Abstract

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.

The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.

First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.

Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.

There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.

Keywords: Micronucleus assay; Mouse splenocyte; Human lymphocyte; Nocodazole; Paclitaxel

Article Outline

1. Introduction

2. Materials and methods

2.1. Chemicals

2.2. Cytokinesis-blocked micronucleus (HL-CBMN) assay with human lymphocytes

2.2.1. Blood sample collection

2.2.2. Lymphocyte cultures

2.3. Cytokinesis-blocked micronucleus (MS-CBMN) assay with mouse splenocytes

2.3.1. Animals

2.3.2. Splenocyte cultures

2.3.3. Mitogen and cytochalasin B time evaluations

2.4. Harvesting and slide preparation of HL-CBMN and MS-CBMN

2.5. Analysis of HL-CBMN and MS-CBMN

2.6. Statistical analysis

2.6.1. Cell proliferation

2.6.2. Genotoxic responses

3. Results

3.1. Choice of mitogen for mouse splenocytes

3.2. Optimization of cytochalasin B in mouse splenocytes

3.3. Effects of nocodazole and paclitaxel on cell proliferation and genotoxic responses in human lymphocytes and mouse splenocytes

3.3.1. Effect of aneugens on cell proliferation

3.3.2. Genotoxic responses

4. Discussion

Acknowledgements

References