Premature Avian Melanocyte Death Due to Low Antioxidant Levels of Protection: Fowl Model for Vitiligo

Feather melanocytes in the Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens die prematurely in vivo when compared to the wild type Jungle Fowl (JF) chicken. Since these mutant melanocytes live in vitro, an environmental factor in the feather must precipitate their death. Results show that the addition of selected antioxidants, glutathione (GSH) and superoxide dismutase (SOD), can rescue these mutant melanocytes in vitro that have been placed under stress conditions that cause their premature cell death. Measurements of in vivo levels of GSH, catalase, and SOD show no significant difference in catalase activity between the JF, BPR, and WL feathers but do show a significant reduction in GSH activity in both the BPR and WL feathers to approximately 66% of the GSH concentration found in JF feathers. SOD activity in the BPR tissue is reduced significantly to approximately 50% of the JF activity and the WL SOD activity is reduced significantly to approximately 50% of the BPR SOD activity. Preliminary results of measurements of glutathione peroxidase activity indicate there is no difference in the levels of this enzyme in JF, BPR and WL feathers. A working hypothesis, based on current results, is proposed for premature cell death in BPR and WL feather melanocytes. The BPR melanocytes are genetically sensitive due to a defect in their SOD and GSH levels caused by the barring gene (B) and their death, due to reactive species of oxygen radicals, is precipitated in the poorly vascularized feather by the accumulation of oxygen radicals due to the low turnover of tissue fluids. The WL chicken carries the dominant white gene (I) in addition to the B gene. This gene directs the further reduction of the level of SOD and, when combined with the cell death mechanism already present in the BPR chicken, causes the WL feather melanocytes to die much earlier than the BPR feather melanocytes which in turn die much earlier than the wild type JF melanocytes. This same mechanistic hypothesis could apply as a cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their oxygen radical protection system.     

Modulation of melanocytic activity by acetylcholine

Acetylcholine esterase (AChE) activity is lowered in vitiliginous skin. The AChE activity in 52 cases of vitiligo during repigmentation and depigmentation has been observed in this study. The cases with marginal dendritic melanocytes show that AChE is negative in these cells during depigmentation but positive on repigmentation. There is little variation in activity in the cases showing nondendritic marginal melanocytes. Acetylcholine (ACh) has an inhibitory effect on dopa oxidase activity in both types of marginal melanocytes in vitiligo. ACh modulates pigment production by the melanocytes, its role being inhibitory. From the present results, it is evident that a fall in AChE activity in the melanocytes leads to greater inhibition by ACh aggravating the process of depigmentation in vitiligo.     

Smyth Chicken Melanocyte Autoantibodies: Cross-Species Recognition,In Vivo Binding,and Plasma Membrane Reactivity of the Antiserum

Smyth line (SL) chickens, which develop a depigmenting disorder similar to human vitiligo, produce circulating anti-melanocyte antibodies (Austin, L.M. et al., (1992) The detection of melanocyte autoantibodies in the Smyth chicken model for vitiligo. Clin. Immunol. Immunopathol., 64:112–120). In order to characterize these autoantibodies, we studied the reactivity of cultured chicken, mouse, and human melanocytes, as well as frozen sections of chicken feather follicles and embryonic eyes, against SL serum, employing indirect immunofluorescence. Light Brown Leghorn (LBL) serum was used as a negative control. Chicken (SL and LBL), mouse, and human melanocytes exhibited greater fluorescence with SL serum than with LBL serum (up to a 1:60,000 dilution). The fluorescent pattern was predominant along the perimeter of the cells, suggesting plasma membrane staining. Fluorescence-activated flow cytometry analysis and immunocytochemical localization at the ultrastructural level using intact chicken cells supported this hypothesis. Melanocytes were readily stained in cryosections of regenerating feather follicles and embryonic eyes incubated with SL, but not LBL, serum. In addition, amelanotic melanocytes in albino chicken feathers reacted with SL serum. SL serum also preferentially stained cells emigrating from cultured avian neural tubes and within the dermis of the proliferative germ of regenerating feather follicles suggesting that melanoblasts express the antigens. We conclude that Smyth line serum contains melanocyte autoantibodies that cross-react with mouse and human melanocytes, are able to bind to pigment cells within tissues, and recognize antigens expressed in the cytoplasm and on the surface of melanocytes and melanoblasts.     

Exposure to a competitive social environment activates an epigenetic mechanism that limits pheomelanin synthesis in zebra finches

Competitive environments promote high testosterone levels, produce oxidative stress and, consequently, impair cellular homeostasis. The regulation of genes involved in the synthesis of the pigment pheomelanin in melanocytes seems to help to maintain homeostasis against environmental oxidative stress. Here, we experimentally increased social interactions in some zebra finch (Taeniopygia guttata) males by keeping them in groups of six birds during feather growth, while others were kept alone, to test if melanocytes show epigenetic lability under a competitive social environment. As these changes may depend on the oxidative status, we administrated buthionine sulfoximine (BSO) to decrease the antioxidant capacity of some birds. The competitive environment downregulated a gene involved in pheomelanin synthesis (Slc7a11) by changing the level of DNA methylation in feather melanocytes. In other genes involved in pheomelanin synthesis (Slc45a2, MC1R and AGRP), DNA methylation was also affected, but no changes in expression were detected. Exposure to the competitive environment did not affect systemic oxidative stress and damage, indicating that a protective epigenetic mechanism that changes the expression of Slc7a11 may have been activated. However, no changes to the pigmentation phenotype of birds were found, probably due to the short duration or low intensity of the competitive environment. BSO treatment did not affect the epigenetic mechanism, suggesting that the antioxidant capacity of birds was high enough to deal with the competitive environment. An epigenetic mechanism limiting pheomelanin synthesis therefore becomes activated under exposure to a competitive environment in male zebra finches, which may help to avoid damage caused by competitive interactions.     

Effect of the barring gene on eye pigmentation in the fowl

Pigment cells of the iris, pecten, retinal pigment epithelium, and choroid of the wild-type jungle fowl (JF) and the barred Plymouth rock (BPR) breeds of adult chickens were studied at both light and electron microscopic levels. BPR choroidal tissues had 2.8 times fewer melanophores than the JF choroid, and BPR melanophores also contained 2.4 times fewer melanosomes, which tended to clump together in variously sized clusters. The melanosomes were often irregular in shape, smaller in diameter, and less mature (stage III) than those granules in the JF. The retinal pigment epithelium of both JF and BPR breeds contained a single epithelial layer of columnar cells. Rod-shaped melanosomes were present in the more apical regions of this cell type in both breeds. Both JF and BPR irides contained a multilayered posterior pigmented epithelium of columnar shaped cells that were densely filled with large spherical granules. Intercellular spaces with interdigitating cytoplasmic projections were present between pigment cells of both breeds. The pecten melanophores of both breeds were dendritic with melanosomes that were larger and fewer in numbers than those pigment cells of the iris and choroid. Intercellular spaces were present between cells in both breeds, with numerous villous-like pigment cell extensions. Choroid melanophores contained very little, if any, acid phosphatase activity. Approximately one-half of the retinal pigment epithelial cells observed contained small amounts of diffuse acid phosphatase activity in both breeds. The iris and pecten melanophores of both breeds contained profuse acid phosphatase activity scattered throughout their cytoplasms. Sparse tyrosinase activity was seen in iris and pecten pigment cells, whereas no tyrosine activity was observed in choroid melanophores or in retinal pigment epithelial cells in the two breeds, indicating that little new melanogenesis occurs in adult pigmented eye tissues. The results show that the barring gene reduces the number and melanin content of the choroidal melanophores in homozygous male BPR chickens as compared to the wild-type JF chickens. Whether this gene prevents the initial migration of embryonic neural crest cells (future melanophores) to the choroid or whether some of the choroidal melanophores prematurely degenerate in the embryo of young birds is yet to be determined. If the latter is the case, this choroid system may serve as a model for a genetic hypomelanotic disease such as vitiligo.     

Repigmentation of vitiliginous skin by cultured cells

Epidermal cells from pigmented areas of a patient with vitiligo were cultured in MCDB-153 medium, which supports the clonal growth of undifferentiated keratinocytes and melanocytes. The cells were grown on collagen-coated substrata. After the cells reached semiconfluence, the composite of substratum and cells was emplaced onto dermabraded vitiliginous areas as a graft. Re-epithelialization of the grafted areas was complete after 2 weeks. Repigmentation was evident after 1 month and continued over the observation period of several months. There was complete and normal differentiation of the graft, including a normal distribution of melanocytes in the basal layer. Ultrastructural studies showed a normal distribution of melanosomes in the melanocytes and showed keratinocytes that were indistinguishable from the uninvolved skin.     

Polynitroxyl alphaalpha-hemoglobin (PNH) inhibits peroxide and superoxide-mediated neutrophil adherence to human endothelial cells.

Experimental hemoglobin-based O2 carriers e.g. cross-linked alphaalpha-hemoglobin (alphaalpha-Hb), are under investigation as potential blood substitutes. However, some Hb-based products form strong oxidant species in vivo that may cause adverse clinical effects. We report the prototype of a new class of modified Hb-based O2 carrier, polynitroxylated alphaalpha-Hb (PNH), which has antioxidant activities that may reduce inflammatory effects mediated by oxidant formation. We compared the effects of alphaalpha-Hb and PNH on xanthine oxidase and H2O2-induced neutrophil-endothelial adhesion in vitro. Both peroxide (>0.1 mM), and superoxide/peroxide generated by xanthine oxidase (XO) (> 10 mU/ml) + 0.1 mM xanthine (X), increased endothelial-neutrophil adhesion. At 30 microM, alphaalpha-Hb significantly increased X/XO-mediated adhesion, while PNH inhibited peroxide or X/XO induced adhesion, with maximal inhibition at 10 microM PNH. These data indicate that PNH has antioxidant-anti-inflammatory properties that suggest its use as a potentially safer blood substitute in reperfusion injury, stroke, myocardial infarction and other forms of inflammation.     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果     

Antioxidant and xanthine oxidase inhibitory activities of total polyphenols from onion

Onions (Allium cepa L.) comprise a valuable vegetable crop in many countries. Modern scientific research has shown that onions possess many biological activities, including antibacterial, anticancer, hypoglycemic, hypolipidemic, antiplatelet aggregation, and antioxidant activities. The goal of this study was to investigate the impact of total onion polyphenols on antioxidant and xanthine oxidase (XO) inhibitory activities. Total onion polyphenols showed significant antioxidant activity in DPPH, FRAP, and OH-assays (IC₅₀ [µg/mL]), 43.24, 560.61, and 12.97, respectively). In a X/XO system, antioxidant properties of these polyphenols significantly inhibited XO activity (IC₅₀ [µg/mL], 17.36). These results indicated that total onion polyphenols showed promising antioxidant and anti-gout properties and might be used as potential, natural drugs against oxidative diseases after successful studies in vivo as well as clinical trials.     

Effects of Greek legume plant extracts on xanthine oxidase,catalase and superoxide dismutase activities

Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered.     

Xanthine oxidase activates pro-matrix metalloproteinase-2 in cultured rat vascular smooth muscle cells through non-free radical mechanisms

Reactive oxygen species (ROS) have been implicated in the regulation of matrix metalloproteinases (MMPs). The xanthine/xanthine oxidase (X/XO) reaction has been widely used as a source of exogenous ROS in studying MMPs, but commercial XO has also been known to be contaminated by proteolytic activity, and MMPs are protease sensitive substrate. We have investigated the activation of proMMP-2 by X/XO in cultured vascular smooth muscle cells (SMCs). SMCs were incubated with X/XO (unpurified or purified) or XO alone for 24h. X/XO activated proMMP-2 in a dose-dependent manner. A similar profile was observed using XO. Purified XO produced lower amounts of active MMP-2 compared to unpurified XO. EPR study showed that X/XO, not XO itself, produced superoxide anion, which was completely scavenged by SOD. However, X/XO-induced proMMP-2 activation could not be inhibited by combination of SOD and catalase. Incubation with XO either in cell-free conditioned media or in cells resulted in similar amounts of active MMP-2, suggesting that membrane-type-MMPs were not involved in proMMP-2 activation. This was further confirmed by the lack of inhibitory effect of hydroxamate MMP inhibitor, BB1101. Aprotinin blocked unpurified XO-induced proMMP-2 activation in a dose-dependent manner, demonstrating the proteolytic activity contained in XO is essential. We conclude that proteolytic activity contained in XO, rather the ROS derived from X/XO, is responsible for proMMP-2 activation in cultured SMCs. The results also suggest that caution needs to be taken when interpreting the reported results on activation of MMPs where X/XO had been used as an "authentic" source of superoxide anion.     

Functional defects of peripheral regulatory T lymphocytes in patients with progressive vitiligo

Auto-reactive cytotoxic T lymphocytes play a key role in the progressive loss or destruction of melanocytes in vitiligo but the mechanism underlying the loss of self-tolerance is unknown. A deregulation of regulatory T-cell biology has recently been suggested. The analysis of the suppressive effects of peripheral T regulatory cells in vitiligo patients revealed a functional defect in seven of 15 cases. This defect was strongly correlated with disease activity. The evaluation of the percentage of peripheral regulatory T lymphocytes did not reveal any intrinsic quantitative defect. Yet, a decrease in the percentage of such cells was noted in patients with progressive forms, suggesting a recruitment of regulatory T cells from the peripheral blood to the site of injury. This was further corroborated by the significant increase of Forkhead box P3 expression in the vitiliginous skin of patients. Our data support the involvement of a functional defect of peripheral regulatory T cells in the pathogenesis of vitiligo and open new possibilities to advance therapeutic approaches.     

Lipid Peroxidation-Induced Inhibition of γ-Aminobutyric Acid Uptake in Rat Brain Synaptosomes: Protection by Glucocorticoids

Incubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) resulted in an inhibition of gamma-aminobutyric acid (GABA) uptake. The inhibitory effects of X/XO were temperature- and time-dependent, and were characterized by an increased Km for GABA and a decreased Vmax. Inhibition of GABA uptake by X/XO was associated with both the formation of malonyldialdehyde (MDA) and conjugated dienes, indicating that lipid peroxidation was involved. Studies with catalase, superoxide dismutase (SOD), mannitol, and chelated iron suggested that hydroxyl radical (OH X) was probably responsible for the initiation of lipid peroxidation. Both the peroxidation of synaptosomal membranes and the inhibition of GABA uptake by X/XO were enhanced by the addition of ADP and FeCl2. The X/XO-induced inhibition of GABA uptake by synaptosomes could be prevented by preincubation of synaptosomes with certain glucocorticoids prior to X/XO exposure. Methylprednisolone sodium succinate (MPSS), dexamethasone sodium phosphate (DMSP), and prednisolone sodium succinate (PSS) all prevented the inhibition of GABA uptake by X/XO. MPSS was most effective at concentrations around 100 microM, DMSP was slightly more potent, and PSS was optimal at around 300 microM. On the other hand, hydrocortisone sodium succinate (HCSS) was ineffective at preventing X/XO-induced inhibition of GABA uptake at concentrations up to 3 mM. The steroids are presumed to work through a mechanism that blocked the formation of lipid peroxides, as MPSS inhibited the formation of conjugated dienes in synaptosomes exposed to X/XO at a concentration that also protected GABA uptake.     

Strain preference in donor and recipient for production of W-bearing sperm in mixed-sex germline chimeric chickens

To elucidate the strain preference in donor and recipient for the production of W-bearing sperm, mixed-sex germline chimeric chickens were produced. The combination of donor and recipient was White Leghorn (WL) and Barred Plymouth Rock (BPR), and vice versa. Four sets of mixed-sex chimeras that had the male phenotype at sexual maturity were subjected to analysis: group 1, a female WL donor and a male BPR recipient; group 2, a male WL donor and a female BPR recipient; group 3, a female BPR donor and a male WL recipient; group 4, a male BPR donor and a female WL recipient. The mean number of W-bearing sperm detected by in situ hybridization among 10000 sperm observed was 135, 158, 26 and 71 in groups 1, 2, 3 and 4, respectively. The number in group 1 was significantly higher than that of group 3 (P<0.05). And the number in group 2 was significantly higher than those of groups 3 and 4 (P<0.05). It is suggested that the combination of a WL donor and a BPR recipient produced W-bearing sperm more efficiently than the reverse combination.     

Calcium Enhances In Vitro Free Radical-Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons

Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.     

Hyperoxia and xanthine dehydrogenase/oxidase activities in rat lung and heart

Cell injury from hyperoxia is associated with increased formation of superoxide radicals (O2-). One potential source for O2- radicals is the reduction of molecular O2 catalyzed by xanthine oxidase (XO). Physiologically, this reaction occurs at a relatively low rate, because the native form of the enzyme is xanthine dehydrogenase (XD) which produces NADH instead of O2-. Reports of accelerated conversion of XD to XO, and increased formation of O2- formation in ischemia-reperfusion injury, led us to examine whether hyperoxia, which is known to increase O2- radical formation, is associated with increased lung XO activity, and accelerated conversion of XD to XO. We exposed 3-month-old rats either to greater than 98% O2 or room air. After 48 h, we sacrificed the rats and measured XD and XO activities and uric acid contents of the lungs. We also measured the activities of the two enzymes in the heart as a control organ. We found that the activity of XD was not altered significantly by hyperoxia in rat lungs or hearts, but XO activity was markedly lower in the lung, whether expressed per whole organ or per milligram protein, and remained unchanged in the heart. Lung uric acid content was also significantly lower with hyperoxia. The decrease in lung XO activity may reflect inactivation of the enzyme by reactive O2 metabolites, possibly as a negative feedback mechanism. The concomitant decrease in uric acid content suggests either decreased production mediated by XO due to its inactivation or greater utilization of uric acid as an antioxidant. We examined these postulates in vitro using a xanthine/xanthine oxidase system and found that H2O2, but not uric acid, has an inhibitory effect on O2- formation in the system. We therefore conclude that hyperoxia is not associated with increased conversion of XD to XO, and that the exact contribution of XO to hyperoxic lung injury in vivo remains unclear.     

Synthesis and evaluation of hydroxychalcones as multifunctional non-purine xanthine oxidase inhibitors for the treatment of hyperuricemia

A series of hydroxychalcone derivatives have been designed, synthesized and evaluated for human xanthine oxidase (XO) inhibitory activity. Most of the tested compounds acted moderate XO inhibition with IC₅₀ values in the micromolar rang. Molecular docking and kinetic studies have been performed to explain the binding modes of XO with the selected compounds. In addition, in vitro antioxidant screening results indicated that some of the hydroxychalcones possessed good anti-free radical activities. Furthermore, the preferred compounds 16 and 18 were able to significantly inhibit hepatic xanthine oxidase activity and reduced serum uric acid level of hyperuricemic mice in vivo. In summary, compounds 16 and 18 with balanced activities of antioxidant, XO inhibition and serum uric acid reduction, which are promising candidates for the treatment of hyperuricemia and gout.     

Role of neutrophils in xanthine/xanthine oxidase-induced oxidant injury in isolated rabbit lungs.

Reactive oxygen species have been shown to play an important role in the pathogenesis of lung injury. This study was designed to clarify the role of intrapulmonary neutrophils in the development of xanthine/xanthine oxidase (X/XO)-induced lung injury in isolated buffer-perfused rabbit lungs. We measured microvascular fluid filtration coefficient (K(f)) and wet-to-dry weight ratio to assess lung injury. X/XO induced a significant increase in K(f) and wet-to-dry weight ratio in neutrophil-replete lungs, whereas the lung injury was attenuated in neutrophil-depleted lungs. A neutrophil elastase inhibitor, ONO-5046, also attenuated the lung injury. In addition, X/XO induced a transient pulmonary arterial pressure (P(pa)) increase. The thromboxane inhibitor OKY-046 attenuated the P(pa) increase but did not alter the increase in permeability. Neutrophil depletion reduced the K(f) increase but had no effect on the P(pa) increase. These results suggest that intrapulmonary neutrophils activated by X/XO play a major role in development of the lung injury, that neutrophil elastase is involved in the injury, and that the X/XO-induced vasoconstriction is independent of intrapulmonary neutrophils.     

Repair of DNA damage induced by oxygen radicals in human non-proliferating and proliferating lymphocytes.

Repair of DNA lesions induced by oxygen radicals, generated by xanthine/xanthine oxidase (X/XO), was studied in human peripheral blood lymphocytes and in PHA-stimulated proliferating lymphocytes from 4 healthy subjects. The lesions included DNA-strand breaks (SSB) and other lesions that are converted to SSB under alkaline conditions. The frequencies of SSB were estimated by fluorometric analysis of DNA unwinding. Maximum production of SSB occurred within 10 min of incubation with X/XO at 22 degrees C; with 0.5 mM or higher concentrations of xanthine; and with 0.1-0.5 units/ml of xanthine oxidase. Proliferating lymphocytes repaired X/XO-induced SSB about 4 times more rapidly than lymphocytes. Lymphocytes repaired X/XO-induced SSB more slowly than SSB caused by gamma-radiation. These findings are consistent with the evidence that a number of DNA-repair enzymes have greater activity in proliferating cells than in resting cells. These findings also support the view that there are differences between the DNA damage due to oxygen radicals and that due to ionizing radiation.     

Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes

Exposure of red blood cells to oxygen radicals can induce hemoglobin damage and stimulate protein degradation, lipid peroxidation, and hemolysis. To determine if these events are linked, rabbit erythrocytes were incubated at 37 degrees C with various oxygen radical-generating systems and antioxidants. Protein degradation, measured by the production of free alanine, increased more than 11-fold in response to xanthine (X) + xanthine oxidase (XO). A similar increase in proteolysis occurred when the cells were incubated with acetaldehyde plus XO, with ascorbic acid plus iron (Asc + Fe), or with hydrogen peroxide (H2O2) alone. Upon addition of XO, increased proteolysis was evident within 5 min and was linear for up to 5 h. In contrast, lipid peroxidation, as shown by the production of malonyldialdehyde, conjugated dienes, or lipid hydroperoxides was observed only after 2 h of incubation with X + XO, acetaldehyde + XO, or H2O2. Ascorbate plus Fe2+ induced both protein degradation and lipid peroxidation; however, the addition of various antioxidants (urate, xanthine, glucose, or butylated hydroxytoluene) decreased lipid peroxidation without affecting proteolysis. Thus, these processes seem to occur by distinct mechanisms. Furthermore, at low concentrations of XO, protein degradation was clearly increased in the absence of detectable lipid peroxidation products. Hemolysis occurred only in a small number of cells (9%) and followed the appearance of lipid peroxidation products. Thus, an important response of red cells to oxygen radicals is rapid degradation of damaged cell proteins. Increased proteolysis seems to occur independently of membrane damage and to be a more sensitive indicator of cell exposure to oxygen radicals than is lipid peroxidation.