Establishment of the micronucleus assay in the human hepatoma cell line HepG2 to identify genotoxic natural products

Alternative therapies become more and more popular. Indeed a lot of herbal preparations are hardly toxicologically analysed. According to the OECD guideline [1] the cytokinesis-block micronucleus assay (CBMN assay) has been established in the human hepatoma cell line HepG2 to identify genotoxic natural products. The CBMN assay is used to detect chemicals, which generate structural (clastogenic) or numeric (aneugenic) chromosome aberrations that are manifested in the formation of so-called micronuclei. Micronuclei originate from chromosome fragments or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division.

To examine the test system methyl-methanesulfonate (MMS) and cyclophosphamide have been used as positive control. The clastogen MMS induced at non-cytotoxic concentrations a significant increase in micronuclei. The effect was dose dependent. In contrast cyclophosphamide, a clastogen that requires metabolic activation, did not show an enhancement of the frequency of micronuclei at concentrations up to 10µM. Therefore it can be assumed that the HepG2 are not metabolically competent contrary to references [2].

At first aristolochic acid, a component of the plant extract of Aristolochia, has been tested for their genotoxicity in the CBMN assay. Aristolochia plants are used in Traditional Chinese Medicine. Aristolochic acid did not cause an increase in micronuclei at non-cytotoxic concentrations, probably due to the inadequate endogenous metabolic capacity of HepG2. Hence all further studies will be executed with and without an exogenous metabolising system.

References: 1. OECD (2006) OECD Guideline for the Testing of Chemicals: Draft Proposal for a New Guideline 487, In Vitro Micronucleus Test [available at: www.oecd.org]

2. Kevekordes, S. et al. (2001) Anticancer Res. 21: 461–469