Fullerene derivatives protect against oxidative stress in RAW 264.7 cells and ischemia-reperfused lungs

Fullerene derivatives have often been used as effective scavengers for reactive oxygen species (ROS). This study was designed to test whether polyhydroxylated fullerene derivatives [C(60)(OH)(7+/-2)] protect against oxidative stress in cultured RAW 264.7 cells and ischemia-reperfused (IR) lungs. In RAW 264.7 cells, sodium nitroprusside (SNP, 1 mM) and H(2)O(2) (400 microM) caused a marked (90%) decrease in cell viability, and this decrease was dose dependently reversed by pretreatment with C(60)(OH)(7+/-2) (10-50 microM). The increase in ROS production induced by SNP and H(2)O(2) was significantly suppressed by C(60)(OH)(7+/-2). Also, the decrease in mitochondrial membrane potential induced by SNP and H(2)O(2) was significantly reversed by C(60)(OH)(7+/-2). However, high concentration of C(60)(OH)(7+/-2) (1 and 1.5 mM) lead to cell death (apoptosis or necrosis). In the isolated rat lung, the increases in pulmonary artery pressure and capillary filtration pressure induced by SNP during IR were reversed significantly by C(60)(OH)(7+/-2) (10 mg/kg). These results indicate that polyhydroxylated fullerene derivatives C(60)(OH)(7+/-2) at low concentrations protect against oxidative stress in RAW 264.7 cells and IR lungs.     

Procyanidins protect Fao cells against hydrogen peroxide-induced oxidative stress

In this paper, we evaluate the extent to which flavonoids in red wine (catechin, epicatechin, quercetin and procyanidins) protect against hydrogen peroxide-induced oxidative stress in Fao cells. When cells were exposed to H(2)O(2), malondialdehyde (MDA) levels, oxidized glutathione (GSSG) levels and lactate dehydrogenase (LDH) release increased, indicating membrane damage and oxidative stress. All the flavonoids studied, and in particular epicatechin and quercetin, protected the plasma membrane. Only procyanidins lowered MDA levels and LDH leakage, maintained a higher reduced/oxidized glutathione ratio, and increased catalase/superoxide dismutase and glutathione peroxidase/superoxide dismutase ratios, and glutathione reductase and glutathione transferase activities. These results show that the procyanidin mixture has a greater antioxidant effect than the individual flavonoids studied, probably due to its oligomer content and/or the additive/synergistic effect of its compounds. This suggests that the mixture of flavonoids found in wine has a greater effect than individual phenols, which may explain many of the healthy effects attributed to wine.     

Synthesis and anticancer activity of benzyloxybenzaldehyde derivatives against HL-60 cells

A series of benzyloxybenzaldehyde derivatives were prepared and tested against the HL-60 cell line for anticancer activity. Preliminary structure-activity relationships were established. It was discovered that 2-(benzyloxy)benzaldehyde (17), 2-(benzyloxy)-4-methoxybenzaldehyde (26), 2-(benzyloxy)-5-methoxybenzaldehyde (27), 2-(benzyloxy)-5-chlorobenzaldehyde (28), 2-[(3-methoxybenzyl)oxy]benzaldehyde (29), 2-[(2-chlorobenzyl)oxy]benzaldehyde (30), and 2-[(4-chlorobenzyl)oxy]benzaldehyde (31) exhibited significant activity at 1-10 microM. Among them, compound 29 was the most potent one. The morphological assessment and DNA fragmentation analysis indicated that these compounds arrested cell cycle progression at G2/M phase and induced cell apoptosis. They resulted in the loss of mitochondrial membrane potential after 12h of treatment.     

Quercetin modifies reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells

Neutron specular reflection has been used to study the structure of a monolayer of dimyristoylphosphatidylcholine (DMPC) deposited using the Langmuir-Blodgett technique onto a silicon oxide substrate. A self-assembled monolayer of octadecyltrichlorosilane with a deuterated alkyl chain (d-OTS) had been previously bonded onto this silicon oxide substrate which rendered it hydrophobic. In the system under study, the alkyl chains of the phospholipid were found to penetrate extensively into the d-OTS layer with the mixed chain region (d-OTS and DMPC) having a total thickness of 30.5 A. This mixed region was divided into two halves for analysis; the 'lower half' (nearest to the substrate surface) was found to comprise anchored d-OTS chains mixed with the lipid chains in the volume ratio approx. 0.60:0.35. The corresponding volume ratio in the 'upper half' of this region was determined to be approx. 0.50:0.40. The thicknesses of these regions were found to be 17.9 A (incorporating approx. 6% solvent) and 12.6 A (incorporating approx. 9% solvent) for the lower and upper halves respectively. The DMPC head groups were found to be confined to the most external layer (furthest away from the silicon substrate). This layer was found to have a thickness of 9.4 A and included a small fraction of the lipid alkyl chains with approx. 47% solvent.     

Tyrphostins protect neuronal cells from oxidative stress

Tyrphostins are a family of tyrosine kinase inhibitors originally synthesized as potential anticarcinogenic compounds. Because tyrphostins have chemical structures similar to those of the phenolic antioxidants, we decided to test the protective efficacy of tyrphostins against oxidative stress-induced nerve cell death (oxytosis). Many commercially available tyrphostins, at concentrations ranging from 0.5 to 200 microm, protect both HT-22 hippocampal cells and rat primary neurons from oxytosis brought about by treatment with glutamate, as well as by treatment with homocysteic acid and buthionine sulfoximine. The tyrphostins protect nerve cells by three distinct mechanisms. Some tyrphostins, such as A25, act as antioxidants and eliminate the reactive oxygen species that accumulate as a result of glutamate treatment. These tyrphostins also protect cells from hydrogen peroxide and act as antioxidants in an in vitro assay. In contrast, tyrphostins A9 and AG126 act as mitochondrial uncouplers, collapsing the mitochondrial membrane potential and thereby reducing the generation of reactive oxygen species from mitochondria during glutamate toxicity. Finally, the third group of tyrphostins does not appear to be effective as antioxidants but rather protects cells by increasing the basal level of cellular glutathione. Therefore, the effects of tyrphostins on cells are not limited to their ability to inhibit tyrosine kinases.     

Cytotoxicity and oxidative stress induced by the glyceraldehyde-related Maillard reaction products for HL-60 cells

We demonstrated the cytotoxicity of glyceraldehyde-related Maillard reaction products for HL-60 cells. Glyceraldehyde-modified bovine serum albumin and glyceraldehyde-modified casein inhibited the proliferation of HL-60 cells. The reaction products formed from glyceraldehyde and Nalpha-acetyllysine had also a cytotoxic effect on HL-60 cells. The cytotoxic effect was prevented by N-acetylcysteine or pyrrolidinedithiocarbamate as the antioxidants. In addition, the reaction products depressed the intracellular glutathione level, and induced the reactive oxygen species (ROS) production. These results suggested that the glyceraldehyde-related advanced glycation end products (AGEs) induced the cytotoxicity and the oxidative stress.We previously reported that the glyceraldehyde-related AGE was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named GLAP (glyceraldehyde-derived pyridinium compound), formed from glyceraldehyde and Nalpha-acetyllysine (Biosci. Biotechnol. Biochem., 67, 930-932 (2003)). In this study, GLAP inhibited the proliferation of HL-60 cells, and the inhibitory effect was prevented by the antioxidants. Furthermore, GLAP depressed the intracellular glutathione level, and induced the ROS production.This work indicated the possibility that the cytotoxicity and the oxidative stress in the progression of diabetic complications and chronic renal disease might be induced by GLAP.     

Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.     

Managanese stimulates the oxidative brust of differentiated HL-60 cells

Incubation with manganese results in a twofold increase in the oxidative burst of differentiated HL-60 cells. This stimulation was characterized by examining the dose response, length of incubation time, and specifity of manganese. Managese only stimulated the burst in cells induced to differentiated with retinoic acid and not in undifferentiated HL-60 cells. Incubation with manganese did not result in a greater number of differentiated cells. The maximum stimulation occurred at 0.2 μmol/L manganese. Stimulation of the oxidative burst required 96 h of incubation with manganese, since cells incubated with the same levels of manganese for the last 24 h of culture did not result in any stimulation. Magnesium, present in the incubation medium at physiological serum levels (820 μmol/L) also stimulated the oxidative burst, whereas iron (0.3 μmol/L), zinc (18 μmol/L), and copper (12 μmol/L) had no effect. To determine whether manganese and magnesium stimulated the burst differently, the initial rates of superoxide anion production was determined. The initial rate of the reaction proceeded rapidly in cells incubated with managnese, whereas there appeared to be a lag before magnesiumtreated cells produced superoxide anion. Thus, manganese seems to stimulate the oxidative burst differently than magnesium.     

星形胶质细胞通过谷胱甘肽保护MN9D细胞抵抗鱼藤酮所致氧化应激

星形胶质细胞维持神经元微环境,给予营养和代谢支持,并调节其对损伤的反应。鱼藤酮特异阻断线粒体复合物Ⅰ,长期暴露于鱼藤酮可能增加患帕金森病的几率,并引起帕金森综合征。然而,星形胶质细胞在鱼藤酮所致多巴胺能神经元损伤过程中的作用尚无报道。本研究采用多巴胺能神经元细胞系MN9D细胞模型,将经过或未经过星形胶质细胞条件培养基处理的MN9D细胞暴露于不同浓度的鱼藤酮中,用计数法测生长曲线,MTT法测细胞活性,DCFH染色流式细胞仪测氧化应激水平,比色法测还原型谷胱甘肽含量。结果显示,MN9D细胞在条件和普通培养基培养条件下生长曲线无明显差别;鱼藤酮浓度依赖性地降低细胞活性;不同浓度鱼藤酮作用24、48h后,经条件培养基处理的细胞其活性显著高于普通培养基培养的细胞：不同浓度的条件培养基都有保护作用,纯的条件培养基保护作用稍弱：预先24h条件培养基处理或同时给予鱼藤酮和条件培养基处理都有保护作用,鱼藤酮作用12h后再给予条件培养基则无保护作用;经条件培养基处理的细胞氧化应激水平降低：另外,条件培养基提高了细胞内还原型谷胱甘肽含量,缓解了鱼藤酮所致的谷胱甘肽耗竭。结果提示,星形胶质细胞可保护MN9D细胞抵抗鱼藤酮所致的氧化应激,还原型谷胱甘肽可能参与了该保护过程。     

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.     

Iron chelators can protect against oxidative stress through ferryl heme reduction

Iron chelators such as desferrioxamine have been shown to ameliorate oxidative damage in vivo. The mechanism of this therapeutic action under non-iron-overload conditions is, however, complex, as desferrioxamine has properties that can impact on oxidative damage independent of its capacity to act as an iron chelator. Desferrioxamine can act as a reducing agent to remove cytotoxic ferryl myoglobin and hemoglobin and has recently been shown to prevent the formation of a highly cytotoxic heme-to-protein cross-linked derivative of myoglobin. In this study we have examined the effects of a wide range of iron chelators, including the clinically used hydroxypyridinone CP20 (deferriprone), on the stability of ferryl myoglobin and on the formation of heme-to-protein cross-linking. We show that all hydroxypyridinones, as well as many other iron chelators, are efficient reducing agents of ferryl myoglobin. These compounds are also effective at preventing the formation of cytotoxic derivatives of myoglobin such as heme-to-protein cross-linking. These results show that the use of iron chelators in vivo may ameliorate oxidative damage under conditions of non-iron overload by at least two mechanisms. The antioxidant effects of chelators in vivo cannot, therefore, be attributed solely to iron chelation.     

Lignan compounds and 4,4'-dihydroxybiphenyl protect C2C12 cells against damage from oxidative stress

Lignan compounds are known to have various biological activities, especially antioxidative effects. We investigated whether lignan compounds show antioxidative activity in myoblast C2C12 cells. Among 14 lignan compounds investigated, two lignans containing two phenolic functional groups, namely Gomisin J and GR-12, prevented hydrogen peroxide (H(2)O(2))-induced cell death. A simple compound, 4,4'-dihydroxybiphenyl, which was found to be a common component of Gomisin J and GR-12, also largely prevented H(2)O(2)-induced cell death and almost completely prevented H(2)O(2)-induced increases in p38 MAPK phosphorylation. Our present results provide a useful in vitro system for clarifying the molecular mechanisms of lignan-mediated antioxidative effects and evaluating lead molecules toward the development of therapeutic drugs.     

Isotope-reinforced polyunsaturated fatty acids protect yeast cells from oxidative stress

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4 h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D₂-linoleic or 11,11,14,14-D₄-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress.     

Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress.

Mitochondria are subcellular organelles with an essentially oxidative type of metabolism. The production of reactive oxygen species (ROS) in it increases under stress conditions and causes oxidative damage. In the present study, effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on both the ROS metabolism in mitochondria and functions of plasma membrane (PM) and tonoplast were studied in cucumber seedlings treated with 100mM NaCl. NaCl treatment induced significant accumulation of H(2)O(2) and led to serious lipid peroxidation in cucumber mitochondria, and the application of 50muM SNP stimulated ROS-scavenging enzymes and reduced accumulation of H(2)O(2) in mitochondria of cucumber roots induced by NaCl. As a result, lipid peroxidation of mitochondria decreased. Further investigation showed that application of SNP alleviated the inhibition of H(+)-ATPase and H(+)-PPase in PM and/or tonoplast by NaCl. While application of sodium ferrocyanide (an analog of SNP that does not release NO) did not show the effect of SNP, furthermore, the effects of SNP were reverted by addition of hemoglobin (a NO scavenger).     

Interspecific protection against oxidative stress: green algae protect harmful cyanobacteria against hydrogen peroxide

Oceanographic studies have shown that heterotrophic bacteria can protect marine cyanobacteria against oxidative stress caused by hydrogen peroxide (H₂O₂). Could a similar interspecific protection play a role in freshwater ecosystems? In a series of laboratory experiments and two lake treatments, we demonstrate that freshwater cyanobacteria are sensitive to H₂O₂ but can be protected by less-sensitive species such as green algae. Our laboratory results show that green algae degrade H₂O₂ much faster than cyanobacteria. Consequently, the cyanobacterium Microcystis was able to survive at higher H₂O₂ concentrations in mixtures with the green alga Chlorella than in monoculture. Interestingly, even the lysate of destructed Chlorella was capable to protect Microcystis, indicating a two-component H₂O₂ degradation system in which Chlorella provided antioxidant enzymes and Microcystis the reductants. The level of interspecific protection provided to Microcystis depended on the density of Chlorella. These findings have implications for the mitigation of toxic cyanobacterial blooms, which threaten the water quality of many eutrophic lakes and reservoirs worldwide. In several lakes, H₂O₂ has been successfully applied to suppress cyanobacterial blooms. Our results demonstrate that high densities of green algae can interfere with these lake treatments, as they may rapidly degrade the added H₂O₂ and thereby protect the bloom-forming cyanobacteria.     

Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin

Enhanced endoplasmic reticulum (ER) stress leads to cell death in various pathophysiological situations. During a search for compounds that regulate ER stress, we identified methoxyflavones, a group of flavonoids, as strong protective agents against ER stress. Analysis in mouse insulinoma MIN6 cells revealed that methoxyflavones mildly activated the eukaryotic initiation factor 2 and nuclear factor erythroid 2-related factor pathways, but not the XBP1 pathway, and induced downstream genes, including glucose-regulated protein (GRP) 78, a molecular chaperone in the ER. The protective effect of methoxyflavones was enhanced by agents that increase intracellular cAMP levels such as forskolin, dibutyryl-cAMP and IBMX, but suppressed by the protein kinase A (PKA) inhibitor H-89, suggesting involvement of the PKA pathway in the regulation of ER stress by methoxyflavones. Consistent with the results in cultured cells, pretreatment of mice with tangeretin, a methoxyflavone, enhanced expression of GRP78 and HO-1 without causing ER stress in renal tubular epithelium and prevented tunicamycin-induced cell death. Furthermore, preadministration of tangeretin in mice enhanced expression of GRP78 in the substantia nigra pars compacta and protected dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin that induces both oxidative and ER stress. These results suggest that methoxyflavones play an important role in the regulation of ER stress and could be a therapeutic target for the ER stress-related diseases. neuronal degeneration; flavonoids     

Lipotropes promote immunobiochemical plasticity and protect fish against low-dose pesticide-induced oxidative stress

An experiment was conducted to evaluate the role of different lipotropes in modulating immunity and biochemical plasticity under conditions of sublethal low-dose pesticide-induced stress in fish. Labeo rohita fish fingerlings were divided in two sets with one set of fish continuously exposed to low-dose endosulfan (1/10th of 96-h LC₅₀) for 21 days, the other was unexposed, and both sets of fish were fed with practical diets supplemented with either 2 % lecithin, 0.5 % betaine, or 0.1 % choline and compared against unsupplemented diet. Low-dose endosulfan exposure had adverse effects (P < 0.05/P < 0.01) on hematological profile (erythrocyte count, hemoglobin, and hematocrit), serum protein (total protein, albumin, and globulin) and lipid profile (cholesterol and triglyceride), anti-oxidative status (ascorbic acid content of muscle, liver, brain, and kidney and activity of anti-oxidative enzymes: catalase and superoxide dismutase), neurotransmission (acetylcholinesterase activity in muscle and brain), immunological attributes (WBC count, albumin to globulin ratio, phagocytic activity, and serum cortisol), and metabolic plasticity as revealed from enzyme activities (muscle lactate dehydrogenase, liver and kidney glucose-6-phosphatase dehydrogenase-G6PDH activity). Dietary lipotropes prevented these effects completely or partially and the effects were lipotrope dependent. Kinetics (maximum velocity value V_max, catalytic efficiency and Michaelis constant K_m) of G6PDH enzyme from crude extracts of liver and kidney indicated inhibition due to endosulfan but lipotropes could protect enzyme and showed a stabilizing effect. The supplements also helped maintain integrity of histoarchitecture of the hepatocytes in endosulfan-exposed fish to a great extent. Feeding lipotropes to fish reared in endosulfan-free water also improved hematological and serum protein and lipid profiles and were immunostimulatory. In conclusion, dietary lipotropes, especially betaine and lecithin at the levels used, improve erythropoiesis, serum protein and lipid profile, anti-oxidant status, immunocompetence, neurotransmission, and protect the livers of L. rohita fingerlings even when continuously exposed to low-dose endosulfan.     

Autotaxin protects microglial cells against oxidative stress

Oxidative stress occurs when antioxidant defenses are overwhelmed by oxygen-reactive species and can lead to cellular damage, as seen in several neurodegenerative disorders. Microglia are specialized cells in the central nervous system that act as the first and main form of active immune defense in the response to pathological events. Autotaxin (ATX) plays an important role in the modulation of critical cellular functions, through its enzymatic production of lysophosphatidic acid (LPA). In this study, we investigated the potential role of ATX in the response of microglial cells to oxidative stress. We show that treatment of a microglial BV2 cell line with hydrogen peroxide (H(2)O(2)) stimulates ATX expression and LPA production. Stable overexpression of ATX inhibits microglial activation (CD11b expression) and protects against H(2)O(2)-treatment-induced cellular damage. This protective effect of ATX was partially reduced in the presence of the LPA-receptor antagonist Ki16425. ATX overexpression was also associated with a reduction in intracellular ROS formation, carbonylated protein accumulation, proteasomal activity, and catalase expression. Our results suggest that up-regulation of ATX expression in microglia could be a mechanism for protection against oxidative stress, thereby reducing inflammation in the nervous system.     

Low concentrations of methamphetamine can protect dopaminergic cells against a larger oxidative stress injury: mechanistic study

Mild stress can protect against a larger insult, a phenomenon termed preconditioning or tolerance. To determine if a low intensity stressor could also protect cells against intense oxidative stress in a model of dopamine deficiency associated with Parkinson disease, we used methamphetamine to provide a mild, preconditioning stress, 6-hydroxydopamine (6-OHDA) as a source of potentially toxic oxidative stress, and MN9D cells as a model of dopamine neurons. We observed that prior exposure to subtoxic concentrations of methamphetamine protected these cells against 6-OHDA toxicity, whereas higher concentrations of methamphetamine exacerbated it. The protection by methamphetamine was accompanied by decreased uptake of both [(3)H] dopamine and 6-OHDA into the cells, which may have accounted for some of the apparent protection. However, a number of other effects of methamphetamine exposure suggest that the drug also affected basic cellular survival mechanisms. First, although methamphetamine preconditioning decreased basal pERK1/2 and pAkt levels, it enhanced the 6-OHDA-induced increase in these phosphokinases. Second, the apparent increase in pERK1/2 activity was accompanied by increased pMEK1/2 levels and decreased activity of protein phosphatase 2. Third, methamphetamine upregulated the pro-survival protein Bcl-2. Our results suggest that exposure to low concentrations of methamphetamine cause a number of changes in dopamine cells, some of which result in a decrease in their vulnerability to subsequent oxidative stress. These observations may provide insights into the development of new therapies for prevention or treatment of PD.