Macular pigment and macular volume in eyes of patients with cystic fibrosis.

BACKGROUND: Because patients with cystic fibrosis (CF) are living longer, chronic malabsorption of carotenoids associated with CF resulting in decreased macular pigment (MP) may affect macular long-term health in later-life pathology. This study compared the macular pigment optical density (MPOD) and corresponding central macular volume (MV) of adult CF subjects and age-matched normal controls subjects to determine whether chronic malabsorption associated with CF could adversely affect macular photoreceptor anatomy. OBJECTIVE: Our aim was to compare MPOD with measurements of central MV in CF patients with age-matched controls. Design. In nine adult CF patients (ages: 29-46) without a history of carotenoid supplementation or known retinal or optic nerve disease MPOD and MV were measured by heterochromatic flicker photometry (HFP) and optical coherence tomography (OCT), respectively, and compared to results obtained from 14 age-matched controls. RESULTS: MPOD was significantly reduced at 15' and 30' eccentricities in CF subjects compared to normal subjects (mean difference -0.21 at 15', -0.25 at 30', p < 0.005). No significant difference, in MV noted at any of the eccentricities tested between CF and normal subjects (CF: normal MV ratios ranged from 0.94 to 1.1 for all eccentricities with p > 0.1 at all eccentricities). Best corrected vision acuity and fundus examination were normal in all subjects. CONCLUSIONS: Unsupplemented CF patients have markedly lower levels of macular carotenoids (e.g., lutein and zeaxanthin), but well-maintained visual function and no significant reductions in central MV primarily composed of macular photoreceptors. Future studies are needed to determine whether the lifelong decrease in protective central retinal carotenoids predisposes CF patients to later-life retinal pathology.

Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats.

This study isolated the effects of maternal hypoxia independent of changes in maternal nutrition on maternal circulatory and placental molecular indices of oxidative stress and determined whether maternal antioxidant treatment conferred protection. Pregnant rats were subjected to normoxic pregnancy or 13% O2 chronic hypoxia for most of gestation with and without maternal treatment with vitamin C in the drinking water. Maternal hypoxia with and without vitamin C did not affect maternal food or water intake and led to a significant increase in maternal and fetal haematocrit. At gestational day 20, maternal plasma urate and L-cysteine concentrations, and placental levels of 4-hydroxynonenal and heat shock protein 70 were increased while placental heat shock protein 90 levels were decreased in hypoxic pregnancy. The induction of maternal circulatory and placental molecular indices of oxidative stress in hypoxic pregnancies was prevented by maternal treatment with vitamin C. Maternal hypoxia during pregnancy with or without vitamin C increased placental weight, but not total or compartmental volumes. Maternal treatment with vitamin C increased birth weight in both hypoxic and normoxic pregnancies. The data show that maternal hypoxia independent of maternal undernutrition promotes maternal and placental indices of oxidative stress, effects that can be prevented by maternal treatment with vitamin C in hypoxic pregnancy. While vitamin C may not be the ideal candidate of choice for therapy in pregnant women, and taking into consideration differences in ascorbic acid metabolism between rats and humans, the data do underlie that antioxidant treatment may provide a useful intervention to improve placental function and protect fetal growth in pregnancy complicated by fetal hypoxia.

Nitroxide radical TEMPO reduces ozone-induced chemokine IL-8 production in lung epithelial cells.

Exposure to high levels of ozone (O(3)) damages respiratory tract epithelial cells. This research evaluated the ability of TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a stable nitroxide free radical, to decrease O(3)-mediated injury to a respiratory tract-derived cell line (A549 cells) by monitoring in this cell system the interleukine-8 (IL-8) production. TEMPO reduced O(3)-induced IL-8 production in A549 cells, as evidenced by PCR analysis, Western blot and ELISA assays. This behaviour is explainable on the basis of the reactivity between TEMPO with O(3) and/or O(3)-derived free radicals in biological systems. The study provides evidence that TEMPO reacts with O(3) and/or its cytotoxic products and may provide protections against O(3)-induced biotoxicities.

Dimensions of postpartum psychiatric distress: preliminary evidence for broadening clinical scope.

BACKGROUND: Perinatal psychiatric disturbances afflict a significant number of women sometimes with tragic consequence. Yet, the range and characteristics of these disturbances are poorly understood. The goals of this research were to characterize a broader range of postpartum psychiatric symptoms and to identify their inherent structure using exploratory factor analysis (EFA). METHODS: An Internet-based survey with 142 Likert-type questions, covering nine dimensions of postpartum mental health was constructed and posted on women's health websites. Data collected from 215 respondents was analyzed in three steps: (1) inter-item correlations were used to reduce the total number of variables by eliminating items that provided redundant information; (2) an EFA using a principal components extraction and VARIMAX rotation was performed and factors loading with Eigenvalues >1.0 were retained; (3) internal consistency was measured with Cronbach's alpha. RESULTS: The 10 factors retained accounted for 58% of the variance and included: mental status (28%), psychoticism/morbid thoughts (6%), generalized anxiety (6%), panic (3%), guilt/self-criticism (3%), compulsive behavior (3%), hyper-vigilance (2%), contentment (2%), negative body-image (2%), and manic behavior (2%). There was strong (>0.8) internal consistency in all but the mania factor (0.6). LIMITATIONS: The study was retrospective and respondent demographics were homogeneous. CONCLUSION: Postpartum psychiatric disturbances are not limited to depressive symptoms. In the current study, cognitive difficulties, psychotic-morbid thoughts and anxiety symptoms accounted for the preponderance of variance while depressive symptoms did not form a cohesive factor and accounted for minimal variance. These results suggest postpartum screening tools should assess a broader array of symptoms.

Tocopherol transfer protein deficiency modifies nuclear receptor transcriptional networks in lungs: modulation by cigarette smoke in vivo.

Dietary factors and environmental pollutants initiate signaling cascades that converge on AhR:Nrf2:NF-kappaB transcription factor (TF) networks and, in turn, affect the health of the organism through its effects on the expression of numerous genes. Reactive oxygen metabolites (ROMs) have been hypothesized to be common mediators in these pathways. alpha-Tocopherol (AT) is a potent, lipophilic, scavenger of ROMs in vitro and has been hypothesized to be a major chain-breaking anti-oxidant in lipoproteins and biological membranes in vivo. The lung offers a vital organ to test the various postulated actions of AT in vivo. Lung AT concentrations can be manipulated by several methods that include dietary and genetic techniques. In this study we have used mice with severe AT deficiency inflicted at birth by the deletion of AT transfer protein (ATTP) which is abundantly expressed in the liver and regulates systemic concentrations of AT. Mice and humans deficient in ATTP are AT deficient. Female ATTP-deficient (ATTP-KO) mice and their congenic ATTP normal (WT) mice fed a diet containing 35 IU AT/kg diet were used to test our hypothesis. The mice (n=5/group) were exposed to either air or cigarette smoke (CS, total suspended particles 60 mg/m(3), 6h/day), a source of ROM, for 3 or 10 days. Post-exposure lung tissue was dissected, RNA extracted from each lung and it was pooled group-wise and processed for GeneChip analysis (Affymetrix 430A 2.0). Differential analysis of the transcriptomes ( approximately 16,000 mRNAs) identified CS sensitive genes that were modulated by lung AT-concentration. CS activated AhR driven genes such as cyp1b1 whose induction was augmented in CS-exposed, AT-deficient lungs. However, CS-induced expression of some of the Nrf2 driven genes was not potentiated in the AT-deficient lungs. Largest clusters of CS-AT sensitive genes were lymphocyte and leukocyte specific genes. These gene-clusters included those encoding cytokines and immunoglobulins, which were repressed by CS and were modulated by lung AT concentrations. Our genome-wide analysis suggests reciprocal regulation of xenobiotic and immune response genes by CS and a modulatory role of lung AT concentration on the expression of these clusters of genes. These data suggest that in vivo network of AT, AT-metabolites and ATTP affects the transcription of genes driven by AhR, Nrf2 and NF-kappaB, transcription factor networks that transduce cellular metabolic signals and orchestrate adaptive responses of lungs to inhaled environmental pollutants.

Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells. Modulation by alpha-tocopherol and ascorbic acid.

Acrolein is a highly reactive unsaturated hazardous air pollutant of human health concern, particularly as a component of cigarette smoke. In this study, the mechanisms of acrolein-induced cytotoxicity in human bronchial epithelial cells (HBE1) and the modulating effects of antioxidants were examined. Our results show that acrolein induces a cell death pathway in human bronchial epithelial cells, which retain key features of apoptosis, as indicated by phosphatidylserine (PS) externalization and DNA fragmentation. Acrolein-induced apoptosis was associated with depletion of cellular GSH and intracellular generation of oxidants. Supplementation of cells with either alpha-tocopherol or ascorbic acid was found to strongly inhibit acrolein-induced apoptosis and to prevent the increase in the generation of intracellular oxidants, although GSH depletion was unaffected. Moreover, recovery of cellular GSH levels after acrolein exposure was enhanced following either alpha-tocopherol or ascorbic acid supplementation. The intracellular generation of oxidants following acrolein exposure seems to be an important event triggering the apoptotic response in this model system.

Vitamin E kinetics in smokers and nonsmokers.

Does cigarette smoking increase vitamin E utilization in vivo? A trial was carried out in 6 smokers and 5 nonsmokers of comparable ages and serum lipids. Subjects consumed 75 mg each d(3)-RRR and d(6)-all rac-alpha-tocopheryl acetates (natural and synthetic vitamin E, respectively) daily for 7 d with a standardized breakfast. Fasting blood samples were drawn on days -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 9, 14, 21 (negative days indicate supplementation). In both groups, plasma d(3)-alpha-tocopherol concentrations were approximately double of d(6)-alpha-tocopherol. At day 0, the %d(3) alpha-tocopherols (d(3)-alpha-tocopherol/total-alpha-tocopherol x 100) were similar in both smokers and nonsmokers. Subsequently, there was a trend toward a faster exponential disappearance of the plasma %d(3) alpha-tocopherol in smokers compared with nonsmokers (0.30 +/- 0.04 compared with 0.24 +/- 0.05, p =.0565). The calculated %d(3) half-lives were 55.6 +/- 7.4 h in smokers and 72.1 +/- 17.3 h in nonsmokers (p =.0630). By day 21, the %d(3) in smokers had decreased to 1.4% +/- 0.3% while it was 2.2% +/- 0.7% (p =.0418) in the nonsmokers. These data suggest that smoking increases plasma vitamin E disappearance, but further studies are needed to confirm this finding and to assess its cause.

Direct activation of dendritic cells by the malaria parasite, Plasmodium chabaudi chabaudi.

A primary infection of mice with Plasmodium chabaudi chabaudi (AS) is characterized by a rapid and marked inflammatory response. Typically, IL-12, TNF-alpha and IFN-gamma are produced in the spleen, and are transiently present in plasma. The cells involved in this early response are unknown. Here we show that dendritic cells derived from GM-CSF-stimulated mouse bone marrow cultures produce TNF-alpha within 30 min of exposure to P.c.chabaudi schizonts. IL-6, IL-12p40 and p70 follow this. The production of these cytokines was not dependent on the presence of T cells or NK cells and did not require CD40. Incubation of dendritic cells with P.c.chabaudi schizonts also resulted in up-regulation of MHC class II, CD40 and CD86 but not CD80. In contrast to some strains of the human parasite, P. falciparum, P.c. chabaudi (AS) did not inhibit the up-regulation of MHC class II, CD86 or CD40 induced by LPS. Therefore, the erythrocytic stages of P.c.chabaudi are able to activate dendritic cells directly. The consequences of such an interaction could be rapid activation of TH1 cells and induction of immunity, and in the event of a large response also induction of TNF-alpha associated pathology.

Cigarette smoke impairs neutrophil respiratory burst activation by aldehyde-induced thiol modifications.

Exposure to airborne pollutants such as tobacco smoke is associated with increased activation of inflammatory-immune processes and is thought to contribute to the incidence of respiratory tract disease. We hypothezised that cigarette smoke (CS) could synergize with activated inflammatory/immune cells to cause oxidative injury or result in the formation of unique reactive oxidants. Isolated human neutrophils were exposed to gas-phase CS, and the production of nitrating and chlorinating oxidants following neutrophil stimulation was monitored using the substrate 4-hydroxyphenylacetate (HPA). Stimulation of neutrophils in the presence of CS resulted in a reduced oxidation and chlorination of HPA, suggesting inhibition of NADPH oxidase or myeloperoxidase (MPO), the two major enzymes involved in inflammatory oxidant formation. Peroxidase assays demonstrated that neutrophil MPO activity was not significantly affected after CS-exposure, leaving the NADPH oxidase as a likely target. The inhibition of neutrophil oxidant formation was found to coincide with depletion of cellular GSH, and a similar modification of critical cysteine residues, such as those in NADPH oxidase components, might be involved in reduced respiratory burst activity. As alpha,beta-unsaturated aldehydes such as acrolein have been implicated in thiol modifications by CS, we exposed neutrophils to acrolein prior to stimulation, and observed inhibition of NADPH oxidase activation in relation to GSH depletion. Additionally, translocation of the cytosolic components of NADPH oxidase to the membrane, a necessary requirement for enzyme activation, was inhibited. Protein adducts of acrolein (or related aldehydes) could be detected in several neutrophil proteins, including NADPH oxidase components, following neutrophil exposure to either CS or acrolein. Alterations in neutrophil function by exposure to (environmental) tobacco smoke may affect inflammatory/infectious conditions and thereby contribute to tobacco-related disease.

Phagocyte oxidants and nitric oxide in cystic fibrosis: new therapeutic targets?

Chronic persistent respiratory tract (RT) infection and overly exuberant activation of host inflammatory-immune processes represent the paramount pathobiologic consequences of cystic fibrosis (CF). The host inflammatory-immune system activation includes the production of reactive oxygen species (ROS) and nitric oxide (NO*), which are helpful in RT antimicrobial defenses but under conditions of excess are believed to be harmful to host tissues. However, the genetic defect underlying CF is recognized to uniquely affect inflammatory-immune (oxidative) processes, including alterations in cytokine release, phagocyte activation, antioxidant mechanisms, and NO* synthesis and metabolism, and these appear to contribute importantly to the persistent inflammation and infection in patients with CF. The dysregulation of inflammatory-immune responses has also been observed in CF epithelial cells and in some mouse models of CF, and marked advances in our understanding of these processes can be expected from future studies in such model systems. The emerging ideas of how ROS may influence molecular events that control inflammatory protease/antiprotease homeostasis and RT epithelial cell signaling and gene expression, and the role that augmentation of local antioxidant micronutrients (aerosolized and/or systemically administrated) might play in these effects, still remain to be further established and clarified. Further understanding of these processes can be expected to play an important role in future treatment directed toward the devastating effects of CF on the RT of patients with this disorder.

Oxidants, nitrosants, and the lung.

The respiratory tract is subjected to a variety of environmental stresses, including oxidizing gases, particulates, and airborne microorganisms, that together, may injure structural and functional lung components and thereby jeopardize the primary lung function of gas exchange. To cope with such various environmental threats, the lung has developed elaborate defense mechanisms that include inflammatory-immune pathways as well as several antioxidant systems. These defense systems operate largely in extracellular spaces, thus protecting underlying bronchial and alveolar epithelial cells from injury, although these cells themselves are also active participants in such (inflammatory) defense mechanisms. Although potentially harmful, oxidants are increasingly recognized as pathophysiologic mediators produced primarily by inflammatory-immune cells as a host defense mechanism, but also by various other cell types as an intracellular mediator in various cell responses, thus affecting inflammatory-immune processes or inducing resistance. The molecular mechanisms and signaling pathways involved in such processes are the focus of much current investigation. Nitric oxide, a messenger molecule produced by many lung cell types, also modulates oxidant-mediated processes, thereby giving rise to a new family of reactive nitrogen species ("nitrosants") with potentially unique signaling properties. The complex role of oxidants and nitrosants in various pathophysiologic processes in the lung have confounded the design of therapeutic approaches with antioxidant substrates. This review discusses current knowledge regarding extracellular antioxidant defenses in the lung, and oxidant/nitrosant mechanisms operating under inflammatory-immune conditions and their potential contribution to common lung diseases. Finally, some recent developments in antioxidant therapeutic strategies are discussed.

Myeloperoxidase and protein oxidation in cystic fibrosis.

Cystic fibrosis (CF) is associated with chronic pulmonary inflammation and progressive lung dysfunction, possibly associated with the formation of neutrophil myeloperoxidase (MPO)-derived oxidants. Expectorated sputum specimens from adult CF patients were analyzed for MPO characteristic protein modifications and found to contain large amounts of active MPO as well as high levels of protein-associated 3-chlorotyrosine and 3,3'-dityrosine, products that result from MPO activity, compared with expectorated sputum from non-CF subjects. Sputum levels of nitrite (NO(2)(-)) and nitrate (NO(3)(-)), indicating local production of nitric oxide (NO. ), were not elevated but in fact were slightly reduced in CF. However, there was a slight increase in protein-associated 3-nitrotyrosine in CF sputum compared with controls, reflecting the formation of reactive nitrogen intermediates, possibly through MPO-catalyzed oxidation of NO(2)(-). CF sputum MPO was found to contribute to oxidant-mediated cytotoxicity toward cultured tracheobronchial epithelial cells; however, peroxidase-dependent protein oxidation occurred primarily within sputum proteins, suggesting scavenging of MPO-derived oxidants by CF mucus and perhaps formation of secondary cytotoxic products within CF sputum. Our findings demonstrate the formation of MPO-derived oxidizing and possibly nitrating species within the respiratory tract of subjects with CF, which collectively may contribute to bronchial injury and respiratory failure in CF.

Ultraviolet B-mediated phosphorylation of the small heat shock protein HSP27 in human keratinocytes.

Exposure of human keratinocytes to environmental stress is known to induce changes in the expression, phosphorylation, and subcellular relocalization of the 27 kDa heat shock protein. This study demonstrates that ultraviolet B (280-320 nM) irradiation with physiologic doses induces a dose-dependent phosphorylation of 27 kDa heat shock protein, generating the more acidic 27 kDa heat shock protein B, C, and D isoforms. Ultraviolet B also induces perinuclear cytoplasmic relocation and nuclear translocation of 27 kDa heat shock protein and caused aggregation of cytoplasmic actin filaments into a broad perinuclear distribution. The ultraviolet B-induced phosphorylation is reversible, returning to baseline levels 4 h after exposure, and this coincides with the reversal of ultraviolet B-induced actin reorganization. The ultraviolet B-induced phosphorylation is not affected by the protein kinase C inhibitor, GF 109203X, is partially inhibited by epidermal growth factor receptor tyrosine kinase inhibitor, PD 153035, and is substantially inhibited by the specific p38 mitogen-activated protein kinase inhibitor, SB 203580. In addition, pretreatment of cells with the anti-oxidant N-acetyl cysteine partially inhibits ultraviolet B-and oxidant-induced 27 kDa heat shock protein phosphorylation. The p38 mitogen-activated protein kinase cascade is thus the major transduction pathway for ultraviolet B-induced 27 kDa heat shock protein phosphorylation, and reactive oxygen species generated in response to ultraviolet B also contribute to this phosphorylation. As 27 kDa heat shock protein phosphorylation and relocalization has been associated with increased cell survival after environmental insult, our data suggest that ultraviolet B, in addition to initiating recognized cytotoxic events in keratinocytes, also initiates a signaling pathway that may provide cellular protection against this ubiquitous environmental insult.

Association between the level of ERCC-1 expression and the repair of cisplatin-induced DNA damage in human ovarian cancer cells.

Nucleotide excision repair (NER) is responsible for the repair of platinum-DNA lesions. ERCC-1 is a critical gene within the NER pathway, and cells without a functional ERCC-1 do not repair cisplatin-caused DNA damage. The present study was therefore designed to evaluate the relationship between the expression of ERCC-1 and the repair of cisplatin-induced DNA adducts in human ovarian cancer cells in vitro. One hour exposure of MCAS cells to cisplatin yielded an approximately two-fold increment in the levels of ERCC-1 mRNA and ERCC-1 protein, as determined, respectively, by Northern and Western blottings. In addition, nuclear run-on assay showed that ERCC-1 gene transcription rate was increased to about the same extent as steady-state ERCC-1 mRNA and protein, in response to cisplatin treatment. However, the levels of ERCC-1 mRNA, ERCC-1 protein, and ERCC-1 transcript in MCAS cells are two-fold lower than those in A2780/CP70 cells, as previously reported. Furthermore, the repair of cisplatin-DNA adducts in MCAS cells, as measured by atomic absorption spectrometry, is also nearly two-fold less than that in A2780/CP70 cells, indicating a strong association between the level of ERCC-1 expression and the activity of excision repair in these two human ovarian tumor cell lines. These results suggest that ERCC-1 may be a useful marker to monitor the repair of platinum-DNA damage in tumor cells, and further highlight that potential pharmacological approaches which specifically inhibit ERCC-1 expression may increase cellular sensitivity to cisplatin.

Tobacco-related diseases. Is there a role for antioxidant micronutrient supplementation?

It is clear that smoking causes an increase in free radicals, reactive nitrogen and oxygen species (RNS and ROS, respectively), and that cigarette smoking is associated with increases in the incidence and severity of several diseases including atherosclerosis, cancer, and chronic obstructive lung disease. Although there is still no unequivocal evidence that oxidative stress is a contributor to these diseases or that an increased intake of antioxidant nutrients is beneficial, the observation that smokers have lower circulating levels of some of these nutrients, raises concern. This article discusses the possible links between the observed oxidant-induced damage related to tobacco smoking, effects on cellular mechanisms, and their potential involvement in the causation and enhancement of disease processes.

Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue.

Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys(283)) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.

Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum.

As the outermost layer of the skin, the stratum corneum is exposed to environmental oxidants. To investigate putative synergisms of environmental oxidative stressors in stratum corneum, hairless mice were exposed to ultraviolet radiation (UV) and ozone (O(3)) alone and in combination. Whereas a significant depletion of alpha-tocopherol was observed after individual exposure to either a 0.5 minimal erythemal dose of UV or 1 ppm O(3) for 2 h, the combination did not increase the effect of UV alone. However, a dose of 0.5 ppm O(3) x 2 h, which had no effect when used alone, significantly enhanced the UV-induced depletion of vitamin E. We conclude that concomitant exposure to low doses of UV and O(3) at levels near those that humans can be exposed to causes additive oxidative stress in the stratum corneum.

Vitamin C, uric acid, and glutathione gradients in murine stratum corneum and their susceptibility to ozone exposure.

The stratum corneum has been recognized as the main cutaneous oxidation target of atmospheric ozone (O3), a major part of photochemical smog. This study reports the presence and distribution of vitamin C, glutathione, and uric acid in murine stratum corneum, and evaluates their susceptibility to acute environmental exposure to O3. Based on tape stripping and a modified extraction method with high performance liquid chromatography electrochemical analysis, we detected vitamin C (208.0 +/- 82.5 pmol per 10 consecutive pooled tapes), glutathione (283.7 +/-96.3), and uric acid (286.4 +/-47.1) in murine stratum corneum as compared with only 16.5 +/- 1.4 pmol alpha-tocopherol. Vitamin C, glutathione (both p < 00.001), and urate (p < 0.01) were found to exhibit a gradient with the lowest concentrations in the outer layers and a steep increase in the deeper layers. To investigate the effect of O3 exposure on hydrophilic antioxidants, we exposed SKH-1 hairless mice to O3 concentrations of 0, 0.8, 1, and 10 p.p.m., and stratum corneum was analyzed before and after exposure. Whereas mock exposure with 0 p.p. m. for 2 h had no significant effect, O3 doses of 1 p.p.m. for 2 h and above showed depletion of all three antioxidants. Vitamin C was decreased to 80% +/- 15% of its pretreatment content (p < 0.05), GSH to 41% +/- 24% (p < 0.01), and uric acid to 44% +/- 28% (p < 0.01). This report demonstrates the previously unrecognized role of hydrophilic antioxidants in the stratum corneum and provides further evidence that O3 induces oxidative stress in this outer skin layer.