In vitro evaluation of the genotoxicity of a naturally occurring crosslinking agent (genipin) for biologic tissue fixation

The objective of the present study was to evaluate in vitro, using Chinese hamster ovary (CHO-K1) cells, the genotoxicity of genipin, a naturally occu rring crosslinking agent. Glutaraldehyde, the most commonly used crosslinki ng agent fur biologic tissue fixation, was employed as a reference chemical . The selected procedures for this evaluation were the micronucleus (MN) an d sister chromatid exchange (SCE) assays with or without the addition of a metabolic activation system (S9 mix). Before starting the genotoxicity assa ys, the maximum noncytotoxic amounts of glutaraldehyde and genipin were det ermined using the MTT assay. The results obtained in the MTT assay revealed that the cytotoxicity of genipin was significantly lower than that of glut araldehyde with or without S9 mix. The frequencies or MN observed in the ca ses drugged with varying concentrations of glutaraldehyde or genipin were n ot statistically different from those seen in the negative controls (blank) in the presence or absence of S9 mix. However, it was noted that glutarald ehyde significantly inhibited the cell-cycle progression while the cells dr ugged with genipin did not result in cell-cycle delay. In the SCE assay, th e numbers of SCE per cell observed in the cases drugged with varying concen trations of glutaraldehyde were significantly greater than those found in t he negative controls with or without S9 mix. Nevertheless, these numbers we re still low compared to the numbers of SCE induced by the strong mutagens used as our positive control substances. This suggests that glutaraldehyde may produce a weakly clastogenic response in CHO-K1 cells. In contrast, the numbers of SCE per cell obtained in the cases drugged with genipin were co mparable to those observed in the negative controls in those that were exce pt drugged with the highest dose (50 ppm). This suggests that genipin does not cause clastogenic response in CHO-K1 cells provided its concentration i s lower than 50 ppm. in conclusion, as far as cytotoxicity and genotoxicity are concerned, genipin is a promising crosslinking agent for biologic tiss ue fixation. (C) 2000 John Wiley & Sons, Inc.