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Significance: After approximatelty three decades of research, two Mn(III) porphyrins (MnPs), MnTE-2-PyP5+ (BMX-010, AEOL10113) and MnTnBuOE-2-PyP5+ (BMX-001), have progressed to five clinical trials. In parallel, another similarly potent metal-based superoxide dismutase (SOD) mimic—Mn(II)pentaaza macrocycle, GC4419—has been tested in clinical trial on application, identical to that of MnTnBuOE-2-PyP5+—radioprotection of normal tissue in head and neck cancer patients. This clearly indicates that Mn complexes that target cellular redox environment have reached sufficient maturity for clinical applications. Recent Advances: While originally developed as SOD mimics, MnPs undergo intricate interactions with numerous redox-sensitive pathways, such as those involving nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), thereby impacting cellular transcriptional activity. An increasing amount of data support the notion that MnP/H2O2/glutathione (GSH)-driven catalysis of S-glutathionylation of protein cysteine, associated with modification of protein function, is a major action of MnPs on molecular level. Critical Issues: Differential effects of MnPs on normal versus tumor cells/tissues, which support their translation into clinic, arise from differences in their accumulation and redox environment of such tissues. This in turn results in different yields of MnP-driven modifications of proteins. Thus far, direct evidence for such modification of NF-κB, mitogen-activated protein kinases (MAPK), phosphatases, Nrf2, and endogenous antioxidative defenses was provided in tumor, while indirect evidence shows the modification of NF-κB and Nrf2 translational activities by MnPs in normal tissue. Future Directions: Studies that simultaneously explore differential effects in same animal are lacking, while they are essential for understanding of extremely intricate interactions of metal-based drugs with complex cellular networks of normal and cancer cells/tissues. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Antioxidants in translational medicine" by Harald H.H.W. Schmidt and colleague in the December 10, 2013 issue.

Significance: Superoxide dismutase (SOD) enzymes are indispensable and ubiquitous antioxidant defenses maintaining the steady-state levels of O2·−; no wonder, thus, that their mimics are remarkably efficacious in essentially any animal model of oxidative stress injuries thus far explored. Recent Advances: Structure-activity relationship (half-wave reduction potential [E1/ 2] versus log kcat), originally reported for Mn porphyrins (MnPs), is valid for any other class of SOD mimics, as it is dominated by the superoxide reduction and oxidation potential. The biocompatible E1/2 of ∼+300 mV versus normal hydrogen electrode (NHE) allows powerful SOD mimics as mild oxidants and antioxidants (alike O2·−) to readily traffic electrons among reactive species and signaling proteins, serving as fine mediators of redox-based signaling pathways. Based on similar thermodynamics, both SOD enzymes and their mimics undergo similar reactions, however, due to vastly different sterics, with different rate constants. Critical Issues: Although log kcat(O2·−) is a good measure of therapeutic potential of SOD mimics, discussions of their in vivo mechanisms of actions remain mostly of speculative character. Most recently, the therapeutic and mechanistic relevance of oxidation of ascorbate and glutathionylation and oxidation of protein thiols by MnP-based SOD mimics and subsequent inactivation of nuclear factor κB has been substantiated in rescuing normal and killing cancer cells. Interaction of MnPs with thiols seems to be, at least in part, involved in up-regulation of endogenous antioxidative defenses, leading to the healing of diseased cells. Future Directions: Mechanistic explorations of single and combined therapeutic strategies, along with studies of bioavailability and translational aspects, will comprise future work in optimizing redox-active drugs. Antioxid. Redox Signal. 20, 2372-2415. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative stress, a redox imbalance between the endogenous reactive species and antioxidant systems, is common to numerous pathological conditions such as cancer, central nervous system injuries, radiation injury, diabetes etc. Therefore, compounds able to reduce oxidative stress have been actively sought for over 3 decades. Superoxide is the major species involved in oxidative stress either in its own right or through its progeny, such as ONOO-, H2O2, •OH, CO3 • -, and •NO2. Hence, the very first compounds developed in the late 1970-ies were the superoxide dismutase (SOD) mimics. Thus far the most potent mimics have been the cationic meso Mn(III) N-substituted pyridylporphyrins and N,N’-disubstituted imidazolylporphyrins (MnPs), some of them with k cat(O2 .-) similar to the k cat of SOD enzymes. Most frequently studied are ortho isomers MnTE-2-PyP5+, MnTnHex-2-PyP5+, and MnTDE-2-ImP5+. The ability to disproportionate O2 .- parallels their ability to remove the other major oxidizing species, peroxynitrite, ONOO-. The same structural feature that gives rise to the high k cat(O2 .-) and k red(ONOO-), allows MnPs to strongly impact the activation of the redox-sensitive transcription factors, HIF-1α, NF-κB, AP-1, and SP-1, and therefore modify the excessive inflammatory and immune responses. Coupling with cellular reductants and other redox-active endogenous proteins seems to be involved in the actions of Mn porphyrins. While hydrophilic analogues, such as MnTE-2-PyP5+ and MnTDE-2-ImP5+ are potent in numerous animal models of diseases, the lipophilic analogues, such as MnTnHex-2-PyP5+, were developed to cross blood brain barrier and target central nervous system and critical cellular compartments, mitochondria. The modification of its structure, aimed to preserve the SOD-like potency and lipophilicity, and diminish the toxicity, has presently been pursued. The pulmonary radioprotection by MnTnHex-2-PyP5+ was the first efficacy study performed successfully with non-human primates. The Phase I toxicity clinical trials were done on amyotrophic lateral sclerosis patients with N,N’-diethylimidazolium analogue, MnTDE-2-ImP5+ (AEOL10150). Its aggressive development as a wide spectrum radioprotector by Aeolus Pharmaceuticals has been supported by USA Federal government. The latest generation of compounds, bearing oxygens in pyridyl substituents is presently under aggressive development for cancer and CNS injuries at Duke University and is supported by Duke Translational Research Institute, The Wallace H. Coulter Translational Partners Grant Program, Preston Robert Tisch Brain Tumor Center at Duke, and National Institute of Allergy and Infectious Diseases. Metal center of cationic MnPs easily accepts and donates electrons as exemplified in the catalysis of O2 .- dismutation. Thus such compounds may be equally good anti- and pro-oxidants; in either case the beneficial therapeutic effects may be observed. Moreover, while the in vivo effects may appear antioxidative, the mechanism of action of MnPs that produced such effects may be pro-oxidative; the most obvious example being the inhibition of NF-κB. The experimental data therefore teach us that we need to distinguish between the mechanism/s of action/s of MnPs and the effects we observe. A number of factors impact the type of action of MnPs leading to favorable therapeutic effects: levels of reactive species and oxygen, levels of endogenous antioxidants (enzymes and low-molecular compounds), levels of MnPs, their site of accumulation, and the mutual encounters of all of those species. The complexity of in vivo redox systems and the complex redox chemistry of MnPs challenge and motivate us to further our understanding of the physiology of the normal and diseased cell with ultimate goal to successfully treat human diseases. [Copyright &y& Elsevier]

Increased expression of Na+/H+ exchanger (NHE) and Na+,K+-ATPase activity have been demonstrated in diabetic nephropathy and are implicated in the development of hypertension. The aim of this study was to investigate the effect of a synthetic manganese porphyrin SOD mimic and peroxynitrite scavenger, Mn(III) 5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP) on the expression of NHE and Na+,K+-ATPase activity in the kidneys of streptozotocin (STZ) diabetic rats. MnTM-2-PyP administration (1 mg/kg/day) started immediately after STZ and lasted 2 months. Glucose and glycosylated hemoglobin levels were measured in blood. NHE-1 and NHE-3 isoform expression, Na+,K+-ATPase activity, and markers of ROS/RNS-induced damage were determined in kidney homogenates. Diabetes caused lipid peroxidation, inactivation of aconitase, and increase of nitrotyrosine, which paralleled an increase in NHE-1 and NHE-3 expression and Na+,K+-ATPase activity. MnTM-2-PyP treatment had no effect on blood glucose and glycosylated hemoglobin, but suppressed lipid peroxidation and nitrotyrosine, protected aconitase against inactivation, and reversed the induction of NHE-1 and NHE-3 isoforms. Na+/H+ exchanger is under the control of redox-based cellular transcriptional activity, including members of the SP family of transcription factors. Mn(III) alkylpyridylporphyrins were previously found to inhibit activation of major transcription factors, including SP-1 via scavenging of signaling ROS/RNS. Therefore, our data suggest that, by reducing the levels of ROS/RNS, MnTM-2-PyP might interfere with signaling pathways responsible for NHE up-regulation. [ABSTRACT FROM AUTHOR]

The isocitrate dehydrogenases (ICDs) catalyse the oxidative decarboxylation of isocitrate to alpha-ketoglutarate and can use either NAD+ or NADP+ as a cofactor. Recent studies demonstrate that the NADP+-dependent isocitrate dehydrogenase, as a source of electrons for cellular antioxidants, is important for protection against oxidative damage. ICD, however, is susceptible to oxidative inactivation, which in turn compromises cellular antioxidant defense. This study investigates the effect of a superoxide dismutase (SOD) mimic, MnTM-2-PyP5+, on the inactivation of NADP+-dependent ICD in SOD-deficient Escherichia coli and in diabetic rats. The findings show that E. coli ICD is inactivated by superoxide, but the inactivated enzyme is replaced by de novo protein synthesis. Statistically significant decrease of ICD activity was found in the hearts of diabetic rats. MnTM-2-PyP5+ protected ICD in both models. [ABSTRACT FROM AUTHOR]

Abstract: Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP5+), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP5+ in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 μg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-β subunit as a key protein induced by MnTnHex-2-PyP5+ treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP5+ protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury. [Copyright &y& Elsevier]

Purpose: To determine whether administration of a catalytic antioxidant, Mn(III) tetrakis(N,N'-diethylimidazolium-2-yl) porphyrin, AEOL 10150, with superoxide dismutase (SOD) mimetic properties, reduces the severity of radiation-induced injury to the lung from single-dose irradiation (RT) of 28 Gy.Methods and Materials: Rats were randomly divided into four different dose groups (0, 1, 10, and 30 mg/kg/day of AEOL 10150), receiving either short-term (1 week) or long-term (10 weeks) drug administration via osmotic pumps. Rats received single-dose irradiation (RT) of 28 Gy to the right hemithorax. Breathing rates, body weights, blood samples, histopathology, and immunohistochemistry were used to assess lung damage.Results: There was no significant difference in any of the study endpoints between the irradiated controls and the three groups receiving RT and short-term administration of AEOL 10150. For the long-term administration, functional determinants of lung damage 20 weeks postradiation were significantly worse for RT + phosphate-buffered saline (PBS) and RT + 1 mg/kg/day of AEOL 10150 as compared with the irradiated groups treated with higher doses of AEOL 10150 (10 or 30 mg/kg/day). Lung histology at 20 weeks revealed a significant decrease in structural damage and collagen deposition in rats receiving 10 or 30 mg/kg/day after radiation in comparison to the RT + PBS and 1 mg/kg/day groups. Immunohistochemistry demonstrated a significant reduction in macrophage accumulation, oxidative stress, and hypoxia in rats receiving AEOL 10150 (10 or 30 mg/kg/day) after lung irradiation compared with the RT + PBS and 1 mg/kg/day groups.Conclusions: The chronic administration of a novel catalytic antioxidant, AEOL 10150, demonstrates a significant protective effect from radiation-induced lung injury. AEOL 10150 has its primary impact on the cascade of events after irradiation, and adding the drug before irradiation and its short-term administration have no significant additional benefits. [ABSTRACT FROM AUTHOR]

Purpose: To determine the effect of the superoxide dismutase mimetic Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+) on tumor radioresponsiveness. Methods and Materials: Various rodent tumor (4T1, R3230, B16) and endothelial (SVEC) cell lines were exposed to MnTE-2-PyP5+ and assayed for viability and radiosensitivity in vitro. Next, tumors were treated with radiation and MnTE-2-PyP5+ in vivo, and the effects on tumor growth and vascularity were monitored. Results: In vitro, MnTE-2-PyP5+ was not significantly cytotoxic. However, at concentrations as low as 2 μmol/L it caused 100% inhibition of secretion by tumor cells of cytokines protective of irradiated endothelial cells. In vivo, combined treatment with radiation and MnTE-2-PyP5+ achieved synergistic tumor devascularization, reducing vascular density by 78.7% within 72 h of radiotherapy (p < 0.05 vs. radiation or drug alone). Co-treatment of tumors also resulted in synergistic antitumor effects, extending tumor growth delay by 9 days (p < 0.01). Conclusions: These studies support the conclusion that MnTE-2-PyP5+, which has been shown to protect normal tissues from radiation injury, can also improve tumor control through augmenting radiation-induced damage to the tumor vasculature. [Copyright &y& Elsevier]

Radiation therapy (RT) is an important therapeutic modality in the treatment of thoracic tumors. The maximum doses to these tumors are often limited by the radiation tolerance of lung tissues. Lung injury from ionizing radiation is believed to be a consequence of oxidative stress and a cascade of cytokine activity. Superoxide dismutase (SOD) is a key enzyme in cellular defenses against oxidative damage. The objective of this study was to determine whether the SOD mimetic AEOL 10113 [manganese (III) mesotetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP5+)] increases the tolerance of lung to ionizing radiation. AEOL 10113 was able to significantly reduce the severity of RT-induced lung injury. This was strongly supported with histopathology results and measurements of collagen deposition (hydroxyproline content). There was a significant reduction in the plasma level of the profibrogenic cytokine transforming growth factor-β (TGF-β) in the group of rats receiving RT + AEOL 10113. In conclusion, the novel SOD mimetic, AEOL 10113, demonstrates a significant protective effect from radiation-induced lung injury. [Copyright &y& Elsevier]

Characterizes a manganese(III) complex of biliverdin IX dimethyl ester. Attachment of each trivalent manganese to four pyrrolic nitrogen of one ester molecule; Occupant of the fifth coordination site of the manganese.

Examines the relationship among redox potentionals, proton dissociation constants of pyrrolic nitrogens, abd superoxide dismutating activities of Mn(III) and Fe(III) water-soluble porphyrins. Comparisons of meso-substituted ortho, meta and para groups; Anionic and cationic iron and Mn porphyrins; Use of metalloporphyrin as photosensitizer.

Examines the synthesis and superoxide dismuting activities of partially-chlorinated derivatives of manganese (III) meso-tetrakis(N-ethylpyridium-2-yl)porphyrin. Metal centered redox potentials; Ethylation and metallation; Relationship between redox properties and superoxide dismuting-like activities.

Examines the interaction of ferrioxamine B (FeHDFB+) with various host ionophore structures to form a host-guest complex in the second-sphere coordination shell. Method used in the extraction of ferrioxamine B and other cations; Effects of the host-guest complex on the nature of the counterion.

Examines the influence of the anion X- on the stability of second-sphere coordination of ferrioxamine B with cis-dicyclohexano-18-crown-6 in Chloroform. Distribution equilibria of the complexes; Extraction equilibria; Host-guest equilibria.

Reports on the hydrolysis of ferrioxamine B in aqueous micellar solution. Fundamental principles governing the complex behavior of natural systems; Investigation of the iron(III) exchange between the hydrophilic siderephore deferriferrioxamine B and benzohydroxamic acid in aqueous micellar solution.

This Editorial has the intention to stress the complex chemistry and biology of redox-active compounds, regarded as SOD mimics. It further aims to caution the researchers of the importance of being up-to-date with the present knowledge on such compounds and their cellular redox biology when coming up with their conclusions, based on the particular species involved in their studies. Antioxid. Redox Signal. 20, 2323-2325. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate how modifications at the periphery of the porphyrin ring affect the anticancer activity of Mn porphyrins (MnPs)-based SOD mimics. Six compounds: MnTE-2-PyP with a short ethyl chain on the pyridyl ring; MnTnHexOE-2-PyP and MnTnOct-2-PyP with linear 8-atom alkyl chains, but the former with an oxygen atom within the alkyl chain; MnTE-2-PyPhP and MnTPhE-2-PyP with pyridyl and phenyl substituents, were investigated. Cytotoxicity was studied using pII and MDA-MB-231 cancer cell lines. Viability was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide) assay and cell proliferation was determined by the sulforhodamine B assay. Cellular uptake was increased with the increase of the lipophilicity of the compounds, whereas reduction potential (E½) of the Mn(III)/Mn(II) redox couple shifted away from the optimal value for efficient redox cycling with ascorbate, necessary for ROS production. Amphiphilic MnPs, however, exerted anticancer activity by a mechanism not involving ROS. Two different processes account for MnPs cytotoxicity. MnPs with appropriate E½ act via a ROS-dependent mechanism. Amphiphilic MnPs with suitable structure damage sensitive cellular constituents, leading to the suppression of proliferation and loss of viability. Design of compounds interacting directly with sensitive cellular targets is highly promising in the development of anticancer drugs with high selectivity and specificity. [ABSTRACT FROM AUTHOR]

Uterine serous carcinoma is a highly aggressive non-endometrioid subtype of endometrial cancer with poor survival rates overall, creating a strong need for new therapeutic strategies to improve outcomes. High-dose ascorbate (vitamin C) has been shown to inhibit cell proliferation and tumor growth in multiple preclinical models and has shown promising anti-tumor activity in combination with chemotherapy, with a favorable safety profile. We aimed to study the anti-tumor effects of ascorbate and its synergistic effect with carboplatin on uterine serous carcinoma cells. Cell proliferation was evaluated by MTT and colony formation assays in ARK1, ARK2 and SPEC2 cells. Cellular stress, antioxidant ability, cleaved caspase 3 activity and adhesion were measured by ELISA assays. Cell cycle was detected by Cellometer. Invasion was measured using a wound healing assay. Changes in protein expression were determined by Western immunoblotting. High-dose ascorbate significantly inhibited cell proliferation, caused cell cycle arrest, induced cellular stress, and apoptosis, increased DNA damage, and suppressed cell invasion in ARK1 and SPEC2 cells. Treatment of both cells with 1 mM N -acetylcysteine reversed ascorbate-induced apoptosis and inhibition of cell proliferation. The combination of ascorbate and carboplatin produced significant synergistic effects in inhibiting cell proliferation and invasion, inducing cellular stress, causing DNA damage, and enhancing cleaved caspase 3 levels compared to each compound alone in both cells. Ascorbate has potent antitumor activity and acts synergistically with carboplatin through its pro-oxidant effects. Clinical trials of ascorbate combined with carboplatin as adjuvant treatment of uterine serous carcinoma are worth exploring. • High-dose ascorbate exhibits antiproliferative activity in uterine serous carcinoma cells through its pro-oxidant activity. • High-dose ascorbate effectively reduces adhesive and invasive abilities in uterine serous carcinoma cells. • High-dose ascorbate synergistically increases the sensitivity to carboplatin in uterine serous carcinoma cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Oxidative stress affects several features of cancer, including those related to metastases formation. Non-small cell lung cancer (NSCLC) is commonly detected in advanced stages when metastases have already developed. MnTnBuOE-2-PyP5+ (MnBuOE, BMX-001) is a redox-active Mn porphyrin-based drug, and a mimic of superoxide dismutase, that is currently in clinical trials for several types of cancer. Nevertheless, there is still insufficient information regarding the effect of this compound on lung cancer. Here, we aim at filling this gap by assessing the impact of MnBuOE alone or combined with cisplatin—a frequently used platinum-based drug in chemotherapy—on the viability and migration of two NSCLC cell lines. Although MnBuOE only displayed moderate effects on suppressing cancer cell viability, it significantly reduced collective and individual migration and invasion in both types of cells, especially when combined with cisplatin. The data thus support the therapeutic potential of MnBuOE as an anti-metastatic drug for the treatment of NSCLC. Manganese(III) porphyrin MnTnBuOE-2-PyP5+ (MnBuOE, BMX-001) is a third-generation redox-active cationic substituted pyridylporphyrin-based drug with a good safety/toxicity profile that has been studied in several types of cancer. It is currently in four phase I/II clinical trials on patients suffering from glioma, head and neck cancer, anal squamous cell carcinoma and multiple brain metastases. There is yet an insufficient understanding of the impact of MnBuOE on lung cancer. Therefore, this study aims to fill this gap by demonstrating the effects of MnBuOE on non-small cell lung cancer (NSCLC) A549 and H1975 cell lines. The cytotoxicity of MnBuOE alone or combined with cisplatin was evaluated by crystal violet (CV) and/or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-Tetrazolium (MTS) reduction assays. Intracellular ROS levels were assessed using two fluorescent probes. Furthermore, the impact of MnBuOE alone or in combination with cisplatin on collective cell migration, individual chemotactic migration and chemoinvasion was assessed using the wound-healing and transwell assays. The expression of genes related to migration and invasion was assessed through RT-qPCR. While MnBuOE alone decreased H1975 cell viability at high concentrations, when combined with cisplatin it markedly reduced the viability of the more invasive H1975 cell line but not of A549 cell line. However, MnBuOE alone significantly decreased the migration of both cell lines. The anti-migratory effect was more pronounced when MnBuOE was combined with cisplatin. Finally, MnBuOE alone or combined with cisplatin significantly reduced cell invasion. MnBuOE alone or combined with cisplatin downregulated MMP2, MMP9, VIM, EGFR and VEGFA and upregulated CDH1 in both cell lines. Overall, our data demonstrate the anti-metastatic potential of MnBuOE for the treatment of NSCLC. [ABSTRACT FROM AUTHOR]

Sulfite is generated in vivo as a result of metabolism of sulfur containing amino acids methionine and cysteine. Sulfite is consumed as antioxidant/preservative in food, beverages and medications. We are also consuming it through exposure to major environmental pollutant - sulfur dioxide. The enzyme sulfite oxidase (SO) is responsible for the detoxification of sulfite to sulfate, which is subsequently excreted into urine. The SO deficiency is one of the most common causes of sulfite hypersensitivity and toxicity. Herein we report for the first time the ability of cationic Mn(III) N-substituted pyridyl(imidazolyl)-porphyrins (MnPs) to catalyze sulfite oxidation. Originally developed as SOD mimics, MnPs operate at Mn III P/Mn II P reduction potential that is highly compatible with reduction potentials of numerous biomolecules and reactive species, among them superoxide, peroxynitrite, oxygen, hydrogen peroxide, hypochlorite, nitric oxide, thiols, and ascorbate. At least 4 oxidation states of Mn in MnPs (+2, +3, +4 and +5) are involved in their actions which may occur via one- or two-electron transfers. We are here providing evidence, based on spectrophotometric measurements, that several analogs of MnP are able to catalyze sulfite oxidation one-electronically via employing Mn III P/Mn II P redox couple. The reaction proceeds through generation of sulfite radical anion as an intermediate species characterized by electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Clark electrode measurements of the MnP/sulfite system indicated that molecular oxygen is also involved, both in regeneration of Mn III P and further oxidation of sulfite radical to sulfate. Such redox further substantiates tight connections of MnP bioeffects to sulfur biology and indicates that we are still in infancy with regards to fully understanding the plethora of reactions of redox-active drugs. The MnP/sulfite cycling is the reminiscence of redox behavior of MnP/ascorbate system, which has been used successfully in tumor models, suggesting its therapeutic potential. Support: NC Biotechnology# 2016-BIG-6518 [ABSTRACT FROM AUTHOR]

Coordination of metal ions by the tetrapyrrolic macrocyclic ring of porphyrin-based photosensitizers (PSs) affects their photophysical properties and consequently, their photodynamic activity. Diamagnetic metals increase the singlet oxygen quantum yield while paramagnetic metals have the opposite effect. Since singlet oxygen is considered the main cell-damaging species in photodynamic therapy (PDT), the nature of the chelated cation would directly affect PDT efficacy. This expectation, however, is not always supported by experimental results and numerous exceptions have been reported. Understanding the effect of the chelated metal is hindered because different chelators were used. The aim of this work was to investigate the effect of the nature of chelated cation on the photophysical and photodynamic properties of metalloporphyrins, using the same tetrapyrrole core as a chelator of Ag(II), Cu(II), Fe(III), In(III), Mn(III), or Zn(II). Results demonstrated that with the exception of Ag(II), all paramagnetic metalloporphyrins were inefficient as generators of singlet oxygen and did not act as PSs. In contrast, the coordination of diamagnetic ions produced highly efficient PSs. The unexpected photodynamic activity of the Ag(II)-containing porphyrin was attributed to reduction of the chelated Ag(II) to Ag(I) or to demetallation of the complex, caused by cellular reductants and/or by exposure to light. Our results indicate that in biological systems, where PSs localize to various organelles and are subjected to the action of enzymes, reactive metabolites, and reducing or oxidizing agents, their physicochemical and photosensitizing properties change. Consequently, the photophysical properties alone cannot predict the anticancer efficacy of a PS. [ABSTRACT FROM AUTHOR]

Increased interest in clinical application of photodynamic therapy (PDT) in various medical fields poses a demand for better understanding of processes triggered by photo-treatment. Most of the work on PDT performed so far has focused on the immediate effects of photo-treatment. It is generally accepted that cellular damage occurs during light exposure and within a short period thereafter. If cells are not killed during the PDT, they might recover, depending on the extent of the photo-induced damage. Little is known, however, about the relationship between the properties of photosensitizers (PSs) and the delayed consequences of PDT. The aim of this work was to investigate cellular responses to sub-lethal photodynamic treatment and how toxicogenic potency may be affected by molecular features of the PS. Results demonstrated that for cationic porphyrin-based PSs, lipophilicity is the main factor determining the fate of the cells in the 24-hour post-illumination period. PSs with amphiphilic properties initiated oxidative reactions that continued in the dark, long after light exposure, and caused suppression of metabolism and loss of cell viability with concomitant changes in electrophoretic mobility of proteins, including caspases. Apoptotic activity was not stimulated in the post-illumination period. This study demonstrated that in PDT mediated by amphiphilic cationic metalloporphyrin PSs, even when immediate photo-damage is relatively mild, destructive oxidative processes initiated during PDT continue in the absence of light to substantially impair metabolism, and that post-illumination protein modification may modify utilization of cell death pathways. [ABSTRACT FROM AUTHOR]

Aims: The susceptibility of CA1 over CA3 to damage from cerebral ischemia may be related to the differences in reactive oxygen species (ROS) production/removal between the two hippocampal subfields. We aimed to measure CA1/CA3 differences in net ROS production in real time in the first 30 min of reperfusion in pyramidal cells. We aimed to determine the underlying cause of the differential vulnerability of CA1 and CA3. Results: Real-time determinations of mitochondrial H2O2 and, independently, glutathione (GSH) redox status from roGFP-based probes in individual pyramidal cells in organotypic hippocampal cultures during oxygen-glucose deprivation (OGD)-reperfusion (RP) demonstrate a significantly more oxidizing environment during RP in CA1 than CA3 mitochondria. Protein levels (immunohistochemistry and Western blots), roGFP2-based probe measurements during controlled mitochondrial production of ROS, and thioredoxin reductase (TrxR) inhibition by auranofin are consistent with a more effective mitochondrial thioredoxin (Trx) system in CA3. Inhibition of TrxR eliminates the differences in redox status and cell death between the regions. Overexpression of cytosolic Trx1 does not influence mitochondrial H2O2 production. Innovation: Real-time changes of mitochondrial H2O2 and GSH in tissue cultures during early RP, and also during controlled production of superoxide and peroxide, reveal significant differences between CA1 and CA3. The mitochondrial Trx system is responsible for the observed differences during RP as well as for delayed cell death 18 h afterward. Conclusion: Greater mitochondrial Trx efficacy in CA3 pyramidal cells results in less vulnerability to ischemia/reperfusion because of the less oxidizing environment in CA3 mitochondria during RP. Antioxid. Redox Signal. 27, 534-549. [ABSTRACT FROM AUTHOR]

Myocardial ischemia-reperfusion injury (I/R) causes damage to cardiomyocytes through oxidative stress and apoptosis. We investigated the cardioprotective effects of MnTnBuOE-2-PyP5+ (BMX-001), a superoxide dismutase mimic, in an in vitro model of I/R injury in H9c2 cardiomyocytes. We found that BMX-001 protected against hypoxia/reoxygenation (H/R)-induced oxidative stress, as evident by a significant reduction in intracellular and mitochondrial superoxide levels. BMX-001 pre-treatment also reduced H/R-induced cardiomyocyte apoptosis, as marked by a reduction in TUNEL-positive cells. We further demonstrated that BMX-001 pre-treatment significantly improved mitochondrial function, particularly O2 consumption, in mouse adult cardiomyocytes subjected to H/R. BMX-001 treatment also attenuated cardiolipin peroxidation, 4-hydroxynonenal (4-HNE) level, and 4-HNE adducted proteins following H/R injury. Finally, the pre-treatment with BMX-001 improved cell viability and lactate dehydrogenase (LDH) activity in H9c2 cells following H/R injury. Our findings suggest that BMX-001 has therapeutic potential as a cardioprotective agent against oxidative stress-induced H/R damage in H9c2 cardiomyocytes. [ABSTRACT FROM AUTHOR]

Simple Summary: Although heart failure is a long-term chronic disease, it can also lead to arrhythmia, triggering cardiac arrest, and eventually death. Our study is a randomized and blinded preclinical assessment of the efficacy of the Mn porphyrin compound, MnTnBuOE-2-PyP5+ (BMX-001), commonly known as a superoxide dismutase mimic, in both mouse and rat models of cardiac arrest and resuscitation. Our study indicates that MnTnBuOE-2-PyP5+ improves survival, neurologic function recovery, and behavioral performance in animals suffering from cardiac arrest. The results support the development of Mn Porphyrin-based therapeutics for the treatment of heart failure and cardiac arrest. Introduction Cardiac arrest (CA) and resuscitation induces global cerebral ischemia and reperfusion, causing neurologic deficits or death. Manganese porphyrins, superoxide dismutase mimics, are reportedly able to effectively reduce ischemic injury in brain, kidney, and other tissues. This study evaluates the efficacy of a third generation lipophilic Mn porphyrin, MnTnBuOE-2-PyP5+, Mn(III) ortho meso-tetrakis (N-n-butoxyethylpyridinium-2-yl)porphyrin (MnBuOE, BMX-001), in both mouse and rat models of CA. Methods Forty-eight animals were subjected to 8 min of CA and resuscitated subsequently by chest compression and epinephrine infusion. Vehicle or MnBuOE was given immediately after resuscitation followed by daily subcutaneous injections. Body weight, spontaneous activity, neurologic deficits, rotarod performance, and neuronal death were assessed. Kidney tubular injury was assessed in CA mice. Data were collected by the investigators who were blinded to the treatment groups. Results Vehicle mice had a mortality of 20%, which was reduced by 50% by MnBuOE. All CA mice had body weight loss, spontaneous activity decline, neurologic deficits, and decreased rotarod performance that were significantly improved at three days post MnBuOE daily treatment. MnBuOE treatment reduced cortical neuronal death and kidney tubular injury in mice (p < 0.05) but not hippocampus neuronal death (23% MnBuOE vs. 34% vehicle group, p = 0.49). In rats, they had a better body-weight recovery and increased rotarod latency after MnBuOE treatment when compared to vehicle group (p < 0.01 vs. vehicle). MnBuOE-treated rats had a low percentage of hippocampus neuronal death (39% MnBuOE vs. 49% vehicle group, p = 0.21) and less tubular injury (p < 0.05) relative to vehicle group. Conclusions We demonstrated the ability of MnBuOE to improve post-CA survival, as well as functional outcomes in both mice and rats, which jointly account for the improvement not only of brain function but also of the overall wellbeing of the animals. While MnBuOE bears therapeutic potential for treating CA patients, the females and the animals with comorbidities must be further evaluated before advancing toward clinical trials. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) cause oxidative stress by generating reactive aldehydes known as 4-hydroxynonenal (4-HNE). 4-HNE modifies protein via covalent adduction; however, little is known about the degradation mechanism of 4-HNE-adducted proteins. Autophagy is a dynamic process that maintains cellular homeostasis by removing damaged organelles and proteins. In this study, we determined the role of a superoxide dismutase (SOD) mimetic MnTnBuOE-2-PyP5+ (MnP, BMX-001) on rotenone-induced 4-HNE aggresome degradation in HL-1 cardiomyocytes. A rotenone treatment (500 nM) given for 24 h demonstrated both increased ROS and 4-HNE aggresome accumulation in HL-1 cardiomyocytes. In addition, cardiomyocytes treated with rotenone displayed an increase in the autophagy marker LC3-II, as shown by immunoblotting and immunofluorescence. A pre-treatment with MnP (20 µM) for 24 h attenuated rotenone-induced ROS formation. An MnP pre-treatment showed decreased 4-HNE aggresomes and LC3-II formation. A rotenone-induced increase in autophagosomes was attenuated by a pre-treatment with MnP, as shown by fluorescent-tagged LC3 (tfLC3). Rotenone increased tubulin hyperacetylation through the ROS-mediated pathway, which was attenuated by MnP. The disruption of autophagy caused HL-1 cell death because a 3-methyladenine inhibitor of autophagosomes caused reduced cell death. Yet, rapamycin, an inducer of autophagy, increased cell death. These results indicated that a pre-treatment with MnP decreased rotenone-induced 4-HNE aggresomes by enhancing the degradation process. [ABSTRACT FROM AUTHOR]

The manganese porphyrin, manganese (III) meso -tetrakis N -ethylpyridinium-2-yl porphyrin (MnTE-2-PyP 5+ ), acts as a pro-oxidant in the presence of intracellular H 2 O 2 . Mitochondria are the most prominent source of intracellular ROS and important regulators of the intrinsic apoptotic pathway. Due to the increased oxidants near and within the mitochondria, we hypothesized that the mitochondria are a target of the pro-oxidative activity of MnTE-2-PyP 5+ and that we could exploit this effect to enhance the chemotherapeutic response in lymphoma. In this study, we demonstrate that MnTE-2-PyP 5+ modulates the mitochondrial redox environment and sensitizes lymphoma cells to antilymphoma chemotherapeutics. MnTE-2-PyP 5+ increased dexamethasone-induced mitochondrial ROS and oxidation of the mitochondrial glutathione pool in lymphoma cells. The combination treatment induced glutathionylation of Complexes I, III, and IV in the electron transport chain, and decreased the activity of Complexes I and III, but not the activity of Complex IV. Treatment with the porphyrin and dexamethasone also decreased cellular ATP levels. Rho(0) malignant T-cells with impaired mitochondrial electron transport chain function were less sensitive to the combination treatment than wild-type cells. These findings suggest that mitochondria are important for the porphyrin’s ability to enhance cell death. MnTE-2-PyP 5+ also augmented the effects of 2-deoxy- D- glucose (2DG), an antiglycolytic agent. In combination with 2DG, MnTE-2-PyP 5+ increased protein glutathionylation, decreased ATP levels more than 2DG treatment alone, and enhanced 2DG-induced cell death in primary B-ALL cells. MnTE-2-PyP 5+ did not enhance dexamethasone- or 2DG-induced cell death in normal cells. Our findings suggest that MnTE-2-PyP 5+ has potential as an adjuvant for the treatment of hematologic malignancies. [ABSTRACT FROM AUTHOR]

Abstract: Resistance to therapy-mediated apoptosis in inflammatory breast cancer, an aggressive and distinct subtype of breast cancer, was recently attributed to increased superoxide dismutase (SOD) expression, glutathione (GSH) content, and decreased accumulation of reactive species. In this study, we demonstrate the unique ability of two Mn(III) N-substituted pyridylporphyrin (MnP)-based SOD mimics (MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+) to catalyze oxidation of ascorbate, leading to the production of excessive levels of peroxide, and in turn cell death. The accumulation of peroxide, as a consequence of MnP+ascorbate treatment, was fully reversed by the administration of exogenous catalase, showing that hydrogen peroxide is essential for cell death. Cell death as a consequence of the action of MnP+ascorbate corresponded to decreases in GSH levels, prosurvival signaling (p-NF-κB, p-ERK1/2), and in expression of X-linked inhibitor of apoptosis protein, the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and annexin V staining, administration of a pan-caspase inhibitor, Q-VD-OPh, did not reverse the observed cytotoxicity. MnP+ascorbate-treated cells showed nuclear translocation of apoptosis-inducing factor, suggesting the possibility of a mechanism of caspase-independent cell death. Pharmacological ascorbate has already shown promise in recently completed phase I clinical trials, in which its oxidation and subsequent peroxide formation was catalyzed by endogenous metalloproteins. The catalysis of ascorbate oxidation by an optimized metal-based catalyst (such as MnP) carries a large therapeutic potential as an anticancer agent by itself or in combination with other modalities such as radio- and chemotherapy. [Copyright &y& Elsevier]

Abstract: Peroxynitrite (ONOO−) formation in mitochondria may be favored due to the constant supply of superoxide radical () by the electron transport chain plus the facile diffusion of nitric oxide ( NO) to this organelle. Herein, a model system of submitochondrial particles (SMP) in the presence of succinate plus the respiratory inhibitor antimycin A (to increase rates) and the NO-donor NOC-7 was studied to directly establish and quantitate peroxynitrite by a multiplicity of methods including chemiluminescence, fluorescence and immunochemical analysis. While all the tested probes revealed peroxynitrite at near stoichiometric levels with respect to its precursor radicals, coumarin boronic acid (a probe that directly reacts with peroxynitrite) had the more straightforward oxidation profile from -forming SMP as a function of the NO flux. Interestingly, immunospintrapping studies verified protein radical generation in SMP by peroxynitrite. Substrate-supplemented SMP also reduced Mn(III)porphyrins (MnP) to Mn(II)P under physiologically-relevant oxygen levels (3–30μM); then, Mn(II)P were capable to reduce peroxynitrite and protect SMP from the inhibition of complex I-dependent oxygen consumption and protein radical formation and nitration of membranes. The data directly support the formation of peroxynitrite in mitochondria and demonstrate that MnP can undergo a catalytic redox cycle to neutralize peroxynitrite-dependent mitochondrial oxidative damage. [Copyright &y& Elsevier]

Abstract: Cell proliferation is notably dependent on energy supply and generation of reducing equivalents in the form of NADPH for reductive biosynthesis. Blockage of pathways generating energy and reducing equivalents has proved successful for cancer treatment. We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP4+) can act as photosensitizers, preventing cell proliferation and causing cell death in vitro. The present study demonstrates that upon illumination, ZnTM-3-PyP inactivates glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, NADP+ -linked isocitrate dehydrogenase, aconitase, and fumarase in adenocarcinoma LS174T cells. ZnTM-3-PyP4+ was significantly more effective than hematoporphyrin derivative (HpD) for inactivation of all enzymes, except aconitase and isocitrate dehydrogenase. Enzyme inactivation was accompanied by aggregation, presumably due to protein cross-linking of some of the enzymes tested. Inactivation of metabolic enzymes caused disruption of cancer cells'' metabolism and is likely to be one of the major reasons for antiproliferative activity of ZnTM-3-PyP. [Copyright &y& Elsevier]

We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP4 + ) 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-PyP4 + 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-PyP4 + -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. [ABSTRACT FROM AUTHOR]

Normal tissue injury poses a major limitation to the success of radiation therapy (RT) in the treatment of solid tumors. We propose that radiation-induced lung injury is a result of chronic oxidative stress propagated by hypoxia-induced macrophage activation and cytokine production. Therefore, the objective of our study was two-fold. First, in vivo studies were conducted to support our hypothesis suggesting radiation injury is characterized by chronic hypoxia associated with increased macrophage infiltration/activation and pro-fibrogenic/angiogenic cytokine production. Second, we investigated the proposed mechanism of radiation injury in vitro. We demonstrate that hypoxia (0.5% O2) elicits macrophages to produce higher levels of , TGF-β, and VEGF than normoxia. Our hypothesis that is contributing to increased macrophage cytokine production was supported by a significant reduction in TGF-β and VEGF when redox signaling was minimized using a small molecular weight metalloporphyrin antioxidant, MnTE-2-PyP5+ . [ABSTRACT FROM AUTHOR]

Abstract: The reduction of manganese(III) meso-tetrakis((N-ethyl)pyridinium-2-yl)porphyrin (MnTE-2-PyP) to manganese(II) was catalyzed by flavoenzymes such as xanthine oxidase and glucose oxidase, and by Complex I and Complex II of the mitochondrial electron transport chain. The reduced manganese porphyrin has been previously shown to react rapidly with superoxide and carbonate radical anion. Herein, we describe the reaction of a reduced manganese porphyrin with peroxynitrite that proceeds as a two-electron process, has a rate constant greater than 7 × 106 M−1 s−1 (at pH 7.25 and 37°C), and produces nitrite and the Mn(IV)Porphyrin. The Mn(II)/Mn(IV) redox cycle was used to divert peroxynitrite from the inactivation of succinate dehydrogenase. In a typical experiment, 5 μM MnTE-2-PyP in the presence of excess succinate was able to protect the succinate dehydrogenase and succinate oxidase activities of submitochondrial particles challenged with a cumulative dose of 140 μM peroxynitrite infused in the course of 2 h. Other MnPorphyrins that are reduced more slowly do not provide as much protection underscoring the rate limiting character of the reduction step. The data presented here serve to rationalize the pharmacological action of MnPorphyrins as peroxynitrite reduction catalysts in vivo and opens avenues for the development of MnPorphyrins to protect mitochondria from oxidative damage. [Copyright &y& Elsevier]

The success of photodynamic therapy (PDT), as a minimally invasive approach, in treating both neoplastic and non-neoplastic diseases has stimulated the search for new compounds with potential application in PDT. We have previously reported that Zn(II) N -alkylpyridylporphyrins (ZnTM-2(3,4)-PyP 4+ and ZnTE-2-PyP 4+ ) can act as photosensitizers and kill antibiotic-resistant bacteria. This study investigated the photosensitizing effects of the isomers of ZnTMPyP 4+ (ZnTM-2(3,4)-PyP 4+ ) and respective ligands on a human colon adenocarcinoma cell line. At 10 µM and 30 min of illumination the isomeric porphyrins completely inhibited cell growth, and at 20 µM killed approximately 50% of the cancer cells. All these effects were entirely light-dependent. The isomers of the ZnTMPyP 4+ and the respective ligands show high photosensitizing efficiency and no toxicity in the dark. Their efficacy as photosensitizers is comparable to that of hematoporphyrin derivative (HpD). [ABSTRACT FROM AUTHOR]

Aims: We aim here to demonstrate that radiation (RT) enhances tumor sensitization by only those Mn complexes that are redox active and cycle with ascorbate (Asc), thereby producing H2O2 and utilizing it subsequently in protein S-glutathionylation in a glutathione peroxidase (GPx)-like manner. In turn, such compounds affect cellular redox environment, described by glutathione disulfide (GSSG)/glutathione (GSH) ratio, and tumor growth. To achieve our goal, we tested several Mn complexes of different chemical and physical properties in cellular and animal flank models of 4T1 breast cancer cell. Four other cancer cell lines were used to substantiate key findings. Results: Joint administration of cationic Mn porphyrin (MnP)-based redox active compounds, MnTE-2-PyP5+ or MnTnBuOE-2-PyP5+ with RT and Asc contributes to high H2O2 production in cancer cells and tumor, which along with high MnP accumulation in cancer cells and tumor induces the largest suppression of cell viability and tumor growth, while increasing GSSG/GSH ratio and levels of total S-glutathionylated proteins. Redox-inert MnP, MnTBAP3− and two other different types of redox-active Mn complexes (EUK-8 and M40403) were neither efficacious in the cellular nor in the animal model. Such outcome is in accordance with their inability to catalyze Asc oxidation and mimic GPx. Innovation: We provided here the first evidence how structure-activity relationship between the catalytic potency and the redox properties of Mn complexes controls their ability to impact cellular redox environment and thus enhance the radiation and ascorbate-mediated tumor suppression. Conclusions: The interplay between the accumulation of cationic MnPs and their potency as catalysts for oxidation of Asc, protein cysteines, and GSH controls the magnitude of their anticancer therapeutic effects. [ABSTRACT FROM AUTHOR]

Abstract Cationic manganese(III) ortho N -substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. Although an LC-MS/MS-based method for MnP quantitation and subcellular distribution has been reported, a direct method capable of evaluating both the uptake and the redox state of MnP in living cells has not yet been developed. In the present work we applied resonance Raman (RR) spectroscopy to analyze the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+ within endothelial cells. RR experiments with isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. To assess the combined impact of reactivity and bioavailability, we studied the kinetics of Mn(III)TnBuOE-2-PyP5+ with peroxynitrite and evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems. Graphical abstract fx1 Highlights • Raman Resonance was utilized to reveal the redox state of Mn-porphyrins (MnP) in cells. • Mn(III)P are readily reduced intracellularly to the Mn(II) state. • Intramitochondrial oxidation of a peroxynitrite-sensitive probe is inhibited by MnP. • The cytotoxicity of peroxynitrite is neutralized by MnP via a catalytic redox cycle. [ABSTRACT FROM AUTHOR]

We investigated the cytogenetic and gene expression responses of peripheral blood cells of non-human primates (NHP, Macaca mulatta) that were whole-thorax irradiated with a single dose of 10 Gy. In this model, partial irradiation of NHPs in the thoracic region (Whole Thorax Lung Irradiation, WTLI) allows the study of late radiation-induced lung injury, while avoiding acute radiation syndromes related to hematopoietic and gastrointestinal injury. A transient drop in circulating lymphocytes and platelets was seen by 9 days, followed by elevations in respiratory rate, circulating neutrophils, lymphocytes, and monocytes at 60–100 days, corresponding to computed tomography (CT) and histologic evidence of pneumonitis, and elective euthanasia of four animals. To evaluate long-term DNA damage in NHP peripheral blood lymphocytes after 10 Gy WTLI, we used the cytokinesis-block micronucleus (CBMN) assay to measure chromosomal aberrations as post-mitotic micronuclei in blood samples collected up to 8 months after irradiation. Regression analysis showed significant induction of micronuclei in NHP blood cells that persisted with a gradual decline over the 8-month study period, suggesting long-term DNA damage in blood lymphocytes after WTLI. We also report transcriptomic changes in blood up to 30 days after WTLI. We isolated total RNA from peripheral blood at 3 days before and then at 2, 5 and 30 days after irradiation. We identified 1187 transcripts that were significantly changed across the 30-day time course. From changes in gene expression, we identified biological processes related to immune responses, which persisted across the 30-day study. Response to oxygen-containing compounds and bacteria were implicated by gene-expression changes at the earliest day 2 and latest, day 30 time-points. Gene expression changes suggest a persistent altered state of the immune system, specifically response to infection, for at least a month after WTLI. [ABSTRACT FROM AUTHOR]

Aims: Cationic manganese (Mn)-substituted N-pyridylporphyrin-based potent mimics of the family of superoxide dismutases (SODs) protect normal tissues from injury related to ionizing radiation (IR) by reducing levels of reactive oxygen and nitrogen species (ROS/RNS). Furthermore, Mn-porphyrins have demonstrated antitumor and radiosensitizing effects on cancer cells by promoting IR-induced tumor vasculature damage and apoptotic processes. In this study, we explored the underlying mechanisms of Mn-porphyrin-mediated tumor radiosensitization using murine mammary carcinoma 4T1 and melanoma B16 cells in vitro and in vivo. Results: Combination treatment with MnTnHex-2-PyP and IR substantially reduced cell viability, clonogenic cell survival, and DNA damage repair and synergistically increased IR-induced apoptosis of 4T1 and B16 cells. MnTnHex-2-PyP in combination with IR caused a significant delay in growth of 4T1 and B16 xenograft tumors. MnTnHex-2-PyP dose-dependently enhanced IR-mediated production of H2O2-derived species, but not superoxide. Catalase overexpression reversed MnTnHex-2-PyP-enhanced ROS production and apoptosis. Demonstrated suppression of phosphorylation of several mitogen-activated protein (MAP) kinases and activation of NF-κB by MnTnHex-2-PyP/IR, which presumably inhibited activation of the antiapoptotic pathway, are in agreement with our other data on the apoptosis of cancer cells. Innovation and Conclusions: MnTnHex-2-PyP exerted a radiosensitizing effect on 4T1 and B16 tumor models in vitro and in vivo via pro-oxidative actions and therefore bears a large therapeutic potential. When combined with IR, it attenuated DNA damage repair and triggered a shift from prosurvival pathways to apoptotic cell death, likely due to increased ROS production and disturbed cellular redox balance, acting at the level of nuclear factor κB (NF-κB). Antioxid. Redox Signal. 27, 1067-1082. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is a promising alternative approach particularly attractive for treatment of localized fungal infections. It is based on compounds, photosensitizers (PSs), which when excited with visible light, generate reactive species that ultimately cause cell death. Such species have short lifespans; as a consequence, efficiency and selectivity of the PDT treatment depend mainly on the properties of the PSs. This study is the first to explore the effect of cationic porphyrin-based photosensitizers on Saccharomyces cerevisiae, a member of the fungus kingdom. The study investigates which properties of the PS are essential for efficient antifungal PDT. Cationic Zn(ii) meso-tetrakis(N-alkylpyridinium-2-yl)porphyrins (ZnP) with identical tetrapyrrole core and photo-physical properties, but with different substituents at the meso positions of the porphyrin ring were studied. Attaching six-carbon aliphatic chains to the four pyridyl nitrogens at all meso positions to the porphyrin ring produced a highly photo-efficient amphiphilic, water soluble PS, with minimal dark toxicity. It was taken up by the yeast cells and upon illumination suppressed metabolism by inactivating cytoplasmic and mitochondrial enzymes, and compromising plasma membrane barrier function. At low concentrations (up to 5 μM) the tetrahexyl derivative was a much more powerful antifungal agent than the commercially available chlorin e6. The more lipophilic tetraoctyl analog was also highly photo-efficient but displayed strong dark toxicity, presumably due to higher lipophilicity which might affect the lipid bilayer of membranes. Results presented here can assist the design of antifungal agents whose biological action depends on efficient and rapid uptake by the cells. [ABSTRACT FROM AUTHOR]

We disclose here the studies that preceded and guided the preparation of the metal-based, redox-active therapeutic Mn(III) meso -tetrakis( N -n-butoxyethylpyridyl)porphyrin, MnTnBuOE-2-PyP 5 + (BMX-001), which is currently in Phase I/II Clinical Trials at Duke University (USA) as a radioprotector of normal tissues in cancer patients. N -substituted pyridylporphyrins are ligands for Mn(III) complexes that are among the most potent superoxide dismutase mimics thus far synthesized. To advance their design, thereby improving their physical and chemical properties and bioavailability/toxicity profiles, we undertook a systematic study on placing oxygen atoms into N -alkylpyridyl chains via alkoxyalkylation reaction. For the first time we show here the unforeseen structural rearrangement that happens during the alkoxyalkylation reaction by the corresponding tosylates. Comprehensive experimental and computational approaches were employed to solve the rearrangement mechanism involved in quaternization of pyridyl nitrogens, which, instead of a single product, led to a variety of mixed N -alkoxyalkylated and N -alkylated pyridylporphyrins. The rearrangement mechanism involves the formation of an intermediate alkyl oxonium cation in a chain-length-dependent manner, which subsequently drives differential kinetics and thermodynamics of competing N -alkoxyalkylation versus in situ N -alkylation. The use of alkoxyalkyl tosylates, of different length of alkyl fragments adjacent to oxygen atom, allowed us to identify the set of alkyl fragments that would result in the synthesis of a single compound of high purity and excellent therapeutic potential. [ABSTRACT FROM AUTHOR]

The persistent problem of antibiotic resistance has created a strong demand for new methods for therapy and disinfection. Photodynamic inactivation (PDI) of microbes has demonstrated promising results for eradication of antibiotic-resistant strains. PDI is based on the use of a photosensitive compound (photosensitizer, PS), which upon illumination with visible light generates reactive species capable of damaging and killing microorganisms. Since photogenerated reactive species are short lived, damage is limited to close proximity of the PS. It is reasonable to expect that the larger the number of damaged targets is and the greater their variety is, the higher the efficiency of PDI is and the lower the chances for development of resistance are. Exact molecular mechanisms and specific targets whose damage is essential for microbial inactivation have not been unequivocally established. Two main cellular components, DNA and plasma membrane, are regarded as the most important PDI targets. Using Zn porphyrin-based PSs and Escherichia coli as a model Gram-negative microorganism, we demonstrate that efficient photoinactivation of bacteria can be achieved without detectable DNA modification. Among the cellular components which are modified early during illumination and constitute key PDI targets are cytosolic enzymes, membrane-bound protein complexes, and the plasma membrane. As a result, membrane barrier function is lost, and energy and reducing equivalent production is disrupted, which in turn compromises cell defense mechanisms, thus augmenting the photoinduced oxidative injury. In conclusion, high PDI antimicrobial effectiveness does not necessarily require impairment of a specific critical cellular component and can be achieved by inducing damage to multiple cellular targets. [ABSTRACT FROM AUTHOR]