The Potential Contribution of Hexavalent Chromium to the Carcinogenicity of Chrysotile Asbestos.

Chrysotile asbestos is a carcinogenic mineral that has abundantly been used in industrial and consumer applications. The carcinogenicity of the fibers is partly governed by reactive Fe surface sites that catalyze the generation of highly toxic hydroxyl radicals (HO•) from extracellular hydrogen peroxide (H2O2). Chrysotile also contains Cr, typically in the low mass permille range. In this study, we examined the leaching of Cr from fibers at the physiological lung pH of 7.4 in the presence and absence of H2O2. Furthermore, we investigated the potential of cells from typical asbestos-burdened tissues and cancers to take up Cr leached from chrysotile in PCR expression, immunoblot, and cellular Cr uptake experiments. Finally, the contribution of Cr to fiber-mediated H2O2 decomposition and HO• generation was studied. Chromium readily dissolved from chrysotile fibers in its genotoxic and carcinogenic hexavalent redox state upon oxidation by H2O2. Lung epithelial, mesothelial, lung carcinoma, and mesothelioma cells expressed membrane-bound Cr(VI) transporters and accumulated Cr up to 10-fold relative to the Cr(VI) concentration in the spiked medium. Conversely, anion transporter inhibitors decreased cellular Cr(VI) uptake up to 45-fold. Finally, chromium associated with chrysotile neither decomposed H2O2 nor contributed to fiber-mediated HO• generation. Altogether, our results support the hypothesis that Cr may leach from inhaled chrysotile in its hexavalent state and subsequently accumulate in cells of typically asbestos-burdened tissues, which could contribute to the carcinogenicity of chrysotile fibers. However, unlike Fe, Cr did not significantly contribute to the adverse radical production of chrysotile.

Antileishmanial Anthracene Endoperoxides: Efficacy In Vitro, Mechanisms and Structure-Activity Relationships.

Leishmaniasis is a vector-borne disease caused by protozoal Leishmania parasites. Previous studies have shown that endoperoxides (EP) can selectively kill Leishmania in host cells. Therefore, we studied in this work a set of new anthracene-derived EP (AcEP) together with their non-endoperoxidic analogs in model systems of Leishmania tarentolae promastigotes (LtP) and J774 macrophages for their antileishmanial activity and selectivity. The mechanism of effective compounds was explored by studying their reaction with iron (II) in chemical systems and in Leishmania. The correlation of structural parameters with activity demonstrated that in this compound set, active compounds had a LogPOW larger than 3.5 and a polar surface area smaller than 100 Å2. The most effective compounds (IC50 in LtP < 2 µM) with the highest selectivity (SI > 30) were pyridyl-/tert-butyl-substituted AcEP. Interestingly, also their analogs demonstrated activity and selectivity. In mechanistic studies, it was shown that EP were activated by iron in chemical systems and in LtP due to their EP group. However, the molecular structure beyond the EP group significantly contributed to their differential mitochondrial inhibition in Leishmania. The identified compound pairs are a good starting point for subsequent experiments in pathogenic Leishmania in vitro and in animal models.

Identifying the reactive sites of hydrogen peroxide decomposition and hydroxyl radical formation on chrysotile asbestos surfaces.

BACKGROUND: Fibrous chrysotile has been the most commonly applied asbestos mineral in a range of technical applications. However, it is toxic and carcinogenic upon inhalation. The chemical reactivity of chrysotile fiber surfaces contributes to its adverse health effects by catalyzing the formation of highly reactive hydroxyl radicals (HO•) from H2O2. In this Haber-Weiss cycle, Fe on the fiber surface acts as a catalyst: Fe3+ decomposes H2O2 to reductants that reduce surface Fe3+ to Fe2+, which is back-oxidized by H2O2 (Fenton-oxidation) to yield HO•. Chrysotile contains three structural Fe species: ferrous and ferric octahedral Fe and ferric tetrahedral Fe (Fe3+tet). Also, external Fe may adsorb or precipitate onto fiber surfaces. The goal of this study was to identify the Fe species on chrysotile surfaces that catalyze H2O2 decomposition and HO• generation. RESULTS: We demonstrate that at the physiological pH 7.4 Fe3+tet on chrysotile surfaces substantially contributes to H2O2 decomposition and is the key structural Fe species catalyzing HO• generation. After depleting Fe from fiber surfaces, a remnant fiber-related H2O2 decomposition mode was identified, which may involve magnetite impurities, remnant Fe or substituted redox-active transition metals other than Fe. Fe (hydr)oxide precipitates on chrysotile surfaces also contributed to H2O2 decomposition, but were per mole Fe substantially less efficient than surface Fe3+tet. Fe added to chrysotile fibers increased HO• generation only when it became incorporated and tetrahedrally coordinated into vacancy sites in the Si layer. CONCLUSIONS: Our results suggest that at the physiological pH 7.4, oxidative stress caused by chrysotile fibers largely results from radicals produced in the Haber-Weiss cycle that is catalyzed by Fe3+tet. The catalytic role of Fe3+tet in radical generation may also apply to other pathogenic silicates in which Fe3+tet is substituted, e.g. quartz, amphiboles and zeolites. However, even if these pathogenic minerals do not contain Fe, our results suggest that the mere presence of vacancy sites may pose a risk, as incorporation of external Fe into a tetrahedral coordination environment can lead to HO• generation.

Essential Oil from Melaleuca leucadendra: Antimicrobial, Antikinetoplastid, Antiproliferative and Cytotoxic Assessment.

Essential oils (EOs) are known for their use in cosmetics, food industries, and traditional medicine. This study presents the chemical composition and therapeutic properties against kinetoplastid and eukaryotic cells of the EO from Melaleucaleucadendra (L.) L. (Myrtaceae). Forty-five compounds were identified in the oil by GC-MS, containing a major component the 1,8-cineole (61%). The EO inhibits the growth of Leishmania amazonensis and Trypanosoma brucei at IC50 values <10 µg/mL. However, 1,8 cineole was not the main compound responsible for the activity. Against malignant (22Rv1, MCF-7, EFO-21, including resistant sublines MCF-7/Rap and MCF-7/4OHTAMO) and non-malignant (MCF-10A, J774A.1 and peritoneal macrophage) cells, IC50 values from 55 to 98 µg/mL and from 94 to 144 µg/mL were obtained, respectively. However, no activity was observed on Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Candida parapsilosis, Microsporum canis, or Trypanosoma cruzi. The EO was able to control the lesion size and parasite burden in the model of cutaneous leishmaniasis in BALB/c mice caused by L. amazonensis compared to untreated animals (p < 0.05) and similar with those treated with Glucantime® (p > 0.05). This work constitutes the first evidence of antiproliferative potentialities of EO from M. leucadendra growing in Cuba and could promote further preclinical investigations to confirm the medical value of this plant, in particular for leishmaniasis treatment.

Molecular Mechanisms Responsible for Pharmacological Effects of Genipin on Mitochondrial Proteins.

Genipin, a natural compound from Gardenia jasminoides, is a well-known compound in Chinese medicine that is used for the treatment of cancer, inflammation, and diabetes. The use of genipin in classical medicine is hindered because of its unknown molecular mechanisms of action apart from its strong cross-linking ability. Genipin is increasingly applied as a specific inhibitor of proton transport mediated by mitochondrial uncoupling protein 2 (UCP2). However, its specificity for UCP2 is questionable, and the underlying mechanism behind its action is unknown. Here, we investigated the effect of genipin in different systems, including neuroblastoma cells, isolated mitochondria, isolated mitochondrial proteins, and planar lipid bilayer membranes reconstituted with recombinant proteins. We revealed that genipin activated dicarboxylate carrier and decreased the activity of UCP1, UCP3, and complex III of the respiratory chain alongside with UCP2 inhibition. Based on competitive inhibition experiments, the use of amino acid blockers, and site-directed mutagenesis of UCP1, we propose a mechanism of genipin's action on UCPs. At low concentrations, genipin binds to arginine residues located in the UCP funnel, which leads to a decrease in UCP's proton transporting function in the presence of long chain fatty acids. At concentrations above 200 µM, the inhibitory action of genipin on UCPs is overlaid by increased nonspecific membrane conductance due to the formation of protein-genipin aggregates. Understanding the concentration-dependent mechanism of genipin action in cells will allow its targeted application as a drug in the above-mentioned diseases.

The Effect of pH and Biogenic Ligands on the Weathering of Chrysotile Asbestos: The Pivotal Role of Tetrahedral Fe in Dissolution Kinetics and Radical Formation.

Chrysotile asbestos is a soil pollutant in many countries. It is a carcinogenic mineral, partly due to its surface chemistry. In chrysotile, FeII and FeIII substitute Mg octahedra (Fe[6]), and FeIII substitutes Si tetrahedra (Fe[4]). Fe on fiber surfaces can generate hydroxyl radicals (HO. ) in Fenton reactions, which damage biomolecules. To better understand chrysotile weathering in soils, net Mg and Si dissolution rates over the pH range 3.0-11.5 were determined in the presence and absence of biogenic ligands. Also, HO. generation and Fe bulk speciation of pristine and weathered fibers were examined by EPR and Mössbauer spectroscopy. Dissolution rates were increased by ligands and inversely related to pH with complete inhibition at cement pH (11.5). Surface-exposed Mg layers readily dissolved at low pH, but only after days at neutral pH. On longer timescales, the slow dissolution of Si layers became rate-determining. In the absence of ligands, Fe[6] precipitated as Fenton-inactive Fe phases, whereas Fe[4] (7 % of bulk Fe) remained redox-active throughout two-week experiments and at pH 7.5 generated 50±10 % of the HO. yield of Fe[6] at pristine fiber surfaces. Ligand-promoted dissolution of Fe[4] (and potentially Al[4]) labilized exposed Si layers. This increased Si and Mg dissolution rates and lowered HO. generation to near-background level. It is concluded that Fe[4] surface species control long-term HO. generation and dissolution rates of chrysotile at natural soil pH.

The leishmanicidal activity of artemisinin is mediated by cleavage of the endoperoxide bridge and mitochondrial dysfunction.

Endoperoxides kill malaria parasites via cleavage of their endoperoxide bridge by haem or iron, leading to generation of cytotoxic oxygen-centred radicals. In view of the Leishmania parasites having a relatively compromised anti-oxidant defense and high iron content, this study aims to establish the underlying mechanism(s) accounting for the apoptotic-like death of Leishmania promastigotes by artemisinin, an endoperoxide. The formation of reactive oxygen species was confirmed by flow cytometry and was accompanied by inhibition of mitochondrial complexes I-III and II-III. However, this did not translate into a generation of mitochondrial superoxide or decrease in oxygen consumption, indicating minimal impairment of the electron transport chain. Artemisinin caused depolarization of the mitochondrial membrane along with a substantial depletion of adenosine triphosphatase (ATP), but it was not accompanied by enhancement of ATP hydrolysis. Collectively, the endoperoxide-mediated radical formation by artemisinin in Leishmania promastigotes was the key step for triggering its antileishmanial activity, leading secondarily to mitochondrial dysfunction indicating that endoperoxides represent a promising therapeutic strategy against Leishmania worthy of pharmacological consideration.

Techniques to study phagocytosis and uptake of Leishmania tarentolae by J774 macrophages.

Leishmaniasis is a vector borne parasitic disease affecting millions of people worldwide and is spreading into further areas because of global warming. The development of new active substances against these single-cell eukaryotic parasites is of great importance. Leishmania tarentolae promastigotes (LtP) are non-pathogenic for mammals and serve as model organisms for pathogenic Leishmania in basic research. However, it is important to refine methods to study the process of the infection of mammalian macrophages by LtP and pathogenic Leishmania. Important stages of the infection are phagocytosis by macrophages and multiplication of Leishmania amastigotes in the phagolysosome of macrophages. In this study, advanced methods using electron spin resonance (ESR) spectroscopy and genetically manipulated LtP were used to monitor the infection of adherent J774 macrophages with LtP. An ESR method was established to detect the formation of superoxide radicals directly in adherent J774 cells and to investigate the effect of LtP on this activity. J774 cells responded with a burst of superoxide radicals in the presence of phorbol myristate acetate as positive control. In contrast, challenging J774 cells with LtP resulted in a much lower burst of superoxide radicals. To facilitate LtP detection in the phagolysosome of J774 macrophages, LtP expressing enhanced green fluorescent protein (EGFP-LtP) were constructed. After different infection times with EGFP-LtP, the J774 cells were visualized by phase contrast microscopy and the cell number was determined. The intramacrophage Leishmania tarentolae amastigotes (LtA) expressing EGFP were detected by fluorescence microscopy and then counted with ImageJ. These experiments showed that LtP are taken up by J774 cells and form intraphagolysosomal amastigotes. LtA under our conditions multiplied intracellularly and were able to persist about 48 h in J774 cells. These experiments showed that ESR spectroscopy of attached macrophages and the use of the EGFP-LtP are suitable methods to study the initial phase of Leishmania infection in vitro.

Berberine chloride mediates its antileishmanial activity by inhibiting Leishmania mitochondria.

Berberine chloride, a plant-derived isoquinoline alkaloid, has been demonstrated to have leishmanicidal activity, which is mediated by generation of a redox imbalance and depolarization of the mitochondrial membrane, resulting in a caspase-independent apoptotic-like cell death. However, its impact on mitochondrial function remains to be delineated and is the focus of this study. In UR6 promastigotes, berberine chloride demonstrated a dose-dependent increase in generation of reactive oxygen species and mitochondrial superoxide, depolarization of the mitochondrial membrane potential, a dose-dependent inhibition of mitochondrial complexes I-III and II-III, along with a substantial depletion of ATP, collectively suggesting inhibition of parasite mitochondria. Accordingly, the oxidative stress induced by berberine chloride resulting in an apoptotic-like cell death in Leishmania can be exploited as a potent chemotherapeutic strategy, mitochondria being a prime contributor.

Interaction of ascaridole, carvacrol, and caryophyllene oxide from essential oil of Chenopodium ambrosioides L. with mitochondria in Leishmania and other eukaryotes.

The antileishmanial activity of the essential oil (EO) from Chenopodium ambrosioides L. has been demonstrated in vitro and in animal models, attributed to the major components of the EO. This study focused on the effects of the three major EO compounds carvacrol, caryophyllene oxide (Caryo), and the antileishmanial endoperoxide ascaridole (Asc) on mitochondrial functions in Leishmania tarentolae promastigotes (LtP). EO and Caryo were able to partially inhibit the leishmanial electron transport chain, whereas other components failed to demonstrate a direct immediate effect. Caryo demonstrated inhibition of complex III activity in LtP and in isolated complex III from other species. The formation of superoxide radicals was studied in Leishmania by electron spin resonance spectroscopy in the presence of iron chelators wherein selected compounds failed to trigger a significant immediate additional superoxide production in LtP. However, upon prolonged incubation of Leishmania with Asc and especially in the absence of iron chelators (allowing the activation of Asc), an increased superoxide radical production and significant impairment of mitochondrial coupling in Leishmania was observed. Prolonged incubation with all EO components resulted in thiol depletion. Taken together, the major components of EO mediate their leishmanicidal activity via different mitochondrial targets and time profiles. Further studies are required to elucidate possible synergistic effects of carvacrol and Asc and the influence of minor compounds.

Activation of Anthracene Endoperoxides in Leishmania and Impairment of Mitochondrial Functions.

Leishmaniasis is a vector-borne disease caused by protozoal Leishmania. Because of resistance development against current drugs, new antileishmanial compounds are urgently needed. Endoperoxides (EPs) are successfully used in malaria therapy, and experimental evidence of their potential against leishmaniasis exists. Anthracene endoperoxides (AcEPs) have so far been only technically used and not explored for their leishmanicidal potential. This study verified the in vitro efficiency and mechanism of AcEPs against both Leishmania promastigotes and axenic amastigotes (L. tarentolae and L. donovani) as well as their toxicity in J774 macrophages. Additionally, the kinetics and radical products of AcEPs' reaction with iron, the formation of radicals by AcEPs in Leishmania, as well as the resulting impairment of parasite mitochondrial functions were studied. Using electron paramagnetic resonance combined with spin trapping, photometry, and fluorescence-based oximetry, AcEPs were demonstrated to (i) show antileishmanial activity in vitro at IC50 values in a low micromolar range, (ii) exhibit host cell toxicity in J774 macrophages, (iii) react rapidly with iron (II) resulting in the formation of oxygen- and carbon-centered radicals, (iv) produce carbon-centered radicals which could secondarily trigger superoxide radical formation in Leishmania, and (v) impair mitochondrial functions in Leishmania during parasite killing. Overall, the data of different AcEPs demonstrate that their structures besides the peroxo bridge strongly influence their activity and mechanism of their antileishmanial action.

Expansion of BCR/ABL1+ cells requires PAK2 but not PAK1.

The p21-activated kinases (PAKs) are key nodes in oncogenic signalling pathways controlling growth, survival, and motility of cancer cells. Their activity is increased in many human cancers and is associated with poor prognosis. To date, PAK deregulation has mainly been studied in solid tumours, where PAK1 and PAK4 are the main isoforms deregulated. We show that PAK1 and PAK2 are the critical isoforms in a BCR/ABL1+ haematopoietic malignancy. In suspension, leukaemic cells deficient for PAK1 and PAK2 undergo apoptosis, while the loss of either protein is well tolerated. Transfer of medium conditioned by shPAK2- but not shPAK1-expressing leukaemic cells interferes with endothelial cell growth. We found that leukaemic cells produce exosomes containing PAK2. Transfer of isolated exosomes supports endothelial cell proliferation. In parallel, we found that leukaemic cells explicitly require PAK2 to grow towards an extracellular matrix. PAK2-deficient cells fail to form colonies in methylcellulose and to induce lymphomas in vivo. PAK2 might therefore be the critical isoform in leukaemic cells by controlling tumour growth in a dual manner: vascularization via exosome-mediated transfer to endothelial cells and remodelling of the extracellular matrix. This finding suggests that the PAK2 isoform represents a promising target for the treatment of haematological diseases.

Degradation of the Cellulosic Key Chromophore 5,8-Dihydroxy-[1,4]-naphthoquinone by Hydrogen Peroxide under Alkaline Conditions.

5,8-Dihydroxy-[1,4]-naphthoquinone (DHNQ) is one of the key chromophores in cellulosic materials. Its almost ubiquitous presence in cellulosic materials makes it a target molecule of the pulp and paper industry's bleaching efforts. In the presented study, DHNQ was treated with hydrogen peroxide under alkaline conditions at pH 10, resembling the conditions of industrial hydrogen peroxide bleaching (P stage). The reaction mechanism, reaction intermediates, and final degradation products were analyzed by UV/vis, NMR, GC-MS, and EPR. The degradation reaction yielded C1-C4 carboxylic acids as the final products. Highly relevant for pulp bleaching are the findings on intermediates of the reaction, as two of them, 2,5-dihydroxy-[1,4]-benzoquinone (DHBQ) and 1,4,5,8-naphthalenetetrone, are potent chromophores themselves. While DHBQ is one of the three key cellulosic chromophores and its degradation by H2O2 is well-established, the second intermediate, 1,4,5,8-naphthalenetetrone, is reported for the first time in the context of cellulose discoloration.

The antileishmanial activity of xanthohumol is mediated by mitochondrial inhibition.

Xanthohumol (Xan) is a natural constituent of human nutrition. Little is known about its actions on leishmanial parasites and their mitochondria as putative target. Therefore, we determined the antileishmanial activity of Xan and resveratrol (Res, as alternative compound with antileishmanial activity) with respect to mitochondria in Leishmania amazonensis promastigotes/amastigotes (LaP/LaA) in comparison with their activity in peritoneal macrophages from mouse (PMM) and macrophage cell line J774A.1 (J774). Mechanistic studies were conducted in Leishmania tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Xan and Res demonstrated antileishmanial activity in LaA [half inhibitory concentration (IC50): Xan 7 µ m, Res 14 µ m]; while they had less influence on the viability of PMM (IC50: Xan 70 µ m, Res >438 µ m). In contrast to Res, Xan strongly inhibited oxygen consumption in Leishmania (LtP) but not in J774 cells. This was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan, which was less pronounced with Res. Neither Xan nor Res increased mitochondrial superoxide release in LtP, while both decreased the mitochondrial membrane potential in LtP. Bioenergetic studies showed that LtP mitochondria have no spare respiratory capacity in contrast to mitochondria in J774 cells and can therefore much less adapt to stress by mitochondrial inhibitors, such as Xan. These data show that Xan may have antileishmanial activity, which is mediated by mitochondrial inhibition.

Role of mitochondria in the leishmanicidal effects and toxicity of acyl phloroglucinol derivatives: nemorosone and guttiferone A.

Nemorosone (Nem) and guttiferone A (GutA) are acyl phloroglucinol derivatives (APD) that are present in different natural products. For both compounds anti-cancer and anti-microbial properties have been reported. In particular, an anti-leishmanial activity of both compounds was demonstrated. The aim of this study was to explore the possible role of mitochondria in the anti-leishmanial activity of Nem and GutA in comparison with their action on mammalian mitochondria. Both APD inhibited the growth of promastigotes of Leishmania tarentolae (LtP) with half maximal inhibitory concentration (IC50) values of 0·67 ± 0·17 and 6·2 ± 2·6 µ m; while IC50 values for cytotoxicity against peritoneal macrophages from BALB/c mice were of 29·5 ± 3·7 and 9·2 ± 0·9 µ m, respectively. Nemorosone strongly inhibited LtP oxygen consumption, caused species-specific inhibition (P < 0·05) of succinate:ubiquinone oxidoreductase (complex II) from LtP-mitochondria and significantly increased (P < 0·05) the mitochondrial superoxide production. In contrast, GutA caused only a moderate reduction of respiration in LtP and triggered less superoxide radical production in LtP compared with Nem. In addition, GutA inhibited mitochondrial complex III in bovine heart submitochondrial particles, which is possibly involved in its mammalian toxicity. Both compounds demonstrated at low micromolar concentrations an effect on the mitochondrial membrane potential in LtP. The present study suggests that Nem caused its anti-leishmanial action due to specific inhibition of complexes II/III of mitochondrial respiratory chain of Leishmania parasites that could be responsible for increased production of reactive oxygen species that triggers parasite death.

Tocopheramine succinate and tocopheryl succinate: mechanism of mitochondrial inhibition and superoxide radical production.

Tocopherols (TOH) are lipophilic antioxidants which require the phenolic OH group for their redox activity. In contrast, non-redox active esters of α-TOH with succinate (α-TOS) were shown to possess proapoptotic activity in cancer cells. It was suggested that this activity is mediated via mitochondrial inhibition with subsequent O2(-) production triggering apoptosis and that the modification of the linker between the succinate and the lipophilic chroman may modulate this activity. However, the specific mechanism and the influence of the linker are not clear yet on the level of the mitochondrial respiratory chain. Therefore, this study systematically compared the effects of α-TOH acetate (α-TOA), α-TOS and α-tocopheramine succinate (α-TNS) in cells and submitochondrial particles (SMP). The results showed that not all cancer cell lines are highly sensitive to α-TOS and α-TNS. In HeLa cells α-TNS did more effectively reduce cell viability than α-TOS. The complex I activity of SMP was little affected by α-TNS and α-TOS while the complex II activity was much more inhibited (IC50=42±8µM α-TOS, 106±8µM α-TNS, respectively) than by α-TOA (IC50 >1000µM). Also the complex III activity was inhibited by α-TNS (IC50=137±6µM) and α-TOS (IC50=315±23µM). Oxygen consumption of NADH- or succinate-respiring SMP, involving the whole electron transfer machinery, was dose-dependently decreased by α-TOS and α-TNS, but only marginal effects were observed in the presence of α-TOA. In contrast to the similar inhibition pattern of α-TOS and α-TNS, only α-TOS triggered O2(-) formation in succinate- and NADH-respiring SMP. Inhibitor studies excluded complex I as O2(-) source and suggested an involvement of complex III in O2(-) production. In cancer cells only α-TOS was reproducibly able to increase O2(-) levels above the background level but neither α-TNS nor α-TOA. Furthermore, the stability of α-TNS in liver homogenates was significantly lower than that of α-TOS. In conclusion, this suggests that α-TNS although it has a structure similar to α-TOS is not acting via the same mechanism and that for α-TOS not only complex II but also complex III interactions are involved.

Essential oil from Chenopodium ambrosioides and main components: activity against Leishmania, their mitochondria and other microorganisms.

Chenopodium ambrosioides is an aromatic herb used by native people to treat parasitic diseases. The aim of this work is to compare the in vitro anti-leishmanial activity of the essential oil (EO) from C. ambrosioides and its major components (ascaridole, carvacrol and caryophyllene oxide) and study their mechanism of action and activity against a panel of microorganism. Antileishmanial activity and cytotoxicity of the EO and major components was study. In addition, experiments to elucidate the mechanism of action were perform and activities against other microorganisms (bacteria, fungi and protozoa) were evaluate. All products were active against promastigote and amastigote forms of Leishmania. Ascaridole exhibited the better antileishmanial activity and the EO the highest selectivity index. The exploration of the mechanism suggests that the products cause a breakdown of mitochondrial membrane potential and a modification of redox indexes. Only EO showed antiprotozoal effect against Plasmodium falciparum and Trypanosoma brucei; while no activity against bacteria and fungi was observed. Our results demonstrate the potentialities of EO in cellular and molecular system, which could be consider in future studies to develop new antileishmanial drugs with a wide anti-parasitic spectrum.

Natural endoperoxides as promising anti-leishmanials.

BACKGROUND: The discovery of artemisinin, an endoperoxide, encouraged the scientific community to explore endoperoxides as potential anti-parasitic molecules. Although artemisinin derivatives are rapidly evolving as potent anti-malarials, their potential as anti-leishmanials is emerging gradually. The treatment of leishmaniasis, a group of neglected tropical diseases is handicapped by lack of effective vaccines, drug toxicities and drug resistance. The weak antioxidant defense mechanism of the Leishmania parasites due to lack of catalase and a selenium dependent glutathione peroxidase system makes them vulnerable to oxidative stress, and this has been successful exploited by endoperoxides. PURPOSE: The study aimed to review the available literature on the anti-leishmanial efficacy of natural endoperoxides with a view to achieve insights into their mode of actions. METHODS: We reviewed more around 110 research and review articles restricted to the English language, sourced from electronic bibliographic databases including PubMed, Google, Web of Science, Google scholar etc. RESULTS: Natural endoperoxides could potentially augment the anti-leishmanial drug library, with artemisinin and ascaridole emerging as potential anti-leishmanial agents. Due to higher reactivity of the cyclic peroxide moiety, and exploiting the compromised antioxidant defense of Leishmania, endoperoxides like artemisinin and ascaridole potentiate their leishmanicidal efficacy by creating a redox imbalance. Furthermore, these molecules minimally impair oxidative phosphorylation; instead inhibit glycolytic functions, culminating in depolarization of the mitochondrial membrane and depletion of ATP. Additionally, the carbon-centered free radicals generated from endoperoxides, participate in chain reactions that can generate even more reactive organic radicals that are toxic to macromolecules, including lipids, proteins and DNA, leading to cell cycle arrest and apoptosis of Leishmania parasites. However, the precise target(s) of the toxic free radicals remains open-ended. CONCLUSION: In this overview, the spectrum of natural endoperoxide molecules as major anti-leishmanials and their mechanism of action has been delineated. In view of the substantial evidence that natural endoperoxides (e.g., artemisinin, ascaridole) exert a noxious effect on different species of Leishmania, identification and characterization of other natural endoperoxides is a promising therapeutic option worthy of further pharmacological consideration.

Effect of Salts on Laccase-Catalyzed Polymerization of Lignosulfonate.

Enzymatic polymerization of lignosulfonate (LS) has a high potential for various applications ranging from coatings to adhesives. Here, the effect of different ions in low concentrations on enzymatic polymerization of LS was investigated, including salt solutions consisting of mono- and dicarboxylic acids, sulfate, phosphate and chloride with sodium as counter ion. LS polymerization was followed by viscometry and size exclusion (SEC) chromatography. Interestingly, there was only a small effect of ions on the activity of the laccase on standard substrate ABTS, while the effect on polymerization of LS was substantially different. The presence of acetate led to a 39 % higher degree of polymerization (DP) for LS. Small angle X-ray scattering (SAXS) revealed that the structure of the enzyme was largely unaffected by the ions, while the determination of the zeta potential showed that those ions conveying higher negative surface charges onto LS particles showed lower DPs, than those not affecting the surface charge. Further, electron paramagnetic resonance (EPR) spectroscopy showed 5-times higher intensity in phenoxyl radicals for the monovalent ions compared to the divalent ones. It was concluded that the DPs of LS could be tuned in the presence of certain ions, by facilitating the interaction between the laccase substrate-binding site and the LS molecules.

Tocopheramines and tocotrienamines as antioxidants: ESR spectroscopy, rapid kinetics and DFT calculations.

Tocopheramines (TNH2) and tocotrienamines (T3NH2) are analogues of tocopherols (TOH) and tocotrienols in which phenolic OH is replaced by NH2. It was shown in previous studies that TNH2 and T3NH2 act as potent antioxidants. In this study we compared the one-electron oxidation of TNH2/T3NH2 by diphenyl picryl hydrazyl (DPPH) and galvinoxyl (GOX) radicals with the one of α-TOH as a reference compound using ESR spectroscopy, stopped flow spectrophotometry and density functional theory (DFT) calculations. ESR spectroscopy revealed the presence of tocopheramine radicals during electrochemical oxidation of α-TNH2. Kinetic measurements demonstrated that in apolar n-hexane TNH2/T3NH2 derivatives reacted two to three orders of magnitude slower than α-TOH with the model radicals. DFT calculations indicated that this correlates well with the higher bond dissociation energy (BDE) for N-H in TNH2 than for O-H in α-TOH in pure H-atom transfer (HAT). In the more polar medium ethanol TNH2/T3NH2 derivatives partially reacted faster than α-TOH depending on the reaction partner. DFT calculations suggest that this is due to reaction mechanisms alternative to HAT. According to thermochemistry data sequential proton loss and electron transfer (SPLET) is more favored for α-TOH in ethanol than for TNH2. Therefore, for TNH2 a contribution of the alternative mechanism of sequential electron transfer-proton transfer (SET-PT) could be a possible explanation. These data show that the antioxidant reactivity strongly depends on the structure, reaction partners and environment. According to these findings TNH2/T3NH2 should be superior as antioxidants over α-TOH in polar head group regions of membranes but not in the apolar core of lipid bilayers.