Involvement of oxidative stress in hydroquinone-induced cytotoxicity in catalase-deficient Escherichia coli mutants

Hydroquinone is a benzene-derived metabolite. To clarify whether the reactive oxygen species (ROS) are involved in hydroquinone-induced cytotoxicity, we constructed transformants of Escherichia coli (E. coli) strains that express mammalian catalase gene derived from catalase mutant mice (Cs^b, Cs^c) and the wild-type (Cs^a) using a catalase-deficient E. coli UM255 as a recipient. Specific catalase activities of these tester strains were in order of Cs^a > Cs^c > Cs^b > UM255, and their susceptibility to hydrogen peroxide (H₂O₂) showed UM255 > Cs^b > Cs^c > Cs^a. We found that hydroquinone exposure reduced the survival of catalase-deficient E. coli mutants in a dose-dependent manner significantly, especially in the strains with lower catalase activities. Hydroquinone toxicity was also confirmed using zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition, followed by Cs^b, Cs^c and Cs^a. Furthermore, we found that hydroquinone-induced cell damage was inhibited by the pretreatment of catalase, ascorbic acid, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA), and augmented by superoxide dismutase (both CuZnSOD and MnSOD). The present results suggest that H₂O₂ is probably involved in hydroquinone-induced cytotoxicity in catalase-deficient E. coli mutants and catalase plays an important role in protection of the cells against hydroquinone toxicity.     

Effect of endocrine disruptor para-nonylphenol on the cell growth and oxygen radical generation in Escherichia coli mutant cells deficient in catalase and superoxide dismutase

para-Nonylphenol (NP) had previously been found to have strong suppressive effects of growth of bacterial and yeast cells, and these effects were associated with NP-induced generation of radical oxygen species (ROS). In the present study, we determined that wild-type strains of Escherichia coli (CSH 7, SY-11, and IFO-3545) were resistant to NP compared with other sensitive microorganisms reported previously. To elucidate the relationship between NP-induced ROS generation and cell growth inhibition in more detail, we analyzed the effect of NP on cell growth and survival of wild-type and mutant E. coli strains deficient in ROS-scavenging enzymes such as catalase and superoxide dismutase (SOD). The SOD-deficient strain QC 774 (sod A- and sod B-) was much more sensitive to NP than wild-type (CSH 7) and catalase-deficient (UM 1 kat E- and kat G-) strains. As a comparative experiment, when hydrogen peroxide was applied to the same growth and survival assays, UM 1 cells were more sensitive to hydrogen peroxide than QC 774 and CSH 7. A chemiluminescence (CHL) experiment using MCLA (2-methyl-6-Lf-methylphenyl]-3,7-dihydroimidazc [1,2-alpha] pyrazin-3-one) reflecting predominantly superoxide generation showed that NP caused marked CHL generation in QC 774 cells, but not in CSH 7 and UM 1 cells. However, the CHL experiment using L-012 reflecting predominantly hydroxyl radical and hypochlorite did not exhibit significant CHL generation in QC 774 cells at the same concentrations of NP. Furthermore, supplementation with SOD prevented NP-induced ROS generation and cell survival inhibition of QC 774 cells, but the catalase and metal-chelating agent deferoxamine did not have significant effects. These results suggest that one of the primary actions of NP in cells is the generation of superoxide which may be responsible for NP-induced cell growth inhibition.     

The distinct role of catalase and DNA repair systems in protection against hydrogen peroxide in Escherichia coli

The katEkatG mutant of E. coli, UM1, had no assayable catalase activities in the extract and showed increased (about 20 fold) sensitivity to killing by H2O2 when compared with its parental strain CSH7. The mutant strain was able to reactivate H2O2-damaged lambda phage. On the other hand, recA and polA mutants were also highly sensitive to H2O2, but they had normal level of catalase activities. RecA derivatives of UM1 were much more sensitive to H2O2 than UM1 and recA strains. The induction of umu operon occurred in UM1 at lower (1/10-1/20) doses of H2O2 than in CSH7. From the results it is concluded that the lethal effect of H2O2 is due to DNA damage induced by it and that catalase and DNA repair systems have a distinct role in protection against H2O2 in E. coli.     

Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide.

Pseudomonas aeruginosa is an obligate aerobe that is virtually ubiquitous in the environment. During aerobic respiration, the metabolism of dioxygen can lead to the production of reactive oxygen intermediates, one of which includes hydrogen peroxide. To counteract the potentially toxic effects of this compound, P. aeruginosa possesses two heme-containing catalases which detoxify hydrogen peroxide. In this study, we have cloned katB, encoding one catalase gene of P. aeruginosa. The gene was cloned on a 5.4-kb EcoRI fragment and is composed of 1,539 bp, encoding 513 amino acids. The amino acid sequence of the P. aeruginosa katB was approximately 65% identical to that of a catalase from a related species, Pseudomonas syringae. The katB gene was mapped to the 71- to 75-min region of the P. aeruginosa chromosome, the identical region which harbors both sodA and sodB genes encoding both manganese and iron superoxide dismutases. When cloned into a catalase-deficient mutant of Escherichia coli (UM255), the recombinant P. aeruginosa KatB was expressed (229 U/mg) and afforded this strain resistance to hydrogen peroxide nearly equivalent to that of the wild-type E. coli strain (HB101). The KatB protein was purified to homogeneity and determined to be a tetramer of approximately 228 kDa, which was in good agreement with the predicted protein size derived from the translated katB gene. Interestingly, KatB was not produced during the normal P. aeruginosa growth cycle, and catalase activity was greater in nonmucoid than in mucoid, alginate-producing organisms. When exposed to hydrogen peroxide and, to a greater extent, paraquat, total catalase activity was elevated 7- to 16-fold, respectively. In addition, an increase in KatB activity caused a marked increase in resistance to hydrogen peroxide. KatB was localized to the cytoplasm, while KatA, the "housekeeping" enzyme, was detected in both cytoplasmic and periplasmic extracts. A P. aeruginosa katB mutant demonstrated 50% greater sensitivity to hydrogen peroxide than wild-type bacteria, suggesting that KatB is essential for optimal resistance of P. aeroginosa to exogenous hydrogen peroxide.     

Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity

A number of catalase-deficient mutants of Escherichia coli which exhibit no assayable catalase activity were isolated. The only physiological difference between the catalase mutants and their parents was a 50- to 60-fold greater sensitivity to killing by hydrogen peroxide. For comparison, mutations in the xthA and recA genes of the same strains increased the sensitivity of the mutants to hydrogen peroxide by seven- and fivefold, respectively, showing that catalase was the primary defense against hydrogen peroxide. One class of mutants named katE was localized between pfkB and xthA at 37.8 min on the E. coli genome. A second class of catalase mutants was found which did not map in this region.     

An electron spin resonance study of oxyradical generation in superoxide dismutase- and catalase-deficient mutants of Escherichia coli K-12

The response of superoxide dismutase- and catalase-deficient strains of Escherichia coli to redox active compounds was examined by electron spin resonance. Levels of radicals formed in response to pyocyanine in situ were extremely low and were found to be predominantly extracellular, even in a strain completely deficient in both superoxide dismutase and catalase. In cell-free extracts of superoxide dismutase-minus strains incubated with NADPH and pyocyanine, the primary accumulating radical was the superoxide anion (O2-), although low levels of the hydroxyl radical (.OH) were also detected. In contrast, extracts from strains lacking catalase were found to accumulate higher levels of hydroxyl radicals.     

Cloning,characterization and phenotypic expression in Escherichia coli of catF,which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae

The phytopathogenic, gram-negative bacterium Pseudomonas syringae pv. syringae 61 contains three isozymes of catalase (EC 1.11.1.6), which have been proposed to play a role in the bacterium's responses to various environmental stresses. To study the role of individual isozymes, the gene coding for the catalytic subunit of one catalase isozyme was cloned from a cosmid library hosted in Escherichia coli DH5 by using a designed catalase-specific DNA probe for the screening. One out of four clones with a catalase-positive genotype was subcloned and a pUC19-based 2.7 × 10³-base (2.7-kb) insert subclone, pMK3E5, was used to transform catalase-deficient E. coli strain UM255 (HPI^–, HPII^–). The transformants contained a single isozyme of catalase that had electrophoretic and enzymic properties similar to catalase isozyme CatF from P. syringae pv. syringae 61. Analysis of the sequenced 2.7-kb insert DNA revealed six putative open-reading frames (ORF). The 1542-base-pair DNA sequence of ORF2, called catF, encodes a peptide of 513 amino acid residues with a calculated molecular mass of 66.6 kDa. The amino acid sequence deduced from catF had homology to the primary structure of true catalases from mammals, plants, yeasts and bacteria. The activity of the recombinant catalase was inhibited by 3-amino-1,2,4-triazole and azide and stimulated by chloramphenicol. The N terminus contained a signal sequence of 26 amino acids necessary for secretion into the periplasm, a so-far unique property of Pseudomonas catalases.Paper no. 4552 of the Utah Agricultural Experiment station     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

Cloning and characterization of monofunctional catalase from photosynthetic bacterium Rhodospirillum rubrum S1

In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from 20 degrees C to 60 degrees C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's K(m) value and V(max) of the catalase for H2O2 were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of A406 to A280 for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.     

In vivo formation of transmissible resistance factor by recombination between nontransmissible resistance factor and Col B factor.

Germ-free swine were artificially contaminated with tetracycline (TC) sensitive strains of Escherichia coli and Klebsiella pneumoniae. One of these strains, E. coli 3306, was infected with a plasmid carrying kanamycin (KM) resistance, i.e., T-kan factor. Another strain, E. coli P-5, carried a conjugally transferable Col B factor. Among the nine strains used, only E. coli P-38 became TC-resistant after TC administration. Three types of TC-resistance E. coli P-38 strains were found; (a) one strain carried nontransferable TC resistance and could not produce colicin, (b) one strain carried TC resistance with a high transmission frequency which could not produce colicin, and (c) one strain carried TC resistance with a low transmission frequency that could produce colicin B. Genetic studies disclosed that the transmissible TC resistance factors, i.e., Rms105 (group b) and Rms104 (group c), were formed by recombination between Col B factor and nontransmissible TC-resistance (tet) determinant which appeared in E. coli P-38 mutants.     

Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains

The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E. coli strains, AB1157 and B/r. Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157. However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2. Pretreatment also induced resistance to broad-band NUV radiation in these strains. The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains. The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E. coli UM1, is more resistant to NUV radiation than B/r. Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect. Two hypotheses are put forward to account for the sensitivity of exponential phase B/r. Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.     

Phenyl hydroquinone, an Ames test-negative carcinogen, induces Hog1-dependent stress response signaling

Recently, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae. We further found that phenyl hydroquinone arrested the cell cycle at G(1) and G(2)/M. In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G(2)/M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1/Cdc2 homolog). Furthermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by phenyl hydroquinone, indicating that the Mec1/Tel1 DNA-damage checkpoint was not functional. Mutations of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at the G(2)/M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development. These data suggest that a phenyl hydroquinone-induced G(2)/M transition checkpoint that is activated by the Hog1-Swe1 pathway plays a role in the development of aneuploidy.     

Biophysical radiosensitization

Experimental studies on normal and tumor-bearing rats revealed that chronic treatment with hydroquinone (5 mg/kg/day) inhibited catalase activity in liver, spleen, blood, and H 18R tumor. 3H-hydroquinone (1.5 microCi/g body weight) showed tumor specificity, with maximum radioactivity in the tumor at 1 h after administration. The biological half-time of 3H-hydroquinone in the tumor was 2 h, but there seems to exist a longer component, since 24 h after administration, some 30% of the maximum radioactivity could be detected in the tumor. Hydroquinone treatment produces a specific inhibition of catalase in the tumor and a higher degree of oxygenation at this level. These findings support the assumption that the mechanism of action of hydroquinone as an anticancer agent is achieved mainly via peroxide production. The oxygenation of the hypoxic tumoral tissue is done at non-toxic levels of hydroquinone, through a natural and specific biophysical pathway, recommending hydroquinone for combined anticancer treatment (radiotherapy and chemotherapy).     

Transglucosylation of hydroquinone catalysed by α-glucosidase from baker's yeast

Hydroquinone α-isomaltoside and hydroquinone α-glucoside were synthesized by transglucosylation in an aqueous system with baker's yeast α-glucosidase from hydroquinone and maltose as a glucosyl donor. Only one phenolic group was glucosylated, with α-selectivity, and the nature of the reaction products was governed by the concentration of hydroquinone. The optimal conditions for synthesis of glycosides were 9 mM hydroquinone and 1.5 M maltose in a 100 mM sodium citrate/phosphate buffer at pH 5.0 and 30 °C for 20 h. Under these conditions both hydroquinone α-glycosides were obtained in nearly equimolar amounts with a total molar yield of 28% with respect to hydroquinone and a total glycoside concentration of 1 mg/mL in the reaction mixture.     

Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium

We identified Shiga toxin gene (stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic-uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)-hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains, and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS.     

Hydroquinone, a control agent of agglutination and adherence of Streptococcus mutans induced by sucrose

Hydroquinone was found to alter agglutination of Streptococcus mutans induced by sucrose. The newly formed agglutination product produced by hydroquinone does not kill this cariogenic bacterium and the formation is reversible. The agglutination altering activity of hydroquinone seems to be specific for strains of S. mutans. As a result, hydroquinone inhibits sucrose-induced adherence of S. mutans.     

Examination of recovery in vitro and in vivo of nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7 (strains ATCC 43895 and FO46) became nonculturable in sterile, distilled, deionized water or after exposure to chlorine. Recovery of nonculturable E. coli O157:H7 was examined by in vitro and in vivo methods. The decline in culturability of starved E. coli O157:H7 was measured by plate count on rich medium. Recovery in vitro of nonculturable cells was conducted with media amended with catalase or sodium pyruvate; however, there was no apparent increase over culturable cell counts on amended versus nonamended media. Although nonculturable E. coli O157:H7 did not recover under in vitro conditions, a mouse model was used to determine if in vivo conditions would provide sufficient conditions for recovery of nonculturable E. coli O157:H7. In separate studies, mice were orally challenged with starvation-induced nonculturable cells (FO46) or chlorine-induced nonculturable cells (43895 and FO46). Passage through the mouse gastrointestinal tract had no effect on recovery of nonculturable (starvation or chlorine induced) E. coli O157:H7 (43895 or FO46), based on analysis of fecal samples. Mouse kidneys were assayed for the presence of Shiga toxin using the Vero cell assay. Differences in cytotoxicity towards Vero cells from kidney samples of mice receiving nonculturable cells and control mice were not significant, suggesting a loss of virulence.     

Oxidative stress effects on conjugational recombination and mutation in catalase-deficient Escherichia coli

The objective of the present investigation was to determine the effects on genetic recombination and mutation in Escherichia coli of either endogenous increases in oxygen radicals resulting from catalase deficiencies, or exogenous increases resulting from H2O2 treatment. Using the classical paradigm of Escherichia coli bacterial conjugation, strains deficient in the production of hydroperoxidase I (HPI) and/or hydroperoxidase II (HPII) were used as recipients in Hfr x F- matings. 'Background' recombination rates, measured by the rate of appearance of threonine prototrophs, was similar to wild-type levels in the HPI-deficient (katG) strain, but were significantly decreased in HPII- (katE) mutants. The addition of relatively nontoxic H2O2 concentrations (0.25 mmoles dm-3) to the mating mixtures stimulated recombination rates in wild-type and katE strains, but decreased rates in katG and katEkatG strains. A 0.5 mmoles dm-3 concentration of H2O2 inhibited recombination rates in all strains. In order to gauge the level of recA-dependent 'SOS' processes occurring under the experimental conditions, 'background' mutation rates were determined in both fluctuation and forward mutation (thyA) assays. Mutation rates in aerobically-grown cultures were increased up to 2.2-fold in katG and katEkatG strains. Treatment with relatively nontoxic H2O2 concentrations elevated the thyA mutagenesis up to 8-fold in catalase-deficient cultures. Furthermore, these studies along with data presented elsewhere show that the SOS phenotype of katEkatG is more resistant than that of katG strains. These studies clearly show that cellular oxidative stress occurring from catalase deficiency interferes with normal DNA metabolism.     

N-terminal methionine in recombinant proteins expressed in two different Escherichia coli strains

Two genes coding for chloramphenicol acetyltransferase and human interferon gamma, respectively, were overexpressed constitutively in two different strains of Escherichia coli (E. coli LE392 and E. coli XL1). The N-terminal amino acid analysis of the purified proteins showed that: (a) the N-terminal methionine is processed more efficiently in E. coli LE392 rather than in E. coli XL1 cells; (b) the N-terminal methionine is removed better from the heterologous human interferon gamma in comparison with the homologous chloramphenicol acetyltransferase protein: and (c) there is no strong correlation between the efficiency of N-terminal procession and the yield of recombinant protein.