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Respirable crystalline silica has been classified as a human lung carcinogen. Ultrafine (diameter < 100 nm) silica particles may be important in carcinogenesis, although the mechanisms remain unclear. In the present study, WIL2-NS cells were incubated for 6, 24, and 48 hr with 0, 30, 60, and 120 microg/ml ultrafine crystalline SiO(2) (UF-SiO(2)). The cytotoxic and genotoxic effects caused by UF-SiO(2) in cultured human cells were investigated via a set of bioassays. Significant dose- dependent decreases in percent cell viability were seen with increasing dose of UF-SiO(2) in the methyl tetrazolium assay. Significant decreases were seen at 120 microg/ml (58, 38, and 57% for 6, 24, and 48-hr exposure, respectively). During 4 days growth in the flasks, there was a slight recovery observed after washing off UF-SiO(2) as measured by the population growth assay. Significant dose-dependent reduction in the cytokinesis block proliferation index was observed by the cytokinesis block micronucleus assay. Treatment with 120 microg/ml UF-SiO(2) for 24 hr produced a fourfold increase in the frequency of micronucleated binucleated cells (MNBNC). The increase in MNBNC was dose-dependent. The lowest dose that gave a statistically significant increase in MNBNC was 30 microg/ml (24-hr treatment), which had cytotoxicity of less than 10%. There was no significant difference in DNA strand breakage as measured by the Comet assay. A significant increase in induced mutant frequency was found at 120 microg/ml as detected by the hypoxanthine guanine phosphoribosyltransferase mutation assay. The results indicate that UF-SiO(2) is cytotoxic and genotoxic in cultured human cells.

The issue of appropriate testing strategies has been raised for the genotoxicity assessment of nanomaterials. Recently, efforts have been made to evaluate the adequacy of Organisation for Economic Co-operation and Development-standardised tests to assess the genotoxicity of nanomaterials. The aim of this review was to examine whether the current guideline for the in vitro micronucleus (MN) assay is applicable for testing nanomaterials. From a Pubmed literature search, 21 available studies were identified for analysis. We reviewed all protocols used for testing nanomaterials with the in vitro MN assay. All studies were categorised based on the particle type and size. Different aspects of the protocols were evaluated such as the exposure (duration and doses), the cytochalasin-B treatment, serum levels and cytotoxicity assessment. Sixteen of the 21 studies demonstrated increased frequencies of MN. Some recommendations regarding the protocol were formulated to maximise sensitivity and avoid false negatives. Determination of the cellular dose was advised for a better interpretation of MN frequency results. The level of serum can modulate the cellular response, therefore the serum percentage used should enable cell growth and proliferation and a maximal sensitivity of the assay. Furthermore, different types of cytochalasin-B treatment were used, co-treatment, post-treatment and delayed co-treatment. In order to avoid decreased cellular uptake as a consequence of actin inhibition, post-treatment or delayed co-treatment is suggested. Exposure during mitosis should be recommended to allow contact with the chromatin or mitotic apparatus for nanomaterials that are unable to cross the nuclear membrane. With these adaptations, the in vitro MN assay can be recommended for genotoxicity testing of nanomaterials.

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Somatic mutations, either spontaneous or produced by identifiable mutagens, are thought to be important in the aetiology of cancer and in the ageing process. The study of somatic mutations in human cells in vivo has recently been made possible by the development of techniques for enumeration and clonal expansion of lymphocytes mutated at the chromosome X-linked hypoxanthine phosphoribosyl transferase (HPRT) locus. We have studied the molecular basis of in vivo hprt mutations in human lymphocytes and report here that a surprisingly high proportion (57%) involve substantial gene alterations which are not evident cytogenetically. These major gene alterations include deletions, exon amplifications and novel, sometimes amplified, bands on Southern analysis. Such changes emphasize the fluid nature of information in DNA and may be indicative of general mechanisms by which functional gene loss is involved in the aetiology of cancer and the homeostatic failure of ageing.

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