Coffee drinking increases levels of urinary hydrogen peroxide detected in healthy human volunteers

Freshly-voided human urine contains significant concentrations of hydrogen peroxide (H₂O₂). This H₂O₂ appears to arise in whole or in part by superoxide-dependent autoxidation of urinary biomolecules. Since instant coffee also contains high levels of H₂O₂, we examined the effect of coffee drinking on urinary levels of H₂O₂. Studies on healthy human volunteers showed that coffee drinking is rapidly and reproducibly followed by increased levels of H₂O₂ detectable in the urine for up to 2 h after drinking the coffee. The levels of H₂O₂ detected in urine suggest that exposure of human tissues to H₂O₂ may be greater than is commonly supposed. It is possible that H₂O₂ in urine could act as an antibacterial agent, and that H₂O₂ is involved in the regulation of glomerular function.     

Caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid up-regulate NQO1 expression and prevent H₂O₂-induced apoptosis in primary cortical neurons

Neurodegenerative disorders are strongly associated with oxidative stress, which is induced by reactive oxygen species including hydrogen peroxide (H₂O₂). Epidemiological studies have suggested that coffee may be neuroprotective, but the molecular mechanisms underlying this effect have not been clarified. In this study, we investigated the protective effects of caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid (5-O-caffeoylquinic acid), which is present in both caffeinated and decaffeinated coffee, against oxidative neuronal death. H₂O₂-induced apoptotic nuclear condensation in neuronal cells was strongly inhibited by pretreatment with caffeinated coffee, decaffeinated coffee, or chlorogenic acid. Pretreatment with caffeinated coffee, decaffeinated coffee, or chlorogenic acid inhibited the H₂O₂-induced down-regulation of anti-apoptotic proteins Bcl-2 and Bcl-X_L while blocking H₂O₂-induced pro-apoptotic cleavage of caspase-3 and pro-poly(ADP-ribose) polymerase. We also found that caffeinated coffee, decaffeinated coffee, and chlorogenic acid induced the expression of NADPH:quinine oxidoreductase 1 (NQO1) in neuronal cells, suggesting that these substances protect neurons from H₂O₂-induced apoptosis by up-regulation of this antioxidant enzyme. The neuroprotective efficacy of caffeinated coffee was similar to that of decaffeinated coffee, indicating that active compounds present in both caffeinated and decaffeinated coffee, such as chlorogenic acid, may drive the effects.     

Antioxidant System Differential Regulation is Involved in Coffee Ripening Time at Different Altitudes

Fruit ripening can be seen as an oxidative phenomenon that, depending on its intensity, may directly influence fruit quality. At relatively higher altitudes, coffee fruit ripening takes place through an extended period of time, which positively affects coffee quality. However, little is known about the oxidative processes and antioxidant metabolism of coffee fruits grown at these altitudes. Thus, this study aimed to characterise coffee fruit development from trees grown at two contrasting altitudes (965 m and 1310 m) through phenological analysis and antioxidant metabolism evaluation (Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents; superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activity and gene expression). Phenological analysis showed that altitude extended coffee reproductive cycle by a month and promoted a higher ripening uniformity, with 100% of fruits at the ideal ripening stage for harvest (cherry stage) in the last evaluation time. H₂O₂ and malondialdehyde contents revealed that in both altitudes fruits went through oxidative damage, though in an early manner at the lower altitude. Although gene expression and enzyme activity did not well correlate, the delay in the oxidative damage in fruits of the higher altitude was probably a result of an increased efficiency in H₂O₂ neutralisation due to the higher activity levels of the APX and CAT enzymes, mainly in green fruits. Thus, a better removal of reactive oxygen species in coffee fruits from plants grown at higher altitudes is involved in the extension of the coffee reproductive cycle, contributing to the production of a higher cup quality coffee.     

Urinary desmosine as a biomarker in acute lung injury

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/α₁-antiprotease complex concentration or P_aO₂/F_iO₂ ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.     

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H₂O₂) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H₂O₂ formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H₂O₂ formation during acidic conditions (pH: 3.5–5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H₂O₂ concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H₂O₂ accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H₂O₂ formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H₂O₂. Ascorbic acid/Cu(II)-induced H₂O₂ formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.     

Investigation of the effect of naringenin on oxidative stress‐related alterations in testis of hydrogen peroxide‐administered rats

Testis tissue is prone to oxidation because its plasma membrane contains many polyunsaturated fatty acids. Naringenin is a plant‐derived natural flavonoid. We investigated the possible ameliorative role of naringenin on the hydrogen peroxide (H₂O₂)‐induced testicular damage in Wistar rats. Animals received 12 mg/kg H₂O₂ by intraperitoneal injection, and 50 mg/kg naringenin via orogastric gavage for 4 weeks. In the H₂O₂ group, the testis malondialdehyde level increased, while the amount of reduced glutathione, glutathione transferase activities, and the testis weight decreased. There were severe testicular damages in the H₂O₂ group otherwise their grade were less in the naringenin + H₂O₂ group. However, the serum testosterone concentrations decreased in both the H₂O₂ and the naringenin + H₂O₂ groups. The testicular zinc and calcium levels reduced in the H₂O₂‐treated rats. In conclusion, the administration of H₂O₂ caused oxidative stress in the testes and naringenin supplementation decreased the H₂O₂‐induced effects, except for changes in testosterone levels.     

Bioavailability of chlorogenic acids following acute ingestion of coffee by humans with an ileostomy

The intestinal absorption and metabolism of 385 μmol chlorogenic acids following a single intake of 200 mL of instant coffee by human volunteers with an ileostomy was investigated. HPLC-MS³ analysis of 0-24 h post-ingestion ileal effluent revealed the presence of 274 ± 28 μmol of chlorogenic acids and their metabolites accounting for 71 ± 7% of intake. Of the compounds recovered, 78% comprised parent compounds initially present in the coffee, and 22% were metabolites including free and sulfated caffeic and ferulic acids. Over a 24 h period after ingestion of the coffee, excretion of chlorogenic acid metabolites in urine accounted for 8 ± 1% of intake, the main compounds being ferulic acid-4-O-sulfate, caffeic acid-3-O-sulfate, isoferulic acid-3-O-glucuronide and dihydrocaffeic acid-3-O-sulfate. In contrast, after drinking a similar coffee, urinary excretion by humans with an intact colon corresponded to 29 ± 4% of chlorogenic acid intake [23]. This difference was due to the excretion of higher levels of dihydroferulic acid and feruloylglycine together with sulfate and glucuronide conjugates of dihydrocaffeic and dihydroferulic acids. This highlights the importance of colonic metabolism. Comparison of the data obtained in the current study with that of Stalmach et al. [23] facilitated elucidation of the pathways involved in post-ingestion metabolism of chlorogenic acids and also helped distinguish between compounds absorbed in the small and the large intestine.     

Endogenous H2O2 produced by Streptococcus pneumoniae controls FabF activity

FabF elongation condensing enzyme is a critical factor in determining the spectrum of products produced by the FASII pathway. Its active site contains a critical cysteine-thiol residue, which is a plausible target for oxidation by H₂O₂. Streptococcus pneumoniae produces exceptionally high levels of H₂O₂, mainly through the conversion of pyruvate to acetyl-P via pyruvate oxidase (SpxB). We present evidence showing that endogenous H₂O₂ inhibits FabF activity by specifically oxidizing its active site cysteine-thiol residue. Thiol trapping methods revealed that one of the three FabF cysteines in the wild-type strain was oxidized, whereas in an spxB mutant, defective in H₂O₂ production, none of the cysteines was oxidized, indicating that the difference in FabF redox state originated from endogenous H₂O₂. In vitro exposure of the spxB mutant to various H₂O₂ concentrations further confirmed that only one cysteine residue was susceptible to oxidation. By blocking FabF active site cysteine with cerulenin we show that the oxidized cysteine was the catalytic one. Inhibition of FabF activity by either H₂O₂ or cerulenin resulted in altered membrane fatty acid composition. We conclude that FabF activity is inhibited by H₂O₂ produced by S. pneumoniae.     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

Hydrogen peroxide enhanced Ca(2+)-activated BK currents and promoted cell injury in human dermal fibroblasts

AimsRecent studies have shown that dermal fibroblasts possess multiple types of voltage-dependent K⁺ channels, and the activation of these channels induces apoptosis. In the present study, we aimed to investigate whether hydrogen peroxide (H₂O₂), an oxidative stress inducer, could modulate these channels or induce human dermal fibroblasts injury.Main methodsThe effects of H₂O₂ on K⁺ currents were studied using a whole-cell recording. Intracellular PKC levels were measured with a direct human PKC enzyme immunoassay kit. Cell viability was assessed using PI staining and apoptotic nuclei were detected with TdT-mediated digoxigenin-dUTP nick-end labelling assay (TUNEL) assay.Key findingsTreatment of cells with 100 μM H₂O₂ resulted in a partially reversible increase in non-inactivating outward K⁺ currents and an alteration in the steady-state activation property of the channels. The H₂O₂-induced increase in K⁺ currents was mimicked by a PKC activator, and was blocked by the PKC inhibitor or the large conductance Ca²⁺-activited K⁺ (BK) channel blockers. The intracellular PKC levels were significantly enhanced by H₂O₂ treatment in a concentration-dependent manner. After exposure to H₂O₂, evaluation of fibroblasts survival rate and damaged cell number with TUNEL-positive nuclei revealed an increased cell injury. Blocking the K⁺ channels with blockers significantly decreased the H₂O₂-induced human dermal fibroblasts injury.SignificanceOur results revealed that H₂O₂ could enhance BK currents by PKC pathway. Increased K⁺ currents might be related to H₂O₂-induced human dermal fibroblasts injury. The results reported here contribute to our understanding of the mechanism underlying H₂O₂-induced human dermal fibroblasts injury.     

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH₄⁺ and/or H⁺ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH₄Cl-induced acidosis were prepared. These extracts were tested for their effect on NH₄⁺ excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH₄⁺ excretion in the toad urinary bladder. We found no effect on H⁺ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH₄⁺. The factor could act as a permease in the bladder cell or as a stimulator of an NH₄⁺ transport system.     

Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

Background

Hydrogen sulfide (H₂S) has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer''s disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂) induced endothelial cells damage.

Methodology and Principal Findings

H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm) and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE), diphenyl-l-pyrenylphosphine (DPPP) and malonaldehyde (MDA) levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG), a selective inhibitor of cystathionine γ-lyase (CSE), abolished the protective effects of H₂S donors.

Innovation

This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences.

Significance

H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.     

Pistacia lentiscus fruit oil reduces oxidative stress in human skin explants caused by hydrogen peroxide

We investigated the efficacy of Pistacia lentiscus fruit oil (PLFO) for protecting human skin from damage due to oxidative stress. PLFO contains natural antioxidants including polyphenols, sterols and tocopherols. We compared the antioxidant potential of PLFO with extra virgin olive oil (EVOO). Explants of healthy adult human skin were grown in culture with either PLFO or EVOO before adding hydrogen peroxide (H₂O₂). We also used cultured skin explants to investigate the effects of PLFO on lipid oxidation and depletion of endogenous antioxidant defense enzymes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) one day after 2 h exposure to H₂O₂. We found that PLFO scavenged radicals and protected skin against oxidative injury. PLFO exhibited greater antioxidant and free radical scavenging activity than EVOO. Skin explants treated with PLFO inhibited H₂O₂ induced MDA formation by inhibition of lipid oxidation. In addition, the oil inhibited H₂O₂ induced depletion of antioxidant defense enzymes including GPx, SOD and CAT. We found that treatment with PLFO repaired skin damage owing to its antioxidant properties.     

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15–44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-l-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H₂O₂ which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H₂O₂ significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H₂O₂ significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H₂O₂. NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H₂O₂.     

Fish Oil and Antipsychotic Drug Risperidone Modulate Oxidative Stress in PC12 Cell Membranes Through Regulation of Cytosolic Calcium Ion Release and Antioxidant System

Oxidative stress is a critical route of damage in various psychological disorders such as schizophrenia, although fish oil and risperidone (RISP) induce antioxidant effects in the human body. However, the mechanisms behind these effects remain elusive. We investigated the effects of fish oil and RISP in the PC12 cell line by evaluating Ca²⁺ mobilization, lipid peroxidation (LP) and antioxidant levels. PC12 cells were divided into eight flasks: control, fish oil, RISP, H₂O₂, fish oil + H₂O₂, RISP + H₂O₂, fish oil + RISP and fish oil + RISP + H₂O₂. Cells were incubated with fish oil and RISP for 24 and 48 h, respectively. Then, cells were exposed to H₂O₂ for 15 min before analysis. Ca²⁺ release and LP levels were higher in the H₂O₂ group than in the control, RISP and fish oil groups, although their levels were decreased by incubation of cells in fish oil and RISP. Glutathione peroxidase activity, reduced glutathione and vitamin C levels in the cells were lower in the H₂O₂ group than in the control, RISP and fish oil groups, although levels were higher in cells incubated with fish oil and RISP than in those in the H₂O₂ groups. In conclusion, these results indicate that RISP and fish oil induced protective effects on oxidative stress in PC12 cells by modulating cytosolic Ca²⁺ release and antioxidant levels.     

On the mechanism of apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> production during lignin formation and elicitation in cultured spruce cells—peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H₂O₂ generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H₂O₂ were present in the culture medium during lignin formation. Elicitation induced a burst of H₂O₂, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H₂O₂ from O₂, NADH stimulated H₂O₂ production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H₂O₂, but usually increased or prolonged elicitor-induced H₂O₂ formation. Culture medium peroxidases were not able to generate H₂O₂ in vitro with Cys or GSH as reductants. These thiols, however, generated H₂O₂ non-enzymically at pH 4.5. [³⁵S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H₂O₂ formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H₂O₂ generation but strongly delayed the elicitor-induced H₂O₂ accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H₂O₂ production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H₂O₂ generation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chlorogenic acid ameliorates intestinal mitochondrial injury by increasing antioxidant effects and activity of respiratory complexes

Dietary polyphenols are thought to be beneficial for human health by acting as antioxidants. Chlorogenic acid (CGA) is abundant in plant-based foods as an ester of caffeic acid and quinic acid. In this study, we investigated the effects of CGA on mitochondrial protection. Our results demonstrated that pretreatment with CGA ameliorated the intestinal mitochondrial injury induced by H₂O₂; membrane potential was increased, mitochondrial swelling, levels of reactive oxygen species, contents of 8-hydroxy-2-deoxyguanosine, and cytochrome c released were decreased. The beneficial effects of CGA were accompanied by an increase in antioxidant and respiratory-chain complex I, IV, and V activities. In trinitrobenzene-sulfonic acid-induced colitic rats indicated that CGA supplementation improved mitochondria ultrastructure and decreased mitochondrial injury. Our results suggest a promising role for CGA as a mitochondria-targeted antioxidant in combating intestinal oxidative injury. Daily intake of diets containing CGA, such as coffee and honeysuckle, may be useful for prevention of intestinal diseases.     

Clonal growth of lymphoid cells in serum-free media requires elimination of H2O2 toxicity

We have recently described the development of a serum-free medium that contains casein, insulin, testosterone, transferrin, and linoleic acid and that supports the long-term growth of a wide variety of lymphoid cells. A problem of culturing cells in this medium is the difficulty of cloning cells or growing cells at low density. We now describe the formulation of a chemically defined medium that supports the clonal growth of the murine S49 T lymphoma cell line. This medium contains catalase, insulin, transferrin, testosterone, Na₂SeO₃, and dilinoleoyl phosphatidylcholine and contains less than 50 μg/ml total protein. The two novel additions in this medium are catalase, which replaces casein and dilinoleoyl phosphatidylcholine, which substitutes for linoleic acid in this defined medium. In addition to S49 cells, the medium described above supports the long-term growth of other lymphoid cells, including human and murine hybridomas. We propose that catalase functions to degrade H₂O₂ that is present in the cultures and that casein, bovine serum albumin, and other proteins commonly included in media for cultured cells may also scavenge H₂O₂. Na₂SeO₃ also partially protects against the death of cells at clonal density and this protection may, like catalase, be due to removal of H₂O₂. Our results suggest that H₂O₂ is an important cytotoxic agent that prevents growth of lymphoid cells during culture in serum-free media and perhaps in serum-containing media as well.