Acrylamide and glycidamide: genotoxic effects in V79-cells and human blood.

Acrylamide (AA) can be formed in certain foods by heating, predominantly from the precursor asparagine. It is a carcinogen in animal experiments, but the relevance of dietary exposure for humans is still under debate. There is substantial evidence that glycidamide (GA), metabolically formed from AA by Cyp 2E1-mediated epoxidation, acts as ultimate mutagenic agent. We compared the mutagenic potential of AA and GA in V79-cells, using the hprt mutagenicity-test with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) as positive control. Whereas MNNG showed marked mutagenic effectivity already at 0.5 microM, AA was inactive up to a concentration of 10 mM. In contrast, GA showed a concentration dependent induction of mutations at concentrations of 800 microM and higher. Human blood was used as model system to investigate genotoxic potential in lymphocytes by single cell gel electrophoresis (comet assay) and by measuring the induction of micronuclei (MN) with bleomycin (BL) as positive control. AA did not induce significant genotoxicity or mutagenicity up to 6000 microM. With GA, concentration dependent DNA damage was observed in the dose range of 300-3000 microM after 4 h incubation. Significant MN-induction was not observed with AA (up to 5000 microM) and GA (up to 1000 microM), whereas BL (4 microM) induced significantly enhanced MN frequencies. Thus, in our systems GA appears to exert a rather moderate genotoxic activity.

Acrylamide and glycidamide: approach towards risk assessment based on biomarker guided dosimetry of genotoxic/mutagenic effects in human blood.

Acrylamide (AA) is a carcinogen as demonstrated in animal experiments, but the relevance for the human situation is still unclear. AA and its metabolite glycidamide (GA) react with nucleophilic regions in biomolecules. However, whereas AA and GA react with proteins, DNA adducts are exclusively formed by GA under conditions simulating in vivo situations. For risk assessment it is of particular interest to elucidate whether AA or GA within the plasma concentration range resulting from food intake are "quenched" by preferential reaction with non-critical blood constituents or whether DNA in lymphocytes is damaged concomitantly under such conditions. To address this question dose- and time-dependent induction of hemoglobin (Hb) adducts as well as genotoxic and mutagenic effects by AA or GA were studied in human blood as a model system.

Micronuclei induced by aneugens and clastogens in mononucleate and binucleate cells using the cytokinesis block assay.

The human in vitro micronucleus (MN) test has become a fast and reliable assay for mutagenicity testing. Currently, this assay is mostly performed with cytochalasin B, which prevents cytokinesis, resulting in polynucleated cells. The number of nuclei per cell indicates the number of nuclear divisions that have occurred since the addition of cytochalasin B. It is recommended that MN are only counted in binucleated lymphocytes, because these cells have finished one nuclear division. Therefore, almost no attention has been paid to MN in mononucleated cells. However, recent studies have indicated that aneugens, but not clastogens, also induce MN in mononucleates. In order to evaluate mononucleates to distinguish between aneugenic and clastogenic effects, we tested some typical aneugens and clastogens in whole blood lymphocyte cultures of four donors with the cytokinesis block micronucleus (CBMN) assay. Results showed that the aneugens diethylstilbestrol (80 microM), griseofulvin (25 microg/ml) and vincristine sulphate (15 microg/ml) increased MN frequencies in mononucleated and binucleated cells, whilst the clastogens mitomycin C (500 ng/ml), bleomycin (6 microg/ml) and doxorubicin (20 microg/ml) increased MN frequency only in binucleates. We also tested the Y heterochromatin decondensing drug berenil (300 microg/ml). Berenil induced an extremely high number of MN in mononucleated as well as in binucleated cells, indicating an aneugenic action. This was confirmed by centromere labelling. The results suggest that MN in mononucleates may be an interesting additional parameter in the CBMN assay. Future studies should clarify whether the micronucleated mononucleate cells have escaped the cytokinesis block and become polyploid.