Cell death by reactive oxygen species generated from water-soluble cationic metalloporphyrins as superoxide dismutase mimics.

We investigated the effect on cell death of reactive oxygen species induced by water-soluble cationic metalloporphyrins with superoxide dismutase (SOD) activity. The SOD activity of 5,10,15,20-tetrakis(4-N-methylpyridyl)]porphine (MPy(4)P) containing Fe, Mn or Cu was measured using a cytochrome c assay by the xanthine/xanthine oxidase system and stopped-flow kinetic analysis. Cell viability of four cell lines treated with metalloporphyrins, mitomycin c (MMC), or cisplatin was estimated by a trypan blue exclusion assay. FeMPy(4)P with a high SOD activity showed a significant cytotoxicity compared with MMC and cisplatin, while CuMPy(4)P without SOD activity exhibited no cytotoxicity. However, MnMPy(4)P showing an SOD activity as high as that of FeMPy(4)P did not indicate cytotoxicity. These findings suggest that FeMPy(4)P as SOD mimic converts intracellular O2(*-) to H(2)O(2) and that it rapidly reacts with H(2)O(2) to form *OH, causing DNA damage and inducing cell death. On the other hand, MnMPy(4)P did not participate in the Fenton reaction, so that DNA damage in the cells treated with MnMPy(4)P was not observed. In addition, the cytotoxicity by the metalloporphyrin was inversely correlated with the SOD activity of the cells and the selective damage at cellular and DNA levels was confirmed. We believe that for an anticancer drug with antioxidant ability O(2)(*-) is useful as a target molecule to induce selective cell death between cancer and normal cells and that metalloporphyrins showing SOD activity and Fenton-like reaction are a new class of anticancer agents.     

Superoxide dismutase mimetics: synthesis and structure-activity relationship study of MnTBAP analogues

Carboxylic ester and amide-substituted analogues of [5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrinato]manganese(III) chloride (MnTBAP) were synthesized and assayed as potential superoxide dismutase (SOD) mimetics. The tetraester analogues 4a and 4b were found to have comparable SOD activity to the known SOD mimetic MnTBAP, while amides 4c-4e exhibited reduced SOD activity. In the substituted methyl benzoate/acid and disubstituted porphyrin series, analogues 12c, 12f, and 12m were found to have comparable to improved SOD activity relative to MnTBAP and analogues 12j, 13a, and 13d exhibited improved activity in both the SOD and thiobarbituric acid reactive species (TBARS) assays relative to MnTBAP.     

Design of metalloporphyrin-carbohydrate conjugates for a new superoxide dismutase mimic with cellular recognition

Metalloporphyrin-carbohydrate conjugates have been synthesized as superoxide dismutase (SOD) mimics with cellular recognition. To synthesize the conjugates, aliphatic primary amino groups for conjugation were introduced, with the cationic pyridyl groups for the SOD activity of porphyrin preserved. The reductive amination between introduced amino groups and the reducing end of lactose was then carried out. The resulting conjugates consisting of manganese (Mn)-porphyrin surrounded by several lactose molecules possessed significant SOD activity and low cytotoxicity. Compared with metalloporphyrins having no lactose molecule, the recognition of the resulting conjugates by human hepatoma HepG2 cells increased. The cellular recognition was inhibited by competitors of beta-galactose. These results suggest that the Mn-porphyrin-lactose conjugates recognized the hepatic lectin on the cell surface.     

Differential Activation of Staphylococcus aureus Heme Detoxification Machinery by Heme Analogues

The reactive nature of heme enables its use as an enzymatic cofactor while rendering excess heme toxic. The importance of heme detoxification machinery is highlighted by the presence of various types of these homeostatic systems in Gram-positive and Gram-negative microorganisms. A number of pathogens possess orthologs of the HssRS/HrtAB heme detoxification system, underscoring a potential role this system plays in the survival of bacteria in heme-rich environments such as the vertebrate host. In this work, we sought to determine the role of this system in protection against metalloporphyrin heme analogues identified by previous studies as antimicrobial agents. Our findings demonstrate that only toxic metalloporphyrins maximally activate expression of the Staphylococcus aureus heme detoxification system, suggesting that the sensing mechanism of HssRS might require a component of the associated toxicity rather than or in addition to the metalloporphyrin itself. We further establish that only a subset of toxic metalloporphyrins elicit the oxidative damage previously shown to be a significant component of heme toxicity whereas all toxic noniron metalloporphyrins inhibit bacterial respiration. Finally, we demonstrate that, despite the fact that toxic metalloporphyrin treatment induces expression of S. aureus heme detoxification machinery, the HrtAB heme export pump is unable to detoxify most of these molecules. The ineffectiveness of HrtAB against toxic heme analogues provides an explanation for their increased antimicrobial activity relative to heme. Additionally, these studies define the specificity of HssRS/HrtAB, which may provide future insight into the biochemical mechanisms of these systems.     

Hemodynamic effects of metalloporphyrin catalytic antioxidants: structure-activity relationships and species specificity

Superoxide plays a role in blood pressure regulation in certain vascular diseases, however, its involvement in regulating basal blood pressure is uncertain. Vascular superoxide concentrations are limited by extracellular superoxide dismutase (EC-SOD), which is highly expressed in the vasculature of most animal species. Metalloporphyrins are low molecular weight, synthetic, redox-active, catalytic antioxidants that act as SOD mimetics. We evaluated the effects of metalloporphyrins on blood pressure in different animal species. The metalloporphyrin AEOL10113 (5–10 μg/kg iv), but not native or polyethylene glycol-CuZnSOD, caused a dose-dependent reduction in blood pressure in anesthetized rats. AEOL10113 had no effect on blood pressure in mice (wild-type or EC-SOD knockouts), guinea pigs, dogs, or baboons at doses up to 5 mg/kg iv Structure-activity studies indicated that metalloporphyrins with high SOD activity were more effective in lowering rat blood pressure than low-activity analogs. The blood pressure effect of AEOL10113 was not attributable to the release of manganese, nor was it affected by inhibitors of nitric oxide synthase (L-NAME) and guanylate cyclase (ODQ, 8-bromo-cGMP, and methylene blue) or nitric oxide scavengers (HbA_o). Chlorpheniramine attenuated the effect, suggesting that the blood pressure response in rats is related to histamine release rather than the protection of nitric oxide.     

Metalloporphyrins are potent inhibitors of lipid peroxidation

The objectives of these studies were to determine whether metalloporphyrins could inhibit lipid peroxidation, characterize factors that influence their potency and compare their potency to prototypical antioxidants. Lipid peroxidation was initiated with iron and ascorbate in rat brain homogenates and the formation of thiobarbituric acid reactive species was used as an index of lipid peroxidation. Metalloporphyrins were found to be a novel and potent class of lipid peroxidation inhibitors. Inhibition of lipid peroxidation by metalloporphyrins was dependent on the transition metal ligated to the porphyrin, indicating that metal centered redox chemistry was important to the mechanism of their antioxidant activities. Manganese porphyrins with the highest superoxide dismutase (SOD) activities, MnOBTM-4-PyP and MnTM-2-PyP (charges are omitted throughout text for clarity), were the most potent inhibitors of lipid peroxidation with calculated IC50s of 1.3 and 1.0 microM, respectively. These manganese porphyrins were 2 orders of magnitude more potent than either trolox (IC50 = 204 microM) or rutin (IC50 = 112 microM). The potencies of the manganese porphyrins were related not only to their redox potentials and SOD activities, but also to other factors that may contribute to their ability to act as electron acceptors. The broad array of antioxidant activities possessed by metalloporphyrins make them attractive therapeutic agents in disease states that involve the overproduction of reactive oxygen species.     

Oxidative cleavage of DNA mediated by hybrid metalloporphyrin-ellipticine molecules and functionalized metalloporphyrin precursors

The nuclease activity of functionalized metalloporphyrins 1-8 and hybrid metalloporphyrin-ellipticine molecules 10-16 in the presence of potassium monopersulfate (KHSO5) or magnesium monoperoxyphthalate (MMPP), water-soluble oxygen atom donors at physiological pH, toward double-stranded phi X174 DNA is reported. The DNA cleavage efficiency as a function of the nature of functionalized metalloporphyrins, the length of the linkage between the two parts of the hybrid molecule, viz., metalloporphyrin and 9-methoxyellipticine, the nature of the central metal atom (Mn, Fe, or Zn) the ionic strength, and the nature of the oxygen donor has been studied. Single-strand breaks (SSBs) are observed on double-stranded DNA with a short incubation time of 2 min in the presence of manganese derivatives of both metalloporphyrins and hybrid molecules. Owing to their cytotoxic and nuclease activity, these new water-soluble hybrid molecules may be considered as efficient bleomycin models based on cationic metalloporphyrins.     

Chloroplast biogenesis: Biosynthesis and accumulation of Mg-protoporphyrin IX monoester and other metalloporphyrins by isolated etioplasts and developing chloroplasts

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester as well as longer wavelength metalloporphyrins when incubated in the dark, in the presence of air, δ-aminolevulinic acid, and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium, and phosphate). The putative metalloporphyrins exhibited distinct fluorescence emission and excitation properties and were detected by spectrofluorometry in situ and after extraction in organic solvents. The cofactors were previously shown to be required for protochlorophyll, and chlorophyll biosynthesis and grana assembly in vitro. The putative long wavelength metalloporphyrins were suggested earlier to represent intermediates between Mg-protoporphyrin IX monomethyl ester and protochlorophyllide. The isolated plastids were similar in this aspect of their biosynthetic activity to etiolated cotyledons greening in distilled H₂O. In contrast to greening cotyledons, however, the biosynthetic activity of the isolated plastids depended on the addition of exogenous cofactors and δ-aminolevulinic acid. This was interpreted as an indication that the isolated plastids were not capable of generating their own δ-aminolevulinic acid and cofactors under the present incubation conditions. Light was not required for the conversion of added ALA to metalloporphyrins in vitro. The metalloporphyrins synthesized in vitro were more highly fluorescent in situ than those of greening cotyledons. In addition to Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins, isolated etioplasts synthesized and accumulated Zn-protoporphyrin and Zn-protoporphyrin IX monoesterlike compounds.     

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalis hmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.     

Peroxide metabolism enzymes in diabetic children: relationship to duration and control of diabetes

Enzyme activities of erythrocyte superoxide dismutase (SOD), peroxidase and catalase in groups of diabetic children, the duration of the disease and control qualities were compared with respective values obtained for healthy children. Generally the disease duration was clearly found to affect SOD activity, and catalase activity only a little. No similar influence of either diabetes duration or control was noticed in the case of peroxidase. SOD activity was diminished in diabetics when compared with control subjects, and in turn peroxidase and catalase activities were generally elevated. The diminution in SOD activity may constitute a reason for enhanced pancreatic cells susceptibility to deterioration, followed by the augmentation of peroxidase activity with an 'elaborated' mechanism compensating for the loss of erythrocyte SOD activity.     

Synthesis and biological evaluation of a C5-biphenyl thiolactomycin library

Fifteen novel C5 analogues of thiolactomycin (13 biphenyl analogues and two biphenyl mimics) have been synthesised and assessed for their in vitro mtFabH and whole cell Mycobacterium bovis BCG activity, respectively. Analysis of the 15 compounds revealed that six possessed enhanced in vitro activity in a direct mtFabH assay. Encouragingly analogues 11, 12 and 13 gave a significant enhancement in in vitro activity against mtFabH. Analogue 13 (5-(4-methoxycarbonyl-biphenyl-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one) gave an IC(50) value of 3 microM compared to the parent drug thiolactomycin (75 microM) against mtFabH. The biological analysis of this library reaffirms the requirement for a linear pi-rich system containing hydrogen bond accepting substituents attached to the para-position of the C5 biphenyl analogue to generate compounds with enhanced activity.     

Dependence of excitotoxic neurodegeneration on mitochondrial aconitase inactivation

Using the inactivation of mitochondrial and cytosolic aconitases as markers of compartment-specific superoxide (O2(-)) production, we show that oxygen-glucose deprivation (OGD) or excitotoxin exposure produce a time-dependent inactivation of mitochondrial, but not cytosolic, aconitase in cortical cultures. To determine if mitochondrial O2(-) production was an important determinant in neuronal death resulting from OGD, metalloporphyrins with varying superoxide dismutase (SOD) activity were tested for their ability to protect against mitochondrial aconitase inactivation and cell death. OGD-induced mitochondrial aconitase inactivation and cell death was inhibited by manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP), manganese tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) and NMDA receptor antagonists. By contrast, NMDA- or kainate (KA)-induced mitochondrial aconitase inactivation and cell death was inhibited by MnTBAP, but not MnTE-2-PyP. Moreover, both MnTBAP and MnTE-2-PyP penetrated mitochondrial fractions of cortical cells. These data suggest that mitochondrial aconitase inactivation closely correlates with subsequent neuronal death following excitotoxicity produced by OGD or NMDA/KA exposure. Assessment of biological rather biochemical antioxidant activities better predicted neuroprotection by metalloporphyrins. Moreover, antioxidants that protect oxidant-sensitive mitochondrial targets such as aconitase may be useful as therapies for disease states involving excitotoxicity.     

Enhancement of superoxide dismutase activity in the leaves of white clover (Trifolium repens L.) in response to polyethylene glycol-induced water stress

Effects of polyethylene glycol (PEG)-induced water stress on the activities of total leaf superoxide dismutase (SOD) and chloroplast SOD (including thylakoid-bound SOD and stroma SOD) are described in white clover (Trifolium repens L.) grown in solution culture from rooted cuttings. Both leaf SOD and chloroplast SOD activities were markedly enhanced with increasing concentration of PEG stress, generating osmotic potentials around the roots 0, −0.5, −1.0, −1.5 MPa. The effects increased with time up to 72 h. Chloroplast Fe-containing SOD represented about 30% of the total leaf SOD activity in the control plants and a significant increase in chloroplast SOD activity was found during the stress period. This accounted for about 35.5–71.1% of the total leaf SOD activity. The proportion of chloroplast SOD in total leaf SOD not only increased with the decreasing of osmotic potential, but also increased with incubation time. Furthermore, the increase in thylakoid-bound SOD activity was much higher than that of stroma SOD in chloroplast of plants under water stress. The enhanced chloroplastic SOD activity, especially thylakoid-bound SOD activity, demonstrated in Trifolium repens suggests that Fe-SOD located in chloroplasts play a more important role than cytosolic Cu/Zn-containing SODs in scavenging O₂ ⁻.     

Acetylhomocysteine thiolactone protection against phosphamidon-induced alteration of regional superoxide dismutase activity in the central nervous system and its correlation with altered lipid peroxidation.

Phosphamidon intoxication (2 mg/kg body wt./day for 7 days) inhibited SOD activity, but enhanced the lipid peroxidation in various CNS regions. Administration of cithiolone (8 mg/kg body wt./day, ip for 7 days), however, elevated SOD activity and depleted lipid peroxidation. Interestingly, no significant change was observed either in SOD activity or in lipid peroxidation following simultaneous administration of phosphamidon and cithiolone.     

紫外辐射对菜豆不同叶位叶片光合及保护酶活性的影响

以菜豆为实验材料,用紫外辐射处理,测定植物各叶位叶片的生理活性。结果表明,紫外辐射条件下,叶片的光合、蒸腾速率先升高,随后迅速降低,辐射处理3 h对光合作用的抑制程度随叶位升高而增加;第1叶气孔导度和胞间CO2浓度随辐射处理略有增加,而第2、3叶的则先升高随后降低;辐射处理使叶绿素含量先降低随后有所增加,但明显增加叶片丙二醛(MDA)含量;紫外辐射处理后,叶片过氧化物酶(POD)活性先升高随后降低,紫外辐射3 h第1叶POD活性比对照降低26.9%,辐射使第1叶超氧化物歧化酶(SOD)活性略有升高,但明显降低第2、3叶的SOD活性。     

Properties of human kidney heme oxygenase: inhibition by synthetic heme analogues and metalloporphyrins

Heme oxygenase activities in human kidney microsomes were found to be from 0.238 to 0.620 nmol of bilirubin/mg/hr (mean 0.375, SD 0.134), which represent approximately 30% of activities determined for human adult liver. There was interindividual variation in heme oxygenase activity of a 2-5-fold difference. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from human kidney was identified on Western blots by its reaction with the anti-heme oxygenase liver antibody similar to the purified enzyme protein. Thus, a homology exists between human hepatic and kidney heme oxygenase. The enzyme activity was sensitive to inhibition by metalloporphyrins, such as tin-protoporphyrin IX and, to a lesser degree, by zinc and cobalt protoporphyrin IX. In a study of different synthetic heme analogues for in vitro inhibition of heme oxygenase, we found that replacement of iron by zinc in deuteroporphyrin IX 2,4 bis glycol dramatically potentiated the inhibition of heme oxygenase activity. This finding demonstrated that zinc deuteroporphyrin IX 2,4 bis glycol is a most potent inhibitor of heme oxygenase activity.     

Semisynthetic analogues of the marine cembranoid sarcophine as prostate and breast cancer migration inhibitors

Sarcophine (1) is a bioactive cembranoid diterpene isolated from the Red Sea soft coral Sarcophyton glaucum. Previous semisynthesis attempts resulted in decreased or complete loss of 1's anticancer activity. Sarcophine and analogues showed antimigratory activity against breast and prostate cancer cell lines. This encouraged further semisynthestic optimizations to improve its activity and establish a preliminary structure-activity relationship. Eight new and five known semisynthetic analogues were generated. These compounds were evaluated for their ability to inhibit growth, proliferation, and migration of the prostate and breast metastatic cancer cell lines PC-3 and MDA-MB-231, respectively. Most analogues exhibited enhanced antimigratory activity.     

Structural requirements for porphyrin inhibition of the hemin-controlled protein kinase and maintenance of protein synthesis in reticulocytes

The role of hemin in the maintenance of protein synthesis in reticulocyte lysates was examined by comparing the effects of various porphyrins and metalloporphyrins on the protein kinase activity of the hemin-controlled repressor and on protein synthesis. The porphyrin requirements for maintenance of protein synthesis were relatively specific. Iron and cobalt metalloporphyrins sustained protein synthesis whereas other metalloporphyrins, metal-deficient porphyrins, and non-porphyrin precursor and degradation products of protoporphyrin IX were ineffective. These same compounds were examined for their effectiveness in inhibiting the protein kinase activity of the hemin-controlled repressor with initiation factor 2 (eIF-2). Most of the metalloporphyrins and porphyrins tested were inhibitory. The presence of the iron atom in the porphyrin was not essential for inhibition, but the maintenance of the integrity of the porphyrin ring was imperative. The porphyrins which inhibited the hemin-regulated protein kinase contained vinyl groups or ethyl groups, or were protonated in the 2- and 4-positions of the porphyrin ring, whereas those with bulky or acidic groups in these positions were ineffective. Precursor and degradation products of protoporphyrin IX and synthetic porphyrins modified at other positions had no effect on the enzyme. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 exogenously added to a reticulocyte lysate; however, hemin sustained protein synthesis in the lysate, whereas protoporphyrin IX did not. These results suggest that regulation of the protein kinase phosphorylating the alpha subunit of eIF-2 is not the only point at which hemin modulates protein synthesis in reticulocytes and reticulocyte lysates, since a correlation between inhibition of protein synthesis, inhibition of protein kinase activity, and phosphorylation of eIF-2 is not observed with all porphyrins.     

Bicarbonate enhances peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical and scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme mimetics.

Much evidence exists for the increased peroxidase activity of copper, zinc superoxide dismutase (SOD1) in oxidant-induced diseases. In this study, we measured the peroxidase activity of SOD1 by monitoring the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF). Bicarbonate dramatically enhanced DCFH oxidation to DCF in a SOD1/H(2)O(2)/DCFH system. Peroxidase activity could be measured at a lower H(2)O(2) concentration ( approximately 1 microm). We propose that DCFH oxidation to DCF is a sensitive index for measuring the peroxidase activity of SOD1 and familial amyotrophic lateral sclerosis SOD1 mutants and that the carbonate radical anion (CO(3)) is responsible for oxidation of DCFH to DCF in the SOD1/H(2)O(2)/bicarbonate system. Bicarbonate enhanced H(2)O(2)-dependent oxidation of DCFH to DCF by spinal cord extracts of transgenic mice expressing SOD1(G93A). The SOD1/H(2)O(2)/HCO(3)(-)-dependent oxidation was mimicked by photolysis of an inorganic cobalt carbonato complex that generates CO(3). Metalloporphyrin antioxidants that are usually considered as SOD1 mimetic or peroxynitrite dismutase effectively scavenged the CO(3) radical. Implications of this reaction as a plausible protective mechanism in inflammatory cellular damage induced by peroxynitrite are discussed.     

SOD2-mediated effects induced by WR1065 and low-dose ionizing radiation on micronucleus formation in RKO human colon carcinoma cells

RKO36 cells exposed to either WR1065 or 10 cGy X rays show elevated SOD2 gene expression and SOD2 enzymatic activity. Cells challenged at this time with 2 Gy exhibit enhanced radiation resistance. This phenomenon has been identified as a delayed radioprotective effect or an adaptive response when induced by thiols or low-dose radiation, respectively. In this study we investigated the relative effectiveness of both WR1065 and low-dose radiation in reducing the incidence of radiation-induced micronucleus formation in binucleated RKO36 human colon carcinoma cells. The role of SOD2 in this process was assessed by measuring changes in enzymatic activity as a function of the inducing agent used, the level of protection afforded, and the inhibitory effects of short interfering RNA (SOD2 siRNA). Both WR1065 and 10 cGy X rays effectively induced a greater than threefold elevation in SOD2 activity 24 h after exposure. Cells irradiated at this time with 2 Gy exhibited a significant resistance to micronucleus formation (P < 0.05; Student's two-tailed t test). This protective effect was significantly inhibited in cells transfected with SOD2 siRNA. SOD2 played an important role in the adaptive/delayed radioprotective response by inhibiting the initiation of a superoxide anion-induced ROS cascade leading to enhanced mitochondrial and nuclear damages.