3-Amino-1,2,4-triazole Limits the Oxidative Damage in UVA-Irradiated Dysplastic Keratinocytes.

Reactive oxygen species (ROS) generated by UVA irradiation affect the keratinocyte cell membrane, DNA, and proteins and may cause serious injury to the skin. Treating human dysplastic keratinocytes (DOK) with 3-amino-1,2,4-triazole (AMT), a common catalase inhibitor, induced a compensatory mechanism for the hydrogen peroxide detoxification, which included a rise in glutathione peroxidase and glutathione reductase activities. Here, we examined a possible role of AMT in protecting a human DOK cell line against UVA-induced damage. In DOK cells exposed to UVA irradiation, we observed a substantial decrease in antioxidant enzymatic activities, such as catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase and an increase in lipid peroxidation and protein oxidation levels. Treating DOK cells with AMT prior to UVA exposure enhanced the activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase, relative to nontreated cells. The enhanced antioxidant activities were correlated with decreased protein oxidation levels. Based on these results, we suggest that AMT may protect dysplastic keratinocytes against the harmful effects of UVA radiation.

Silica Nanoparticles Induce Oxidative Stress and Autophagy but Not Apoptosis in the MRC-5 Cell Line.

This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO NPs) on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The nanoparticles' morphology, composition, and structure were investigated using high resolution transmission electron microscopy, selected area electron diffraction and X-ray diffraction. Our study showed a decreased cell viability and the induction of cellular oxidative stress as evidenced by an increased level of reactive oxygen species (ROS), carbonyl groups, and advanced oxidation protein products after 24, 48, and 72 h, as well as a decreased concentration of glutathione (GSH) and protein sulfhydryl groups. The protein expression of Hsp27, Hsp60, and Hsp90 decreased at all time intervals, while the level of protein Hsp70 remained unchanged during the exposure. Similarly, the expression of p53, MDM2 and Bcl-2 was significantly decreased for all time intervals, while the expression of Bax, a marker for apoptosis, was insignificantly downregulated. These results correlated with the increase of pro-caspase 3 expression. The role of autophagy in cellular response to SiO2NPs was demonstrated by a fluorescence-labeled method and by an increased level of LC3-II/LC3-I ratio. Taken together, our data suggested that SiO2 NPs induced ROS-mediated autophagy in MRC-5 cells as a possible mechanism of cell survival.

Magnetite nanoparticles induced adaptive mechanisms counteract cell death in human pulmonary fibroblasts.

Magnetite nanoparticles (MNP) have attracted great interest for biomedical applications due to their unique chemical and physical properties, but the MNP impact on human health is not fully known. Consequently, our study proposes to highlight the biochemical mechanisms that underline the toxic effects of MNP on a human lung fibroblast cell line (MRC-5). The cytotoxicity generated by MNP in MRC-5 cells was dose and time-dependent. MNP-treated MRC-5 cells accumulated large amount of iron and reactive oxygen species (ROS) and exhibited elevated antioxidant scavenger enzymes. Reduced glutathione (GSH) depletion and enhanced lipid peroxidation (LPO) processes were also observed. The cellular capacity to counteract the oxidative damage was sustained by high levels of heat shock protein 60 (Hsp60), a protein that confers resistance against ROS attack and inhibition of cell death. While significant augmentations in nitric oxide (NO) and prostaglandine E2 (PGE2) levels were detected after 72 h of MNP-exposure only, caspase-1 was activated earlier starting with 24h post-treatment. Taken together, our results suggest that MRC-5 cells have the capacity to develop cell protection mechanisms against MNP. Detailed knowledge of the mechanisms induced by MNP in cell culture could be essential for their prospective use in various in vivo biochemical applications.

Interaction of silicon-based quantum dots with gibel carp liver: oxidative and structural modifications.

Quantum dots (QDs) interaction with living organisms is of central interest due to their various biological and medical applications. One of the most important mechanisms proposed for various silicon nanoparticle-mediated toxicity is oxidative stress. We investigated the basic processes of cellular damage by oxidative stress and tissue injury following QD accumulation in the gibel carp liver after intraperitoneal injection of a single dose of 2 mg/kg body weight Si/SiO2 QDs after 1, 3, and 7 days from their administration.QDs gradual accumulation was highlighted by fluorescence microscopy, and subsequent histological changes in the hepatic tissue were noted. After 1 and 3 days, QD-treated fish showed an increased number of macrophage clusters and fibrosis, while hepatocyte basophilia and isolated hepatolytic microlesions were observed only after substantial QDs accumulation in the liver parenchyma, at 7 days after IP injection.Induction of oxidative stress in fish liver was revealed by the formation of malondialdehyde and advanced oxidation protein products, as well as a decrease in protein thiol groups and reduced glutathione levels. The liver enzymatic antioxidant defense was modulated to maintain the redox status in response to the changes initiated by Si/SiO2 QDs. So, catalase and glutathione peroxidase activities were upregulated starting from the first day after injection, while the activity of superoxide dismutase increased only after 7 days. The oxidative damage that still occurred may impair the activity of more sensitive enzymes. A significant inhibition in glucose-6-phosphate dehydrogenase and glutathione-S-transferase activity was noted, while glutathione reductase remained unaltered.Taking into account that the reduced glutathione level had a deep decline and the level of lipid peroxidation products remained highly increased in the time interval we studied, it appears that the liver antioxidant defense of Carassius gibelio does not counteract the oxidative stress induced 7 days after silicon-based QDs exposure in an efficient manner.

UVA irradiation of dysplastic keratinocytes: oxidative damage versus antioxidant defense.

UVA affects epidermal cell physiology in a complex manner, but the harmful effects have been studied mainly in terms of DNA damage, mutagenesis and carcinogenesis. We investigated UVA effects on membrane integrity and antioxidant defense of dysplastic keratinocytes after one and two hours of irradiation, both immediately after exposure, and 24 h post-irradiation. To determine the UVA oxidative stress on cell membrane, lipid peroxidation was correlated with changes in fatty acid levels. Membrane permeability and integrity were assessed by propidium iodide staining and lactate dehydrogenase release. The effects on keratinocyte antioxidant protection were investigated in terms of catalase activity and expression. Lipid peroxidation increased in an exposure time-dependent manner. UVA exposure decreased the level of polyunsaturated fatty acids, which gradually returned to its initial value. Lactate dehydrogenase release showed a dramatic loss in membrane integrity after 2 h minimum of exposure. The cell ability to restore membrane permeability was noted at 24 h post-irradiation (for one hour exposure). Catalase activity decreased in an exposure time-dependent manner. UVA-irradiated dysplastic keratinocytes developed mechanisms leading to cell protection and survival, following a non-lethal exposure. The surviving cells gained an increased resistance to apoptosis, suggesting that their pre-malignant status harbors an abnormal ability to control their fate.

Ethanol-induced redox imbalance in rat kidneys.

This study reports the effects of long-term ethanol consumption on kidney redox status, in terms of enzymatic mechanisms involved in regulating the cytosolic [NADH]/[NAD(+) ] balance. Wistar rats were treated with ethanol (2 g/kg body weight/24 h) via intragastric intubation for 10 and 30 weeks, respectively. Ethanol administration induced an enhancement of alcohol dehydrogenase activities and affected the capacity of the kidney to prevent NADH accumulation in the cytosol. After 10 weeks, the excess of NADH was balanced by increased activities of malate dehydrogenase and aspartate transaminase. In the event of a longer period of ethanol intake, the kidney was not able to balance the NADH excess, even though an increase in malate dehydrogenase, lactate dehydrogenase, aspartate transaminase, and alanine transaminase activities was noted. The electrophoretic analysis of alcohol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase isoforms revealed differences between control and ethanol-treated animals. The results suggest that rat kidneys have a multicomponent metabolic response to the same daily dose of ethanol that functions to maintain the redox status and which varies with the length of the administration period.

Adapted response of the antioxidant defense system to oxidative stress induced by deoxynivalenol in Hek-293 cells.

The mycotoxin deoxynivalenol (DON), a contaminant of certain foods and feeds, is cytotoxic and genotoxic to mammalians cells. Exposure of human embryonic kidney (Hek-293) cells to DON led to a dose- and time-dependent decrease in cell viability, with an IC(50) about 7.6 µM. The DON effects on Hek-293 morphology, reactive oxygen species, lipid peroxidation and antioxidative system and caspase 3 and bcl-2 expression were studied. Cells became round and in some are progressive loss of cell attachment appeared. These biochemical parameters were assessed after 6, 12 and 24 h of treatment with 2.5 and 5 µM DON. An increase in superoxide dismutase activity within the interval 6-12 h and almost complete recovery by the end of experiment for both concentrations was observed, whereas the profile of catalase activity was the same with the superoxide dismutase one for 2.5 µM and decreased in a time-dependent manner for 5 µM. A temporary activation of glutathione peroxidase and glutathione reductase was recorded at 12 h post-exposure, while the glutathione-S-transferase activity was unchanged for both concentrations. The NADP(+)-dependent isocitrate dehydrogenase activity showed a transient increase at the 12 h post-exposure. The caspase 3 expression remained unchanged and the bcl-2 one decreased after 24 h of exposure for the two concentrations. Our results showed the dose- and time specific changes in the antioxidants system of Hek-293 cells, which could not counteract efficiently the effects DON exposure. The different types of cell death which could be activated by this DON induced changes are mentioned.

Depletion of intracellular glutathione and increased lipid peroxidation mediate cytotoxicity of hematite nanoparticles in MRC-5 cells.

Particles generated from numerous anthropogenic and/or natural sources, such as crystalline α-Fe2O3 nanoparticles, have the potential to damage lung cells. In our study we investigated the effects of these nanoparticles (12.5 µg/ml) on lipid peroxidation and the antioxidative system in MRC-5 lung fibroblast cells following exposure for 24, 48 or 72h. Exposure to α-Fe2O3 nanoparticles increased lipid peroxidation by 81%, 189% and 110% after 24, 48 and 72h, respectively. Conversely, the reduced glutathione concentration decreased by 23.2% and 51.4% after 48 and 72h of treatment, respectively. In addition, an augmentation of the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase and glutathione reductase within the interval between 48-72h was noticed. Taking into account that the reduced glutathione level decreased and the malondialdehyde level, a lipid peroxidation product, remained highly increased up to 72h of exposure, it would appear that the MRC-5 antioxidant defense mechanisms did not efficiently counteract the oxidative stress induced by exposure to hematite nanoparticles.

Modulatory effects of deltamethrin on antioxidant defense mechanisms and lipid peroxidation in Carassius auratus gibelio liver and intestine.

Pyrethroids, such as deltamethrin, are toxic substances that lead to generation of reactive oxygen species, which harm living organisms. We assessed the level and patterns of imbalance evolved by a single dose of 2 microg/L deltamethrin on the lipid peroxidation (LPO) and the antioxidant defense system of Carassius auratus gibelio liver and intestine, and monitored the recovery dynamics of these parameters during a 14-day post-exposure period. LPO and antioxidative defense mechanisms displayed different responses in the investigated tissues. Sudden increase of LPO in the liver, persisting at this elevated level throughout the test period, was observed on the third day post-exposure, while in the intestine significant enhancement of this parameter was recorded from the seventh day. Reduced glutathione (GSH) showed a transient increase in the liver, and was depleted in the intestine by the second day of exposure, with signs of recovery by the end of the experimental tenure. In the liver of fish a temporary inhibition of superoxide dismutase (SOD) and catalase (CAT) activity, and activation of glutathione peroxidase (GPX), glutathione transferase (GST), and glutathione reductase (GR) enzymes was observed, with maximum thresholds recorded on the third and second days, respectively. In the intestine a relevant increase in CAT and GST activity up to the second day and almost complete recovery by the end of the experiment was recorded, while for GR a continuous enhancement was apparent.

Malathion-induced alteration of the antioxidant defence system in kidney, gill, and intestine of Carassius auratus gibelio.

Pesticides such as malathion, commonly used in agriculture and households, are toxic substances that lead to reactive oxygen species generation, which harms organisms. Ecotoxicological consequences of malathion, particularly its effects on antioxidants in fish, are not well understood. Thus, we investigated the effects of malathion (0.05 mg/L) on lipid peroxidation and antioxidant systems in Carassius auratus gibelio kidney, intestine, and gills following exposure times of 1, 2, 3, and 6 days. The lipid peroxidation and antioxidative defense mechanisms display different responses in investigated tissues. The lipid peroxidation was increased in all investigated tissues, especially after 1 day of malathion administration. Changes in reduced glutathione levels have been registered, mainly after 6 days of pesticide exposure. The modulation in the activities of antioxidant enzymes, catalase, gluthatione peroxidase, glutathione reductase, and glutathione-S-transferase was time and tissue specific. The investigated parameters can be used as biomarkers of fish exposure to malathion.

Enzymes with new biochemical properties in the pectinolytic complex produced by Aspergillus niger MIUG 16.

A comprehensive study on the purification and characterization of pectinolytic enzymes produced by Aspergillus niger MIUG 16 on raw materials solid-state fermentation is reported. Five pectinolytic enzymes were purified using a combination of chromatographic techniques. The properties of these homogenous enzymes were analyzed. The purified enzymes were classified with respect to their biochemical properties and substrate specificity. Among these proteins, one revealed polygalacturonase activity, another appeared to be a pectin methylesterase and three were identified as pectate lyases. The capacity of the fungus A. niger to produce pectate lyases with optimum pH in acidic domain was reported for the first time.

Biochemical changes in rat testis induced in vitro by reactive oxygen species.

We report the effects of reactive oxygen species generated by ultraviolet-A radiation on some biochemical parameters specific for oxidative stress, in rat testis homogenates. Results show an increase in lipid peroxidation products under ultraviolet-A exposure, and suggest that the involved mechanism is typical for a radical-mediated chain reaction. The amount of SH groups also increases during irradiation, probably as a consequence of conformational changes in proteins. Electrophoresis results revealed protein pattern changes mainly in the low molecular weight domain. The catalytic activities of alkaline phosphatase and gamma-glutamil transpeptidase are modified under the oxidative conditions generated by reactive oxygen species. The changes of the enzymatic activities are UVA exposure time-dependent, suggesting that conformational modifications are responsible for enzymatic activities enhancement.

Ethanol-induced alterations of the antioxidant defense system in rat kidney.

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and gamma-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, gamma-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration.