Molecular Targets Implicated in the Antiparasitic and Anti-Inflammatory Activity of the Phytochemical Curcumin in Trichomoniasis.

Trichomoniasis, is the most prevalent non-viral sexually transmitted disease worldwide. Although metronidazole (MDZ) is the recommended treatment, several strains of the parasite are resistant to MDZ, and new treatments are required. Curcumin (CUR) is a polyphenol with anti-inflammatory, antioxidant and antiparasitic properties. In this study, we evaluated the effects of CUR on two biochemical targets: on proteolytic activity and hydrogenosomal metabolism in Trichomonas vaginalis. We also investigated the role of CUR on pro-inflammatory responses induced in RAW 264.7 phagocytic cells by parasite proteinases on pro-inflammatory mediators such as the nitric oxide (NO), tumor necrosis factor α (TNFα), interleukin-1beta (IL-1ß), chaperone heat shock protein 70 (Hsp70) and glucocorticoid receptor (mGR). CUR inhibited the growth of T. vaginalis trophozoites, with an IC50 value between 117 ± 7 µM and 173 ± 15 µM, depending on the culture phase. CUR increased pyruvate:ferredoxin oxidoreductase (PfoD), hydrogenosomal enzyme expression and inhibited the proteolytic activity of parasite proteinases. CUR also inhibited NO production and decreased the expression of pro-inflammatory mediators in macrophages. The findings demonstrate the potential usefulness of CUR as an antiparasitic and anti-inflammatory treatment for trichomoniasis. It could be used to control the disease and mitigate the associated immunopathogenic effects.

Evidence for the role of extrusomes in evading attack by the host immune system in a scuticociliate parasite.

Like other ciliates, Philasterides dicentrarchi, the scuticociliate parasite of turbot, produces a feeding-only or growing stage called a trophont during its life cycle. Exposure of the trophonts to heat-inactivated serum extracted from the turbot host and containing specific antibodies that induce agglutination/immobilization leads to the production of a mucoid capsule from which the trophonts later emerge. We investigated how these capsules are generated, observing that the mechanism was associated with the process of exocytosis involved in the release of a matrix material from the extrusomes. The extruded material contains mucin-like glycoproteins that were deposited on the surface of the cell and whose expression increased with time of exposure to the heat-inactivated immune serum, at both protein expression and gene expression levels. Stimulation of the trophonts with the immune serum also caused an increase in discharge of the intracellular storage compartments of calcium necessary for the exocytosis processes in the extrusomes. The results obtained suggest that P. dicentrarchi uses the extrusion mechanism to generate a physical barrier protecting the ciliate from attack by soluble factors of the host immune system. Data on the proteins involved and the potential development of molecules that interfere with this exocytic process could contribute to improving the prevention and control of scuticociliatosis in turbot.

New data on flatfish scuticociliatosis reveal that Miamiensis avidus and Philasterides dicentrarchi are different species.

Scuticociliatosis is a severe disease in farmed flatfish. However, the causative agent is not always accurately identified. In this study, we identified two isolates of scuticociliates from an outbreak in cultured fine flounder Paralichthys adspersus. Scuticociliate identification was based on morphological data, examination of life stages and the use of molecular approaches. The isolates were compared with a strain of Philasterides dicentrachi from turbot Scophthalmus maximus and with a strain deposited in the American Type Culture Collection as Miamiensis avidus ATCC® 50180™. The use of morphological, biological and molecular methods enabled us to identify the isolates from the fine flouder as P. dicentrarchi. Comparison of P. dicentrachi isolates and M. avidus revealed some differences in the buccal apparatus. Unlike P. dicentrarchi, M. avidus has a life cycle with three forms: macrostomes (capable of feeding on P. dicentrarchi), microstomes and tomites. Additionally, we found differences in the 18S rRNA and α- and ß-tubulin gene sequences, indicating that P. dicentrarchi and M. avidus are different species. We therefore reject the synonymy/conspecificity of the two taxa previously suggested. Finally, we suggest that a combination of morphological, biological, molecular (by multigene analysis) and serological techniques could improve the identification of scuticociliates parasites in fish.

Enzymes Involved in Pyrophosphate and Calcium Metabolism as Targets for Anti-scuticociliate Chemotherapy.

Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 µM and 80 ± 8 µM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 µM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis.

Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences.

H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi.

Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment.

Database on eukaryotic symbionts of native and invasive gammarids (Crustacea, Amphipoda) in the Baltic region of Poland with information on water parameters for sampling sites.

This dataset documents the diversity of eukaryotic endo- and epibiotic organisms from 612 host individuals of seven gammarid (Amphipoda) species (Gammarus pulex, Gammarus zaddachi, Gammarus roeselii, Gammarus tigrinus, Dikerogammarus villosus, Pontogammarus robustoides, Echinogammarus ischnus) of native and invasive origin in the Baltic region of Poland. We identify 60 symbiotic species of nine phyla from 16 localities of freshwater and brackish habitats. Twenty-nine symbiotic species belonged to the Ciliophora, 12 to Apicomplexa, 8 to Microsporidia, 3 to Platyhelminthes, 2 to Acanthocephala, 2 to Nematoda, 2 to Rotifera, 1 to Choanozoa and 1 to Nematomorha. The material in this Data in Brief paper is composed of three Microsoft® Excel files. The first file represents the raw data on the number of individuals (infrapopulation size) of each eukaryotic symbiont taxa recorded in each host individual and location. The data set contains information on the assemblage of symbionts per host individual in one table-matrix; macro- (host) and symbiont taxa name, host length, the date of collection, the geographic coordinates and locality name in columns; and amphipod host specimens in lines. The second file reports the symbiont species list (the species breakdown by phyla in spreadsheets) with information on host species, sampling date, locality and geographic coordinates, infection site, obtained sequences (if the case), brief morphological characteristics and microphotographs. The third file reports measured water parameters, habitat features and host density per sample. We generate the present dataset to evaluate the richness, diversity, population and community features of symbiotic organisms in native and invasive gammarid hosts in Poland. Biological sciences: ParasitologyEnvironmental Science: Ecology; Hydrology and Water Quality.

Plant- and Bacteria-Derived Compounds with Anti-Philasterides dicentrarchi Activity.

Philasterides dicentrarchi is a scuticociliate that causes high mortalities in farmed fish. Although vaccination is an effective method to prevent scuticociliatosis caused by the homologous serotype, a universal vaccine has not been developed yet. Many compounds have been shown to be toxic to this ciliate species; moreover, most of them are toxic to aquatic life and cannot be used to prevent the disease. We have evaluated the toxicity to P. dicentrarchi of several compounds of natural origin to be used to reduce parasite levels in the seawater. Ciliates were exposed to several compound concentrations, and the mortality was determined at several incubation times. Tomatine, plumbagin and 2',4'-dihydroxychalcone displayed the highest anticiliate activity, with a dose-dependent response. The effects of these compounds on the EPC cell line were also evaluated, finding that 2',4'-dihydroxychalcone displayed the lowest toxicity to fish cells. At 7.54 µM, 2',4'-dihydroxychalcone inhibited 50% parasite growth but only killed about 10% of EPC cells after 24 h incubation. Finally, we evaluated the toxicity of Pseudomonas H6 surfactant (PS) to P. dicentrarchi, finding that PS was toxic to the ciliate but showed lower toxicity to EPC cells. At a concentration of 7.8 µg/mL (LC50 for the ciliate after 3 h incubation), PS killed 14.9% of EPC cells. We conclude that 2',4'-dihydroxychalcone, and PS could be used to reduce parasite levels in seawater, thus decreasing the risk of scuticociliatosis infection in cultured fish.

Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors.

Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). In addition to a cytochrome pathway (CP), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AOX) in the mitochondrion. Although AOX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AOX in the ciliate has not previously been reported. In this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene (size 1,106 bp) contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AOX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with EXXH motifs associated with the diiron active centers. The aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AOX in respiration under conditions of low levels of O2 and in protecting against oxidative stress generated during infection in the host.

Identification and Molecular Characterization of Superoxide Dismutases Isolated From A Scuticociliate Parasite: Physiological Role in Oxidative Stress.

Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fish. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity capable of inactivating the superoxide anion (•O2-) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34-44 kDa and with very different AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50 kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60 kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2- is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stress.

Increased frequency of micronuclei in peripheral blood lymphocytes of subjects infected with Helicobacter pylori.

Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori and the development of gastric carcinoma and mucosa-associated lymphoid tissue lymphomas in humans. The cytokinesis-block micronucleus assay was performed on peripheral blood lymphocytes of H. pylori-infected patients in order to investigate the possible induction of genotoxic damage. The study group consisted of 70 infected subjects including 33 women and 37 men, and 66 healthy controls (37 females and 29 males). Our results indicate that in the infected group the overall frequency of binucleated micronucleated cells (BNMN) per 1000 cells was higher (17.65+/-1.55) than in the controls (7.39+/-0.66), this difference being statistically significant. No differences were found between the infected and control groups regarding the cytokinesis-block proliferation index (CBPI). When the effect of different counfounding factors was evaluated, mutivariate statistical analysis revealed that age and alcohol consumption modulated the frequency of BNMN in infected people, and the interaction between alcohol use-smoking-infection also affected the BNMN frequency in H. pylori patients. Our results indicate that infection by H. pylori is associated with an increased level of cytogenetic damage in the cells of the host.

Study on mutagenic effects of bisphenol A diglycidyl ether (BADGE) and its derivatives in the Escherichia coli tryptophan reverse mutation assay.

The di-epoxy compound bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (BADGE.H2O and BADGE.2H2O, respectively) and its bis-chlorohydrin derivative (BADGE.2HCl) were examined for their mutagenicity in the Escherichia coli tryptophan reverse mutation test with strains WP2, WP2uvrA and IC3327. The assays were performed in the presence and absence of exogenous metabolic activation (S9 fraction from rat liver). The di-epoxy compound BADGE was able to induce mutagenic effects in strains WP2uvrA and IC3327 and the epoxy-diol BADGE.H2O also showed a positive response with these strains, although the latter was less potent than the former. On the other hand, the lack of mutagenic activity of BADGE.2H2O and BADGE.2HCl was also demonstrated.

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems.

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.

Sister chromatid exchanges and micronuclei analysis in lymphocytes of men exposed to simazine through drinking water.

In some cities of the autonomous community of Extremadura (south-west of Spain), levels of simazine from 10 to 30 ppm were detected in tap water. To analyse the possible effect of this herbicide, two biomarkers, sister chromatid exchanges (SCE) and micronuclei (MN), were used in peripheral blood lymphocytes from males exposed to simazine through drinking water. SCE and MN analysis failed to detect any statistically significant increase in the people exposed to simazine when compared with the controls. With respect to high frequency cells (HFC), a statistically significant difference was detected between exposed and control groups.

Mutagenic assessment of Prestige fuel oil spilled on the shore and submitted to field trials of bioremediation.

The mutagenicy of the slightly weathered fuel oil from the Prestige oil spill and the effects of different bioremediation products (nutrients and/or microorganisms and biodiesel) on the potential mutagenic activity of this heavy fuel oil spilled on the shore were evaluated for a period of 1 year using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537 in the absence and presence of exogenous metabolic activation (S9 fraction from rat liver). The in situ bioremediation experiment was performed using tiles located in the supra-littoral and intertidal zones of a beach seriously affected by the fuel oil spill. The results obtained showed the mutagenic activity of the slightly weathered fuel oil extracts at the beginning of the experiment in strain TA98 that persisted for more than 150 days in both the untreated control and treated tiles independently of the zone of the beach considered. However, after 360 days neither the control nor the treated tiles in the intertidal zone showed mutagenic activity and a weak positive response in strain TA98 was detected for the control fuel oil extracts from supra-littoral tiles. The application of biodiesel to accelerate the biodegradation of this type of fuel oil may constitute a further genotoxic hazard to the environment, since the mutagenic response achieved from the biodiesel-fuel oil mixture in the first samplings (days 0 and 30) was more potent than that obtained from the control tiles. The mutagenic activity was detected along the study with S. typhimurium TA98 in both the presence and absence of S9 microsomal fraction, but the addition of S9 fraction in the assay always increased the number of revertants induced. In general, these findings suggest that the bioremediation strategies used were not effective in eliminating the genotoxic hazard associated with this heavy fuel oil attached to rocky substrate since they did not achieve a decrease in the mutagenic response with respect to the untreated control tiles. These data also confirm that genotoxicity assays should be used to evaluate the effectiveness of bioremediation efforts associated with oil spills for a better risk assessment.