Differential effects of α-tocopherol and N-acetyl-cysteine on advanced glycation end product-induced oxidative damage and neurite degeneration in SH-SY5Y cells

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200μM α-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by α-tocopherol with no effects on intracellular GSH concentrations. We observed that 2mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike α-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that α-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while α-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.     

Differential effects of α-tocopherol and N-acetyl-cysteine on advanced glycation end product-induced oxidative damage and neurite degeneration in SH-SY5Y cells

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200 μM α-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by α-tocopherol with no effects on intracellular GSH concentrations. We observed that 2 mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike α-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that α-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while α-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.     

N-methyl bases of ethanolamine prevent apoptotic cell death induced by oxidative stress in cells of oligodendroglia origin

A major reason for brain tissue vulnerability to oxidative damage is the high content of polyunsaturated fatty acids (PUFAs). Oligodendroglia-like OLN 93 cells lack PUFAs and are relatively insensitive to oxidative stress. When grown in serum-free defined medium in the presence of 0.1 mM docosahexaenoic acid (DHA; 22:6 n-3) for 3 days, OLN 93 cells release in the medium 2.6-fold more thiobarbituric acid-reactive substances (TBARS) after a 30-min exposure to 0.1 mM H2O2 and 50 microM Fe2+. Release of TBARS was substantially decreased by approximately 20 and 30% on coincubation with either 1 mM N-monomethylethanolamine or N,N'-dimethylethanolamine (dEa), respectively. The protective effect of dEa was concentration- and time-dependent and was still visible after dEa removal, suggesting a long-lasting mechanism of protection. After 24 h following H2O2-induced stress, cell death monitored by cell sorting showed 16% of the cells in the sub-G1 area, indicative of apoptotic cell death. DHA-supplemented cultures showed 35% cell death, whereas cosupplements with dEa reduced cell death to 12%, indicating cell rescue. Although the exact mechanism for this protection is not known, the nature of the polar head group and the degree of unsaturation may determine the ultimate resistance of nerve cells to oxidative stress.     

Oxidative stress,DNA damage,and the telomeric complex as therapeutic targets in acute neurodegeneration

Oxidative stress has been identified as an important contributor to neurodegeneration associated with acute CNS injuries and diseases such as spinal cord injury (SCI), traumatic brain injury (TBI), and ischemic stroke. In this review, we briefly detail the damaging effects of oxidative stress (lipid peroxidation, protein oxidation, etc.) with a particular emphasis on DNA damage. Evidence for DNA damage in acute CNS injuries is presented along with its downstream effects on neuronal viability. In particular, unchecked oxidative DNA damage initiates a series of signaling events (e.g. activation of p53 and PARP-1, cell cycle re-activation) which have been shown to promote neuronal loss following CNS injury. These findings suggest that preventing DNA damage might be an effective way to promote neuronal survival and enhance neurological recovery in these conditions. Finally, we identify the telomere and telomere-associated proteins (e.g. telomerase) as novel therapeutic targets in the treatment of neurodegeneration due to their ability to modulate the neuronal response to both oxidative stress and DNA damage.     

Water-soluble fractions from defatted sesame seeds protect human neuroblast cells against peroxyl radicals and hydrogen peroxide-induced oxidative stress

Oxidative stress is involved in the development of aging-related diseases, such as neurodegenerative diseases. Dietary antioxidants that can protect neuronal cells from oxidative damage play an important role in preventing such diseases. Previously, we reported that water-soluble fractions purified from defatted sesame seed flour exhibit good antioxidant activity in vitro. In the present study, we investigated the protective effects of white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively) against 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H₂O₂) induced oxidative stress in human neuroblast SH-SY5Y cells. Pretreatment with WS-wsf and GS-wsf did not protect cells against AAPH-induced cytotoxicity, while simultaneous co-treatment with AAPH significantly improved cell viability and inhibited membrane lipid peroxidation. These results suggest that WS-wsf and GS-wsf protect cells from AAPH-induced extracellular oxidative damage via direct scavenging of peroxyl radicals. When oxidative stress was induced by H₂O₂, pretreatment WS-wsf and GS-wsf significantly enhanced cell viability. These results suggest that in addition to radical scavenging, WS-wsf and GS-wsf enhance cellular resistance to intracellular oxidative stress by activation of the Nrf-2/ARE pathway as confirmed by the increased Nrf2 protein level in the nucleus and increased heme oxygenase 1 (HO-1) mRNA expression. The roles of ferulic and vanillic acids as bioactive antioxidants in these fractions were also confirmed. In conclusion, our results indicated that WS-wsf and GS-wsf, which showed antioxidant activity in vitro, are also efficient antioxidants in a cell system protecting SH-SY5Y cells against both extracellular and intracellular oxidative stress.     

Intracellular glutathione protects human monocyte-derived macrophages from hypochlorite damage

AimsMacrophages must function in an inflammatory environment of high oxidative stress due to the production of various oxidants. Hypochlorous acid (HOCl) is a potent cytotoxic agent generated by neutrophils and macrophages within inflammatory sites. This study determines whether glutathione is the key factors governing macrophage resistance to HOCl.Main methodsHuman monocyte derived macrophages (HMDM) were differentiated from human monocytes prepared from human blood. The HMDM cells were exposed to micromolar concentrations of HOCl and the timing of the cell viability loss was measured. Cellular oxidative damage was measured by loss of glutathione, cellular ATP, tyrosine oxidation, and inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Key findingsHOCl causes a rapid loss in HMDM cell viability above threshold concentrations. The cell death occurred within 10 min of treatment with the morphological characteristics of necrosis. The HOCl caused the extensive cellular protein oxidation with the loss of tyrosine residue and inactivation of GAPDH, which was accompanied with the loss of cellular ATP. This cellular damage was only observed after the loss of intracellular GSH from the cell. Removal of intracellular GSH with diethyl maleate (DEM) increased the cells' sensitivity to HOCl damage while protecting the intracellular GSH pool with the antioxidant 7,8-dihydroneopterin prevented the HOCl mediated viability loss. Variations in the HOCl LD₅₀ for inducing cell death were strongly correlated with initial intracellular GSH levels.SignificanceIn HMDM cells scavenging of HOCl by intracellular glutathione is sufficient to protect against oxidative loss of key metabolic functions within the cells.     

Oxidative stress, phospholipid loss and lipid hydrolysis during drying and storage of intermediate seeds

The biochemical and physiological basis of intermediate seed storage behaviour was examined by investigating the effects of equilibrium drying under relative humidities (RHs) of 9–81% and of storage at 20 or 5°C on coffee seed viability and antioxidant, lipid and sugar status. Slow drying induced a significant decrease in the concentrations of the pools of two major antioxidants, glutathione and ascorbate, and an increase in the free fatty acid (FFA) content of seeds, independent of the RH employed. Seeds stored at 81% RH and 20°C lost their viability very rapidly and showed an extensive loss and oxidation of antioxidants, an accumulation of FFA and a selective loss of phospholipids, in particular phosphatidylethanolamine (PE). Interestingly, the changes in PE content were not due to fatty acid de-esterification and the increase in FFA levels resulted from neutral lipid hydrolysis. Decreasing the storage temperature to 5°C considerably slowed both the loss of seed viability and the level of oxidative stress as well as the rates of lipid hydrolysis. No decline in seed viability was observed under storage conditions of 45% RH/20°C. After 1 year under 45% RH/5°C, the loss of seed viability was found to be due to imbibitional damage and could be circumvented by pre-humidifying or pre-heating seeds before sowing.     

Lipid constituents in oligodendroglial cells alter susceptibility to H2O2-induced apoptotic cell death via ERK activation

The present work examines the effect of membrane lipid composition on activation of extracellular signal-regulated protein kinases (ERK) and cell death following oxidative stress. When subjected to 50 microM docosahexaenoic acid (DHA, 22 : 6 n-3), cellular phospholipids of OLN 93 cells, a clonal line of oligodendroglia origin low in DHA, were enriched with this polyunsaturated fatty acid. In the presence of 1 mM N,N-dimethylethanolamine (dEa) a new phospholipid species analog was formed in lieu of phosphatidylcholine. Exposure of DHA-enriched cells to 0.5 mM H2O2, caused sustained activation of ERK up to 24 h. At this time massive apoptotic cell death was demonstrated by ladder and TUNEL techniques. H2O2-induced stress applied to dEa or DHA/dEa co-supplemented cells showed only a transient ERK activation and no cell death after 24 h. Moreover, while ERK was rapidly translocated into the nucleus in DHA-enriched cells, dEa supplements completely blocked ERK nuclear translocation. This study suggests that H2O2-induced apoptotic cell death is associated with prolonged ERK activation and nuclear translocation in DHA-enriched OLN 93 cells, while both phenomena are prevented by dEa supplements. Thus, the membrane lipid composition ultimately modulates ERK activation and translocation and therefore can promote or prevent apoptotic cell death.     

Is oxidative stress a physiological cost of reproduction? An experimental test in house mice

Investment in reproduction is costly and frequently decreases survival or future reproductive success. However, the proximate underlying causes for this are largely unknown. Oxidative stress has been suggested as a cost of reproduction and several studies have demonstrated changes in antioxidants with reproductive investment. Here, we test whether oxidative stress is a consequence of reproduction in female house mice (Mus musculus domesticus), which have extremely high energetic demands during reproduction, particularly through lactation. Assessing oxidative damage after a long period of reproductive investment, there was no evidence of increased oxidative stress, even when females were required to defend their breeding territory. Instead, in the liver, markers of oxidative damage (malonaldehyde, protein thiols and the proportion of glutathione in the oxidized form) indicated lower oxidative stress in reproducing females when compared with non-reproductive controls. Even during peak lactation, none of the markers of oxidative damage indicated higher oxidative stress than among non-reproductive females, although a positive correlation between protein oxidation and litter mass suggested that oxidative stress may increase with fecundity. Our results indicate that changes in redox status occur during reproduction in house mice, but suggest that females use mechanisms to cope with the consequences of increased energetic demands and limit oxidative stress.     

Screening of dietary antioxidants against mitochondria-mediated oxidative stress by visualization of intracellular redox state

Mitochondrial impairment and the resulting generation of reactive oxygen species (ROS) have been associated with aging and its related pathological conditions. Recently, dietary antioxidants have gained significant attention as potential preventive and therapeutic agents against ROS-generated aging and pathological conditions. We previously demonstrated that food-derived antioxidants prevented intracellular oxidative stress under proteasome inhibition conditions, which was attributed to mitochondrial dysfunction and ROS generation, followed by cell death. Here, we further screened dietary antioxidants for their activity as redox modulators by visualization of the redox state using Redoxfluor, a fluorescent protein redox probe. Direct alleviation of ROS by antioxidants, but not induction of antioxidative enzymes, prevented mitochondria-mediated intracellular oxidation. The effective antioxidants scavenged mitochondrial ROS and suppressed cell death. Our study indicates that redox visualization under mitochondria-mediated oxidative stress is useful for screening potential antioxidants to counteract mitochondrial dysfunction, which has been implicated in aging and the pathogenesis of aging-related diseases.     

Association of increased bcl-2 expression with rescue from tumor necrosis factor-alpha-induced cell death in the oligodendrocyte cell line OLN-93

The present study investigated the effects of flupirtine (Katadolon) on tumor necrosis factor (TNF)-alpha-mediated cell death and Bcl-2 expression in the permanent rat oligodendrocyte cell line OLN-93 (OLN cells). TNF-alpha (500 U/ml) induced apoptosis of OLN cells, which was confirmed by DNA fragmentation using an in situ end-labeling technique and ultrastructural analysis. Flupirtine significantly reduced the rate of spontaneous cell death of OLN cells already at low concentrations; TNF-alpha-mediated apoptosis was suppressed only with higher concentrations of flupirtine (100 microM:). Expression of Bcl-2 protein and mRNA in OLN cells was detected by immunocytochemistry, western blot, and RT-PCR. Quantitative analysis of western blots revealed an approximately 2. 5-fold up-regulation of Bcl-2 protein during TNF-alpha treatment. Furthermore, addition of 10 or 100 microM: flupirtine before incubation with TNF-alpha led to an approximately threefold increase of Bcl-2 expression. Exposure of OLN cells to flupirtine alone moderately augmented the expression of Bcl-2 protein. Our data demonstrate that flupirtine up-regulates the expression of Bcl-2 protein in OLN cells; this Bcl-2 induction is associated with a reduced rate of TNF-alpha-induced cell death.     

Sickle cell membranes and oxidative damage.

Sickle erythrocytes and their membranes are susceptible to endogenous free-radical-mediated oxidative damage which correlates with the proportion of irreversibly sickled cells. The suppression of incubation-induced oxidative stress by antioxidants, free radical scavengers and an iron chelator suggest that oxidation products of membrane-bound haemoglobin contribute towards the pathology of the disease.     

Oxidative stress and aberrant signaling in aging and cognitive decline

Brain aging is associated with a progressive imbalance between antioxidant defenses and intracellular concentrations of reactive oxygen species (ROS) as exemplified by increases in products of lipid peroxidation, protein oxidation, and DNA oxidation. Oxidative conditions cause not only structural damage but also changes in the set points of redox-sensitive signaling processes including the insulin receptor signaling pathway. In the absence of insulin, the otherwise low insulin receptor signaling is strongly enhanced by oxidative conditions. Autophagic proteolysis and sirtuin activity, in turn, are downregulated by the insulin signaling pathway, and impaired autophagic activity has been associated with neurodegeneration. In genetic studies, impairment of insulin receptor signaling causes spectacular lifespan extension in nematodes, fruit flies, and mice. The predicted effects of age-related oxidative stress on sirtuins and autophagic activity and the corresponding effects of antioxidants remain to be tested experimentally. However, several correlates of aging have been shown to be ameliorated by antioxidants. Oxidative damage to mitochondrial DNA and the electron transport chain, perturbations in brain iron and calcium homeostasis, and changes in plasma cysteine homeostasis may altogether represent causes and consequences of increased oxidative stress. Aging and cognitive decline thus appear to involve changes at multiple nodes within a complex regulatory network.     

GRP78 Suppresses Lipid Peroxidation and Promotes Cellular Antioxidant Levels in Glial Cells following Hydrogen Peroxide Exposure

Oxidative stress, caused by the over production of reactive oxygen species (ROS), has been shown to contribute to cell damage associated with neurotrauma and neurodegenerative diseases. ROS mediates cell damage either through direct oxidation of lipids, proteins and DNA or by acting as signaling molecules to trigger cellular apoptotic pathways. The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that has been suggested to protect cells against ROS-induced damage. However, the protective mechanism of GRP78 remains unclear. In this study, we used C6 glioma cells transiently overexpressing GRP78 to investigate the protective effect of GRP78 against oxidative stress (hydrogen peroxide)-induced injury. Our results showed that the overexpression of GRP78 significantly protected cells from ROS-induced cell damage when compared to non-GRP78 overexpressing cells, which was most likely due to GRP78-overexpressing cells having higher levels of glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two antioxidants that protect cells against oxidative stress. Although hydrogen peroxide treatment increased lipid peroxidation in non-GRP78 overexpressing cells, this increase was significantly reduced in GRP78-overexpressing cells. Overall, these results indicate that GRP78 plays an important role in protecting glial cells against oxidative stress via regulating the expression of GSH and NQO1.     

Effects of different types of oxidative stress in RPE cells

Oxidative damage to retinal pigmented epithelial (RPE) cells and photoreceptors has been implicated in the pathogenesis of age-related macular degeneration (AMD). In order to develop new treatments, it is necessary to characterize the antioxidant defense system in RPE cells to better define their vulnerabilities and how they can be remedied. In this study, we sought to investigate the effects of three different types of oxidative stress on cultured RPE cells. Carbonyl content in RPE cells increased with increasing concentrations of oxidants or increasing duration of exposure with high reproducibility, validating ELISA for carbonyl content as a valuable quantitative measure of oxidative damage. Compared to other cell types, RPE cells were able to survive exposure to H2O2 quite well and exposure to paraquat extremely well. Comparison of the total amount of oxidative damage at the IC50 for each type of stress showed a rank order of hyperoxia > paraquat > H2O2, and since these stressors primarily target different cellular compartments, it suggests that the endogenous defense system against oxidative damage in RPE cells protects well against damage to mitochondria and endoplasmic reticulum, and is less able to handle oxidative damage at the cell surface. Supplementation of media with ascorbic acid provided significant protection from H2O2-induced oxidative damage, but not that induced by paraquat or hyperoxia. Supplementation with docosahexaenoic acid or alpha-tocopherol significantly reduced oxidative damage from H2O2 or hyperoxia, but not that induced by paraquat. We conclude that exposure to different types of oxidative stress results in different patterns of accrual of oxidative damage to proteins in RPE cells, different patterns of loss of viability, and is differentially countered by antioxidants. This study suggests that multiple types of oxidant stress should be used to probe the vulnerabilities of the retina and RPE in vivo, and that ELISA for carbonyl content provides a valuable tool for quantitative assessment of oxidative damage for such studies.     

Patient-derived fibroblasts indicate oxidative stress status and may justify antioxidant therapy in OXPHOS disorders

Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A>G or m.3302A>G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms.     

The Effect of SOD1 Mutation on Cellular Bioenergetic Profile and Viability in Response to Oxidative Stress and Influence of Mutation-Type

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive degeneration of motor neurons. Substantial evidence implicates oxidative stress and mitochondrial dysfunction as early events in disease progression. Our aim was to ascertain whether mutation of the SOD1 protein increases metabolic functional susceptibility to oxidative stress. Here we used a motor neuron-like cell line (NSC34) stably transfected with various human mutant SOD1 transgenes (G93A, G37R, H48Q) to investigate the impact of oxidative stress on cell viability and metabolic function within intact cells. NSC34 cells expressing mutant SOD1 showed a dose dependent reduction in cell viability when exposed to oxidative stress induced by hydrogen peroxide, with variation between mutations. The G93A transfectants showed greater cell death and LDH release compared to cells transfected with the other SOD1 mutations, and H48Q showed an accelerated decline at later time points. Differences in mitochondrial bioenergetics, including mitochondrial respiration, coupling efficiency and proton leak, were identified between the mutations, consistent with the differences observed in viability. NSC34 cells expressing G93A SOD1 displayed reduced coupled respiration and mitochondrial membrane potential compared to controls. Furthermore, the G93A mutation had significantly increased metabolic susceptibility to oxidative stress, with hydrogen peroxide increasing ROS production, reducing both cellular oxygen consumption and glycolytic flux in the cell. This study highlights bioenergetic defects within a cellular model of ALS and suggests that oxidative stress is not only detrimental to oxygen consumption but also glycolytic flux, which could lead to an energy deficit in the cell.     

Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

Photoprotection by antioxidants against UVB-radiation-induced damage in pig skin organ culture

Topically applied antioxidants constitute an important group of protective agents against skin damage induced by ultraviolet radiation. The current study was performed to investigate whether a recently developed ex vivo pig skin model was suitable for short-term studies of the mechanism(s) of UVB-radiation-induced skin damage; the protective effect of topical application of alpha-tocopherol, l-ascorbic acid, alpha-lipoic acid, glutathione ethylester and N-acetylcysteine was tested. Increasing doses of the antioxidants were applied topically on ex vivo pig skin explants and allowed to penetrate for 60 min. Epidermal antioxidant bioavailability was measured before and 60 min after exposure to an ultraviolet B (UVB) radiation of 7.5 kJ/m2. Cell viability (trypan blue dye exclusion) and apoptosis were measured 48 h later in isolated keratinocytes. UVB-radiation-induced epidermal lipid peroxidation was determined immediately after exposure of the skin to a UVB dose of 28 kJ/m2. All antioxidants tested became bioavailable in pig skin epidermis, and none of them were depleted after UVB-radiation exposure. Increasing doses of the antioxidants tested decreased UVB-radiation-induced cell death and apoptosis. The highest doses of antioxidants prevented UVB-radiation-induced lipid peroxidation; alpha-lipoic acid only tended to decrease lipid peroxidation. In conclusion, a single topical dose of the above antioxidants on ex vivo pig skin can reduce UVB-radiation-induced oxidative stress and lipid peroxidation and thereby reduce apoptotic stimuli and cell death. Furthermore, the ex vivo pig skin model was a useful tool for testing compounds for their antioxidant activity.