gms | German Medical Science

Introduction: To gain representative data that are appropriate to estimate exposure-associated risk in regard to mutagenic effects, a valid mutagenicity test using primary human oropharyngeal epithelial cells (EC) is desirable but was missing so far. Thus a micronucleus (MN) assay according to OECD guideline 487 (draft) was developed for detection of mutagenic effects on EC.

Methods: After receiving patient’s informed consent, mucosa biopsates were taken either during tonsillectomy or tumor dissection from which EC were isolated by tryptic digestion. Thereafter, EC were grown using KSFM (serum-free medium KSFM [Gibco] containing 20 ng/ml of epidermal growth factor) in petri dishes coated with extracellular matrix proteins (EMP). After harvesting passage-2 cells, EC were seeded into EMP-coated 24-well plates and cultured under standard conditions (5% CO2, 36.5°C, 95% relative humidity). At day 3, supernatants were aspirated, and pure medium and mitomycine C (100 nM), which served as negative (NC) and positive controls (PC), respectively, or samples in KSFM were administered in a total volume of 1.00 ml. At 24-h incubation, cytochalasin B was added to block cytokinesis. After further 24 h, supernatants were harvested and EC fixed using ethanol before DNA staining by DAPI. Micronuclei in 1000 bi-nucleated EC (BN) were counted in each case.

Results: Commonly, 15 to 25% of EC were BN (median=21%), and the MN in BN of PC (median=5.0%) were found to be reproducible circa threefold the rate in NC (median=1.8%).

Conclusions: Sufficient BN formation and reliable induction of MN in PC allows for reproducible and valid mutagenicity testing using EC. The data gained using EC should be more convincing than those of cell lines and thus be more substantial regarding risk assessment.

Supported by: Verein Deutscher Zementwerke e.V., Düsseldorf