Micronucleus test using cultured new born rat astrocytes.

Micronuclei is induced in cytoplasm as a consequence of the formation of chromosomal fragments or remaining chromosomes during cell division by the cause of clastogens or spindle poisons, and is used as an indicator of genotoxicity screening tests. There are few short-term genotoxicity screening tests using brain cells. We attempted to establish a new in vitro micronucleus test (MN test) system by use of central nervous system cells. Primary cultured astrocytes were prepared from newborn male Sprague-Dawley (SD) rats. In growth curve of astrocytes, doubling time was determined to be 31 h. In time study, the highest frequency of micronuclei was observed at 48 h, 72 h and 6 h-exposure-66 h-recovery by vincristine (VCR), mitomycin C (MMC) without metabolic activation system and cyclophosphamide (CPM) with metabolic activation system, respectively. Dose-response relationships between micronucleus frequency and concentrations of MMC, VCR and CPM were observed, respectively. It is suggested that the in vitro MN test using new born rat-astrocytes could be used as a screening test of environmental and occupational genotoxic chemicals in the central nervous system cells.

Mutagenicity of p-aminophenol in E. coli WP2uvrA/pKM101 and its relevance to oxidative DNA damage.

It was recently reported that p-aminophenol (p-AP) induces DNA cleavage in mouse lymphoma cells, CHO cells and human lymphoblastoid cells. The mutagenicity of p-AP has not, however, been detected by reverse mutation assays. The purpose of this study was to assess the mutagenicity of p-AP by reverse mutation assay using Escherichia coli WP2uvrA/pKM101, which has a spectrum for detecting mutations different from those of other strains in the family with an AT base pair at the mutation site and has higher sensitivity to certain oxidative mutagens as compared to other strains. We found that p-AP was mutagenic to E. coli WP2uvrA/pKM101. The mutagenic activity of this compound was suppressed with the addition of dimethylsulfoxide or catalase, suggesting the involvement of active oxygen species in the mutagenic process induced by p-AP. To further elucidate the underlying mechanism, we used isolated DNA for the following experiments. It was revealed, by gel electrophoretic analysis, that p-AP induced DNA cleavage in the presence of Fe(III). However, p-AP alone did not induce this cleavage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by p-AP in calf thymus DNA was also detected in the presence of Fe(III) by HPLC with an electrochemical detector. ESR-spin trapping experiments using DMPO detected the production of hydroxyl radical (.OH) in the solution of p-AP with Fe(III). Both p-AP mediated DNA damages and .OH production by p-AP in the presence of Fe(III) were completely inhibited by .OH scavengers (ethanol, mannitol, sodium formate, dimethylsulfoxide) and catalase. These results suggest that .OH derived from the reaction between H2O2 and Fe(III) (Fenton reaction) participates in the oxidative DNA damage. Accordingly, the same mechanism might be working in E. coli WP2uvrA/pKM101 during induction of the mutation by p-AP.