Molecular Characterization and Rescue of Acatalasemic Mutants of Drosophila Melanogaster

The enzyme catalase protects aerobic organisms from oxygen-free radical damage by converting hydrogen peroxide to molecular oxygen and water before it can decompose to form the highly reactive hydroxyl radical. Hydroxyl radicals are the most deleterious of the activated oxygen intermediates found in aerobic organisms. If formed, they can react with biological molecules in their proximity; the ensuing damage has been implicated in the increasing risk of disease and death associated with aging. To study further the regulation and role of catalase we have undertaken a molecular characterization of the Drosophila catalase gene and two potentially acatalasemic alleles. We have demonstrated that a previously existing allele, Cat(n4), likely contains a null mutation, a mutation which blocks normal translation of the encoded mRNA. The Cat(n1) mutation appears to cause a significant change in the protein sequence; however, it is unclear why this change leads to a nonfunctioning protein. Viability of these acatalasemic flies can be restored by transformation with the wild-type catalase gene; hence, we conclude that the lethality of these genotypes is due solely to the lack of catalase. The availability of flies with transformed catalase genes has allowed us to address the effect of catalase levels on aging in Drosophila. Though lack of catalase activity caused decreased viability and life span, increasing catalase activity above wild-type levels had no effect on normal life span.     

Cooperative action of antioxidant defense systems in Drosophila

Molecular oxygen is key to aerobic life but is also converted into cytotoxic byproducts referred to as reactive oxygen species (ROS). Intracellular defense systems that protect cells from ROS-induced damage include glutathione reductase (GR), thioredoxin reductase (TrxR), superoxide dismutase (Sod), and catalase (Cat). Sod and Cat constitute an evolutionary conserved ROS defense system against superoxide; Sod converts superoxide anions to H(2)O(2), and Cat prevents free hydroxyl radical formation by breaking down H(2)O(2) into oxygen and water. As a consequence, they are important effectors in the life span determination of the fly Drosophila. ROS defense by TrxR and GR is more indirect. They transfer reducing equivalents from NADPH to thioredoxin (Trx) and glutathione disulfide (GSSG), respectively, resulting in Trx(SH)(2) and glutathione (GSH), which act as effective intracellular antioxidants. TrxR and GR were found to be molecularly conserved. However, the single GR homolog of Drosophila specifies TrxR activity, which compensates for the absence of a true GR system for recycling GSH. We show that TrxR null mutations reduce the capacity to adequately protect cells from cytotoxic damage, resulting in larval death, whereas mutations causing reduced TrxR activity affect pupal eclosion and cause a severe reduction of the adult life span. We also provide genetic evidence for a functional interaction between TrxR, Sod1, and Cat, indicating that the burden of ROS metabolism in Drosophila is shared by the two defense systems.     

Does Postzygotic Isolation Result from Improper Dosage Compensation?

The X chromosome invariably has the largest effect on postzygotic isolation between animal species. One explanation of this pattern is that inviability and sterility result from a breakdown in the dosage compensation of X-linked genes in hybrids. In Drosophila, such breakdown could result from divergence of the genes used to assess the X/autosomal (X/A) ratio, and thus the sex, of an individual. I test this hypothesis by introducing mutant alleles of the Sex-lethal locus into Drosophila melanogaster-Drosophila simulans hybrids. These mutants "ignore" any perceived anomalous X/A ratio and thus can be used to ensure proper dosage compensation in hybrids. These mutants do not rescue hybrid viability or fertility, implying that postzygotic isolation in this hybridization does not result from a disruption of dosage compensation caused by divergence of the X/A counting system.     

Superoxide and the production of oxidative DNA damage.

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.     

The α-Glycerophosphate in DROSOPHILA MELANOGASTER II. Genetic Aspects

Seven alleles of the alpha-Glycerophosphate dehydrogenase-1 (alphaGpdh-1) locus of Drosophila melanogaster have been described. These include two naturally occurring electrophoretic variants, one EMS-induced electrophoretic variant, and four EMS-induced "null" or "zero" mutants. With the electrophoretic variants, the locus was mapped to II-20.5 +/- 2.5. A complementation matrix was prepared utilizing the null mutants. Three of the four mutants and a deletion of the locus (Grell 1967) exhibit dosage dependency. The dosage independent mutant exhibits complementation with two of the other null alleles. Flies genetically deficient in alpha-glycerophosphate dehydrogenase are fertile, but their relative viability is severely diminished. Such flies also lose the ability to sustain flight, an observation consistent with the enzyme's function in energy production. The levels of mitochondrial alpha-glycerophosphate oxidase, measured in flies genetically deficient in the cytoplasmic enzyme, were normal.     

Mutagenic spectrum resulting from DNA damage by oxygen radicals.

Oxygen free radicals are highly reactive species that damage DNA and cause mutations. We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe2+. The Fe2(+)-treated DNA was transfected into component Escherichia coli, and mutants within the nonessential lac Z alpha gene for beta-galactosidase were identified by decreased alpha-complementation. The frequency of mutants obtained with 10 microM Fe2+ was 20- to 80-fold greater than that obtained with untreated DNA. Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS "error-prone" response. The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals. The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions. Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G----C transversions, followed by C----T transitions and G----T transversions. The clustering of mutations at distinct gene positions suggests that Fe2+/oxygen damage to DNA is nonrandom. This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.     

Characteristics of inserted mutations obtained in the P-M hybrid dysgenesis system in the 9F12-10A7 region of the Drosophila melanogaster X-chromosome

19 new mutations in the 9F12-10A7 region of Drosophila melanogaster X chromosome was obtained in the system of P-M hybrid dysgenesis. They appeared to be lethals, as judged from viability of homo- or hemizygous females. In situ hybridization of P DNA with polytene chromosomes revealed P-element insertion in the 10A1-2 band in the majority of the mutants. As a result of complementation analysis, all these mutations were localized at previously known loci: l(1)BP1, l(1)BP5, l(1)BP8, l(1)BP7. No insertion mutations were found at the vermilion locus. This can imply for non-random distribution of insertion mutations in the region studied. Further comparison of these mutations with previously EMS-induced ones revealed that insertion mutations are predominantly hypomorph lethals which do not influence the viability, morphology and fertility of homozygous males and females, but drastically reduce viability of hemizygous females.     

Mutation spectrum of copper-induced DNA damage.

The ability of metal ions to damage DNA and cause mutagenesis has been analyzed with reversion and forward mutation assays using single-stranded DNA templates. We previously reported that incubation of phi X174 am3 DNA with Fe2+ in vitro results in mutagenesis when the treated DNA is transfected into Escherichia coli spheroplasts (Loeb, L. A., James, E. A., Waltersdorph, A. M., and Klebanoff, S. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3918-3922, 1988). We now extend these studies to other metal ions. Of the metal ions tested, copper ions were the most mutagenic; the frequency of mutants produced was equal to or greater than that produced by Fe2+. Mutagenesis by Cu+ was diminished by catalase, mannitol, and superoxide dismutase suggesting the involvement of H2O2, hydroxyl ions, and superoxide, respectively. However, the findings that Cu+ and Cu2+ are nearly equally mutagenic and that the mutagenic activities are not completely inhibited by oxygen free radical scavengers make it unlikely that the mechanism for mutagenesis is simply the production of hydroxyl free radicals. The spectra of mutations produced by either copper ion using the lacZ gene as a target are very similar and differ from those reported with other agents. The predominant mutagenic sequence changes are single-base substitutions, the most frequent being replacement of a template C by a T. This transition presumably results from mispairing of an altered C with deoxyadenosine. Copper-induced mutations are not randomly distributed. Instead, they are found predominantly in clusters suggesting direct interaction of copper ions with specific nucleotide sequences in DNA. Evidence is considered that the high frequency of C----T transitions may be a common manifestation of DNA damage by oxygen radicals.     

The spectrum of mutations generated by passage of a hydrogen peroxide damaged shuttle vector plasmid through a mammalian host.

Treatment of a plasmid shuttle vector (pZ189) with a combination of hydrogen peroxide and a ferric iron/EDTA complex prior to transfection and passage in simian (CV-1) cells increases the frequency of mutations at the supF locus by up to 60-fold over the spontaneous background. This increase in mutation frequency is abolished when the inhibitors desferrioxamine, superoxide dismutase, catalase or dimethyl sulfoxide are included in the initial reaction or when the iron/EDTA complex is omitted, a strong indication that the premutagenic damage arises as a result of direct attack by hydroxyl radical generated in a superoxide driven Fenton reaction. DNA sequence analysis of the mutated plasmids shows that 1) Deletions occuring in combination with base-substitutions arise in 22.5 percent of the induced mutants compared with only 3 percent of spontaneous mutants 2) Sixty percent of all induced deletion mutations involve the loss of a single base and 77 percent of these (20 out of 26) occur at two adenine-containing sites 3) The base-change spectrum of mutants arising in the treated plasmid population is marked by the predominance of mutants containing a single base-change and by an increase in changes at AT base pairs. These results provide direct information concerning the nature of mutations arising in mammalian cells as a result of hydroxyl radical mediated DNA damage.     

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.     

Catalase T deficient mutants of Saccharomyces cerevisiae

A method for the isolation of catalase T deficient mutants of Saccharomyces cerevisiae is described. Ten mutants lacking catalase T and belonging to 5 complementation groups were isolated. CTT1 locus was identified as the structural gene for catalase T. It is under the control of CTT2, CTT3 and CTT4 loci.     

Antioxidant properties and protective effects of a standardized extract of Hypericum perforatum on hydrogen peroxide-induced oxidative damage in PC12 cells

Free radical scavenging and antioxidant activities of a standardized extract of Hypericum perforatum (SHP) were examined for inhibition of lipid peroxidation, for hydroxyl radical scavenging activity and interaction with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH). Concentrations between 1 and 50 microg/ml of SHP effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe2+/ascorbate or NADPH system. The results showed that SHP scavenged DPPH radical in a dose-dependent manner and also presented inhibitory effects on the activity of xanthine oxidase. In contrast, hydroxyl radical scavenging occurs at high doses. The protective effect of the standardized extract against H2O2-induced oxidative damage on the pheochromocytoma cell line PC 12 was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays, caspase-3-enzyme activity and accumulation of reactive oxygen species [2',7'-dichlorofluorescin (DCF) assay]. Following 8-h cell exposure to H2O2 (300 microM), a marked reduction in cell survival was observed, which was significantly prevented by SHP (pre-incubated for 24 h) at 1-100 microg/ml. In a separate experiment, different concentrations of the standardized extract (0.1-100 microg/ml) also attenuated the increase in caspase-3 activity and suppressed the H2O2 -induced reactive oxygen species generation. Taken together, these results suggest that SHP shows relevant antioxidant activity both in vitro and in a cell system, by means of inhibiting free radical generation and lipid peroxidation.     

Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation

We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway.     

Evaluation of the degree of medical radiation damage with a highly sensitive chemiluminescence method.

A highly sensitive chemiluminescence (CL) method for evaluation of medical radiation damage degree is presented. According to the principle of cell stress response to ionizing radiation, lymphocytes will produce reactive oxygen species (ROS) after irradiation. The ROS produced can react with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one (MCLA), a specific CL probe for superoxide anion (O(.-) (2)) and singlet oxygen ((1)O(2)), to emit light at 465 nm. The CL intensity is positively related to the amount of generated ROS detected 30 min after irradiation. Cell viability, which is inversely related to cell mortality, was determined by MTT assay after 3 days' culture. The results show that both CL intensity and cell mortality of lymphocytes increase with the increase of the radiation dose when the dosage is no more than 3 Gy, suggesting a positive relationship between the degree of lymphocyte cell damage and the amount of ROS generated. In addition, the effects of catalase, Cu-Zn superoxide dismutase (SOD), mannitol, sodium azide (NaN(3)), and D(2)O on MCLA-dependent CL of lymphocytes are discussed. We believe that the MCLA-dependent CL method would potentially provide an easy way for evaluating the degree of lymphocyte damage induced by radiation.     

Effects of scavengers of reactive oxygen and radical species on cell survival following photodynamic treatment in vitro: comparison to ionizing radiation

The effects of various scavengers of reactive oxygen and/or radical species on cell survival in vitro of EMT6 and CHO cells following photodynamic therapy (PDT) or gamma irradiation were compared. None of the agents used exhibited major direct cytotoxicity. Likewise, none interfered with cellular porphyrin uptake, and none except tryptophan altered singlet oxygen production during porphyrin illumination. The radioprotector cysteamine (MEA) was equally effective in reducing cell damage in both modalities. In part, this protection seems to have been induced by oxygen consumption in the system due to MEA autoxidation under formation of H2O2. The addition of catalase, which prevents H2O2 buildup, reduced the effect of MEA to the same extent in both treatments. Whether the remaining protection was due to MEA's radical-reducing action or some remaining oxygen limitation is unclear. The protective action of MEA was not mediated by a doubling of cellular glutathione levels, since addition of buthionine sulfoximine, which prevented glutathione increase, did not diminish the observed MEA protection. The hydroxyl radical scavenger mannitol also afforded protection in both kinds of treatment, but it was approximately twice as effective in gamma irradiation as in PDT. This is consistent with the predominant role of OH radicals in ionizing radiation damage and their presumed minor involvement in PDT damage. Superoxide dismutase, a scavenger of O2, acted as a radiation protector but was not significantly effective in PDT. Catalase, which scavenges H2O2, was ineffective in both modalities. Tryptophan, an efficient singlet oxygen scavenger, reduced cell death through PDT by several orders of magnitude while being totally ineffective in gamma irradiation. These data reaffirm the predominant role of 1O2 in the photodynamic cell killing but also indicate some involvement of free radical species.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Molecular organization of the cut locus of Drosophila melanogaster

Mutations of the cut locus (ct) of Drosophila can be divided into four groups based on their phenotypes and complementation patterns. Each group alters the phenotype of a different set of tissues. Two hundred kilobases of ct DNA, located in 7B1-2, have been cloned by chromosomal walking, and the cloned sequences have been used to analyze more than 40 mutants. Based on the location of transposable element mutations and the extent of deficiencies and an inversion, four cut locus regions can be defined. Mutations in each region affect the phenotype of a different set of tissues. The most centromere proximal region contains mutations that are null for cut locus function. Within individual regions, a higher level of organization can be detected.     

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C₁, xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO₃, an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O₂-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A₂, which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

Alkyl Hydroperoxide Reductase Is the Primary Scavenger of Endogenous Hydrogen Peroxide in Escherichia coli

Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of Escherichia coli that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H(2)O(2) whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H(2)O(2). These mutants excreted substantial amounts of H(2)O(2), and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H(2)O(2) than is catalase and therefore is likely to be the primary scavenger of endogenous H(2)O(2). Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an important role in wild-type cells, because the activity of Ahp is saturated at a low (10(-5) M) concentration of H(2)O(2). In contrast, catalase has a high K(m), and it therefore becomes the predominant scavenger when H(2)O(2) concentrations are high. This arrangement is reasonable because the cell cannot provide enough NADH for Ahp to rapidly degrade large amounts of H(2)O(2). In sum, E. coli does indeed generate substantial H(2)O(2), but damage is averted by the scavenging activity of Ahp.