The impairments of neoblast division in regenerating planarian Polycelis felina (Daly.) caused by in vitro treatment with cadmium sulfate.
ABSTRACT 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.
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ABSTRACT: Objective: Freshwater planarians were used as models for studying metazoan regeneration and stem cell biology. Here a simple, fast and high throughput method for extracting their stem cells (neoblasts) is represented. Materials and Methods: Specimens of the Dugesia sp with an average length of 18 mm were homogenized by a glass Dounce tissue grinder which contained about 1 ml of planarian saline solution. The extracted suspension was serially filtered by 60, 41, 30, 20 and 11 μm nylon meshes. In order to obtain purified neoblasts in the final suspension; this suspension has been compared with a cell suspension from 30 Gy irradiated worms. Hoechst 33342 was used to determine cells from non-cellular particles; methylene blue and propidium iodide were used to detect the number of dead cells in each extraction. Results: About 2.6-3 million cells were extracted from 10-12 worms. Flow cytometry analy-sis showed about 83% of the extracted particles were cells. In suspensions from irradiated animals, about 50% of the cells were absent, the final suspension contained about 62-66% neoblasts and about 17% non-cellular particles. When these extracts were treated with dis-tilled water to destroy the cells, only rabdites and chitinous spines of the parenchyma were observed in the extract. Conclusion: Results show that the purity of neoblasts in the final suspension is about 66%. Non-cellular particles have a carbohydrate nature and, therefore, this extraction method is completely compatible with molecular (e.g. proteomics and transcriptomics) and cellular methods (e.g. neoblast culture).
Article: Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd.[show abstract] [hide abstract]
ABSTRACT: The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10μg/L), B[a]P (10μg/L) or the two combined chemicals (10μg/L+1μg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10μg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10μg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/2011; 723(1):26-35. · 2.85 Impact Factor