Planarian Mucus: A Novel Source of Pleiotropic Cytotoxic and Cytostatic Agents against Cancer Cells.

Biological evolution has generated a vast array of natural compounds produced by organisms across all domains. Among these, secondary metabolites, selected to enhance an organism's competitiveness in its natural environment, make them a reservoir for discovering new compounds with cytotoxic activity, potentially useful as novel anticancer agents. Slime secretions, the first barrier between epithelial surfaces and the surrounding environment, frequently contain cytotoxic molecules to limit the growth of parasitic organisms. Planarians, freshwater Triclads, continuously secrete a viscous mucus with multiple physiological functions. The chemical composition of planarian mucus has been only partially elucidated, and there are no studies reporting its cytotoxic or cytostatic effects. In this study, we developed a protocol for collecting mucus from Dugesia japonica specimens and we demonstrated that it inhibits the growth of cancer cells by activating cytostatic and ROS-dependent cytotoxic mechanisms inducing lipid droplet accumulation and mitochondrial membrane reorganization. Although further research is needed to identify the specific chemicals responsible for the anticancer activity of planarian mucus, this work opens up numerous research avenues aimed at better understanding the mechanisms of action of this product for potential therapeutic applications.

Cotinine influences the effect of high and low nicotine concentrations on planarian motility differently.

Previous work from our laboratory showed that cotinine, a nicotine metabolite, reverses three nicotine-induced behavioral effects in freshwater planarians: motility decrease, seizure-like movements, and withdrawal-like behaviors. The present work explored whether cotinine, a nicotine metabolite, antagonized the nicotine-induced effects on planarian motility in a concentration-dependent manner. We found that nicotine decreased planarian motility at nicotine concentrations above 60 µM but increased planarian velocity at concentrations equal to or below 50 µM, in agreement with previous data. Cotinine did not affect planarian motility at a concentration range between 250 and 2750 µM. Furthermore, we found that cotinine alleviated the 100 µM nicotine-induced motility decrease in a concentration-dependent manner and reversed the low nicotine concentration motility increase, albeit in a concentration-independent manner. The apparent concentration-dependent alleviation of >60 µM nicotine-induced motility decrease by cotinine suggests an orthosteric relationship between nicotine and cotinine. On the other hand, the evident concentration-independent cotinine alleviation of the increase in motility induced by 50 µM nicotine suggests an allosteric relationship. Our data is consistent with the existing literature about the relationship between nicotine and cotinine in various models, reinforcing the case for the usefulness of the planarian model in pharmacological studies.

Differential effect of silver nanoparticles on the microbiome of adult and developing planaria.

Silver nanoparticles (AgNPs) are widely incorporated in household, consumer and medical products. Their unintentional release via wastewaters raises concerns on their environmental impact, particularly for aquatic organisms and their associated bacterial communities. It is known that the microbiome plays an important role in its host's health and physiology, e.g. by producing essential nutrients and providing protection against pathogens. A thorough understanding of the effects of AgNPs on bacterial communities and on their interactions with the host is crucial to fully assess AgNP toxicity on aquatic organisms. Our results indicate that the microbiome of the invertebrate Schmidtea mediterranea, a freshwater planarian, is affected by AgNP exposure at the tested 10 µg/ml concentration. Using targeted amplification of the bacterial 16S rRNA gene V3-V4 region, two independent experiments on the microbiomes of adult worms revealed a consistent decrease in Betaproteobacteriales after AgNP exposure, mainly attributed to a decrease in Curvibacter and Undibacterium. Although developing tissues and organisms are known to be more sensitive to toxic compounds, three independent experiments in regenerating worms showed a less pronounced effect of AgNP exposure on the microbiome, possibly because underlying bacterial community changes during development mask the AgNP induced effect. The presence of a polyvinyl-pyrrolidone (PVP) coating did not significantly alter the outcome of the experiments compared to those with uncoated particles. The observed variation between the different experiments underlines the highly variable nature of microbiomes and emphasises the need to repeat microbiome experiments, within and between physiological states of the animal.

Nicotine chronic tolerance development and withdrawal in the planaria (Schmidtea mediterranea).

Chronic nicotine exposure reduces sensitivity to the effects of nicotine, which then results in behavioural changes and tolerance development. In the planaria, a valuable first-stage preclinical model for addictive behaviour, acute nicotine administration has been shown to steadily alter the motility of the animals, a result that has been interpreted as evidence of tolerance and withdrawal effects; however, chronic exposure - typically regarded as a condition for the development of tolerance - and the role of the contextual cues have not been systematically assessed. The present study assessed the acute and chronic effects of nicotine on the motility of planarians (Schmidtea mediterranea). The animals in the experimental groups received long chronic exposure to nicotine (ten daily 30 min exposures); a control group was exposed to water in the same context but in the absence of the drug. The motility of the animals was closely monitored on every exposure. Following this phase, all the animals were subject to three different tests: in the presence of the exposure context (without the drug, Test 1); in the presence of nicotine in the exposure context (Test 2); and in the presence of the drug in a novel context (Test 3). Exposure to nicotine consistently reduced motility; the motility in the presence of nicotine increased with repeated exposures to the drug, an instance of tolerance development. Tolerance development was dependent on nicotinic receptor activation, because it was blocked by the co-administration of mecamylamine. However, this tolerance was found to be independent of the contextual cues where the effects of the drug had been experienced. The results are discussed by reference to the existent theories of tolerance development to drugs.

Staphylococcus aureus-Derived α-Hemolysin Evokes Generation of Specialized Pro-resolving Mediators Promoting Inflammation Resolution.

Underlying mechanisms of how infectious inflammation is resolved by the host are incompletely understood. One hallmark of inflammation resolution is the activation of specialized pro-resolving mediators (SPMs) that enhance bacterial clearance and promote tissue repair. Here, we reveal α-hemolysin (Hla) from Staphylococcus aureus as a potent elicitor of SPM biosynthesis in human M2-like macrophages and in the mouse peritoneum through selective activation of host 15-lipoxygenase-1 (15-LOX-1). S. aureus-induced SPM formation in M2 is abolished upon Hla depletion or 15-LOX-1 knockdown. Isolated Hla elicits SPM formation in M2 that is reverted by inhibition of the Hla receptor ADAM10. Lipid mediators derived from Hla-treated M2 accelerate planarian tissue regeneration. Hla but not zymosan provokes substantial SPM formation in the mouse peritoneum, devoid of leukocyte infiltration and pro-inflammatory cytokine secretion. Besides harming the host, Hla may also exert beneficial functions by stimulating SPM production to promote the resolution of infectious inflammation.

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products.

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

The regulation of rapamycin in planarian Dugesia japonica Ichikawa & Kawakatsu, 1964 regeneration according to TOR signaling pathway.

The freshwater planarian mostly lives in the upper reaches of springs and rivers. Generally, it is realized as a suitable warning indicator of environmental toxicants. The freshwater planarian Dugesia japonica has a powerful regenerative capability and can regenerate a new individual including a complete central nervous system in one week. Rapamycin is an inhibitor of mammalian TORC1 (target of rapamycin complex-1) and used in the treatment of some diseases like cancer, cardiovascular and neurological diseases. However, the roles of rapamycin in the regulation of planarian regeneration remain to be elucidated. In present study, freshwater planarians D. japonica were firstly treated with 1 µM rapamycin for 18 h exposures and the expression patterns of Djtor was analyzed by the whole-mount in situ hybridization (WISH). Our results indicated rapamycin could strongly inhibit Djtor expression in planarian D. japonica and cause asymmetric blastemas and neuronal defects in planarians. Furthermore, knockdown of Djtor gene in planarians using RNA interference resulted in the suppression of downstream autophagy genes. These findings suggested that rapamycin might regulate freshwater planarian regeneration via Djtor signaling pathway.

Assessment of iron oxide nanoparticle ecotoxicity on regeneration and homeostasis in the replacement model system Schmidtea mediterranea.

Iron oxide nanoparticles (IONs) are used in a number of applications, from food to cosmetics, from medical applications to magnetic storage. In spite of the 550 tons produced each year in Europe alone, no effective dose limit recommendations are established and the overall risks connected to IONs are still debated. The incorporation of IONs in daily life raises a concern about their effects on the environment, on living organisms, and on human health. In this study, we used freshwater planarians to assess the nanoecotoxicity of IONs. Planarians are free-living invertebrates known for their astonishing regenerative ability. Because of their sensitivity to toxicants, they are often used to determine the effects of toxic, genotoxic and carcinogenic environmental compounds with an approach in line with the 3Rs (Reduce, Refine, Replace) principle. Planarians were exposed to IONs at concentrations up to 1 mg/mL and their effects were evaluated at the behavioral, morphofunctional and molecular levels, with a special emphasis on the regeneration process. Our results indicate that IONs did not affect the stem cell population dynamics, nor did they induce substantial changes in either homeostatic or regenerating planarians. As positive controls, gold nanoparticles coated with the pro-apoptotic anti-cancer drug hexadecylmethylammonium bromide, silver nanoparticles and highly concentrated polystyrene nanoparticles were used. These all elicited toxic effects. Therefore, we conclude that IONs at environmental concentrations are safe for planarians, and that the planarian is a powerful model system that can replace vertebrate animal models in nanoecotoxicology research and for nanoecotoxicology studies.

Preliminary evidence from planarians that cotinine establishes a conditioned place preference.

While the psychoactive stimulant nicotine has been the subject of extensive research, considerably less attention has focused on other compounds found in either tobacco smoke or that are nicotine metabolites. Recent papers have suggested that some of the compounds in question may either alter nicotine's effects or have reinforcing properties themselves, although they would only be experienced after consumption of tobacco. The potential for these compounds to function as reinforcers or to potentiate the reinforcing properties of nicotine merits investigation. To pursue this line of inquiry, we examined cotinine in a planarian model of environmental place preference. In the present study, planarians demonstrated that the compound cotinine, which is present in tobacco smoke, and is also the principal nicotine metabolite, establishes a conditioned place preference. These data represent the first ever demonstration that cotinine will establish a conditioned place preference in planarians and possibly contribute to the addictive properties of nicotine.

Blueberry anthocyanin alleviate perfluorooctanoic acid-induced toxicity in planarian (Dugesia japonica) by regulating oxidative stress biomarkers, ATP contents, DNA methylation and mRNA expression.

Blueberry anthocyanin (BA) have strong health benefits as an active natural antioxidant and perfluorooctanoic acid (PFOA) can result in oxidative stress in animals. In our study, the protective effects of BA against stress induced by PFOA was investigated in the planarian Dugesia japonica using oxidative stress biomarkers, ATP contents, ATPase activity, DNA methylation and mRNA expression. PFOA exposure could resulted in malondialdehyde production. At the same time, treatment with BA decreased the production of malondialdehyde in BA-exposed and co-treatment planarians. PFOA caused activities increase in glutathione peroxidase (GPx), glutathione S-transferase (GST) and activities decrease in glutathione reductase (GR). PFOA exposure decreased the GSH and ATP contents. Additionally, it increased the GSSG contents and ATPase activity. BA administration increased the activities of GPx, GST and GR in BA and co-treatment planarians. Meanwhile BA maintained the contents of ATP, ATPase activity, GSH and GSSG by alleviating PFOA toxicity. Moreover, PFOA and BA increased the contents of 5-methylcytosine and decreased 5-hydroxymethylcytosine in all group. In addition, PFOA and BA treated planarians significantly altered the expression of genes associated with above biochemical parameters. The results showed that the mRNA expression of gpx, Djgst, gr, Djnak and dnmt1 were significantly elevated in all groups. Alterations in the mRNA expression levels indicated a stress response to PFOA exposure and anthocyanin protection. These alterations regulated biomarkers of oxidative stress, energy metabolism and DNA methylation levels in planarians. These results indicate that BA attenuated PFOA-induced oxidative stress, energy metabolism, DNA methylation and gene expression disorders.

CuZnSOD and MnSOD from freshwater planarian Dugesia japonica: cDNA cloning, mRNA expression and enzyme activity in response to environmental pollutants.

As an important antioxidant enzyme, the superoxide dismutase (SOD) can protect aerobic organisms from oxidative damage through catalyzing the dismutation of superoxide into hydrogen peroxide and oxygen. The SODs have been cloned in some species and their dynamic expression or enzymatic activity in response to environmental stressors were investigated. In the current study, the full-length cDNA of two SODs from freshwater planarian Dugesia japonica were firstly cloned (named as DjCuZnSOD and DjMnSOD, respectively). The complete cDNA of DjCuZnSOD consists of 661 nucleotides encoding 186 amino acids while the 765 bp DjMnSOD encodes a polypeptide of 226 residues. Sequence analysis and multiple alignment showed that DjCuZnSOD possesses two CuZnSOD family signature motifs and an N-terminal signal peptide suggesting it is an extracellular secretory protein. DjMnSOD possesses the MnSOD family signature sequence and is predicted to be located in mitochondrion with a mitochondrial targeting sequence. Phylogenetic analysis based on CuZnSOD and MnSOD orthologs from representative species further verified that DjCuZnSOD is an extracellular CuZnSOD while DjMnSOD is a mitochondrial MnSOD. For the purpose of studying their potential role against environmental pollutants, D. japonica were exposed to glyphosate or 1-decyl-3-methylimidazolium bromide ([C10mim]Br), and the mRNA expression levels of DjCuZnSOD and DjMnSOD along with total SOD activity were measured. The results showed that DjCuZnSOD exhibited more sensitive expression profiles in response to environmental pollutants in contrast with DjMnSOD, and the total SOD activity in response to both pollutants was more related to the expression level of DjCuZnSOD than to DjMnSOD, indicating that the mRNA expression of CuZnSOD would be a more sensitive biomarker than MnSOD in monitoring the pollution of aquatic environment and CuZnSOD might play more important role than MnSOD in eliminating superoxide anions caused by pollutants in D. japonica.

Planarians as models to investigate the bioactivity of gold(I) complexes in vivo.

Gold(I)-containing complexes are used in drug discovery research for rheumatoid arthritis, cancer, and parasitic infections. In this study, we tested the bioactivity of gold(I) complexes in vivo using planarians. The planarian Schmidtea mediterranea possesses orthologues of tumor suppressor genes, such as p53, that, when silenced, cause deregulation of cell proliferation and apoptosis. In this context, we tested two triethylphosphine-gold(I) complexes (AdO and AdT) to determine if they can attenuate phenotypes that result from p53 inhibition. First, we identified the drug concentration that did not affect survival or regeneration and evaluated the drug's effect on cell division and apoptosis. We found that AdT treatment decreased the number of mitotic cells and that all drug treatments increased the number of apoptotic cells. We then performed p53(RNAi) and drug treatments concomitantly and observed the phenotype progression. Drug treatment increased survival three-fold and decreased apoptosis, which resulted in an attenuated phenotype. Our results indicate that planarians can be treated with gold(I) complexes, and that this treatment can diminish the p53(RNAi) phenotype and extend survival. In this work we show that planarians can be used as a model to study the in vivo effect of gold(I) complexes and to further investigate their mechanisms of action.

Planarians Customize Their Stem Cell Responses Following Genotoxic Stress as a Function of Exposure Time and Regenerative State.

Aiming to in vivo characterize the responses of pluripotent stem cells and regenerative tissues to carcinogenic stress, we employed the highly regenerative organism Schmidtea mediterranea. Its broad regenerative capacities are attributable to a large pool of pluripotent stem cells, which are considered key players in the lower vulnerability toward chemically induced carcinogenesis observed in regenerative organisms. Schmidtea mediterranea is, therefore, an ideal model to study pluripotent stem cell responses with stem cells residing in their natural environment. Including microenvironmental alterations is important, as the surrounding niche influences the onset of oncogenic events. Both short- (3 days) and long-term (17 days) exposures to the genotoxic carcinogen methyl methanesulfonate (50 µM) were evaluated during homeostasis and animal regeneration, two situations that render altered cellular niches. In both cases, MMS-induced DNA damage was observed, which provoked a decrease in proliferation on the short term. The outcome of DNA damage responses following long-term exposure differed between homeostatic and regenerating animals. During regeneration, DNA repair systems were more easily activated than in animals in homeostasis, where apoptosis was an important outcome. Knockdown experiments confirmed the importance of DNA repair systems during carcinogenic exposure in regenerating animals as knockdown of rad51 induced a stem cell-depleted phenotype, after regeneration was completed.

Methylisothiazolinone toxicity and inhibition of wound healing and regeneration in planaria.

Methylisothiazolinone (MIT) is a common biocide used in cosmetic and industrial settings. Studies have demonstrated that MIT is a human sensitizer, to the extent that in 2013 MIT was named allergen of the year. Recently, we showed that MIT exposure in Xenopus laevis (the African clawed frog) inhibits wound healing and tail regeneration. However, it is unknown whether MIT affects these processes in other animals. Here, we investigated the effects of MIT exposure in planaria-non-parasitic freshwater flatworms able to regenerate all tissues after injury. Using a common research strain of Dugesia japonica, we determined that intact planarians exposed to 15µM MIT displayed both neuromuscular and epithelial-integrity defects. Furthermore, regenerating (head and tail) fragments exposed to 15µM MIT failed to close wounds or had significantly delayed wound healing. Planarian wounds normally close within 1h after injury. However, most MIT-exposed animals retained open wounds at 24h and subsequently died, and those few animals that were able to undergo delayed wound healing without dying exhibited abnormal regeneration. For instance, head regeneration was severely delayed or inhibited, with anterior structures such as eyes failing to form in newly produced tissues. These data suggest that MIT directly affects both wound healing and regeneration in planarians. Next, we investigated the ability of thiol-containing antioxidants to rescue planarian wound closure during MIT exposure. The data reveal both n-acetyl cysteine and glutathione were each able to fully rescue MIT inhibition of wound healing. Lastly, we established MIT toxicity levels by determining the LC50 of 5 different planarian species: D. japonica, Schmidtea mediterranea, Girardia tigrina, Girardia dorotocephala, and Phagocata gracilis. Our LC50 data revealed that concentrations as low as 39µM (4.5ppm) are lethal to planarians, with concentrations of just 5µM inhibiting wound healing, and suggest that phylogeny is predictive of species toxicity levels. Together these results indicate MIT may have broad wound healing effects on aquatic species in general and are not limited to X. laevis alone. Future studies should investigate the impact of MIT on wound healing in other organisms, including non-aquatic organisms and mammals.

Planarians as models of cadmium-induced neoplasia provide measurable benchmarks for mechanistic studies.

Bioassays of planarian neoplasia highlight the potential of these organisms as useful standards to assess whether environmental toxins such as cadmium promote tumorigenesis. These studies complement other investigations into the exceptional healing and regeneration of planarians - processes that are driven by a population of active stem cells, or neoblasts, which are likely transformed during planarian tumor growth. Our goal was to determine if planarian tumorigenesis assays are amenable to mechanistic studies of cadmium carcinogenesis. To that end we demonstrate, by examining both counts of cell populations by size, and instances of mitosis, that the activity of the stem cell population can be monitored. We also provide evidence that specific biomodulators can affect the potential of planarian neoplastic growth, in that an inhibitor of metalloproteinases effectively blocked the development of the lesions. From these results, we infer that neoblast activity does respond to cadmium-induced tumor growth, and that metalloproteinases are required for the progression of cancer in the planarian.

The natural compound sanguinarine perturbs the regenerative capabilities of planarians.

The natural alkaloid sanguinarine has remarkable therapeutic properties and has been used for centuries as a folk remedy. This compound exhibits interesting anticancer properties and is currently receiving attention as a potential chemotherapeutic agent. Nevertheless, limited information exists regarding its safety for developing organisms. Planarians are an animal model known for their extraordinary stem cell-based regenerative capabilities and are increasingly used for toxicological and pharmacological studies. Here, we report that sanguinarine, at micromolar concentrations, perturbs the regeneration process in the planarian Dugesia japonica. We show that sanguinarine exposure causes defects during anterior regeneration and visual system recovery, as well as anomalous remodelling of pre-existing structures. Investigating the effects of sanguinarine on stem cells, we found that sanguinarine perturbs the transcriptional profile of early and late stem cell progeny markers. Our results indicate that sanguinarine exposure alters cell dynamics and induces apoptosis without affecting cell proliferation. Finally, sanguinarine exposure influences the expression level of H +, K+-ATPase α subunit, a gene of the P-type-ATPase pump family which plays a crucial role during anterior regeneration in planaria. On the whole, our data reveal that sanguinarine perturbs multiple mechanisms which regulate regeneration dynamics and contribute to a better understanding of the safety profile of this alkaloid in developing organisms.

Cotinine antagonizes the behavioral effects of nicotine exposure in the planarian Girardia tigrina.

Nicotine is one of the most addictive drugs abused by humans. Our laboratory and others have demonstrated that nicotine decreases motility and induces seizure-like behavior in planarians (pSLM, which are vigorous writhing and bending of the body) in a concentration-dependent manner. Nicotine also induces withdrawal-like behaviors in these worms. Cotinine is the major nicotine metabolite in humans, although it is not the final product of nicotine metabolism. Cotinine is mostly inactive in vertebrate nervous systems and is currently being explored as a molecule which possess most of nicotine's beneficial effects and few of its undesirable ones. It is not known whether cotinine is a product of nicotine metabolism in planarians. We found that cotinine by itself does not seem to elicit any behavioral effects in planarians up to a concentration of 1mM. We also show that cotinine antagonizes the aforementioned nicotine-induced motility decrease and also decreases the expression of nicotine-induced pSLMs in a concentration-dependent manner. Also cotinine prevents the manifestation of some of the withdrawal-like behaviors induced by nicotine in our experimental organism. Thus, we obtained evidence supporting that cotinine antagonizes nicotine in this planarian species. Possible explanations include competitive binding of both compounds at overlapping binding sites, at different nicotinic receptor subtypes, or maybe allosteric interactions.

Identification and Actions of a Novel Third Maresin Conjugate in Tissue Regeneration: MCTR3.

Maresin conjugates in tissue regeneration (MCTR) are a new family of evolutionarily conserved chemical signals that orchestrate host responses to promote tissue regeneration and resolution of infections. Herein, we identified the novel MCTR3 and established rank order potencies and matched the stereochemistries of MCTR1, MCTR2 and MCTR3 using material prepared by total organic synthesis and mediators isolated from both mouse and human systems. MCTR3 was produced from endogenous substrate by E. coli activated human macrophages and identified in sepsis patients. Each of the three synthetic MCTR dose-dependently (1-100 nM) accelerated tissue regeneration in planaria by 0.6-0.9 days. When administered at the onset or peak of inflammation, each of the MCTR promoted resolution of E. coli infections in mice. They increased bacterial phagocytosis by exudate leukocytes (~15-50%), limited neutrophil infiltration (~20-50%), promoted efferocytosis (~30%) and reduced eicosanoids. MCTR1 and MCTR2 upregulated human neutrophil and macrophage phagocytic responses where MCTR3 also proved to possess potent actions. These results establish the complete stereochemistry and rank order potencies for MCTR1, MCTR2 and MCTR3 that provide novel resolution moduli in regulating host responses to clear infections and promote tissue regeneration.

Induction of hsp70, hsp90, and catalase activity in planarian Dugesia japonica exposed to cadmium.

The hsp70 and hsp90 expression patterns and catalase (CAT) activity in the freshwater planaria Dugesia japonica exposed to cadmium (Cd) under laboratory conditions were investigated. Planaria were exposed to a range of Cd concentrations (0-150 µg Cd/L) for 24 h. The expression levels of hsp70 and hsp90 were determined by relative quantitative real-time polymerase chain reaction. Within the overall dose range in the experiment, the expression level of hsp70 and the activity of CAT in D. japonica were altered significantly. Hsp70 was induced in D. japonica upon Cd exposure concentrations as low as 9.375 µg Cd/L. No significant effect on the expression level of hsp90 was observed. Our findings demonstrated that stress gene hsp70, but not hsp90, was responsive to Cd contamination in D. japonica CAT activity was significantly induced at concentrations of 18.75, 37.5, and 75 µg Cd/L after 24-h exposure. We recommend that the use of hsp70 as a biomarker should be complemented by evidence of changes in other parameters, such as CAT activity, in D. japonica.

Toxic effects of ionic liquid 1-octyl-3-methylimidazolium bromide on the antioxidant defense system of freshwater planarian, Dugesia japonica.

The activities of antioxidant enzymes and the levels of glutathione (GSH) and malondialdehyde (MDA) were determined when freshwater planarian Dugesia japonica was exposed to different concentrations of 1-octyl-3-methylimidazolium bromide ([C8mim]Br) for one, three, and five days. The results showed that superoxide dismutase (SOD) activity began to increase in all treated groups after three days of exposure, while catalase (CAT) activity was inhibited after the first day, but increased notably on the fifth day except for the lowest concentration group. The activity of glutathione peroxidase (GPX) was induced from the first day of exposure and increased significantly after five days in all treated groups. During the experiment, the levels of intracellular GSH in all treated groups were higher than that of the control group. Changes in MDA suggest that [C8mim]Br is toxic to D japonica and may result in lipid peroxidation in planarian. Our results also indicate that GPX as well as GSH seem to be more sensitive biomarkers of oxidative stress compared with SOD and CAT.