High-content imaging analyses of γH2AX-foci and micronuclei in TK6 cells elucidated genotoxicity of chemicals and their clastogenic/aneugenic mode of action.

BACKGROUND: The in vitro micronucleus (MN) test is an important component of a genotoxicity test battery that evaluates chemicals. Although the standard method of manually scoring micronucleated (MNed) cells by microscope is a reliable and standard method, it is laborious and time-consuming. A high-throughput assay system for detecting MN cells automatically has long been desired in the fields of pharmaceutical development or environmental risk monitoring. Although the MN test per se cannot clarify whether the mode of MN induction is aneugenic or clastogenic, this clarification may well be made possible by combining the MN test with an evaluation of γH2AX, a sensitive marker of DNA double strand breaks (DSB). In the present study, we aimed to establish a high-content (HC) imaging assay that automatically detects micronuclei (MNi) and simultaneously measures γH2AX foci in human lymphoblastoid TK6 cells. RESULTS: TK6 cells were fixed on the bottom of each well in 96-well plates hypotonically, which spreads the cells thinly to detach MNi from the primary nuclei. Then, the number of MNi and immunocytochemically-stained γH2AX foci were measured using an imaging analyzer. The system correctly judged 4 non-genotoxins and 13 genotoxins, which included 9 clastogens and 4 aneugens representing various genotoxic mechanisms, such as DNA alkylation, cross-linking, topoisomerase inhibition, and microtubule disruption. Furthermore, all the clastogens induced both γH2AX foci and MNi, while the aneugens induced only MNi, not γH2AX foci; therefore, the HC imaging assay clearly discriminated the aneugens from the clastogens. Additionally, the test system could feasibly analyze cell cycle, to add information about a chemical's mode of action. CONCLUSIONS: A HC imaging assay to detect γH2AX foci and MNi in TK6 cells was established, and the assay provided information on the aneugenic/clastogenic mode of action.

New DNA probes to detect aneugenicity in rat bone marrow micronucleated cells by a pan-centromeric FISH analysis.

When characterizing the genotoxicity of chemicals that induce micronuclei, it is practical to be able to classify the chemicals as aneugens or clastogens. This classification gives information on the mechanistic properties of chemicals and is indispensable for setting the threshold safety margins for genotoxicity in pharmaceutical development. A widely used method for detecting aneugens is fluorescence in situ hybridization (FISH) but, even though the rat is an experimental animal generally used in preclinical studies in drug development, DNA probes that hybridize to all the centromeres of rat chromosomes have not yet been established. In the present study, in addition to the previously known satellite I sequence, we identified two novel satellite sequences, satellite II and satellite III, from the rat genome database. DNA probes with a mixture of these satellite DNA sequences were used to establish a FISH method for pan-centromeric staining of rat chromosomes. To confirm the feasibility of the method, vinblastine (VBS) and mitomycin C (MMC) were administered to rats as a typical aneugen and clastogen, respectively. Micronucleated polychromatic erythrocytes (MNPCE) from bone marrow were enriched by sorting in flow cytometry and subjected to the FISH method. As a result, the ratio of centromere-positive MNPCE increased in VBS-treated rats but not in MMC-treated ones. Since the FISH method using the novel DNA probes clearly discriminates the aneugens from the clastogens, we suggest this method as a useful tool for providing mechanistic information for micronucleus induction in vivo.

Interlaboratory comparison of quantitative RT-PCR based detection for minimal residual disease in leukemias: a standardization approach in Japan.

Real-time quantitative polymerase chain reaction (RQ-PCR) has been accepted as integral part of the management of patients with hematologic malignancies. Whereas standardization efforts of RQ-PCR, initiated by Europe Against Cancer (EAC) group, have been gradually widespread in the world, Japanese laboratories use their individual protocol for RQ-PCR analysis. Therefore, we assessed the variability of quantitative results obtained from 4 different laboratories in Japan, including 3 companies and Tohoku University Hospital, using identical peripheral blood or bone marrow samples of patients in chronic myeloid leukemia (CML; n = 11) and acute myeloid leukemia (AML; n = 2). RQ-PCR was designed to quantify the copy numbers of disease-specific fusion chimeras; BCR-ABL (CML) and AML1-ETO (AML). In 5 out of 13 samples, the quantitative results from 4 laboratories varied more than 10 times (up to 712 times). Thus, we next sought to determine factors affecting the variability of RQ-PCR results across laboratories, by sending back RNA and cDNA samples from each company to Tohoku University, and they were further proceed to yield quantitative data. The main difference between companies and Tohoku University was probably due to the difference of blood separation method (Blood lysis or Ficoll-Hypaque). On the other hand, the variability among 4 laboratories was the most noticeable in the PCR step, mainly attributable to the difference of primer/probe sequence among laboratories. In conclusion, our analyses indicate the importance to limit both preanalytical (sample processing) and analytical (RQ-PCR) interlaboratory variability for RQ-PCR protocol, and the need of further efforts on standardization program in Japan.

Assessment of quantitative dual-parameter flow cytometric analysis for the evaluation of testicular toxicity using cyclophosphamide- and ethinylestradiol-treated rats.

In the drug discovery process, effects to the human spermatogenesis must be fully evaluated before the first human trial. To estimate testicular toxicity, histopathological evaluation has been recommended in addition to the traditional mating procedure. However, it is laborious and time-consuming. Flow cytometric analysis (FCM) has also been applied to estimate testicular toxicity because of its speed, simplicity, and the objectivity of the data. Using cyclophosphamide (CP)- and ethinylestradiol (EE)-treated rat testis, we attempted to validate our dual-parameter, DNA ploidy and cell-size FCM, in a high-throughput toxicity study. Our results showed that CP damaged some spermatogonia and some early meiotic spermatocytes and EE caused severe decrease of spermatogenic cells except for spermatogonia as well as marked decrease of somatic cells, most probably Leydig cells. This is the first report discriminating between the changes of spermatogonia and that of somatic cells with FCM analysis. These results demonstrate that this method is a very useful and powerful tool to assess testicular toxicity, especially in high-throughput toxicological studies.