Doxorubicin increases intracellular hydrogen peroxide in PC3 prostate cancer cells

We studied the effect of doxorubicin on the production of hydrogen peroxide by PC3 human prostate cancer cells, using a sensitive assay based on aminotriazole-mediated inhibition of catalase. PC3 cells exposed to increasing concentrations of doxorubicin had an increase in intracellular hydrogen peroxide that was concentration-dependent up to 1 microM doxorubicin. The apparent hydrogen peroxide concentration in the PC3 cells was 13 +/- 4 pM under basal steady-state conditions and increased to 51 +/- 13 pM after exposure to 1 microM doxorubicin for 30 min. The level of hydrogen peroxide in the medium as measured by Amplex Red did not increase as a result of doxorubicin treatment. PC3 cells overexpressing catalase were no more resistant to doxorubicin cytotoxicity as compared to non-transduced wild-type cells; therefore, the exact role of hydrogen peroxide in anthracycline cytotoxicity remains unproven. This study demonstrates that a specific oxidative event associated with the exposure of PC3 human prostate cancer cells to anthracyclines results in an increase in intracellular hydrogen peroxide.     

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.     

Oxygen protection of bacteriophage T1 against ionizing radiations

Bacteriophage T1 was suspended in distilled water and in phosphate buffer, saturated with oxygen, nitrogen, hydrogen, and carbon monoxide, and irradiated with gamma rays and x-rays. Under the same conditions phage was exposed to hydrogen peroxide. Oxygen acted as a protective agent against both irradiation and hydrogen peroxide inactivation. As a protective agent against irradiation, oxygen was more efficient in distilled water than in buffer. The phage was much more sensitive to irradiation in the presence of hydrogen or nitrogen than in the presence of oxygen. Survivals of phage irradiated in suspensions saturated with hydrogen and with nitrogen did not differ significantly. From this it was concluded that oxygen did not protect T1 by removing atomic hydrogen from the irradiated medium, since the hydrogen-saturated medium increased the yield of atomic hydrogen but did not increase the yield of inactivated phage. It was presumed, therefore, that phage is sensitive to OH radicals and this was confirmed by irradiating phage with UV in the presence of hydrogen peroxide and comparing this survival with the survivals obtained from hydrogen peroxide alone and from UV alone. The combined effect of hydrogen peroxide and UV acting simultaneously was greater than the effect attributable to hydrogen peroxide and UV acting separately. Evidence for sensitivity to HO₂ radicals was considered, and the effect was attributed chiefly to an oxidizing action since phage sensitivity is greater at higher hydrogen ion concentrations, which favor oxidation by HO₂ radicals. Since the OH radical is a more efficient oxidizing agent than O^-, the former being favored in an acid medium, the latter in an alkaline medium, and since the phage is more sensitive in the first situation than in the second, the present tests proved the importance of oxidation as the mechanism of inactivation. Since some inactivation was encountered when phage was exposed to reducing agents, independently of irradiation, it was concluded that phage is somewhat sensitive to reducing agents, but the inactivation attributable to ionizing radiations is due chiefly to oxidation, against which these reducing agents are very efficient protectors. Under no circumstances did hydrogen peroxide protect T1, whether produced by irradiation in the medium or added beforehand to the medium to be irradiated. The first point was investigated by irradiating T1 in the presence of hydrogen and oxygen combined; this produced a higher yield of hydrogen peroxide but a lower survival of T1. In all these tests phage survival under irradiation was directly correlated with oxygen content of the medium rather than with production of hydrogen peroxide. It is proposed that the protective effect of oxygen is due to a reaction between the phage and oxygen, and this complex confers stability upon the phage.     

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.     

Mitochondrial hydrogen peroxide production alters oxygen consumption in an oxygen-concentration-dependent manner

Metabolic responses of mammalian cells toward declining oxygen concentration are generally thought to occur when oxygen limits mitochondrial ATP production. However, at oxygen concentrations markedly above those limiting to mitochondria, several mammalian cell types display reduced rates of oxygen consumption without energy stress or compensatory increases in glycolytic ATP production. We used mammalian Jurkat T cells as a model system to identify mechanisms responsible for these changes in metabolic rate. Oxygen consumption was 31% greater at high oxygen (150–200 μM) compared to low oxygen (5–10 μM). Hydrogen peroxide was implicated in the response as catalase prevented the increase in oxygen consumption normally associated with high oxygen. Cell-derived hydrogen peroxide, predominately from the mitochondria, was elevated with high oxygen. Oxygen consumption related to intracellular calcium turnover was shown, through EDTA chelation and dantrolene antagonism of the ryanodine receptor, to account for 70% of the response. Oligomycin inhibition of oxygen consumption indicated that mitochondrial proton leak was also sensitive to changes in oxygen concentration. Our results point toward a mechanism in which changes in oxygen concentration influence the rate of hydrogen peroxide production by mitochondria, which, in turn, alters cellular ATP use associated with intracellular calcium turnover and energy wastage through mitochondrial proton leak.     

Hydrogen peroxide: a potent activator of dioxygenase activity of soybean lipoxygenase

Hydrogen peroxide, an ubiquitous biologically occurring peroxide, was found to stimulate the dioxygenase activity of soybean lipoxygenase at the physiologically attainable concentration. The increase in enzyme specific activity was directly proportional to hydrogen peroxide concentration up to 0.5 nM. A decrease in the stimulation of dioxygenase activity was observed at higher concentrations. At low enzyme concentration up to 28-fold stimulation was noted when the formation of lipid hydroperoxide was monitored spectrophotometrically. The stimulation was further confirmed by increased oxygen uptake. It is proposed that the mechanism for in vivo activation involves hydrogen peroxide.     

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol-gel glasses

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase-peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase-peroxidase system was immobilized in a single sol-gel matrix. The enzymatic activity of the encapsulated superoxide dismutase-peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol-gel encapsulation preserved the hierarchy of the enzyme's activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.     

The 2-Cys Peroxiredoxin-Deficient <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> Displays Impaired Growth and Survival in the Presence of Hydrogen Peroxide In Vitro But Not in Mouse Organs

Resistance of Listeria monocytogenes to reactive oxygen radicals may facilitate its survival in phagocytic cells and against some oxidizing sanitizers. The aim of this study was to investigate the function of the 2-cys peroxiredoxin (Prx) homologue in L. monocytogenes, particularly its survival in a hydrogen peroxide–containing environment. An in-frame prx deletion mutant and a complementation strain were constructed and evaluated for their growth and survival either in media containing different concentrations (0, 15, 20, 25, 50, and 294 mmol · L⁻¹) hydrogen peroxide or in macrophages. Bacterial survival in various mouse organs was also investigated after intraperitoneal administration. We found that prx-defective L. monocytogenes was sensitive to hydrogen peroxide in in vitro growth media but not in mouse organs or in macrophages, suggesting that Prx promotes survival in the presence of exogenous hydrogen peroxide but not in mammalian cells or organs.     

Sensitivity to oxidative damage of two Deinococcus radiodurans strains

The action of reactive oxygen species, i.e. hydrogen peroxide, cupric sulphate and ascorbic acid, added at different concentrations to culture media, has been studied in two strains of Deinococcus radiodurans (red-pigmented parental and colourless mutant strains) in relation to their defense antioxidant systems. While the pigmented bacteria were more resistant to elevated concentrations of the different oxidants, the colourless bacteria were more sensitive and their sensitivity was dose-dependent. Reactive oxygen species induced oxidative damage, particularly to the polyethylenic fatty acids, which were more abundant in the mutant strain. Similarly, a significant increase in lipid peroxide levels was observed, whatever the chemical added during the growth of the mutant bacteria. The parental strain required high concentrations of oxidants to shorten its survival. Vitamins A and E, carotenoids and enzymes, largely present in the parental strain, could be responsible for its higher resistance to the lethal effects of radicals generated within the cells.     

The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects.

Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.     

Oxidative stress response in an anaerobe, Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide.

Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after two-dimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide- and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.     

Peroxide sensitivity of cold-shocked Salmonella typhimurium and Escherichia coli and its relationship to minimal medium recovery

Cold-shocked Salmonella typhimurium displayed minimal medium recovery (MMR), viable counts on M9 minimal agar being much higher than those on tryptone soya yeast extract agar (TSYA). The addition of catalase to TSYA restored counts to the level found on M9 agar. Peroxide concentrations between 12 and 30 μmol/1 were measured in TSYB but none was detected in M9 medium. Cold-shocked cells were sensitive to reagent hydrogen peroxide at a concentration similar to that found in TSYB. The minimal medium recovery phenomenon of cold-shocked cells is thus a manifestation of peroxide sensitivity. Changing the composition of growth media affected both cellular catalase activity and the magnitude of the MMR effect but the two properties were not directly related. Factors additional to cellular catalase activity must therefore affect susceptibility to peroxide following cold shock. Muta tional loss of catalase, exonuclease III or recA -dependent DNA repair functions all increased the sensitivity of cold-shocked Escherichia coli to the inhibitory effects of peroxide present in rich medium. The peroxide resistant fraction of a cold-shocked population of Salm. typhimurium (i.e. those cells able to grow on TSYA) was more resistant to gamma radiation than the population as a whole. Cold shock thus sensitizes cells to more than one form of oxidative stress. Prior exposure of growing cells to 30 μ mol/1 hydrogen peroxide abolished their sensitivity to rich medium following cold shock implying that Salm. typhimurium contains an inducible system protecting against oxidative stress.     

Peroxide sensitivity of cold-shocked Salmonella typhimurium and Escherichia coli and its relationship to minimal medium recovery

Cold-shocked Salmonella typhimurium displayed minimal medium recovery (MMR), viable counts on M9 minimal agar being much higher than those on tryptone soya yeast extract agar (TSYA). The addition of catalase to TSYA restored counts to the level found on M9 agar. Peroxide concentrations between 12 and 30 mumol/l were measured in TSYB but none was detected in M9 medium. Cold-shocked cells were sensitive to reagent hydrogen peroxide at a concentration similar to that found in TSYB. The minimal medium recovery phenomenon of cold-shocked cells is thus a manifestation of peroxide sensitivity. Changing the composition of growth media affected both cellular catalase activity and the magnitude of the MMR effect but the two properties were not directly related. Factors additional to cellular catalase activity must therefore affect susceptibility to peroxide following cold shock. Mutational loss of catalase, exonuclease III or recA-dependent DNA repair functions all increased the sensitivity of cold-shocked Escherichia coli to the inhibitory effects of peroxide present in rich medium. The peroxide resistant fraction of a cold-shocked population of Salm. typhimurium (i.e. those cells able to grow on TSYA) was more resistant to gamma radiation than the population as a whole. Cold shock thus sensitizes cells to more than one form of oxidative stress. Prior exposure of growing cells to 30 mumol/l hydrogen peroxide abolished their sensitivity to rich medium following cold shock implying that Salm. typhimurium contains an inducible system protecting against oxidative stress.     

The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-gamma-aspartate to cells in vitro

We have studied the liposome-mediated delivery of methotrexate-gamma-aspartate to five cell lines. The sensitivity of the cells to encapsulated drug varies widely in accordance with their ability to take up the liposomes. CV1-P cells can be 150-times more sensitive to encapsulated methotrexate-gamma-aspartate than to free drug, while AKR/J SL2 cells are only twice as sensitive to the encapsulated drug. Negatively-charged liposomes are much more efficient for delivery than are neutral liposomes, and cholesterol is an essential component of the liposome membrane for optimal drug delivery. The optimal liposome size for drug delivery is 0.1 micron, although the amount of cell-associated lipid is the same for all liposome sizes. The effect of the encapsulated drug is inhibited by NH4Cl, suggesting an endocytic mechanism for delivery. The potency of the encapsulated drug is not affected by wide variations in the drug: lipid ratio.     

Effect of 6-hydroxydopamine on murine hematopoietic stem cells: enhanced cytotoxicity on megakaryocyte colony forming units

We examined the effect of catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on murine committed megakaryocyte progenitor cells, the megakaryocyte-colony forming unit (CFU-Meg). More mature cells of the megakaryocyte series have the capacity for active uptake of catecholamines, and we speculated that the CFU-Meg would also take up 6-OHDA and be selectively killed. CFU-Meg were much more sensitive to the effects of this agent than were granulocyte-macrophage colony forming units (CFU-GM) or spleen-colony forming units. Co-incubation with catalase, which would destroy hydrogen peroxide generated extracellularly by the autoxidation of 6-OHDA, ablated 6-OHDA toxicity towards CFU-GM, but also significantly reduced the effect on CFU-Meg. Mazindol, a selective dopamine uptake inhibitor did not alter 6-OHDA effect on either CFU-Meg or CFU-GM. Finally, CFU-Meg were no more sensitive to incubation with hydrogen peroxide than were CFU-GM. These data suggest that CFU-Meg, unlike their more mature progeny, do not actively concentrate 6-OHDA, and the excess toxicity of this agent towards CFU-Meg is probably due to increased sensitivity to autoxidation products of 6-OHDA, other than hydrogen peroxide, generated extracellularly.     

Simultaneous protective and damaging effects of cysteamine on intracellular DNA of leukocytes

Incubation of human leukocytes with cysteamine can lead to the induction of DNA strand breaks. The induction of breaks is biphasic with increasing concentration of scavenger. The number of breaks increases in a dose-dependent manner to a maximum and then decreases at higher concentrations. Catalase has been shown to prevent the production of breaks, indicating an involvement of hydrogen peroxide. Cysteamine reacts with oxygen to generate hydrogen peroxide but at higher concentrations it also reacts with hydrogen peroxide. Thus, the biphasic effect of cysteamine on leukocyte DNA may be due to the sum of two separate reaction pathways. (i) Cysteamine reacts with oxygen to generate hydrogen peroxide which leads to DNA strand breakage. (ii) At higher concentrations, it eliminates hydrogen peroxide by reacting with it, thereby protecting the cellular DNA. Other antioxidant scavengers such as WR2721, acetylcysteine and ascorbate can also autooxidize to produce strand breaks. Thiourea and tetramethylurea do not. When tested for their ability to protect cells against DNA damage from added H2O2, the agent which most damaging by itself, cysteamine, was also the most protective.     

Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.     

The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-γ-aspartate to cells in vitro

We have studied the liposome-mediated delivery of methotrexate-γ-aspartate to five cell lines. The sensitivity of the cells to encapsulated drug varies widely in accordance with their ability to take up the liposomes. CV1-P cells can be 150-times more sensitive to encapsulated methotrexate-γ-aspartate than to free drug, while AKR/J SL2 cells are only twice as sensitive to the encapsulated drug. Negatively-charged liposomes are much more efficient for delivery than are neutral liposomes, and cholesterol is an essential component of the liposome membrane for optimal drug delivery. The optimal liposome size for drug delivery is 0.1 μm, although the amount of cell-associated lipid is the same for all liposome sizes. The effect of the encapsulated drug is inhibited by NH₄Cl, suggesting an endocytic mechanism for delivery. The potency of the encapsulated drug is not affected by wide variations in the drug:lipid ratio.     

The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase alpha is mediated by hydrogen peroxide

Here, we report the identification and characterization of five ortho-quinone inhibitors of PTPalpha. We observed that the potency of these compounds in biochemical assays was markedly enhanced by the presence of DTT. A kinetic analysis suggested that they were functioning as irreversible inhibitors and that the inhibition was targeted to the catalytic site of PTPalpha. The inhibition observed by these compounds was sensitive to superoxide dismutase and catalase, suggesting that reactive oxygen species may be mediators of their inhibition. We observed that in the presence of DTT, these compounds would produce up to 2.5mM hydrogen peroxide (H(2)O(2)). The levels of H(2)O(2) produced were sufficient to completely inactivate PTPalpha. In contrast, without a reducing agent the compounds did not generate H(2)O(2) and showed little activity towards PTPalpha. In addition, these compounds inhibited PTPalpha-dependent cell spreading in NIH 3T3 cells at concentrations that were similar to their activity in biochemical assays. The biological implications of these results are discussed as they support growing evidence that H(2)O(2) is a key regulator of PTPs.     

Novel antioxidant role of alcohol dehydrogenase E from Escherichia coli

Alcohol dehydrogenase E (AdhE) is an Fe-enzyme that, under anaerobic conditions, is involved in dissimilation of glucose. The enzyme is also present under aerobic conditions, its amount is about one-third and its activity is only one-tenth of the values observed under anaerobic conditions. Nevertheless, its function in the presence of oxygen remained ignored. The data presented in this paper led us to propose that the enzyme has a protective role against oxidative stress. Our results indicated that cells deleted in adhE gene could not grow aerobically in minimal media, were extremely sensitive to oxidative stress and showed division defects. In addition, compared with wild type, mutant cells displayed increased levels of internal peroxides (even higher than those found in a Delta katG strain) and increased protein carbonyl content. This pleiotropic phenotype disappeared when the adhE gene was reintroduced into the defective strain. The purified enzyme was highly reactive with hydrogen peroxide (with a Ki of 5 microM), causing inactivation due to a metal-catalyzed oxidation reaction. It is possible to prevent this reactivity to hydrogen peroxide by zinc, which can replace the iron atom at the catalytic site of AdhE. This can also be achieved by addition of ZnSO4 to cell cultures. In such conditions, addition of hydrogen peroxide resulted in reduced cell viability compared with that obtained without the Zn treatment. We therefore propose that AdhE acts as a H2O2 scavenger in Escherichia coli cells grown under aerobic conditions.