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.

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.

Kinetic profiling an abundantly expressed planarian serotonergic GPCR identifies bromocriptine as a perdurant antagonist.

The diversity and uniqueness of flatworm G protein coupled receptors (GPCRs) provides impetus for identifying ligands useful as tools for studying flatworm biology, or as therapeutics for treating diseases caused by parasitic flatworm infections. To catalyse this discovery process, technologies optimized for mammalian GPCR high throughput screening need be transposed for screening flatworm GPCRs. Here, we demonstrate the utility of a genetically encoded cAMP biosensor for resolving the properties of an abundantly expressed planarian serotonergic GPCR (S7.1R). Application of this methodology resolved the real time kinetics of GPCR modulation by ligands and demonstrated a marked difference in the kinetic action of antagonists at S7.1R. Notably, bromocriptine caused a protracted inhibition of S7.1R activity in vitro and a protracted paralysis of planarian movement, replicating the effect of S7.1R in vivo RNAi. The lengthy inhibition of function caused by bromocriptine at this abundantly expressed GPCR provides a useful tool to ablate serotonergic signaling in vivo, and is a noteworthy feature for exploitation as an anthelmintic vulnerability.

The protective effect of blueberry anthocyanins against perfluorooctanoic acid-induced disturbance in planarian (Dugesia japonica).

The influence of blueberry anthocyanins on perfluorooctanoic acid (PFOA)-induced stress response in planarian mitochondria was investigated. PFOA at 15mg/L and anthocyanins at 10 or 20mg/L were individually and simultaneously administered to planarians for up to 10d. The results showed PFOA treatment induced an increase in mitochondrial permeability transition pore opening and a decrease antioxidant capacity and enzyme activities. In anthocyanin treated animals, the activity of succinate dehydrogenase, cytochrome oxidase and monoamine oxidase increased, but mitochondrial permeability transition pore opening decreased and total antioxidant capacity increased. An improvement in above-mentioned physiological and biochemical parameters was found in the combined PFOA and anthocyanin treated animals, in a dose-dependent manner. Anthocyanins attenuated the PFOA induced toxicity; antioxidant capacity and enzyme activities are involved in the protective mechanism of anthocyanins.

Guarana provides additional stimulation over caffeine alone in the planarian model.

The stimulant effect of energy drinks is primarily attributed to the caffeine they contain. Many energy drinks also contain other ingredients that might enhance the tonic effects of these caffeinated beverages. One of these additives is guarana. Guarana is a climbing plant native to the Amazon whose seeds contain approximately four times the amount of caffeine found in coffee beans. The mix of other natural chemicals contained in guarana seeds is thought to heighten the stimulant effects of guarana over caffeine alone. Yet, despite the growing use of guarana as an additive in energy drinks, and a burgeoning market for it as a nutritional supplement, the science examining guarana and how it affects other dietary ingredients is lacking. To appreciate the stimulant effects of guarana and other natural products, a straightforward model to investigate their physiological properties is needed. The planarian provides such a system. The locomotor activity and convulsive response of planarians with substance exposure has been shown to provide an excellent system to measure the effects of drug stimulation, addiction and withdrawal. To gauge the stimulant effects of guarana we studied how it altered the locomotor activity of the planarian species Dugesia tigrina. We report evidence that guarana seeds provide additional stimulation over caffeine alone, and document the changes to this stimulation in the context of both caffeine and glucose.

Evidence of Nicotine-Induced, Curare-Insensitive, Behavior in Planarians.

Planarians are rapidly developing into very useful research subjects in pharmacology and neuroscience research. Here we report that curare, a cholinergic nicotinic receptor antagonist, alleviates the nicotine-induced planarian seizure-like movements (pSLM) by up to 50 % at equimolar concentrations of nicotine and curare (1 mM), while curare alone does not induce significant pSLMs. The simplest interpretation of our data is that there are nicotine induced behaviors insensitive to curare in our experimental organism. To the best of our knowledge, this is the first report on curare-insensitive, nicotine-induced effects in any organism.

Effects of N,N-dimethylformamide on behaviour and regeneration of planarian Dugesia japonica.

In this study, the toxicity, behavioural and regeneration effects of dimethylformamide (DMF) on planarian Dugesia japonica were investigated. One control and six different concentrations of DMF (10 ppm, 100 ppm, 500 ppm, 1000 ppm, 5000 ppm and 10,000 ppm) were used in triplicate. The results showed that the mortality was directly proportional to the DMF concentration and planarian locomotor velocity (pLMV) was significantly reduced by increasing the exposure time and DMF concentration. pLMV of D. japonica was significantly reduced at a lower concentration of 10 ppm after 7 days of continuous exposure to DMF. The recovery of the motility of planarians pretreated with DMF was found to be time- and dose dependent, all planarians had complete recovery in their motility after 48 h. The appearance of auricles in regenerating animals was easily affected by DMF exposure in comparison with the appearance of eyespot. The present results suggest that the intact adult mobility in the aquatic planarian D. japonica is a more sensitive biomarker than mortality, and the appearance of auricles in regenerating animals is a more sensitive biomarker than eyespot.

Planarians in pharmacology: parthenolide is a specific behavioral antagonist of cocaine in the planarian Girardia tigrina.

Planarians are traditional animal models in developmental and regeneration biology. Recently, these organisms are arising as vertebrate-relevant animal models in neuropharmacology. Using an adaptation of published behavioral protocols, we have described the alleviation of cocaine-induced planarian seizure-like movements (pSLM) by a naturally-occurring sesquiterpene lactone, parthenolide. Interestingly, parthenolide does not prevent the expression of pSLM induced by amphetamines; in vertebrates, amphetamines interact with the same protein target as cocaine. Parthenolide is also unable to prevent pSLM elicited by the cholinergic com-pounds nicotine and cytisine or by the glutamatergic agents L- or D- glutamic acid or NMDA. Thus, we conclude that parthenolide is a specific anti-cocaine agent in this experimental organism.

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.

The impairments of neoblast division in regenerating planarian Polycelis felina (Daly.) caused by in vitro treatment with cadmium sulfate.

The effects of cadmium sulfate on the neoblast mitotic activity in regenerating planarian Polycelis felina (Daly.) were investigated. Mitotic abnormalities and chromosomal aberrations were evaluated after 6-h treatment and 24-h recovery period. The blastema were fixed, and examined cytologically through routine lactoorceine squash preparations. Mitotic indices were also determined. Cadmium sulfate induced a dose-dependent decrease in neoblast mitotic activity, accompanied with disturbances in distribution of cells over mitotic phases. Different cytological abnormalities with varying frequency were observed. Marked mitotic depression was concentration-dependent. Toxic effects of cadmium in regenerating planarian were mainly associated with mitotic spindle disturbances. Immediately after treatment mitotic abnormalities were prevalent over chromosomal and C-mitosis was the most prominent one. After 24-h recovery period a prevalence of mitotic over chromosomal aberrations was still present in animals treated with two higher concentrations of cadmium sulfate. However, the proportions of cells with chromosome stickiness in all treated animals were significantly increased compared to their post-treatment values. Observed mitotic impairments could be related to mitotic arrest contributing to retardations and delays, especially in animals treated with the highest concentration tested. The results obtained indicated usefulness of short term invertebrate assays as an alternative to in vitro pre-screening of toxic chemicals.

In vitro tests to evaluate potential biological activity in natural substances.

The biological activity of polysaccharides from the mycelia of 40 Basidiomycetes was studied using an uncommon toxicity test technique, the planaria bioassay, and the better known potato disk bioassay. The results showed the utility of this duo of 'in vitro' tests as a preliminary screening of the toxicity of substances that are present in aqueous fungal extracts.

Teratological research using in vitro systems. V. Nonmammalian model systems.

In this review of alternative tests to whole-animal rodent studies, the use of sub-mammalian and sub-vertebrate systems is investigated. The history, methodology, known limitations, end points, dose response, and requirements of virus, hydra, planarian, cricket, fish, amphibia, Drosophila, and chicken embryo systems are discussed.

Planarians as a model system for in vitro teratogenesis studies.

Free-living flatworms such as planarians are inexpensive to culture, maintain, and use for toxicologic testing in the laboratory. A considerable number of basic studies by ourselves and others indicate that, in simplified miniature, they possess many features of biochemical and physiologic organization similar to higher animals such as mammals. These include a well-developed brain with a varied behavioral repertoire including complex maneuvers of prey capture and learning, with a number of the same neurotransmitters used in mammalian brain. They are sensitive to a variety of the same toxicants. Undifferentiated totipotent stem cells, i.e., "neoblasts," which are capable of mitosis and differentiation into any of the various specialized cell types, permit regeneration of complete planarians from fragments. They also provide new cells to replace those lost in the normal cellular turnover of nonregenerating planarians. Both regeneration of surgical fragments and aberrant remodeling of whole planarians model important features of embyrogenesis and are potentially useful for assaying teratogens. Results are described from studies in which various representative teratogenic toxicants were tested in these two different planarian paradigms. The potential of planarian cephalic regeneration for behavioral teratogenesis investigations is also indicated.