| Summary: | 碩士 === 國立中興大學 === 動物科學系所 === 104 === Clay is an abundant and inexpensive inorganic mineral. As nano clays show unique physicochemical properties, it has been applied in a variety of fields. The novel physical properties of nano clays draw serious concerns by the public for its biosafety when applied to the organisms, and thus it is critical to evaluate its toxicity. The purpose of this study was to evaluate the cytotoxicity and acute toxicity of micro/nano clays (TB, TBB1, TBI1) and nanosilcate platelets (NSP) in mice. In vitro morphological observations showed that high doses at 500 μg/mL micro/nano clays resulted in cell growth arrest and 100 μg/mL micro/nano clays slightly caused cell apoptosis, but had no significant effect on necrosis. ROS scavenger (N-MPG and PDTC), NADPH oxidase inhibitor (DPI and Apo) and endocytosis inhibitor (Cyto D) were used to investigate the mechanisms of cell apoptosis induced by micro/nano clays. 300 μM N-MPG treatment significantly rescued cell apoptosis induced by NSP, and 2 mM PDTC treatment also significantly ameliorated cell apoptosis by TB or NSP. 10 µM DPI and 30 µM Apo also slightly ameliorated apoptosis by TB, TBB1 and TBI1, but had no effect on apoptosis induced by NSP. Treatment of Cyto D at 1 µg/mL significantly rescued cell apoptosis induced by micro/nano clays or NSP. Both DPI and Cyto D treatment significantly suppressed ROS generation induced by micro/nano clays or NSP, but Apo treatment exacerbated ROS production. Analysis of LDH release suggested that micro/nano clays and NSP could damage cell membranes and lead to a slightly higher LDH activity. TB, TBI1 and NSP had no significant effect on cellular GSH content, but TBB1 significantly reduced GSH content. Neither micro/nano clays nor NSP exerted an effect on SOD activity. Micro/nano clays and NSP at 100 μg/mL significantly caused mitochondrial damage leading to membrane potential loss and treatment of N-MPG failed to rescue the mitochondrial membrane potential loss by micro/nano clays, but NSP add N-MPG or Cyto D treatment significantly rescued the mitochondrial membrane potential loss. Treatment of DPI significantly suppressed caspase-6 activity induced by NSP. Furthermore, treatment of zVAD.fmk, a caspase inhibitor, ameliorated cell apoptosis and necrosis induced by NSP. N-MPG and PDTC treatment failed to affect HSP70 expression by NSP. N-MPG, DPI or Cyto D exerted no effect on gelsolin expression and JNK activation induced by NSP. In acute toxicity of mice with daily oral administration of micro/nano clays for 14 days, body weight and feed intake and histopathology were not different between micro/nano clay treated and control groups. Micronucleus analysis suggested that there was no significant micronucleus formation in the erythrocytes induced by micro/nano clays at various concentrations. Results from the acute toxicity study suggested that micro/nano clays have a lethal dose (LD50) greater than 4 g/kg body weight and further tests suggested that micro/nano clays of LD50 is greater than 4 g/kg body weight for male and female B6 mice. In conclusion, results from this study suggested that micro/nano clays showed slight cytoxicity, but had no significant acute toxicity and genotoxicity in living animals.
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