Case Study of the Response of N6-Methyladenine DNA Modification to Environmental Stressors in the Unicellular Eukaryote Tetrahymena thermophila.

Rediscovered as a potential epigenetic mark, N6-methyladenine DNA modification (6mA) was recently reported to be sensitive to environmental stressors in several multicellular eukaryotes. As 6mA distribution and function differ significantly in multicellular and unicellular organisms, whether and how 6mA in unicellular eukaryotes responds to environmental stress remains elusive. Here, we characterized the dynamic changes of 6mA under starvation in the unicellular model organism Tetrahymena thermophila. Single-molecule, real-time (SMRT) sequencing reveals that DNA 6mA levels in starved cells are significantly reduced, especially symmetric 6mA, compared to those in vegetatively growing cells. Despite a global 6mA reduction, the fraction of asymmetric 6mA with a high methylation level was increased, which might be the driving force for stronger nucleosome positioning in starved cells. Starvation affects expression of many metabolism-related genes, the expression level change of which is associated with the amount of 6mA change, thereby linking 6mA with global transcription and starvation adaptation. The reduction of symmetric 6mA and the increase of asymmetric 6mA coincide with the downregulation of AMT1 and upregulation of AMT2 and AMT5, which are supposedly the MT-A70 methyltransferases required for symmetric and asymmetric 6mA, respectively. These results demonstrated that a regulated 6mA response to environmental cues is evolutionarily conserved in eukaryotes. IMPORTANCE Increasing evidence indicated that 6mA could respond to environmental stressors in multicellular eukaryotes. As 6mA distribution and function differ significantly in multicellular and unicellular organisms, whether and how 6mA in unicellular eukaryotes responds to environmental stress remains elusive. In the present work, we characterized the dynamic changes of 6mA under starvation in the unicellular model organism Tetrahymena thermophila. Our results provide insights into how Tetrahymena fine-tunes its 6mA level and composition upon starvation, suggesting that a regulated 6mA response to environmental cues is evolutionarily conserved in eukaryotes.

Inhibition of Mitochondrial ATP Synthesis and Regulation of Oxidative Stress Based on {SbW8 O30 } Determined by Single-Cell Proteomics Analysis.

The 10-nuclear heteroatom cluster modified {SbW8 O30 } was successfully synthesized and exhibited inhibitory activity (IC50 =0.29 µM). Based on proteomics analysis, Na4 Ni2 Sb2 W2 -SbW8 inhibited ATP production by affecting the expression of 16 related proteins, hindering metabolic functions in vivo and cell proliferation due to reactive oxygen species (ROS) stress. In particular, the low expression of FAD/FMN-binding redox enzymes (relative expression ratio of the experimental group to the control=0.43843) could be attributed to the redox mechanism of Na4 Ni2 Sb2 W2 -SbW8 , which was consistent with the effect of polyoxometalates (POMs) and FMN-binding proteins on ATP formation. An electrochemical study showed that Na4 Ni2 Sb2 W2 -SbW8 combined with FMN to form Na4 Ni2 Sb2 W2 -SbW8 -2FMN complex through a one-electron process of the W atoms. Na4 Ni2 Sb2 W2 -SbW8 acted as catalase and glutathione peroxidase to protect the cell from ROS stress, and the inhibition rates were 63.3 % at 1.77 µM of NADPH and 86.06 % at 10.62 µM of 2-hydroxyterephthalic acid. Overall, our results showed that POMs can be specific oxidative/antioxidant regulatory agents.

Effects of triphenyl phosphate on ciliate protozoa Tetrahymena thermophila following acute exposure and sub-chronic exposure.

Triphenyl phosphate (TPHP) is one of the most widely used organophosphate flame retardants (OPFRs) and is frequently detected in a variety of environmental media. Previous studies reported that TPHP had toxic effects on vertebrates, but little toxic information was available in lower trophic aquatic organisms which were more sensitive to the exposure of many toxic substances. In this study, protozoa Tetrahymena thermophila (T. thermophila) were exposed to 0, 0.01, 0.17 or 2.35 mg/L TPHP for 5 days to study the effects of sub-chronic exposure on theoretical population, cell viability, cell size and number of cilia. Additionally, the effects of TPHP on gene transcription were assessed by transcriptome sequencing technique (RNA-Seq). Cell viability and number of cilia were significantly reduced in all TPHP exposure groups compared with the control. In addition, exposure to 0.17 or 2.35 mg/L TPHP significantly reduced the theoretical population, circumference and body width, and there was a significant decrease in body length in the 2.35 mg/L exposure group. Comparative transcriptome sequencing identified a total of 4105 up- and 4487 down-regulated genes after exposure to 2.35 mg/L TPHP for 5 days compared with the control. KEGG analysis showed that dysfunction of pathways associated with ribosome, spliceosome, phagosome, proteasome and protein processing in endoplasmic reticulum in this study might be responsible for the toxicity of T. thermophila caused by TPHP. In general, the results indicated that TPHP had an adverse effect on the protozoa T. thermophila.

Effects of tris (2-chloroethyl) phosphate (TCEP) on growth, reproduction and gene transcription in the protozoan Tetrahymena thermophila.

As a typical organophosphorus flame retardant, tris (2-chloroethyl) phosphate (TCEP) has been widely detected in various environmental media. Toxicity of TCEP to vertebrates have been investigated, but potential effects on lower trophic level species were unknown to date. In this study, toxic effects and molecular mechanisms of toxic actions of TCEP on the aquatic protozoan Tetrahymena thermophila were evaluated by use of phenotypic observations, transcriptome sequencing analysis and real-time quantitative PCR detection. Exposure to 0.044, 0.411 or 4.26 mg/L TCEP for 5 days decreased the theoretical population, cell viability, number of cilia and cell size of Tetrahymena thermophila in a time- and dose-dependent manner. Meanwhile, RNA-Seq analysis indicated that exposure to 4.26 mg/L TCEP significantly changed expression of 2932 genes (up-regulation: 1228; down-regulation: 1704). Of these, expressions of 9, 10 and 17 genes that were enriched in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) interaction in vesicular transport, proteasome and endocytosis pathway respectively were down-regulated. Data collected during this study suggested that exposure to high concentrations of TCEP might affect growth and reproduction of Tetrahymena thermophila through down-regulating transcriptional levels of genes encoding proteins associated with vesicle trafficking, proteasome and endocytosis.

[Analysis of gene expression of Tetrahymena thermophila treated with Panax japonicas].

Panax japonicus is a traditional Chinese medicine,and its principle components have shown certain pharmacological activities for cell damage,aging and cell apoptosis. In order to clarify the pharmacological mechanism and involved metabolic pathways of P. japonicas,the gene expression of Tetrahymena thermophila under P. japonicus treatment was analyzed through high-throughput transcriptome sequencing in this study. Based on the transcriptome analysis,3 544 differentially expressed genes were identified in control group,of which 1 945 genes showed up-regulated expression and 1 599 genes showed down-regulated expression. Under P. japonicas treatment in the experiment group,3 312 differentially expressed genes were screened,of which 1 `493 genes showed up-regulated expression and 1 819 genes showed down-regulated expression. GO enrichment analysis indicated that in control group,the genes in the cells in a series of fundamental biological process were down-regulated,such as DNA replication and protein synthesis; while the signal transduction process and fatty acids oxidizing process were enriched. Whereas in the experiment group,down-regulated genes were mainly enriched in oxidation-reduction,cofactor metabolic process and vitamin metabolic process; up-regulated genes were enriched in signal transduction process and protein modification process. In the analysis using KEGG database,cell cycle pathway was enhanced and autophagy pathway was inhibited under the condition of P. japonicas treatment. Real-time quantitative polymerase chain reaction( RT-qPCR) was used to detect the expression differences between 6 up-regulated and 4 down-regulated genes in related metabolic pathways. The RT-q PCR results and RNA-Seq data were highly correlated and consistent with each other. This study could provide important direction and basis for further study on the mechanism of cell growth regulation with the treatment of P. japonica.

Occurrence of the fungus mycotoxin, ustiloxin A, in surface waters of paddy fields in Enshi, Hubei, China, and toxicity in Tetrahymena thermophila.

There has been an increasing incidence rate of rice false smut in global rice cultivation areas. However, there is a dearth of studies on the environmental concentrations and hazards of ustiloxin A (UA), which is the major mycotoxin produced by a pathogenic fungus of the rice false smut. Here, the concentrations of UA in the surface waters of two paddy fields located in Enshi city, Hubei province, China, were measured, and its toxicity in T. Thermophila was evaluated. This is the first study to detect UA in the surface waters of the two paddy fields, and the measured mean concentrations were 2.82 and 0.26 µg/L, respectively. Exposure to 2.19, 19.01 or 187.13 µg/L UA for 5 days significantly reduced the theoretical population and cell size of T. thermophila. Furthermore, treatment with 187.13 µg/L UA changed the percentages of T. thermophila cells in different cell-cycle stages, and with an increased malformation rate compared with the control, suggesting the disruption of the cell cycle. The expressions of 30 genes involved in the enriched proteasome pathway, 7 cyclin genes (cyc9, cyc10, cyc16, cyc22, cyc23, cyc26, cyc33) and 2 histone genes (mlh1 and hho1) were significantly down-regulated, which might be the modes of action responsible for the disruption of cell cycling due to UA exposure.

Selenium cytotoxicity in Tetrahymena thermophila: New clues about its biological effects and cellular resistance mechanisms.

Selenium is an essential micronutrient but at high concentrations can produce severe cytotoxicity and genomic damage. We have evaluated the cytotoxicity, ultrastructural and mitochondrial alterations of the two main selenium inorganic species; selenite and selenate, in the eukaryotic microorganism Tetrahymena thermophila. In this ciliate, selenite is more toxic than selenate. Their LC50 values were calculated as 27.65 µM for Se(IV) and 56.88 mM for Se(VI). Significant levels of peroxides/hydroperoxides are induced under low-moderate selenite or selenate concentrations. Se(VI) exposures induce an immediate mitochondrial membrane depolarization. Selenium treated cells show an intense vacuolization and some of them present numerous discrete and small electrondense particles, probably selenium deposits. Mitochondrial fusion, an intense swelling in peripheral mitochondria and mitophagy are detected in selenium treated cells, especially in those exposed to Se (IV). qRT-PCR analysis of diverse genes, encoding relevant antioxidant enzymes or other proteins, like metallothioneins, involved in an environmental general stress response, have shown that they may be crucial against Se(IV) and/or Se (VI) cytotoxicity.

Cheating, facilitation and cooperation regulate the effectiveness of phage-encoded exotoxins as antipredator molecules.

Temperate phage encoded Shiga toxin (Stx) kills the bacterivorous predator, Tetrahymena thermophila, providing Stx+ Escherichia coli with a survival advantage over Stx- cells. Although bacterial death accompanies Stx release, since bacteria grow clonally the fitness benefits of predator killing accrue to the kin of the sacrificed organism, meaning Stx-mediated protist killing is a form of self-destructive cooperation. We show here that the fitness benefits of Stx production are not restricted to the kin of the phage-encoding bacteria. Instead, nearby "free loading" bacteria, irrespective of their genotype, also reap the benefit of Stx-mediated predator killing. This finding indicates that the phage-borne Stx exotoxin behaves as a public good. Stx is encoded by a mobile phage. We find that Stx-encoding phage can use susceptible bacteria in the population as surrogates to enhance toxin and phage production. Moreover, our findings also demonstrate that engulfment and concentration of Stx-encoding and susceptible Stx- bacteria in the Tetrahymena phagosome enhances the transfer of Stx-encoding temperate phage from the host to the susceptible bacteria. This transfer increases the population of cooperating bacteria within the community. Since these bacteria now encode Stx, the predation-stimulated increase in phage transfer increases the population of toxin encoding bacteria in the environment.

Preliminary results from structural systems biology approach in Tetrahymena thermophila reveal novel perspectives for this toxicological model.

Tetrahymena is a unicellular microbial eukaryotic organism that has been used extensively in toxicology and environmental research. This work attempts to model for the first time the wiring of proteins involved in cellular mechanisms of Cd toxicity in Tetrahymena thermophila. 1975 high-confidence PPIs between 68 Cd-binding proteins and 422 partners were inferred through a novel structural systems biology approach that utilizes comparative analysis between Tetrahymena and other eukaryotes for which experimentally supported protein interactomes exist. The PPIs of the potential network were confirmed by known domain interactions in the Protein Data Bank and its topological characteristics were compared with publicly available experimental information for T. thermophila. To experimentally validate the robustness of the proposed PPI network, the interaction between the two most interconnected hub proteins was detected through GST pull-down assay. Potential effects on Tetrahymena's cellular and metabolic processes by PPIs involving Cd-binding proteins were uncovered. Furthermore, 244 PPIs in which Cd-binding proteins or/and their partners are encoded by orthologs of human disease genes in T. thermophila, but not in yeast, were identified and analyzed. The findings suggest that Tetrahymena could be possibly a useful model for an improved understanding of molecular mechanisms of Cd toxicity in human diseases.

Formation and degradation of large extrusion bodies in Tetrahymena thermophila: The role of intramacronuclear microtubules in chromatin segregation.

Large extrusion bodies (EBs) in Tetrahymena thermophila were induced by treatment with aphidiocolin (APH), followed by transfer of the cells to a drug free medium. APH induces over-replication of DNA and reversible cell division arrest (Kaczanowski and Kiersnowska, 2011). After treatment the cells were transferred to a drug free medium, and a central granule of chromatin (a prospective EB) surrounded by microtubules developed inside the macronucleus. Subsequently the chromatin of the central granule appeared in the middle region of the dividing macronucleus, and it was later extruded as an EB. The remaining chromatin segregated to opposite ends of the elongating macronucleus and was separated from the chromatin destined for the EB by intramacronuclear microtubules. TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick end labeling]) and in vivo acridine orange (AO) staining showed that the EBs underwent apoptotic-like degradation and autophagy similar to that observed in programmed nuclear death (PND) of the old macronucleus in conjugant cells. Overall, based on the data obtained in this study we proposed the hypothesis that over- replication of DNA induced by APH results in the appearance of defective DNA copies, that are not segregated to the opposite regions of dividing macronucleus and are destined for EBs.

Stereochemistry of ephedrine and its environmental significance: Exposure and effects directed approach.

Analysis of drugs and pharmaceuticals in the environment is typically performed with non-chiral chromatographic techniques. The environmental risks posed by chiral compounds analysed in this way must therefore be assumed to be independent of chirality, meaning that each enantiomer is equally potent in toxicity and long-lived in stability. This manuscript examines the degradation of each of the four isomers of ephedrine in river simulating microcosms and links this to toxicity data obtained by exposing three different organisms (D. magna, P. subcapitata and T. thermophila) to each of the isomers individually. Microcosms showed that significant degradation only occurred in biotic conditions and that only two isomers (1R,2S-(-)-ephedrine, 1S,2S-(+)-pseudoephedrine) degraded significantly over a period of fourteen days. This is concerning because at least one of the non-degraded isomers (1S,2R-(+)-ephedrine) has been observed in wastewater effluent, which discharges directly into rivers, meaning these isomers could be persistent in the environment. We also observed formation of 1S,2R-(+)-ephedrine in single isomer 1R,2S-(-)-ephedrine river simulating microcosms. Human liver microsome assays and mass spectrometry based data mining revealed that 1S,2R-(+)-ephedrine is not human derived but it could be formed as a results of microbial metabolic processes. Across all three organisms tested the persistent isomers (1S,2R-(+)-ephedrine and 1R,2R-(-)-pseudoephedrine) were more toxic than those that undergo degradation; meaning that if these isomers are entering or formed in the environment they might represent a potentially hazardous contaminant.

Extreme metal adapted, knockout and knockdown strains reveal a coordinated gene expression among different Tetrahymena thermophila metallothionein isoforms.

Metallothioneins (MT) constitute a superfamily of small cytosolic proteins that are able to bind metal cations through numerous cysteine (Cys) residues. Like other organisms the ciliate Tetrahymena thermophila presents several MT isoforms, which have been classified into two subfamilies (Cd- and Cu-metallothioneins). The main aim of this study was to examine the specific functions and transcriptional regulation of the five MT isoforms present in T. thermophila, by using several strains of this ciliate. After a laboratory evolution experiment over more than two years, three different T. thermophila strains adapted to extreme metal stress (Cd2+, Cu2+ or Pb2+) were obtained. In addition, three knockout and/or knockdown strains for different metallothionein (MT) genes were generated. These strains were then analyzed for expression of the individual MT isoforms. Our results provide a strong basis for assigning differential roles to the set of MT isoforms. MTT1 appears to have a key role in adaptation to Cd. In contrast, MTT2/4 are crucial for Cu-adaptation and MTT5 appears to be important for Pb-adaptation and might be considered as an "alarm" MT gene for responding to metal stress. Moreover, results indicate that likely a coordinated transcriptional regulation exists between the MT genes, particularly among MTT1, MTT5 and MTT2/4. MTT5 appears to be an essential gene, a first such report in any organism of an essential MT gene.

Multigenerational effects of tris(1,3-dichloro-2-propyl) phosphate on the free-living ciliate protozoa Tetrahymena thermophila exposed to environmentally relevant concentrations and after subsequent recovery.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is considered a re-emerging environmental pollutant, and exposure to environmentally relevant concentrations has been shown to cause individual developmental toxicity in zebrafish and the water flea (Daphnia magna). However, multigenerational effects during exposure to TDCIPP and after subsequent recovery were unknown. In the present study, individuals of a model aquatic organism, the ciliated protozoan, T. thermophila were exposed to environmentally-relevant concentrations of TDCIPP (0, 300 or 3000 ng/L) for 60 days (e.g., theoretically 372 generations) followed by a 60-day period of recovery, during which T. thermophila were not exposed to TDCIPP. During exposure and after exposure, effects at the molecular, histological, individual and population levels were examined. Multigenerational exposure to 300 or 3000 ng TDCIPP/L for 60 days significantly decreased numbers of individuals, sizes of individuals, expressed as length and width of bodies, number of cilia, and depth and diameter of basal bodies of cilia, and up-regulated expressions of genes related to assembly and maintenance of cilia. Complete or partial recoveries of theoretical sizes of populations as well as sizes of individuals and expressions of genes were observed during the 60-day recovery period. Effects on number of cilia and depth and diameter of basal body of cilia were not reversible and could still be observed long after cease of TDCIPP exposure. Collectedly, and shown for the first time, multigenerational effects to T. thermophila were caused by exposure to environmentally relevant concentrations of TDCIPP. Also, there were multi-generational effects at the population level that were not caused by carry-over exposure to TDCIPP. The "permanent" alterations and their potential significance are discussed.

Ionophoric polyphenols selectively bind Cu(2+), display potent antioxidant and anti-amyloidogenic properties, and are non-toxic toward Tetrahymena thermophila.

Alzheimer's disease (AD) is the most common form of dementia affecting more than 28million people in the world. Only symptomatic treatments are currently available. Anticipated tri-fold increase of AD incidence in the next 50years has established the need to explore new possible treatments. Accumulation of extracellular amyloid-ß (Aß) plaques, intracellular tangles in the brain, and formation of reactive oxygen species (ROS) are the major hallmarks of the disease. The active role of some metal ions, especially Cu(2+), in promoting both Aß aggregation and reactive oxygen species formation has rendered ionophoric drugs as a promising treatment strategy. In this work, a series of 5 disease-modifying and multi-target ionophoric polyphenols (1-5), inspired on the structure of natural resveratrol, have been synthesized and characterized. All compounds bind Cu(2+) selectively over other biologically relevant metal ions. They form 2:1 (compound/Cu(2+)) complexes with association constants logKa 12-14 depending on the molecular design. Our results indicate that compounds 1-5 possess excellent antioxidant properties: they inhibit the Cu(2+)-catalyzed reactive oxygen species production between 47% and 100%, and they scavenge DPPH (1,1-diphenyl-2-picryl-hydrazyl) and AAPH (2,2'-azobis(2-amindino-propane)dihydrochloride) free radicals in general better than clioquinol, resveratrol and ascorbic acid. In addition, compounds 1-5 interact with Aß peptides and inhibit both the Cu(2+)-catalyzed aggregation and the self-assembly of Aß(1-40) up to a ∼92% extent. Interestingly, 1-5 are also able to disaggregate up to ∼91% of pre-formed Aß(1-40) aggregates. Furthermore, cytotoxic studies show remarkably low toxicity of 1-5 toward Tetrahymena thermophila with LD50 values higher than 150µM, comparable to non-toxic natural resveratrol.

The oxidative stress response of myclobutanil and cyproconazole on Tetrahymena thermophila.

Using Tetrahymena thermophila as experimental models, the oxidative stress of triazole fungicides myclobutanil (MYC) and cyproconazole (CYP) was investigated. Results showed that 24-h EC50 values for MYC and CYP were 16.67 (13.37-19.65) and 20.44 (18.85-21.96) mg/L, respectively; 48-h EC50 values for MYC and CYP were 14.31 (13.13-15.42) and 18.76 (17.09-20.31) mg/L, respectively. Reactive oxygen species was significantly induced and cytotoxicity was caused by MYC and CYP by increasing propidium iodide (PI) fluorescence. Damage of regular wrinkles and appearing of small holes on the cell surface were observed by SEM. Furthermore, MYC and CYP also caused notable changes in enzyme activities and mRNA levels. Overall, the present study points out that MYC and CYP lead to oxidative stress on T. thermophila. The information presented in this study will provide insights into the mechanism of triazoles-induced oxidative stress on T. thermophila.

An interlaboratory comparison of nanosilver characterisation and hazard identification: Harmonising techniques for high quality data.

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag(+)-species (the concentration of Ag(+)-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4±6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46-68% of soluble Ag(+)-species in stock, 123.8±12.2 nm mean z-average value in dH2O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50<0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01-1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1-10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10-100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag(+)-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag(+)-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

Two in vitro methods for screening potential parasiticides against Ichthyophthirius multifiliis using Tetrahymena thermophila.

Ichthyophthirius multifiliis (Ich) is a ciliate parasite that infects many species of freshwater fishes worldwide and causes heavy economic losses in aquaculture. Currently, parasiticides for controlling this parasite are limited, and few pond-practical chemical therapies exist. Hence, the search for new parasiticides is urgently needed. One challenge confronting the screening of potential parasiticides is the difficulty in raising enough parasite for efficacy testing as Ich is an obligate parasite. This study used species of Tetrahymena, Ich-related and cultivable ciliate protozoa, to evaluate two in vitro methods to test parasiticides. Plate counting and MTS assays (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay) were used to compare lethal concentrations or median lethal concentrations (LC50) of copper sulphate, formalin and malachite green between T. thermophila and Ich theronts or between T. thermophila and Ich tomonts. The parasiticides that killed T. thermophila have been demonstrated to kill theronts or tomonts. These in vitro methods using T. thermophila can be used to screen novel parasiticides against Ich.

Proteomic analyses of early response of unicellular eukaryotic microorganism Tetrahymena thermophila exposed to TiO2 particles.

Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO2 nanoparticles (TiO2-NPs) and their bulk counterparts (bulk-TiO2). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO2-NPs or bulk-TiO2 particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE + nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO2 particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO2-NPs concentration differed significantly from the response to higher TiO2-NPs concentration and both bulk-TiO2 concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO2; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO2-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.

[Acute Toxicity and Safety Assessment of Three Typical Organic Pollutants to Two Aquatic Organisms].

Acute toxic effects of three typical organic pollutants 1 ,2,4-trichlorobenzene (1,2,4-TCB), nitrobenzene and chlorpyrifos were investigated using Tetrahymena thermophila and Limnodrilus hoffmeisteri as living test organisms under laboratory conditions. The results showed that with the increase of pollutants' concentration and the extension of time, toxicity of the three kinds of pollutants significantly enhanced, and the mortality of two kinds of aquatic organisms also had a rising trend, and an obvious dose-effect relationship. The 96 h-LC50. values of 1 ,2, 4-TCB, nitrobenzene and chlorpyrifos were 71.88, 285.76, and 5.50 mg x L(-1) for L. hoffmeisteri and 15.58, 140.22, and 14.69 mg x L(-1) for T. thermophila. These results showed that the toxicity among the three typical pollutants to T. thermophila was 1 , 2,4-TCB > chlorpyrifos > nitrobenzene. Findings were able to provide more information on water quality criteria and more data on their toxicity to indigenous aquatic organisms in China.

Effects of triclosan and triclocarban on the growth inhibition, cell viability, genotoxicity and multixenobiotic resistance responses of Tetrahymena thermophila.

The information about adverse effects of emerging contaminants on aquatic protozoa is very scarce. The growth inhibition effect, cell viability, genotoxicity and multixenobiotic resistance (MXR) responses of two commonly used antimicrobial agents, triclosan (TCS) and triclocarban (TCC) to protozoan Tetrahymena thermophila were investigated in this study. The results revealed that TCS and TCC can inhibit the growth of T. thermophila with 24h EC50 values of 1063 and 295µgL(-1), respectively. The impairment of plasma membrane was observed after 2h exposure of TCS or TCC at the level of mg/L. Furthermore, it is noticeable that at environmentally relevant concentration (1.0µgL(-1)), both TCS and TCC can lead to statistically significant DNA damage in T. thermophila, while the inhibition of growth and change of cell viability cannot be observed. Our results firstly provide the evidence for genotoxic effects of TCS and TCC on the freshwater protozoan. Additionally, both TCS and TCC were found to inhibit the efflux transporter activities, with the inhibitory potencies of 39% and 40% (using verapamil as a model inhibitor), respectively. Particularly, TCC could significantly down-regulate the expression of MXR related gene Abcb15, which encodes the membrane efflux protein that acting as P-gp in T. thermophila. The results raise the awareness of potential aquatic ecological and human health risks from the exposure of TCS and TCC, as they might potentiate the toxic effects by chemosensitizing with co-existing toxicants.