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.

Schild Analysis of the Interaction between Parthenolide and Cocaine Suggests an Allosteric Relationship for Their Effects on Planarian Motility.

The freshwater planarian is an emerging animal model in neuroscience due to its centralized nervous system that closely parallels closely parallels the nervous system of vertebrates. Cocaine, an abused drug, is the 'founding member' of the local anesthetic family. Parthenolide, a sesquiterpene lactone, acts as a behavioral and physiological antagonist of cocaine in planarians and rats, respectively. Previous work from our laboratory showed that both parthenolide and cocaine reduced planarian motility and that parthenolide reversed the cocaine-induced motility decrease at concentrations where parthenolide does not affect the movement of the worms. However, the exact mechanism of the cocaine/parthenolide antagonism is unknown. Here, we report the results of a Schild analysis to explore the parthenolide/cocaine relationship in the planarian Girardia tigrina. The Schild slopes of a family of concentration-response curves of parthenolide ± a single concentration of cocaine and vice versa were -0.55 and -0.36, respectively. These slopes were not statistically different from each other. Interestingly, the slope corresponding to the parthenolide ± cocaine (but not the cocaine ± parthenolide) data set was statistically different from -1. Our data suggest an allosteric relationship between cocaine and parthenolide for their effect on planarian motility. To the best of our knowledge, this is the first study about the mechanism of action of the antagonism between cocaine and parthenolide. Further studies are needed to determine the specific nature of the parthenolide/cocaine target(s) in this organism.

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.

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.

Theoretical insights, degradation, and sub-lethal toxicity of thiamethoxam to the planarian Girardia tigrina.

Advanced oxidative processes, such as Photo-Fenton, transform organic contaminants due to the attack by radicals. In this context, the lethal and sub-lethal effects of the Cruiser® 350FS (CRZ) with the active ingredient thiamethoxam (TMX) were investigated using the planarian Girardia tigrina. Degradation of thiamethoxam by the Fenton process was also assessed by using theoretical studies and the efficiency of Solar-Fenton versus Fenton. The 48 h LC50 value of CRZ for planarians was 478.6 mg L-1. The regeneration of planarians was significantly affected for concentrations ≥ 17 mg·L-1 of TMX (24 h). The Solar-Fenton showed a high degradation percentage reaching ~70%. The theoretical model showed the atoms of the TMX molecule that will suffer attacks from the formed radicals. Current results open new perspectives concerning the treatment of TMX in the aquatic environment because the 70% degradation seems to be sufficient to reach concentrations that do not induce sub-lethal effects in planarians. Further studies should determine if the by-products generated might be toxic for planaria or other organisms.

Ecotoxicological evaluation and regeneration impairment of planarians by dibutyl phthalate.

Commonly utilized as a plasticizer in the food and chemical sectors, Dibutyl phthalate (DBP) poses threats to the environment and human well-being as it seeps or moves into the surroundings. Nevertheless, research on the harmfulness of DBP to aquatic organisms is limited, and its impact on stem cells and tissue regeneration remains unidentified. Planarians, recognized for their robust regenerative capabilities and sensitivity to aquatic pollutants, are emerging animal models in toxicology. This study investigated the comprehensive toxicity effects of environmentally relevant levels of DBP on planarians. It revealed potential toxicity mechanisms through the use of immunofluorescence, chromatin dispersion assay, Western blot, quantitative real-time fluorescence quantitative PCR (qRT-PCR), chromatin behavioral and histological analyses, immunofluorescence, and terminal dUTP nickel-end labeling (TUNEL). Findings illustrated that DBP caused morphological and motor abnormalities, tissue damage, regenerative inhibition, and developmental neurotoxicity. Further research revealed increased apoptosis and suppressed stem cell proliferation and differentiation, disrupting a balance of cell proliferation and death, ultimately leading to morphological defects and functional abnormalities. This was attributed to oxidative stress and DNA damage caused by excessive release of reactive oxygen species (ROS). This exploration furnishes fresh perspectives on evaluating the toxicity peril posed by DBP in aquatic organisms.

How neurobehavior and brain development in alternative whole-organism models can contribute to prediction of developmental neurotoxicity.

Developmental neurotoxicity (DNT) is not routinely evaluated in chemical risk assessment because current test paradigms for DNT require the use of mammalian models which are ethically controversial, expensive, and resource demanding. Consequently, efforts have focused on revolutionizing DNT testing through affordable novel alternative methods for risk assessment. The goal is to develop a DNT in vitro test battery amenable to high-throughput screening (HTS). Currently, the DNT in vitro test battery consists primarily of human cell-based assays because of their immediate relevance to human health. However, such cell-based assays alone are unable to capture the complexity of a developing nervous system. Whole organismal systems that qualify as 3 R (Replace, Reduce and Refine) models are urgently needed to complement cell-based DNT testing. These models can provide the necessary organismal context and be used to explore the impact of chemicals on brain function by linking molecular and/or cellular changes to behavioural readouts. The nematode Caenorhabditis elegans, the planarian Dugesia japonica, and embryos of the zebrafish Danio rerio are all suited to low-cost HTS and each has unique strengths for DNT testing. Here, we review the strengths and the complementarity of these organisms in a novel, integrative context and highlight how they can augment current cell-based assays for more comprehensive and robust DNT screening of chemicals. Considering the limitations of all in vitro test systems, we discuss how a smart combinatory use of these systems will contribute to a better human relevant risk assessment of chemicals that considers the complexity of the developing brain.

Planarian behavioural endpoints in ecotoxicology: A case study evaluating mercury and salinity effects.

Freshwater planarians can be useful for the evaluation of contaminant stress on behavioral endpoints. In this work, we studied the sensitivity of the freshwater planarian Girardia tigrina in response to two model stressors (Hg and NaCl) by evaluating mortality, feeding rate and locomotion. A simple feeding assay with G. tigrina was devised, and an automated tracking system was used to evaluate locomotion. The estimated 96 h LC50s were 176.8 µg L-1 of Hg and 6.79 g L-1 of NaCl. Acute effects of Hg also included the disintegration of tissues, and loss of pigmentation. Acute effects of NaCl included motionlessness and rupture of the tegument. Hg and NaCl sub-lethal exposures caused feeding inhibition and locomotion impairment. This study demonstrates the usefulness of planarians for ecotoxicological research and that sensitive behavioral endpoints can evaluate the sub-lethal impacts of stressors to freshwater invertebrates.

The negative effect of the PI3K inhibitor 3-methyladenine on planarian regeneration via the autophagy signalling pathway.

As an important PI3K (VPS34) inhibitor, 3-methyladenine (3-MA) can block the formation of autophagic vesicles in animals. Most toxicological studies using 3-MA have shown that 3-MA leads to serious disorders via autophagy suppression in mammals. However, no toxicological research on 3-MA has been performed on individuals undergoing regeneration. The freshwater planarian has powerful regenerative capability, and it can regenerate a new brain in 5 days and undergo complete adult individual remodelling in approximately 14 days. Moreover, it is also an excellent model organism for studies on environmental toxicology due to its high chemical sensitivity and extensive distribution. Here, Dugesia japonica planarians were treated with 3-MA, and the results showed that autophagy was inhibited and Djvps34 expression levels were down-regulated. After exposure to 10 mM 3-MA for 18 h, all the controls showed normal phenotypes, while one-half of the planarians treated with 3-MA showed morphological defects. In most cases, an ulcer appeared in the middle of the body, and a normal phenotype was restored 7 days following 3-MA exposure. During regeneration, disproportionate blastemas with tissue regression were observed. Furthermore, 3-MA treatment suppressed stem cell proliferation in intact and regenerating worms. These findings demonstrate that autophagy is indispensable for tissue homeostasis and regeneration in planarians and that 3-MA treatment is detrimental to planarian regeneration via its effect on the autophagy pathway.

Ecotoxicity Assays Using Freshwater Planarians.

Freshwater planarians are free-living flatworms known for their regenerative ability. Being easily cultured under laboratory conditions, they are recognized test model organisms in regeneration, developmental biology, and neuropharmacological research. Also, they have been recently employed in toxicity testing where they displayed an array of sensitive and reliable responses to environmental stressors. Here, we outline simple and easy-to-follow protocols to evaluate effects of environmental contaminants and other stressors on survival, behavior (feeding and locomotor activity), and regeneration of freshwater planarians. These endpoints are comparable with responses of well-established ecotoxicological model species.

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.

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.

Antioxidant responses and lipid peroxidation can be used as sensitive indicators for the heavy metals risk assessment of the Wei River: a case study of planarian Dugesia Japonica.

PURPOSE: To verify antioxidant responses and lipid peroxidation can be used as sensitive indicators for the risk assessment of the Wei River. MATERIAL AND METHODS: We investigate the effects of the Wei River on oxidative stress of planarian Dugesia japonica by antioxidant parameters, and use ICP-MS to measure the heavy metals in the Wei River. Then, we observe the effects of three common heavy metal ions (Cr3+, Hg2+, Pb2+) on the regeneration of planarians on morphological and histological levels. RESULTS: The significant changes of antioxidant parameters (SOD, CAT, GPx, GST, T-AOC) and MDA content indicate that oxidative stress is induced after the Wei River exposure on planarians, though the heavy metals in the Wei River are not exceeding the standards. Then, the regeneration of planarians shows different degree of morphological and histological damage after Cr3+, Hg2+ and Pb2+ exposure. CONCLUSION: We speculate that the heavy metal ions in the Wei River, especially Cr3+, Hg2+ and Pb2+, may give rise to oxidative damage on planarians. These findings illustrate that planarian can serve as an indicator of aquatic ecosystem pollution, antioxidant responses and lipid peroxidation can also be used as sensitive indicators and provide an excellent opportunity for urban river risk assessment.

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.

5-Fluorouracil-treated planarians, a versatile model system for studying stem cell heterogeneity and tissue aging.

BACKGROUND INFORMATION: Planarians are a sound, well-established model system for molecular studies in the field of stem cells, cell differentiation, developmental biology and translational research. Treated stem cell-less planarians produced by X-ray treatment are commonly used to study stem cell transcriptional profile and their role in planarian biological processes. X-ray induces oxidative and DNA damage to differentiated cells, requires expensive radiation machines that are not available in most of the research centres and demand rigorous risk management and dedicated staff. RESULTS: We tested the use of the well-known antimetabolite genotoxic drug 5-fluorouracil which mainly affects proliferating cells in way to demonstrate its use in replacing X-ray treatment. We succeeded in demonstrating ability of high doses of 5-fluorouracil to deplete Dugesia japonica stem cells and in identifying a 5-fluorouracil transiently resistant population of lineage committed stem cells. CONCLUSIONS AND SIGNIFICANCE: Our results encourage the use of 5-fluorouracil-treated planarians as a model system for studying mechanisms of resistance to genotoxicants, planarian stem cell heterogeneity and molecular cascades of tissue aging.

Stem cell and tissue regeneration analysis in low-dose irradiated planarians treated with cerium oxide nanoparticles.

Owing to the self-renewing reactive oxygen species scavenger capability of cerium oxide nanoparticles (nanoceria), we tested in vivo radioprotective effects on stem cells and tissue regeneration using low-dose irradiated planarians as model system. We treated planarians with nanoceria or gum Arabic, as control, and we analyzed the expression of stem cell molecular markers and tissue regeneration capability, as well as cell death and DNA damage in non-irradiated and in low-dose irradiated animals. Our findings show that nanoceria increase the number of stem cells and tissue regenerative capability, and reduce cell death and DNA damage after low-dose irradiation, suggesting a protective role on stem cells.

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 Occurrence of Potential Harmful Cyanobacteria and Cyanotoxins in the Obrzyca River (Poland), a Source of Drinking Water.

Harmful cyanobacteria and their cyanotoxins may contaminate drinking water resources and their effective control remains challenging. The present study reports on cyanobacterial blooms and associated cyanotoxins in the Obrzyca River, a source of drinking water in Poland. The river was examined from July to October 2019 and concentrations of microcystins, anatoxin-a, and cylindrospermopsin were monitored. The toxicity of water samples was also tested using an ecotoxicological assay. All studied cyanotoxins were detected with microcystins revealing the highest levels. Maximal microcystin concentrations (3.97 µg/L) were determined in September at Uscie point, exceeding the provisional guideline. Extracts from Uscie point, where the dominant species were Dolichospermum flos-aquae (August), Microcystis aeruginosa (September), and Planktothrix agardhii (October), were toxic for Dugesia tigrina Girard. Microcystin concentrations (MC-LR and MC-RR) were positively correlated with cyanobacteria biovolume. Analysis of the chemical indicators of water quality has shown relationships between them and microcystins as well as cyanobacteria abundance.

Biomarkers of Planarian Dugesia japonica in Response to Herbicide Glyphosate Exposure.

As the worldwide top-selling herbicide, glyphosate is ubiquitously distributed in the natural environment, and its influence on the ecological safety and human health has being increasingly concerned. In this study, mRNA expressions of GPX and three heat shock protein genes in freshwater planarian Dugesia japonica in response to glyphosate were determined, and two oxidative stress parameters were measured. The results suggested that GPX activity can be used as a more sensitive biomarker in contrast with GPX gene expression, and mRNA expressions of Hsp70, Hsp90 genes are more sensitive than Hsp40 for planarians in response to glyphosate stress. Besides, the deduced T-AOC as well as varied GPX activity and mRNA expression levels of Hsps also indicated that glyphosate exposure would inhibit antioxidation and induce oxidative stress in D. japonica, while specific antioxidant systems and stress proteins tried to protect cells by their own regulation. The results of this study will be helpful to elucidate the stress response mechanisms of freshwater planarians to herbicide glyphosate.

Dugesia japonica is the best suited of three planarian species for high-throughput toxicology screening.

High-throughput screening (HTS) using new approach methods is revolutionizing toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct comparison with D. japonica. Through quantitative assessments of morphology and multiple behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 concentrations at two time points over a twelve-day period. We obtained two main results: First, G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to D. japonica under HTS conditions, due to decreased health over time and lack of movement under red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. Second, we observed species differences in sensitivity to the solvents, suggesting that care must be taken when extrapolating chemical effects across planarian species. Overall, our data show that D. japonica is best suited for behavioral HTS given the limitations of the other species. Standardizing which planarian species is used in neurotoxicity screening will facilitate data comparisons across research groups and accelerate the application of this promising invertebrate system for first-tier chemical HTS, helping streamline toxicology testing.