[Polyploidy induction by spherical size standard polystyrene particles in a Chinese hamster cell line CHL].

To investigate relationships between particle (as a model of aggregates) size in a nanomaterial test suspension and its cytotoxicity, a series of eleven sizes of polystyrene (PS) particles were tested in the cytotoxicity test and the chromosome aberration test by using a Chinese hamster cell line CHL. The PS particles were spheres with defined diameters ranging from 0.1 to 9.2 µm. A series of eight sizes of particles with diameters ranging from 0.92 to 4.45 µm showed stronger cytotoxicity than the others. There was a marked difference in cytotoxicity between the 4.45- and 5.26-µm particles. The 0.92- to 4.45-µm particles did not induce structural chromosome aberrations but induced a high frequency of polyploidy in the chromosome aberration test. The 5.26-µm particles showed very weak induction of polyploidy. The incorporation of the 4.45-µm particles into CHL cells was observed by scanning electron microscopy (SEM). Some cells incorporated more than 10 particles. The semi-quantitative measurement of incorporation of particles into cells was performed by flow cytometry with a parameter of side scattered light (SSC) intensity. It showed that CHL cells preferably incorporated the 4.45-µm particles to the 5.26-µm particles. These findings suggest that CHL cells may have a kind of size-recognition ability and incorporate a particular size of particles. The particles may prevent a normal cytokinesis resulting in polyploidy induction. Nanomaterials also may show size-dependent toxicity. Data on particle (or aggregate) size distribution in the test suspension should be provided to evaluate properly the results of toxicity tests of nanomaterials.

Development of an in vitro screening method for safety evaluation of nanomaterials.

To evaluate the role of particle size in cytotoxicity tests of nanomaterials (NMs), we exposed Chinese hamster cells to polystyrene (PS) spheres with defined diameters ranging from 0.1 to 9.2 microm. We found that the 4.45-microm PS particles were most cytotoxic while sizes 0.1 and 0.2 microm showed no cytotoxicity up to 1000 microg/ml. In the chromosome aberration test, the 4.45-microm PS particles induced polyploidy in a mass concentration-dependent manner in 24- and 48-h treatments. The 5.26-microm PS particles induced polyploidy only at 1000 microg/ml for 48 h. Next, we performed the cytotoxicity test with as-grown single walled carbon nanohorns (NHas). These were suspended in DMSO and then transferred into the culture medium followed by sonication. Six suspensions differently sonicated showed the same apparent toxicity, although the total particle size distributions differed. However, the sizes of NHas particles predicted to be most toxic from the experiments with PS particles, i.e. 1.01-4.47 microm constituted 40-60% of all particles in all six suspensions. The results suggest that the cytotoxicity of NMs in suspension depends on specific sizes of aggregates and therefore suspensions should be checked with regard to particle size distributions in assays of toxic effects. The uptake of particles into cells was confirmed by confocal microscopy.

Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition.

We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy-trans-stilbene, 3-hydroxy-trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy-trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy-trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.

Uptake of analogs of penetratin, Tat(48-60) and oligoarginine in live cells.

Cell-penetrating peptides are regarded as promising vectors for intracellular delivery of large, hydrophilic molecules, but their mechanism of uptake is poorly understood. Since it has now been demonstrated that the use of cell fixation leads to artifacts in microscopy studies on the cellular uptake of such peptides, much of what has been considered as established facts must be reinvestigated using live (unfixed) cells. In this work, the uptake of analogs of penetratin, Tat(48-60), and heptaarginine in two different cell lines was studied by confocal laser scanning microscopy. For penetratin, an apparently endocytotic uptake was observed, in disagreement with previous studies on fixed cells found in the literature. Substitution of the two tryptophan residues, earlier reported to be essential for cellular uptake, did not alter the uptake characteristics. A heptaarginine peptide, with a tryptophan residue added in the C-terminus, was found to be internalized by cells via an energy-independent, non-endocytotic pathway. Finally, a crucial role for arginine residues in penetratin and Tat(48-60) was demonstrated.

Cell studies of the DNA bis-intercalator Delta-Delta [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+): toxic effects and properties as a light emitting DNA probe in V79 Chinese hamster cells.

Coordination complexes of type [Ru(L)(3)](2+), where L is a nitrogen-containing aromatic bidentate ligand, can often be photolytically reduced, making them useful in studies of DNA- or protein-mediated electron transfer and in artifical photosynthesis model systems. Upon binding to DNA some Ru(L) complexes have been found to display strongly increased fluorescence compared with when free in solution, making those compounds interesting to test as DNA probes. Thus, they are becoming widely used in the chemistry community. Here, asynchronous cultures of V79 Chinese hamster cells were exposed to the DNA bis-intercalator Delta-Delta [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+) at 10(-10)-10(-4) M. The extraordinarily strong binding of the compound to DNA was the reason for testing its possible interference with DNA metabolism in intact mammalian cells. Exposure for 1 h to 10(-10)-10(-4) M did not significantly decrease DNA synthesis. Cells exposed to 10(-5) M for 27 h showed no staining of the nucleus, while DNA was stained in cells electroporated in the presence of the compound. However, the Ru dimer was probably taken up by pinocytosis, because numerous minute precipitates could be observed in the cytoplasm. Treatment for 24 h at concentrations of 10(-10)-10(-5) M did not inhibit growth, as indicated by cell density and mitotic activity. Neither did it affect chromosomal arrangements during mitosis. However, at 10(-4) M the density of cultures was reduced by approximately 45% and apoptotic cells were frequent, as opposed to mitoses. We also investigated the properties of the Ru dimer as a fluorescent DNA stain. The compound appears attractive as a red DNA stain when broad excitation in the visible range is desirable and extremely low background staining is essential. The low toxicity of the compound is a favourable trait in this context.