The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts).

Investigations on potential co-mutagenic effects of formaldehyde.

The genotoxicity and mutagenicity of formaldehyde (FA) has been well-characterized during the last years. Besides its known direct DNA-damaging and mutagenic activity in sufficiently exposed cells, FA at low concentrations might also enhance the mutagenic and carcinogenic effects of other environmental mutagens by interfering with the repair of DNA lesions induced by these mutagens. To further assess potential co-mutagenic effects of FA, we exposed A549 human lung cells to FA in combination with various mutagens and measured the induction and removal of DNA damage by the comet assay and the production of chromosomal mutations by the cytokinesis-block micronucleus assay (CBMN assay).

Insensitivity of the in vitro cytokinesis-block micronucleus assay with human lymphocytes for the detection of DNA damage present at the start of the cell culture.

The cytokinesis-block micronucleus assay (CBMN assay) with cultured human lymphocytes is a well-established assay in genotoxicity testing and human biomonitoring. For both approaches, human lymphocytes are stimulated by phytohaemagglutinin (PHA) and cultured for about 72 h; 44 h after PHA stimulation, cytochalasin B (CytB) is added and micronuclei (MN) are scored in binucleated cells. The main difference between these two applications is the way lymphocytes are exposed to mutagens.

Investigations of potential susceptibility toward formaldehyde-induced genotoxicity.

Blood samples were taken from three groups of volunteers (30 male smokers, 30 female non-smokers, and 30 school children) and tested for ex vivo susceptibility toward formaldehyde (FA)-induced genotoxicity. Blood samples were exposed to 150 μM FA for 2 h, and the induction of DNA-protein crosslinks (DPX) in leukocytes was measured by a modification of the alkaline comet assay (i.e.

Re-evaluation of a reported increased micronucleus frequency in lymphocytes of workers occupationally exposed to formaldehyde.

A replicate evaluation of increased micronucleus (MN) frequencies in peripheral lymphocytes of workers occupationally exposed to formaldehyde (FA) was undertaken to verify the observed effect and to determine scoring variability. May-Grünwald-Giemsa-stained slides were obtained from a previously performed cytokinesis-block micronucleus test (CBMNT) with 56 workers in anatomy and pathology laboratories and 85 controls. The first evaluation by one scorer (scorer 1) had led to a highly significant difference between workers and controls (3.

Does formaldehyde induce aneuploidy?

Formaldehyde (FA) was tested for a potential aneugenic activity in mammalian cells. We employed tests to discriminate between aneugenic and clastogenic effects in accordance with international guidelines for genotoxicity testing. The cytokinesis-block micronucleus test (CBMNT) in combination with fluorescence in situ hybridisation (FISH) with a pan-centromeric probe was performed with cultured human lymphocytes and the human A549 lung cell line.