and pH(i) are coupled, the Cl(-) channel ClC-0 was heterologously expressed. Overexpressing ClC-0 elevates pH(i), decreases intracellular Cl(-) concentration ([Cl(-)](i)), and reduces volume. Acute acidification with butyrate does not activate acid extrusion in ClC-0-expressing or control oocytes. The ClC-0-induced pH(i) change increases after overnight incubation at extracellular pH 8.5 but is unaltered after incubation at extracellular pH 6.5. Membrane depolarization did not change pH(i). In contrast, hyperpolarization elevates pH(i). Thus neither membrane depolarization nor acute activation of acid extrusion accounts for the ClC-0-dependent alkalinization. Overnight incubation in low extracellular Cl(-) concentration increases pH(i) and decreases [Cl(-)](i) in control and ClC-0 expressing oocytes, with the effect greater in the latter. Incubation in hypotonic, low extracellular Cl(-) solutions prevented pH(i) elevation, although the decrease in [Cl(-)](i) persisted. Taken together, our observations suggest that KCl loss leads to oocyte shrinkage, which transiently activates acid extrusion. In conclusion, expressing ClC-0 in oocytes increases pH(i) and decreases [Cl(-)](i). These parameters are coupled via shrinkage activation of proton extrusion. Normal, cyclical changes of oocyte G(Cl) may exert an effect on pH(i) via shrinkage, thus inducing meiotic maturation.

XAFS determination of the bacterial cell wall functional groups responsible for complexation of Cd and U as a function of pH.

Bacteria, which are ubiquitous in near-surface geologic systems, can affect the distribution and fate of metals in these systems through adsorption reactions between the metals and bacterial cell walls. Recently, Fein et al. (1997) developed a chemical equilibrium approach to quantify metal adsorption onto cell walls, treating the sorption as a surface complexation phenomenon. However, such models are based on circumstantial bulk adsorption evidence only, and the nature and mechanism of metal binding to cell walls for each metal system have not been determined spectroscopically. The results of XAFS measurements at the Cd K-edge and U L3-edge on Bacillus subtilis exposed to these elements show that, at low pH, U binds to phosphoryl groups while Cd binds to carboxyl functional groups.

Effect of hyperbaric oxygen treatment on nitric oxide and oxygen free radicals in rat brain.

Oxygen (O(2)) at high pressures acts as a neurotoxic agent leading to convulsions. The mechanism of this neurotoxicity is not known; however, oxygen free radicals and nitric oxide (NO) have been suggested as contributors. This study was designed to follow the formation of oxygen free radicals and NO in the rat brain under hyperbaric oxygen (HBO) conditions using in vivo microdialysis. Male Sprague-Dawley rats were exposed to 100% O(2) at a pressure of 3 atm absolute for 2 h. The formation of 2,3-dihydroxybenzoic acid (2, 3-DHBA) as a result of perfusing sodium salicylate was followed as an indicator for the formation of hydroxyl radicals. 2,3-DHBA levels in hippocampal and striatal dialysates of animals exposed to HBO conditions were not significantly different from controls. However, rats treated under the same conditions showed a six- and fourfold increase in nitrite/nitrate, break down products of NO decomposition, in hippocampal and striatal dialysates, respectively. This increase was completely blocked by the nitric oxide synthase (NOS) inhibitor L-nitroarginine methyl ester (L-NAME). Using neuronal NOS, we determined the NOS O(2) K(m) to be 158 +/- 28 (SD) mmHg, a value which suggests that production of NO by NOS would increase approximately four- to fivefold under hyperbaric O(2) conditions, closely matching the measured increase in vivo. The increase in NO levels may be partially responsible for some of the detrimental effects of HBO conditions.

Effect of keigai-rengyo-to, a Japanese kampo medicine, on neutrophil functions: a possible mechanism of action of keigai-rengyo-to in acne.

On the basis of recent reports that keigai-rengyo-to (TJ-50), an oral Japanese Kampo (herb) medicine, is clinically effective in treating acne, and that tetracyclines are effective against acne by acting directly as an antioxidant on infiltrated neutrophils, we investigated the effect of TJ-50 on the generation of reactive oxygen species (ROS), using human neutrophils and a cell-free, xanthine-xanthine oxidase system. The species investigated were superoxide radical anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH.). In addition, neutrophil chemotaxis, phagocytosis and calcium concentration, [Ca2+]i in neutrophils were also assessed. TJ-50 significantly decreased neutrophil-generated O2-, H2O2 and OH. in a dose-dependent manner. Three kinds of ROS generated in the cell-free system were also reduced in the presence of TJ-50. On the other hand, the medicine did not markedly affect neutrophil chemotaxis, phagocytosis or [Ca2+]i in neutrophils. Our results indicate that the clinical effectiveness of TJ-50 in the treatment of acne may be due partly to its antioxidant action on infiltrated neutrophils.

Role of polymorphonuclear leucocytes and oxygen-derived free radicals in the formation of gastric lesions induced by HCl/ethanol, and a possible mechanism of protection by anti-ulcer polysaccharide.

This study examined the role of oxygen-derived free radicals in the pathogenesis of gastric mucosal lesions induced by HCl/ethanol. Superoxide dismutase, and catalase, and their combination reduced gastric lesion formation in mice. Gastric lesions were also reduced in mice treated with cyclophosphamide or anti-neutrophils, but not in mice treated with allopurinol or desulphated-carrageenan. Cobra venom factor did not reduce lesion formation. These results suggested that oxygen-free radicals may contribute to the formation of gastric mucosal lesions induced by HCl/ethanol, and that oxygen radicals were generated from neutrophils but not from xanthine oxidase. Anti-ulcer pectic polysaccharide, bupleuran 2IIc, which was recently isolated from the roots of Bupleurum falcatum L., showed potent inhibition of HCl/ethanol-induced gastric lesions in mice. Bupleuran 2IIc seemed to scavenge hydroxyl radical effectively. It was suggested that this anti-ulcer polysaccharide may provide protection to the gastric mucosa by scavenging oxygen-free radicals.

Enhancement of merocyanine 540-mediated phototherapy by salicylate.

Merocyanine 540 (MC540) is a photosensitizing dye of potential use in the purging of cancer cells from autologous bone marrow explants. Treatment of marrow with MC540, followed by illumination with visible light, selectively kills neoplastic cells while sparing a sufficient number of stem cells to allow marrow engraftment. The photodynamic action of MC540 is thought to be mediated by reactive oxygen species, particularly singlet oxygen. We have previously shown that salicylic acid (SA) scavenges MC540-generated singlet oxygen. In this work, we sought to abrogate MC540-mediated cell killing of murine L1210 and human K562 leukemia cells with salicylate. Paradoxically, the presence of salicylate during illumination in the presence of MC540 appreciably enhanced cell killing. Enhancement was dependent on salicylate concentration in the range 0.1 to 10 mM, with 1.0 mM SA potentiating the MC540-mediated reduction in survival of L1210 and K562 cells by factors of 2.7 and 1.9, respectively. Neither preincubation with SA followed by washing prior to illumination nor addition of SA following illumination altered MC540-mediated cell killing, indicating that potentiation was dependent on the presence of SA during illumination. Illumination in the presence of salicylate alone did not diminish cell viability. In addition to SA, a number of structurally related compounds including dihydroxybenzoic acids, aspirin, and sodium benzoate also enhanced MC540-mediated cell killing. Potentiation of leukemic cell killing by salicylate could provide a basis for enhancing the clinical efficacy of MC540-mediated phototherapy.

Methylene blue and rose bengal photoinactivation of RNA bacteriophages: comparative studies of 8-oxoguanine formation in isolated RNA.

Several reactive oxygen species, including singlet oxygen (1O2) and hydroxyl free radical (.OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB). Both 1O2 and .OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA. Evidence that MB-or RB-induced bacteriophage (R17 or Q beta) inactivation and 8-oxoGua formation in RNA result from 1O2 rather than .OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37 degrees C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a .OH generating system, the ultraviolet light irradiation of H2O2; and (iii) deuterium oxide (D2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA. The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation. The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O2-mediated lethal lesions in RNA bacteriophages.

Mechanism of synergistic cytotoxic effect between tumor necrosis factor and hyperthermia.

We previously reported that recombinant human tumor necrosis factor (rhTNF) and hyperthermia had a synergistic effect against tumors, in vitro and in vivo. We have now investigated the mechanism of this synergy by measuring the lysosomal enzyme activity and hydroxyl radical production of L-M cells treated with rhTNF and/or hyperthermia. A synergistic activation of lysosomal enzyme and the induction of hydroxyl radical production in L-M cells treated with both rhTNF and hyperthermia was observed. A synergistic cytotoxic effect was observed when rhTNF and hyperthermia were combined, and was inhibited by the addition of a reactive oxygen scavenger, dimethyl sulfoxide or bipyridine. The results show that the augmenting effect of hyperthermia on lysosomal enzyme activation and induction of hydroxyl radical production by rhTNF plays an important role in the synergistic cytotoxic effect.