Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis.

The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test.

We evaluated the suitability of the alkaline comet assay as a screening test in industrial routine testing of new chemicals. Thirty-six pharmaceutical compounds with unknown genotoxic potential were tested comparatively in the comet assay and micronucleus test (MNT) using V79 Chinese hamster cells. The comparison of results is generally based on at least two independent experiments, each with two replicate cultures at a minimum of three concentrations. We found a high degree of concordance between results of the comet assay and MNT. All compounds with negative MNT results were also negative in the comet assay. All positive compounds in the comet assay were also positive in the MNT. However, 16 of 38 positive MNT results were negative in the comet assay. Some of the contrary findings may be due to aneugenic effects, which are detected in the MNT but not in the comet assay. However, the majority of the contrary results may be a consequence of cytotoxicity, which can induce elevated micronucleus frequencies but may not lead to positive effects in the comet assay. Additional data of 39 compounds tested in the Ames test and the comet assay were compared. Four of these compounds that were Ames positive were also positive in the comet assay. However, the comet assay also detected 16 compounds that were negative in the Ames test. We believe that the comet assay in vitro is a useful, fast screening system in mammalian cells that can be used in a test battery during drug development.

Automatic analysis of slides processed in the Comet assay.

In recent years the Comet assay (or single cell gel electrophoresis assay) has been established as a rapid and sensitive method for the detection of DNA damage. For early genotoxicity screening of new chemical entities in industrial toxicology, the Comet assay is more and more used for assessment of the DNA damaging potential of a test compound. In order to increase compound screening throughput, we have established an image analysis system for fully automated measurement of microscope slides processed in the Comet assay. For the comparative investigation various cell types, such as V79 Chinese hamster cells, mouse lymphoma cells and human leukocytes, were treated with several test compounds. Using tail moment as the quantitative parameter for comet formation, we show a very high correlation between our automatic image analysis system and a commercially available, interactive system (Comet Assay II of Perceptive Instruments). The possibility of analyzing 50 samples within 1 day and the high reproducibility of results make automated image processing a powerful tool for automatic analysis of slides processed in the Comet assay.

In vitro models to study mechanisms involved in cyclosporine A-mediated glomerular contraction.

The immunosuppressive drug, cyclosporin A (CsA), which is successfully used to prevent rejection in organ transplantation, induces renal side-effects as shown by a decrease in glomerular filtration rate and ultrafiltration coefficient regulated by the tone of mesangial cells.The aim of the present study was to investigate the effect of CsA on isolated glomeruli and mesangial cells, which constitute appropriate in vitro models for renal vasoreactivity studies. The roles of different intracellular and extracellular mediators such as calcium, endothelin-1 (ET-1), prostaglandins (TXA(2 )and PGI(2)) and reactive oxygen intermediates (ROIs) were analysed. CsA caused a concentration- and time-dependent decrease in the planar cross-sectional areas of isolated glomeruli and mesangial cells as determined by image analysis. Intracytosolic free calcium concentration determined by fluorimetric analysis was significantly increased after 30 min CsA (10 microM) incubation. In the contraction experiment, the calcium antagonist verapamil inhibited the CsA response. ET-1, TXB(2) and keto-PGF(1alpha) were determined directly, however no changes were found statistically significantly different from respective controls. In contrast to these results, the ET-1 specific antibody was able to reduce CsA-mediated cell contraction. In the presence of a prostacyclin agonist iloprost, CsA-induced contraction was also modified. The role of ROIs using a 2'7'-dichlorofluorescein diacetate (DCFdAc) fluorimetric method was directly determined by observing, with 10 microM CsA, a significant production of hydrogen peroxide (H(2)O(2)), which was able alone to induce mesangial cell contraction. Coincubation with the antioxidants led to a significant inhibition of mesangial cell contraction. These results suggest that CsA caused an imbalance in the normal level of all investigated vasoconstrictive and vasodilator mediators, which shifted towards the advantage of vasoconstrictive action.

Automatic analysis of the in vitro micronucleus test on V79 cells.

The in vitro micronucleus test is a well established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and specific parameter. As a measure for numerical and structural chromosome aberrations, the in vitro micronucleus test consists of determining the frequency of micronucleated cells in a representative fraction of cells in a culture. So far, manual counting has been the only method for evaluating microscopic V79 Chinese hamster cell preparations. To replace this tedious and time consuming procedure, a fully automatic system for micronucleus scoring in V79 cells by image analysis has been developed and introduced into the routine genotoxicity screening of drug candidates. The comparison of manual and automatic micronucleus analysis showed a high degree of concordance between the results obtained by the two techniques. For concentration series of cyclophosphamide (CP) and ethyl-methanesulphonate (EMS) as test compounds, the frequency of erroneously missed micronuclei through automatic scoring proved to be below 15% in comparison with manual scoring. Generally, false positive micronucleus decisions could be controlled easily by fast and simple relocation of the automatically detected patterns. The possibility to analyze 24 slides within 1 day by fully automatic overnight analysis and the high reproducibility of the results make automatic image processing a powerful tool for the in vitro micronucleus analysis.

Technical aspects of automatic micronucleus analysis in rodent bone marrow assays.

The mouse bone marrow micronucleus assay is an in vivo test commonly used in the pharmaceutical industry to evaluate the genotoxic potential of new compounds. The test detects agent-induced chromosomal damage or damage of the mitotic spindle apparatus. In this paper the state-of-the-art in automated rodent micronucleus evaluation using computerized image analysis in combination with high-quality slides obtained by the cellulose column fractionation technique is reviewed. The latter allows the effective removal of nucleated cells from rodent bone marrow. It has been found that automatic micronucleus scoring with the Leitz MIAC image analyzer is substantially faster than labor-intensive manual analysis. Automatic scoring can be performed overnight for up to 16 slides. We have been successfully using automatic micronucleus analysis for the testing of new pharmaceutical drugs for more than 3 years.