Kumiko Shimizu

Time-dependent variation in the biodistribution of C60 in rats determined by liquid chromatography–tandem mass spectrometry

We examined the biodistribution of C(60) in rats after tail vein administration using LC-MS/MS. C(60) was detected in various tissues, such as brain, kidneys, liver, lungs, and spleen of rats. On the other hand, no C(60) was found in blood. The highest C(60) concentration was observed in the lungs, followed by spleen, liver, kidneys, and brain. These results suggested that C(60) injected in the tail vein could be filtered by lung capillary vessels and accumulate in the lungs prior to being distributed to other tissues. Moreover, C(60) not being detected in the blood indicates that clearance of C(60) from the blood by filtration might effectively occur in the lungs. The time-dependent variation in the biodistribution of C(60) was evaluated. A time-dependent decrease in C(60) concentrations was observed in all tissues, except spleen. Moreover, a decreasing trend of C(60) levels differed among tissues, which could be due to differences in accumulation. These results suggest that unmodified C(60) and/or C(60) metabolites by metabolic enzymes could be excreted into feces and/or urine. In further studies, the metabolic and excretion pathways of C(60) should be evaluated to understand the toxicokinetics of C(60).

Increase of β2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. β-actin, β-catenin, and β1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but β2-integrin expression markedly increased. Graphical abstract THP-1 cells were seeded in T-75, or CVM, or CVM added PMA 10 ng/mL; and β-actin(A), β-catenin(B), β1-integrin(C) and β2-integrin(D) expression were measured (N = 3).

Effects of the Amino Acid Constituents of Microcystin Variants on Cytotoxicity to Primary Cultured Rat Hepatocytes

Microcystins, which are cyclic heptapeptides produced by some cyanobacterial species from algal blooms, strongly inhibit serine/threonine protein phosphatase and are known as hepatotoxins. Microcystins have many structural variations, yet insufficient information is available on the differences in the cytotoxic potentials among the structural variants. In this study, the cytotoxicities of 16 microcystin variants at concentrations of 0.03–10 μg/mL to primary cultured rat hepatocytes were determined by measuring cellular ATP content, and subsequently determined by their 50% inhibitory concentration (IC50). Differences in the amino acid constituents were associated with differences in cytotoxic potential. [d-Asp3, Z-Dhb7] microcystin-LR exhibited the strongest cytotoxicity at IC50 of 0.053 μg/mL among the microcystin variants tested. Furthermore, [d-Asp3, Z-Dhb7] microcystin-HtyR was also highly cytotoxic. These results suggest that both d-Asp and Z-Dhb residues are important in determining the cytotoxic potential of microcystin variants.

Cytotoxic Effects of Hydroxylated Fullerenes in Three Types of Liver Cells

Fullerenes C60 have attracted considerable attention in the biomedical field due to their interesting properties. Although there has been a concern that C60 could be metabolized to hydroxylated fullerenes (C60(OH)x) in vivo, there is little information on the effect of hydroxylated C60 on liver cells. In the present study, we evaluated the cytotoxic effects of fullerene C60 and various hydroxylated C60 derivatives, C60(OH)2, C60(OH)6–12, C60(OH)12 and C60(OH)36, with three different types of liver cells, dRLh-84, HepG2 and primary cultured rat hepatocytes. C60, C60(OH)2 and C60(OH)36 exhibited little or no cytotoxicity in all of the cell types, while C60(OH)6–12 and C60(OH)12 induced cytotoxic effects in dRLh-84 cells, accompanied by the appearance of numerous vacuoles around the nucleus. Moreover, mitochondrial activity in liver cells was significantly inhibited by C60(OH)6–12 and C60(OH)12. These results indicate that the number of hydroxyl groups on C60(OH)x contribute to the difference of their cytotoxic potential and mitochondrial damage in liver cells.