A manganese porphyrin suppresses oxidative stress and extends the life span of streptozotocin-diabetic rats.

Enhanced oxidative stress due to hyperglycemia has been implicated in diabetic complications and is considered a major cause of cell and tissue damage. The aim of the present study was to investigate whether synthetic manganese porphyrin, Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP5+) can ameliorate diabetes-induced oxidative stress and affect life span of diabetic rats. Diabetes was induced by a single (60 mg/kg) intraperitoneal injection of streptozotocin in male Wistar rats. Oxidative stress was monitored by measuring malondialdehyde levels (MDA) in blood plasma and erythrocytes using HPLC. The antioxidant status was assessed by measuring the total radical-trapping potential (TRAP) of blood plasma. Life span of the animals was used as an indication of the overall effect of MnTM-2-PyP5+. MnTM-2-PyP5+ was administered subcutaneously at 1 mg/kg for the duration of the experiment, five times/week followed by one week of rest. Diabetes increased plasma and erythrocyte levels of MDA and decreased TRAP. MnTM-2-PyP5+ had no effect on blood glucose and glycosylated hemoglobin, but significantly increased TRAP and lowered MDA. This Mn porphyrin decreased mortality and markedly extended the life span of the diabetic animals. MnTM-2-PyP5+ suppressed diabetes-induced oxidative stress, which presumably accounts for its beneficial effect on the life span of the diabetic rats. The results indicate that Mn(III) N-alkylpyridylporphyrins can be used as potent therapeutic agents in diabetes.

How superoxide radical damages the cell.

Superoxide is considered to be poorly reactive, and cell damage has been attributed to HO. generated via the Haber-Weiss reaction. The function of O2- in this reaction is only to reduce Fe3+ to Fe2+. In vivo, however, superoxide could not out-compete cellular reductants such as glutathione, NADPH, and ascorbate, which makes the observed O2- toxicity rather puzzling. Little attention has been paid to the idea that, irrespective of its poor chemical reactivity, superoxide might be capable of interacting directly with specific intracellular targets; and that even the Haber-Weiss reaction might be a consequence of such direct interactions. This paper summarizes latest data that support the concept of such a mechanism.

Polyphosphate accumulation and oxidative DNA damage in superoxide dismutase-deficient Escherichia coli.

Inorganic polyphosphate is a ubiquitous, linear polymer of phosphate residues linked by high-energy phosphoanhydride bonds. In response to starvation, polyP levels are increased up to 100-fold. It has been proposed that chelation of transition metals by polyP might reduce their toxicity, and that polyP accumulation is vital for survival in stationary phase. SOD-deficient E. coli is unable to survive in stationary phase. We found that deletion of the cytoplasmic SODs does not impair the cell's capability of synthesizing polyP. However, transient accumulation of polyphosphate correlated with increased resistance to H(2)O(2) and protection of DNA against oxidative damage. The reason for this protective effect of polyP is the induction of HPII catalase and DNA repair enzymes as members of the rpoS regulon. PolyP did not directly protect DNA against oxidative damage in vitro and acted as a pro-oxidant by stimulating the production of hydroxyl radical in the Fenton reaction. It is thus suggested that accumulation of poly P and rpoS induction cannot compensate for the lack of cytosolic SODs for survival in stationary phase.

Glycerol metabolism in superoxide dismutase-deficient Escherichia coli.

Escherichia coli, which lacks cytoplasmic superoxide dismutases, exhibits various phenotypic deficits if grown aerobically. Here we report that sodAsodB E. coli cannot use glycerol under aerobic conditions. The reason is low activity of glycerol kinase (GK), the rate-limiting enzyme in glycerol metabolism. Superoxide does not inactivate GK, but makes it susceptible to inactivation by a heat-labile factor present in the cell-free extracts. This factor seems to be part of a proteolytic system, which recognizes and degrades oxidatively modified proteins.

Induction of the soxRS regulon of Escherichia coli by superoxide.

The soxRS regulon orchestrates a multifaceted defense against oxidative stress, by inducing the transcription of approximately 15 genes. The induction of this regulon by redox agents, known to mediate O-2 production, led to the view that O-2 is one signal to which it responds. However, redox cycling agents deplete cellular reductants while producing O-2, and one may question whether the regulon responds to the depletion of some cytoplasmic reductant or to O-2, or both. We demonstrate that raising [O-2] by mutational deletion of superoxide dismutases and/or by addition of paraquat, both under aerobic conditions, causes induction of a member of the soxRS regulon and that a mutational defect in soxRS eliminates that induction. This establishes that O-2, directly or indirectly, can cause induction of this defensive regulon.

Why superoxide imposes an aromatic amino acid auxotrophy on Escherichia coli. The transketolase connection.

The lack of superoxide dismutase and the consequent elevation of [O2-] imposes, on Escherichia coli, auxotrophies for branched chain, sulfur-containing, and aromatic amino acids. The former two classes of auxotrophies have already been explained, whereas the third is explained herein. Thus O2- is shown to interfere with the production of erythrose-4-phosphate, which is essential for the first step of the aromatic biosynthetic pathway. It does so by oxidizing the 1, 2-dihydroxyethyl thiamine pyrophosphate intermediate of transketolase and inactivating this enzyme.

Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical.

The fluorogenic oxidation of hydroethidine (HE) to ethidium (E+) has been used as a measure of O2-. Evaluation of this method confirms that O2-, but not O2 or H2O2, rapidly oxidizes HE to E+. However the ratio of E+ produced per O2- introduced decreased as the flux of O2- was increased. This suggested that HE can catalyze the dismutation of O2- and this was affirmed. HE was oxidized to a red product, distinct from E+ by ferricytochrome c and a similar oxidation may occur within Escherichia coli. HE inhibited the growth and killed a SOD-null strain to a greater extent than the SOD-replete parental strain and these effects were much diminished under anaerobic conditions. This indicated that E+ was responsible for the toxicity of HE and indeed E+ was seen to be toxic under both aerobic and anaerobic conditions. In view of the data presented HE can be recommended as a qualitative but not as a quantitative measure of O2(-1).

Superoxide dependence of the toxicity of short chain sugars.

Erythrose inhibited the growth of a sodA sodB strain of Escherichia coli under aerobiosis; but did not inhibit anaerobic growth of the sodA sodB strain, or the aerobic growth of the superoxide dismutase (SOD)-competent parental strain. A SOD mimic protected the sodA sodB strain against the toxicity of erythrose as did the carbonyl-blocking reagents hydrazine and aminoguanidine. Three carbon sugars, such as glyceraldehyde and dihydroxy acetone, and the two carbon sugar glycolaldehyde, were similarly toxic in an O-2-dependent manner. An unidentified dialyzable component in E. coli extract augmented the oxidation of short chain sugars, and this was partially inhibitable by SOD. The toxicity of the short chain sugars appears to be because of an O-2-dependent oxidation to alpha, beta-dicarbonyl compounds. In keeping with this view was the O-2-independent toxicity of methylglyoxal.

The ortho effect makes manganese(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin a powerful and potentially useful superoxide dismutase mimic.

The ortho, meta, and para isomers of manganese(III) 5,10,15, 20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP5+, MnTM-3-PyP5+, and MnTM-4-PyP5+, respectively, were analyzed in terms of their superoxide dismutase (SOD) activity in vitro and in vivo. The impact of their interaction with DNA and RNA on the SOD activity in vivo and in vitro has also been analyzed. Differences in their behavior are due to the combined steric and electrostatic factors. In vitro catalytic activities are closely related to their redox potentials. The half-wave potentials (E1/2) are +0.220 mV, +0.052 mV, and +0.060 V versus normal hydrogen electrode, whereas the rates of dismutation (kcat) are 6.0 x 10(7), 4.1 x 10(6), and 3.8 x 10(6) M-1 s-1 for the ortho, meta, and para isomers, respectively. However, the in vitro activity is not a sufficient predictor of in vivo efficacy. The ortho and meta isomers, although of significantly different in vitro SOD activities, have fairly close in vivo SOD efficacy due to their similarly weak interactions with DNA. In contrast, due to a higher degree of interaction with DNA, the para isomer inhibited growth of SOD-deficient Escherichia coli.

Growth in iron-enriched medium partially compensates Escherichia coli for the lack of manganese and iron superoxide dismutase.

Enrichment of the growth medium with iron partially relieves the phenotypic deficits imposed on Escherichia coli by lack of both manganese and iron superoxide dismutases. Thus iron supplementation increased the aerobic growth rate, decreased the leakage of sulfite, and diminished sensitivity toward paraquat. Iron supplementation increased the activities of several [4Fe-4S]-containing dehydratases, and this was seen even in the presence of 50 microg/ml of rifampicin, an amount which completely inhibited growth. Assessing the O-2 scavenging activity by means of lucigenin luminescence indicated that the iron-enriched sodAsodB cells had gained some means of eliminating O-2, which was not detectable as superoxide dismutase activity in cell extracts. It is noteworthy that iron-enriched cells were not more sensitive toward the lethality of H2O2 despite having the usual amount of catalase activity. This indicates that iron taken into the cells from the medium is not available for Fenton chemistry, but is available for reconstitution of iron-sulfur clusters. We suppose that oxidation of the [4Fe-4S] clusters of dehydratases by O-2 and their subsequent reductive reconstitution provides a mechanism for scavenging O-2 and that speeding this reductive reconstitution by iron enrichment both spared other targets from O-2 attack and maintained adequate levels of these enzymes to meet the metabolic needs of the cells.

Superoxide imposes leakage of sulfite from Escherichia coli.

Escherichia coli, which lacks the cytosolic superoxide dismutases, exhibits several nutritional auxotrophies when growing aerobically. The cysteine/methionine requirement, which is one of these, was previously shown to be due to leakage from the cells, and accumulation in the medium, of a metabolic intermediate on the biosynthetic route to these amino acids. The parental strain does not significantly accumulate this compound. It is now shown that treatment with alkaline cyanide releases sulfite from this compound, a property shared by alpha-hydroxy sulfonic acids (carbonyl-bisulfite adducts). Since E. coli accumulates carbonyl compounds in the growth medium, it appears likely that the sulfitogenic compounds accumulated by the sodA sodB strain are alpha-hydroxy sulfonic acids.

The copper- and zinc-containing superoxide dismutase from Escherichia coli: molecular weight and stability.

The periplasmic Cu,Zn superoxide dismutase (Cu,ZnSOD) from Escherichia coli has been shown by sedimentation equilibrium to be a monomer with a molecular weight of approximately 17,000. The enzyme suffered a reversible inactivation when heated to 70 degrees C. This was minimized by added Cu(II) or Zn(II). Heat lability was greater in phosphate than in Tris buffer. The enzyme exhibited a time-dependent inactivation by Hg(II) and this too was greater in phosphate than in Tris. This behavior can be explained by a modest affinity of the enzyme for Cu(II) and Zn(II) which results in a dissociation/association equilibrium. Elevation of the temperature shifts this equilibrium toward dissociation and phosphate sequesters the released metals making them less available for reinsertion at the active site. Hg(II) competes for occupancy of the active site and there were more unoccupied sites in phosphate than in Tris. A parallel was drawn between the E. coli Cu,ZnSOD and FALS varients of human Cu,ZnSOD, which are also relatively unstable and exhibit low affinity for Cu(II).

The rate of adaptive mutagenesis in Escherichia coli is enhanced by oxygen (superoxide).

Adaptive mutagenesis is that which occurs in non-dividing cells and which allows growth under the selective conditions imposed. We now report that reversion of amino acid auxotrophies in E. coli fits that definition and is enhanced under conditions conducive to oxidative damage to DNA. Thus adaptive mutagenesis was approximately 4-fold more frequent in a sodA sodB strain than in the superoxide dismutase-replete parental strain and this mutagenesis was suppressed under anaerobic conditions. Moreover, a cell permeant manganic porphyrin, capable of catalyzing the dismutation of O2-, diminished the rate of occurrence of these mutations. Repair of oxidative damage to DNA, in the non-dividing cells, appears to provide the opportunity for adaptive mutagenesis.

The mechanism of the auxotrophy for sulfur-containing amino acids imposed upon Escherichia coli by superoxide.

Defects in both of the genes coding for the cytosolic superoxide dismutases (SODs) of Escherichia coli impose an oxygen-dependent nutritional requirement for cysteine. This is now seen to be a bradytrophy, rather than an absolute auxotrophy, since lack of Cys merely imposed a growth lag and escape from this growth lag did not involve genetic reversion. This Cys bradytrophy was not seen in the SOD-competent parental strain, and it was relieved by a cell-permeant mimic of SOD activity; hence, it was due to O2-.. It was also relieved by an osmolyte, such as sucrose; hence, it appears due to leakage from the cell of some component needed for Cys biosynthesis. Medium conditioned by the aerobic growth of the SOD-defective strain relieved the growth lag. Bioassays with Cys mutants suggested that the conditioned medium contained SO3-3 or its equivalent, and sulfite per se was able to eliminate the growth lag. However, some component of the conditioned medium reacted with added sulfite and interfered with attempts to assay for it colorimetrically. These results suggest that the cell envelope of the SOD-defective strain was weakened, directly or indirectly, by O2 and then leaked sulfite. This prevents cysteine biosynthesis until sulfite accumulates in the medium.

Escherichia coli exhibits negative chemotaxis in gradients of hydrogen peroxide, hypochlorite, and N-chlorotaurine: products of the respiratory burst of phagocytic cells.

Escherichia coli can respond to gradients of specific compounds, moving up gradients of attractants and down gradients of repellents. Stimulated phagocytic leukocytes produce H2O2, OCl-, and N-chlorotaurine in a response termed the respiratory burst. E. coli is actively repelled by these compounds. Catalase in the suspending medium eliminated the effect of H2O2. Repulsion by H2O2 could be demonstrated with 1 microM H2O2, which is far below the level that caused overt toxicity. Strains with defects in the biosynthesis of glutathione or lacking hydroperoxidases I and II retained this response to H2O2, and 2.0 mM CN- did not interfere with it. Mutants with defects in any one of the four known methyl-accepting chemotaxis proteins also retained the ability to respond to H2O2, but a "gutted" mutant that was deleted for all four methyl-accepting chemotaxis proteins, as well as for CheA, CheW, CheR, CheB, CheY, and CheZ, did not respond to H2O2. Hypochlorite and N-chlorotaurine were also strongly repellent. Chemotaxis down gradients of H2O2, OCl-, and N-chlorotaurine may contribute to the survival of commensal or pathogenic microorganisms.

Functional significance of the Cu,ZnSOD in Escherichia coli.

Diethyl dithiocarbamate (DDC) was used to inhibit the copper- and zinc- containing superoxide dismutase (Cu,ZnSOD) of Escherichia coli in order to expose its physiological function. DDC inhibited the aerobic growth of a sodA sodB mutant much more than it did the growth of a SOD-replete parental strain and this inhibitory effect was oxygen-dependent. The SOD mimic MnTMPyP markedly diminished the growth inhibitory effect of DDC. Transfer of the sodA sodB strain from anaerobic to aerobic conditions induced fumarase C, which is a member of the soxRS regulon. DDC augmented this induction. These results indicate that the Cu,ZnSOD provides a defense against oxidative stress, which is more important to the sodA sodB mutant than to its SOD-replete parental strain.

Purification and characterization of the Cu,Zn SOD from Escherichia coli.

The periplasmic Cu,Zn superoxide dismutase has been purified to homogeneity by a procedure, which depended upon osmotic shock followed by two chromatographic columns. Its subunit weight, determined by electrospray ionization mass spectrometry, was found to be 15,737 +/- 1.6. The second derivative ultraviolet spectrum indicated a lack of tryptophan. The amino acid composition as well as a partial N-terminal amino acid sequence is reported. The specific activity was 3700 U/mg and the corresponding copper content was 0.77 atoms Cu/subunit. The enzyme was quite unstable and overnight dialysis against EDTA or even prolonged dialysis against neutral phosphate buffer caused partial loss of activity and of copper and visible precipitation. It is likely that some losses occurred during the isolation procedure, and if these could have been prevented the copper content would have been 1.0 Cu/subunit and the specific activity would have been 4800 U/mg. It now appears likely that gram negative bacteria will commonly be found to contain a periplasmic Cu,Zn SOD.

A superoxide dismutase mimic protects sodA sodB Escherichia coli against aerobic heating and stationary-phase death.

Superoxide appears to be a major cause of stationary-phase death and heat kill. In support of this conclusion are the following observations: (a) Stationary-phase death was apparent in the sodA sodB, but not in the superoxide dismutase (SOD)-competent parental strain; (b) Stationary phase death in the sodA sodB strain was dioxygen-dependent; (c) A manganic porphyrin, which catalyzes the dismutation of superoxide, protected the sodA sodB strain against stationary-phase death; (d) Heating the sodA sodB strain to 42 degrees C caused a loss of viability not seen with the SOD-competent parental strain and preventable by the manganic porphyrin. Exposure to aerobic conditions induced antibiotic resistance in the sodA sodB, but not in the parental strain and the manganic porphyrin prevented that induction. This again indicates its ability to substitute for SOD in Escherichia coli.

Superoxide dismutase protects against aerobic heat shock in Escherichia coli.

Exposure of a superoxide dismutase-null (sodA sodB) strain of Escherichia coli to aerobic heat stress (45 to 48 degrees C) caused a profound loss of viability, whereas the same heat stress applied anaerobically had a negligible effect. A superoxide dismutase-competent parental strain was resistant to the lethal effect of the aerobic heating. It follows that aerobic heating imposes an oxidative burden of which O2- must be a major component. This effect is not seen at 53 degrees C, presumably because, at this higher temperature, direct thermolability of vital cell components overrides the effect of superoxide radicals.