ascorbic-acid and resazurin

Biopolymeric films were prepared with gelatin, plasticizer, and three different types of antioxidants (ascorbic acid, phytic acid, and BHA) corresponding to different mechanisms in activity. The antioxidant activity of films was monitored for 14 storage days upon color changes using a pH indicator (resazurin). The instant antioxidant activity of films was measured by a DPPH free radical test. The system using resazurin was composed of an agar, an emulsifier, and soybean oil to simulate a highly oxidative oil-based food system (AES-R). Gelatin-based films (GBF) containing phytic acid showed higher tensile strength and energy to break than all other samples due to the increased intermolecular interactions between phytic acid and gelatin molecules. The oxygen barrier properties of GBF films containing ascorbic acid and phytic acid increased due to the increased polarity, while GBF films containing BHA showed increased oxygen permeability compared to the control. According to "a-value" (redness) of the AES-R system tested with films, films incorporating BHA showed the most retardation of lipid oxidation in the system. This retardation corresponds to 59.8% antioxidation activity at 14 days, compared with the control. Phytic acid-based films did not show antioxidant activity, whereas ascorbic acid-based GBFs accelerated the oxidation process due to its prooxidant activity. The comparison between the DPPH free radical test and the control showed that the ascorbic acid and BHA-based GBFs showed highly effective free radical scavenging behavior (71.7% and 41.7%, respectively). This novel method using a pH indicator system can potentially determine the antioxidation activity of biopolymer films and film-based samples in a food system.

Topics: Antioxidants; Ascorbic Acid; Biofilms; Food Packaging; Gelatin; Oxygen; Phytic Acid

Lactate dehydrogenase (LDH) release is frequently used as an end-point for cytotoxicity studies. We have been unable to measure LDH release during studies using para-aminophenol (PAP) in LLC-PK(1) cells. When LLC-PK(1) cells were incubated with either PAP (0-10 mM) or menadione (0-1000 microM), viability was markedly reduced when assessed by alamar Blue or total LDH activity but not by release of LDH into the incubation medium. In addition, we incubated cells with PAP or menadione and compared LDH activity using two different assays. Both assays confirmed our observation of decreased LDH activity in cell lysates without corresponding increases in LDH activity in incubation media. Using purified LDH and 10 mM PAP, we found that PAP produced loss of LDH activity that was inversely proportional to the amount of LDH initially added. In additional experiments, we incubated 0.5 units of LDH for 1 h with varying concentrations of PAP, menadione, hydrogen peroxide (H(2)O(2)) or cisplatin. All four chemicals produced concentration-dependent decreases in LDH activity. In previous experiments, inclusion of antioxidants such as reduced glutathione (GSH) and ascorbate protected cells from PAP toxicity. GSH (1 mM) preserved LDH activity in the presence of toxicants while ascorbate (1 mM) only prevented LDH loss induced by PAP. These studies suggest that LDH that is released into the incubation medium is susceptible to degradation when reactive chemicals are present.

Topics: Aminophenols; Animals; Antineoplastic Agents; Ascorbic Acid; Cell Survival; Cisplatin; Enzyme Inhibitors; Hydrogen Peroxide; L-Lactate Dehydrogenase; LLC-PK1 Cells; NAD; Oxazines; Oxidants; Oxidation-Reduction; Swine; Toxicity Tests; Vitamin K 3; Xanthenes

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2',7'-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N(G)-monomethyl-L-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK-independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.

Topics: Acetylcysteine; Anthracenes; Antioxidants; Apoptosis; Ascorbic Acid; Butadienes; Cell Line, Tumor; Cell Proliferation; DNA Damage; Enzyme Inhibitors; Fluoresceins; HeLa Cells; Humans; Imidazoles; MAP Kinase Signaling System; Melatonin; Mitochondria; Nitriles; Nitrites; Oxazines; Oxygen; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Radiation Tolerance; Rhodamines; Spectrometry, Fluorescence; Xanthenes

Reactive oxygen and nitrogen species (RO/NS) such as nitric oxide (NO), hydroxyl radical (OH.), and superoxide anion (O(2)(-)) are generated in a variety of neuropathological processes and damage neurons. In the present study, we investigated the neuroprotective effects of rat astrocytes against RO/NS-induced damage using neuron-glia cocultures, and the effects were compared to those of microglial cells. Sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and FeSO(4) were used to generate NO, O(2)(-) and NO, and OH., respectively. Solely cultured neurons, which were transiently exposed to these agents, degenerated, possibly through apoptotic mechanisms as revealed by in situ detection of DNA fragmentation, whereas neurons cocultured with either astrocytes or microglial cells were viable even after exposure to RO/NS. In contrast, most neurons cocultured with meningeal fibroblasts degenerated. Astrocyte-conditioned medium partially attenuated RO/NS-induced neuronal damage. When neurons were cultured on astrocyte-derived extracellular matrix (AsECM), neuronal death induced by SNP and FeSO(4) was almost completely inhibited. AsECM contained significant amounts of laminin and fibronectin, and pure fibronectin and laminin also protected neurons against RO/NS-induced damage in the same manner as AsECM. These results suggest that astrocytes can protect neurons against RO/NS-induced damage by secreting soluble and insoluble factors.

Topics: Animals; Ascorbic Acid; Astrocytes; Cell Culture Techniques; Cell Death; Coloring Agents; Extracellular Matrix; Ferric Compounds; Fibronectins; Free Radical Scavengers; Hydroxyl Radical; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Indicators and Reagents; Laminin; Microtubule-Associated Proteins; Molsidomine; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxazines; Oxidative Stress; Rats; Reactive Oxygen Species; Xanthenes

Topics: Animals; Aqueous Humor; Ascorbic Acid; Half-Life; Humans; Indicators and Reagents; Kinetics; Oxazines; Oxidation-Reduction; Photography; Rabbits; Spectrometry, Fluorescence; Xanthenes