Behavioral and physiological responses of Girardia tigrina exposed to polyethylene microplastics.

Microplastic particles appear in great abundance and variety in freshwater ecosystems across the globe, spanning lakes and rivers, with increasingly frequent exposure of aquatic organisms. Studies on the mechanisms of any effects of plastic particles are still scarce, particularly in relation to the regenerative capacity of biota, for which there is no established model organism; however, planaria have shown sensitivity for assessing these risks to the aquatic environment. Thus, the present study aimed to investigate the behavioral and regeneration responses of the freshwater planaria Girardia tigrina exposed to polyethylene (PE) microplastics (MPs) incorporated into their food source. The greatest effect was observed on planarian regeneration, which was manifested at 10 µg/mg liver. Planaria reproduction and fertility were affected at 50 µg/mg liver; however, planaria locomotion was not affected at the concentrations evaluated. Mid-infrared absorption spectroscopy (FT-IR) was used to identify the constituent polymers, and ingestion of the polyethylene microplastic by the planaria was confirmed by infrared spectroscopy. The results highlight the potential adverse effects of exposure to polyethylene microplastic and show that the reproductive behavior and regeneration of a freshwater organism can be indicators of toxicity resulting from environmental pollution.

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.

Lethal and sub-lethal effects of cyproconazole on freshwater organisms: a case study with Chironomus riparius and Dugesia tigrina.

The fungicide cyproconazole (CPZ) inhibits the biosynthesis of ergosterol, an essential sterol component in fungal cell membrane and can also affect non-target organisms by its inhibitory effects on P450 monooxygenases. The predicted environmental concentration of CPZ is up to 49.05 µg/L and 145.89 µg/kg in surface waters and sediments, respectively, and information about CPZ toxicity towards non-target aquatic organisms is still limited. This study aimed to address the lack of ecotoxicological data for CPZ, and thus, an evaluation of the lethal and sub-lethal effects of CPZ was performed using two freshwater invertebrates (the midge Chironomus riparius and the planarian Dugesia tigrina). The estimated CPZ 48 h LC50 (95% CI) was 17.46 mg/L for C. riparius and 47.38 mg/L for D. tigrina. The emergence time (EmT50) of C. riparius was delayed by CPZ exposure from 0.76 mg/L. On the other hand, planarians showed higher tolerance to CPZ exposure. Sub-lethal effects of CPZ on planarians included reductions in locomotion (1.8 mg/L), delayed photoreceptors regeneration (from 0.45 mg/L), and feeding inhibition (5.6 mg/L). Our results confirm the moderate toxicity of CPZ towards aquatic invertebrates but sub-lethal effects observed also suggest potential chronic effects of CPZ with consequences for population dynamics.

Effect of Donepezil, Tacrine, Galantamine and Rivastigmine on Acetylcholinesterase Inhibition in Dugesia tigrina.

Dugesia tigrina is a non-parasitic platyhelminth, which has been recently utilized in pharmacological models, regarding the nervous system, as it presents a wide sensitivity to drugs. Our trials aimed to propose a model for an in vivo screening of substances with inhibitory activity of the enzyme acetylcholinesterase. Trials were performed with four drugs commercialized in Brazil: donepezil, tacrine, galantamine and rivastigmine, utilized in the control of Alzheimer's disease, to inhibit the activity of acetylcholinesterase. We tested five concentrations of the drugs, with an exposure of 24 h, and the mortality and the inhibition of acetylcholinesterase planarian seizure-like activity (pSLA) and planarian locomotor velocity (pLMV) were measured. Galantamine showed high anticholinesterasic activity when compared to the other drugs, with a reduction of 0.05 µmol·min(-1) and 63% of convulsant activity, presenting screw-like movement and hypokinesia, with pLMV of 65 crossed lines during 5 min. Our results showed for the first time the anticholinesterasic and convulsant effect, in addition to the decrease in locomotion induced by those drugs in a model of invertebrates. The experimental model proposed is simple and low cost and could be utilized in the screening of substances with anticholinesterasic action.

The effect of paraoxon on spermatogenesis in Dugesia gonocephala from the Chilean Altiplano: proliferation and apoptosis.

INTRODUCTION AND AIMS: The Chilean Altiplano ecosystem is conserved free from contaminants and pollutants because of the absence of major local human activities such as agriculture or other industries. We studied the effects of paraoxon on proliferation and apoptosis of testicular cells during active spermatogenesis in Dugesia gonocephala collected from a pristine river (Guacollo) in the Altiplano region nearby Visviri town, Chile. MATERIALS AND METHODS: Adult planarians were incubated in varying concentrations of paraoxon (0.8, 0.4, 0.04, 0.004, and 0.0004 mM) for 4 h. After 3 h of incubation, bromodeoxyuridine (BrdU) was added. Effects on cell proliferation (BrdU) and apoptosis (Apaf-1) were determined by immunohistochemistry. RESULTS: Paraoxon concentrations of 0.4 and 0.8 mM caused 100% mortality in the respective treatment groups. The lowest tested concentration (0.0004 mM) caused a significant increase on cell proliferation in the seminiferous tubules, as well as an increase in the number of apoptotic cells. All other tested concentrations significantly inhibited cell proliferation and induced apoptosis. CONCLUSIONS: Paraoxon inhibits DNA synthesis and induces apoptosis during spermatogenesis in adult planarians from a high-altitude, pollution-free environment. This could suggest its use as a biosensor or biomarker for contamination with agro pesticides.

Removal of methyl parathion in water, by Dugesia dorotocephala.

The aim of this study was to determine the efficiency of Dugesia dorotocephala on Methyl parathion removal. An initial concentration of 1.25 microg mL(-1) of MeP was used to evaluate the removal capacity of planarian. A first-order removal kinetics was obtained with a disappearance rate constant (k(r)) of 0.49 days(-1) and 69% efficiency on contaminant removal. This is significantly different (p < 0.5) from the degradation occurring in control systems, leading us to conclude that D. dorotocephala effectively removes MeP from contaminated water.

Evaluation of copper effects upon Girardia tigrina freshwater planarians based on a set of biomarkers.

Copper is a common environmental contaminant, which is particularly toxic to living organisms when in high concentrations. To monitor environmental contamination by Cu2+ and other heavy metals, well characterized bioindicator organisms and standardized assays are needed. As a first step toward this end, we have analysed Cu2+ effects upon Girardia tigrina freshwater planarians, based on the assessment of mobility, regeneration performance, micronucleus (MN) frequency in regenerating animals, and reproductive performance. These four biomarkers provided complementary information on Cu2+ toxicity, teratogenicity, mutagenicity and chronic (>96 h of exposure) effects, respectively. The LC50 was calculated for newborn, adult and regenerating planarians, and values of 12+/-0.02 mg l(-1), 42+/-0.08 mg l(-1), 48+/-0.13 mg l(-1), respectively, were obtained after 96 h of exposure. Mobility, for intact adults, and time of regeneration and MN frequency, for regenerating animals, were significantly affected by Cu2+ concentrations as low as 0.10 mg l(-1). MN assay for regenerating G. tigrina neoblasts showed higher sensitivities than MN assays performed with other bioindicator freshwater organisms, such as moluscs or fish. Chronic exposure effects were clearly evidenced by assessment of reproductive performance, with significant reduction in fecundity and fertility rates upon exposure to Cu2+ concentrations as low as 0.05 mg l(-1). Therefore, G. tigrina can be regarded as a useful bioindicator species for the detection and evaluation of Cu2+ effects upon freshwater invertebrates, allowing insights on the effects of Cu2+ (and possibly other heavy metals) in a freshwater environment.

Freshwater planarians as novel organisms for genotoxicity testing: Analysis of chromosome aberrations.

Two freshwater species of planarians, Girardia schubarti Marcus and G. tigrina Girard, were used for measuring chromosome aberration (CA) induction under laboratory conditions. Three genotoxicants were tested: methyl methanesulfonate (MMS), a direct-acting genotoxicant; cyclophosphamide, a metabolism-dependent genotoxicant; and gamma-radiation, a clastogenic agent. All three agents produced positive responses in both species. The strongest dose-responses were detected with MMS, and, in general, G. tigrina was somewhat more sensitive to the genotoxicity of the agents than G. schubarti. This difference in sensitivity may be due to: (a) the smaller body mass of G. tigrina; (b) differences in DNA repair, which may be reflected in the marginally higher background CA frequency of G. tigrina; and/or (c) the greater number of chromosomes in G. tigrina (2N = 16) as compared with G. schubarti (2N = 8). The responses induced by gamma-radiation in the planarians were similar to or higher than those induced in cultured human lymphocytes. The CA-planarian assay has advantages for monitoring environmental genotoxicity in natural water resources or urban and industrial wastewater since planarians are characterized by (a) a relatively low number of easily analyzable chromosomes; (b) high regenerating capacity, allowing exposure of replicating cells from different parts of the same organism to different doses; (c) easy maintenance under laboratory conditions; and (d) worldwide distribution, making them available for genotoxicity tests using either in situ or controlled laboratory exposure conditions.

Genotoxic effects of copper sulphate in freshwater planarian in vivo, studied with the single-cell gel test (comet assay).

The alkaline single-cell gel electrophoresis, or comet assay, was used to evaluate the genotoxic potential of copper sulphate in planarians. Concentration-related increase in DNA damage was induced after 2h and 7 days exposure to CuSO4 dissolved in culture water. To study the influence of copper ions on the persistence of mutagen-induced DNA lesions, planarians were treated with methyl methanesulphonate (MMS), and further incubated in the absence (post-incubation) or presence (post-treatment) of CuSO4. After 2h of post-treatment enhanced persistence of DNA effects in relation to the corresponding post-incubation value was detected, which indicate inhibition of DNA repair by CuSO4. At 4h an increase of DNA migration in relation to the 2h value was observed, which is significant for the post-incubation group. After 24h, DNA damage decreased but was still significantly elevated in relation to the control. From our results, we conclude that planarians are suitable organisms for in vivo detection of copper genotoxicity in the comet assay, and can be used to assess both acute and chronic exposure to this chemical in aquatic ecosystems. The inhibition effect of copper ions on repair of MMS-induced DNA damage suggests that copper could modulate the genotoxic effects associated with complex mixture exposure in the environment.