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.     

Glycolaldehyde induces growth inhibition and oxidative stress in human breast cancer cells

Glycolaldehyde (GA) is formed by oxidative degradation of glucose, from glycated proteins, lipid peroxidation, and oxidation of amino acids, and by human neutrophils during phagocytosis. The exact purpose of GA production by phagocytes is unclear, but it is tempting to speculate that it is part of the defense against invading bacteria and tumor cells. We have already reported that GA induces apoptosis in breast cancer cells. Because the GA carbonyl group cannot be blocked by cyclization, it is prone to enolization followed by air oxidation with concomitant production of glyoxal and superoxide. Since both these products can induce oxidative stress, in this work we focused on the ability of GA to cause oxidative cell damage. MCF7 human breast cancer cells were incubated with different GA concentrations and O2*- production, lipid peroxidation, and carbonylated protein were assessed. GA was cytotoxic at 20 microM, inhibiting cell proliferation, and at 100 microM, induced p53 expression and caused apoptosis. These events were accompanied by increases of O2*- production, lipid peroxidation, and accumulation of protein carbonyl. It thus appears that alpha-hydroxy aldehydes can induce oxidative stress. Prevention of oxidative stress, however, did not abolish the effects of GA on cell growth and viability, which appeared to be a direct consequence of glyoxal toxicity.     

Impact of electrostatics in redox modulation of oxidative stress by Mn porphyrins: protection of SOD-deficient Escherichia coli via alternative mechanism where Mn porphyrin acts as a Mn carrier

Understanding the factors that determine the ability of Mn porphyrins to scavenge reactive species is essential for tuning their in vivo efficacy. We present herein the revised structure-activity relationships accounting for the critical importance of electrostatics in the Mn porphyrin-based redox modulation systems and show that the design of effective SOD mimics (per se) based on anionic porphyrins is greatly hindered by inappropriate electrostatics. A new strategy for the beta-octabromination of the prototypical anionic Mn porphyrins Mn(III) meso-tetrakis(p-carboxylatophenyl)porphyrin ([Mn(III)TCPP](3-) or MnTBAP(3-)) and Mn(III) meso-tetrakis(p-sulfonatophenyl)porphyrin ([Mn(III)TSPP](3-)), to yield the corresponding anionic analogues [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-), respectively, is described along with characterization data, stability studies, and their ability to substitute for SOD in SOD-deficient Escherichia coli. Despite the Mn(III)/Mn(II) reduction potential of [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-) being close to the SOD-enzyme optimum and nearly identical to that of the cationic Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (Mn(III)TM-2-PyP(5+)), the SOD activity of both anionic brominated porphyrins ([Mn(III)Br(8)TCPP](3-), E(1/2)=+213 mV vs NHE, log k(cat)=5.07; [Mn(III)Br(8)TSPP](3-), E(1/2)=+209 mV, log k(cat)=5.56) is considerably lower than that of Mn(III)TM-2-PyP(5+) (E(1/2)=+220 mV, log k(cat)=7.79). This illustrates the impact of electrostatic guidance of O(2)(-) toward the metal center of the mimic. With low k(cat), the [Mn(III)TCPP](3-), [Mn(III)TSPP](3-), and [Mn(III)Br(8)TCPP](3-) did not rescue SOD-deficient E. coli. The striking ability of [Mn(III)Br(8)TSPP](3-) to substitute for the SOD enzymes in the E. coli model does not correlate with its log k(cat). In fact, the protectiveness of [Mn(III)Br(8)TSPP](3-) is comparable to or better than that of the potent SOD mimic Mn(III)TM-2-PyP(5+), even though the dismutation rate constant of the anionic complex is 170-fold smaller. Analyses of the medium and E. coli cell extract revealed that the major species in the [Mn(III)Br(8)TSPP](3-) system is not the Mn complex, but the free-base porphyrin [H(2)Br(8)TSPP](4-) instead. Control experiments with extracellular MnCl(2) showed the lack of E. coli protection, indicating that "free" Mn(2+) cannot enter the cell to a significant extent. We proposed herein the alternative mechanism where a labile Mn porphyrin [Mn(III)Br(8)TSPP](3-) is not an SOD mimic per se but carries Mn into the E. coli cell.     

Superoxide-dependence of the short chain sugars-induced mutagenesis

Short chain sugars such as glycolaldehyde are produced at the initial stages of nonenzymatic glycosylation. Because their carbonyl groups cannot be blocked by cyclization, such compounds tautomerize to enediols, which are prone to autoxidation. Superoxide radical serves as an initiator and a propagator of this autoxidation. The biological importance of the involvement of superoxide in sugar autoxidation in vivo was examined using superoxide dismutase (SOD)-deficient and SOD-replete strains of Escherichia coli. Glycolaldehyde, glyceraldehyde, and dihydroxyacetone greatly enhanced the mutation rates in SOD-deficient E. coli. The effect was oxygen-dependent and was suppressed by SOD or by a SOD mimetic. The mutagenic effect of glycolaldehyde coincided with intracellular accumulation of glyoxal, a product of glycolaldehyde autoxidation.     

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.     

Induction of oxidative cell damage by photo-treatment with zinc meta N-methylpyridylporphyrin

We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP^4 + ) can act as photosensitizers with efficacy comparable to that of hematoporphyrin derivative (HpD) in preventing cell proliferation and causing cell death in vitro. To better understand the biochemical basis of this activity, the effects of photo-activated ZnTM-3-PyP^4 +  on GSH/GSSG ratio, lipid peroxidation, membrane permeability, oxidative DNA damage, and the activities of SOD, catalase, glutathione reductase, and glutathione peroxidase were evaluated. Light exposure of ZnTM-3-PyP^4 + -treated colon adenocarcinoma cells caused a wide spectrum of oxidative damage including depletion of GSH, inactivation of glutathione reductase and glutathione peroxidase, oxidative DNA damage and peroxidation of membrane lipids. Cell staining with Hoechst-33342 showed morphological changes consistent with both necrotic and apoptotic death sequences, depending upon the presence of oxygen.     

Bioavailability of metalloporphyrin-based SOD mimics is greatly influenced by a single charge residing on a Mn site

In the cell Mn porphyrins (MnPs) likely couple with cellular reductants which results in a drop of total charge from 5+ to 4+ and dramatically increases their lipophilicity by up to three orders of magnitude depending upon the length of alkylpyridyl chains and type of isomer. The effects result from the interplay of solvation, lipophilicit and stericity. Impact of ascorbate on accumulation of MnPs was measured in E. coli and in Balb/C mouse tumours and muscle; for the latter measurements, the LC/ESI-MS/MS method was developed. Accumulation was significantly enhanced when MnPs were co-administered with ascorbate in both prokaryotic and eukaryotic systems. Further, MnTnHex-2-PyP(5+) accumulates 5-fold more in the tumour than in a muscle. Such data increase our understanding of MnPs cellular and sub-cellular accumulation and remarkable in vivo effects. The work is in progress to understand how coupling of MnPs with ascorbate affects their mechanism of action, in particular with respect to cancer therapy.     

A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrin, combines superb potency and lipophilicity with low toxicity

The Mn porphyrins of k(cat)(O(2)(.-)) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP(5+) is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP(5+) has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH(2) group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP(5+). The high SOD-like activity is preserved and even enhanced: log k(cat)(O(2)(.-))=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP(5+) and MnTnHep-2-PyP(5+), respectively. MnTnBuOE-2-PyP(5+) was tested in an O(2)(.-) -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5-30 μM. MnTnHep-2-PyP(5+) was already toxic at 5 μM, and MnTnHex-2-PyP(5+) became toxic at 30 μM. In a mouse toxicity study, MnTnBuOE-2-PyP(5+) was several-fold less toxic than either MnTnHex-2-PyP(5+) or MnTnHep-2-PyP(5+).