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Lucy Lee

DEVELOPMENT OF A NOVEL FATHEAD MINNOW-DERIVED TESTICULAR (FHMT-W1) CELL LINE AND THEIR USE

ALONG WITH OTHER FISH CELL LINES FOR THE COMPARATIVE IN VITRO TOXICITY EVALUATION OF SILICA NANOPART
Nguyen TK Vo

published: 2010 | Thesis | Honours BSc (Biochemistry)

Nanomaterials and nanoparticles possess unique physicochemical properties that allow a wide range of industrial and biomedical/pharmaceutical applications. However, concerns over the health risks to humans and biota have recently drawn global attention. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivering and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment, yet effects in biota have not been fully evaluated. SiO2 NPs toxicity has been reported in human and mammalian models but seldom in aquatic species models. Twelve fish cell lines derived from six species (rainbow trout, fathead minnow, goldfish, zebrafish, haddock and American eel) were evaluated for cytotoxicity responses using Alamar Blue assay. Toxicity of SiO2 NPs appeared to be size, time, temperature, and dose-dependent as well as tissue and species-specific. However, dosages greater than 100 g/mL were needed to achieve 24-hour EC50 values. Smaller SiO2 NPs (15-20 nm) appeared to be more toxic than larger sized ones (44 nm). Lining epithelial cells (skin, gills, intestine) were shown to be more sensitive than fish cell lines derived from internal tissues (liver, brain, gonads) or embryos. The rainbow trout gill (RTGill-W1) and goldfish skin (GFSk-S1) cell lines were shown to be the most sensitive to SiO2 NPs whereas the zebrafish blastula embryonic (ZEB2J) cell line was the most tolerant. At dosages below the EC50, SiO2 NPs induced morphological changes, possibly as a consequence of oxidative stress. These toxicity findings of SiO2 NPs are in agreement with reported human and mouse studies. Thus fish cell lines are valuable for the quick screening of potential emerging environmental contaminants such as nanomaterials and nanoparticles. Out of twelve fish cell lines used for cytotoxicity evaluation of SiO2 NPs, the newly established FHMT-W1 cell line appeared to show interesting characteristics of spermatogenesis in vitro. In this study, part of the characterization of the FHMT-W1 cell line is reported. FHMT-W1 cells grew at a wide range of physiological temperature of fathead minnows. The optimal growth temperature was 28C at which the doubling time was ca. 3 days. FHMT-W1 cells could be maintained at 5-10% FBS. FHMT-W1 cells have been subcultured over 37 generations over 2 years and successfully cryopreserved and thawed with high cell viability. FHMT-W1 cells displayed various sizes, shapes and nuclear condensation level. Also, FHMT-W1 cells formed gonadal spheres reminiscent of embryoid bodyies both on the adherent tissue culture (TC) surface and in suspension culture. Thus the FHMT-W1 cell line is believed to be derived from testis stem cell lineage and capable of differentiation in vitro. FHMT-W1 cells responded well to model chemicals and toxicants, suggesting this cell line would be of great value in aquatic toxicology and endocrinological studies.


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