Direct comparison of the Ames microplate format (MPF) test in liquid medium with the standard Ames pre-incubation assay on agar plates by use of equivocal to weakly positive test compounds.

The Ames microplate format (MPF™) test, which uses liquid media and in 384-well microplates with a readout based on a colour-change, has been used for over 10 years at several major pharmaceutical companies for screening the genotoxic potential of early drug candidates when compound supply is minimal. Meanwhile, Xenometrix has adapted this screen from the two-strain Ames II test for use with five tester strains, in compliance with OECD Guideline 471. A set of 15 equivocal to weakly positive chemicals selected from the National Toxicology Program (NTP) database was tested simultaneously in the Ames microplate format (MPF) and the standard Ames pre-incubation method on agar plates. Such a direct comparison of the two test methods with the same overnight culture(s), chemicals and S9-mix preparation should exclude external variability factors. Thirteen of the 15 chemicals showed concordant results in both tests despite the choice of chemicals that showed varying inter- and even intra-laboratory results in the NTP studies. These results indicate that the Ames MPF™ assay is a reliable predictive tool that can be used like the regular Ames test to evaluate compounds for mutagenicity.

Comparison of the Ames II and traditional Ames test responses with respect to mutagenicity, strain specificities, need for metabolism and correlation with rodent carcinogenicity.

The Ames II Salmonella mutagenicity assay procedure was used to test 71 chemicals, and the results were compared with those from the traditional Ames Salmonella test using the NTP database as the reference. All Ames II tests were performed using a fluctuation procedure in microplate format, using TAMix for the detection of base pair substitutions and TA98 to detect frameshift mutations. There was 84% agreement between the two procedures in identifying mutagens and non-mutagens, which is equivalent to the intra- and interlaboratory reproducibility of 87% for the traditional test. The two tests also performed similarly in their predictions of rodent carcinogenicity.

Gene expression in two hepatic cell lines, cultured primary hepatocytes, and liver slices compared to the in vivo liver gene expression in rats: possible implications for toxicogenomics use of in vitro systems.

Microarray technology allows the simultaneous analysis of mRNA expression levels of thousands of genes. In the field of toxicogenomics, this technology could help to identify potentially unsafe compounds based on the changes in mRNA expression patterns they induce. Rodent in vivo and in vitro systems are currently the experimental models of choice for predictive toxicology, especially in early phases of development. This study characterizes several hepatic in vitro systems based on mRNA expression profiles, comparing them to gene expression in liver tissue. The in vitro systems investigated comprise two rat liver cell lines (BRL3A and NRL clone 9), primary hepatocytes in conventional monolayer or in sandwich culture, and liver slices. The results demonstrate that liver slices exhibit the strongest similarity to liver tissue regarding mRNA expression, whereas the two cell lines are quite different from the whole liver. We were able to identify genes with strong changes in expression levels in all or at least one of the in vitro systems relative to whole liver. In particular, for some cytochrome P450s the differences observed on the mRNA expression level were paralleled by protein expression and enzymatic activity. In addition, the effect of time in culture was assessed. We were able to show a profound effect of the duration of culture. Expression patterns change most rapidly soon after cell isolation and culture initiation and stabilize with time in culture. The findings are discussed with respect to the usefulness of the various hepatic in vitro systems for microarray-based toxicological testing of compounds.

Comparison of the Salmonella/microsome microsuspension assay with the new microplate fluctuation protocol for testing the mutagenicity of environmental samples.

The objective of this study was to compare the responses of the Salmonella/microsome microsuspension assay with the new microplate fluctuation protocol (MPF) for the evaluation of the mutagenic activity of environmental samples. Organic extracts of total particulate atmospheric air samples, surface waters, and effluents were tested in dose-response experiments. The assays were performed with strain TA98 in the absence and presence of S9 mix. Both protocols produced similar results, despite the fact that the maximum score of the MPF is limited to 48 wells, whereas in the regular plate assay it is possible to count up to 1,500 colonies using an automatic counter. Similar sensitivities based on the lowest dose that resulted in a positive response were obtained for both assays. The MPF procedure is less laborious (e.g., all-liquid format, use of multichannel pipettors) and allows for automation of the pipetting and dispensing steps, thus, reducing time of the analysis which is particularly important in environmental quality monitoring programs or in effect-directed analysis. The results show that the MPF procedure is a promising tool to test environmental samples for mutagenic activity.

Genetic analysis and functional characterization of the Streptococcus pneumoniae vic operon.

The vic two-component signal transduction system of Streptococcus pneumoniae is essential for growth. The vic operon comprises three genes encoding the following: VicR, a response regulator of the OmpR family; VicK, its cognate histidine kinase; and VicX, a putative protein sharing 55% identity to the predicted product (YycJ) of an open reading frame in the Bacillus subtilis genome. We show that not only is vic essential for viability but it also influences virulence and competence. A putative transcriptional start site for the vic operon was mapped 16 bp upstream of the ATG codon of vicR. Only one transcript of 2.9 kb, encoding all three genes, was detected by Northern blot analysis. VicK, an atypical PAS domain-containing histidine kinase, can be autophosphorylated in vitro, and VicR functions in vitro as a phospho-acceptor protein. (PAS is an acronym formed from the names of the proteins in which the domains were first recognized: the Drosophila period clock protein [PER], vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and Drosophila single-minded protein [SIM].) PAS domains are commonly involved in sensing intracellular signals such as redox potential, which suggests that the signal for vic might also originate in the cytoplasm. Growth rate, competence, and virulence were monitored in strains with mutations in the vic operon. Overexpression of the histidine kinase, VicK, resulted in decreased virulence, whereas the transformability of a null mutant decreased by 3 orders of magnitude.