Clastogenicity of the food constituent higenamine in cultured V79 cells.

Higenamine is a catecholic benzyltetrahydroisoquinoline alkaloid occurring in plant-derived foods and food supplements, the latter potentially resulting in substantially higher exposure levels than conventional foods. Because of its catechol and alkylphenol structures, oxidative conversion to electrophilic quinone or quinone methide intermediates is plausible, raising concern about a potential genotoxic hazard. However, experimental data on the genotoxic potential of higenamine are lacking. The present study, therefore, investigated its potential to induce gene mutations and chromosomal damage in cultured V79 cells. Gene mutations were examined at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus after 24 h treatment without metabolic activation and after 4 h in the absence or presence of rat liver S9-mix. Clastogenicity and aneugenicity were assessed using in vitro micronucleus assay with CREST staining after 4 h of treatment, followed by compound-free postincubation periods of 16 and 24 h. Proliferation and mitotic activity were monitored, and higenamine stability was analyzed by HPLC-UV. Higenamine caused a statistically significant increase in the frequency of micronuclei at 300-510 µM, reaching maxima of 51 and 73 micronucleated cells per 1000 cells without and with metabolic activation, respectively. Micronuclei mainly contained chromosomal fragments, indicating a prevalent clastogenic mode of action, although a smaller increase in micronuclei containing whole chromosomes was also observed. Mutant frequencies in the HPRT assay were not significantly increased under any condition tested, despite concentration-dependent cytotoxicity. During incubation, higenamine concentrations decreased while only minor formation of O-methylated metabolites was detected, suggesting conversion into additional reactive products. Overall, these findings provide the first experimental evidence that higenamine exhibits clastogenic potential in vitro and support the hypothesis that oxidative activation of its catechol and/or alkylphenol moiety to electrophilic intermediates capable of reacting with cellular nucleophiles might contribute to its genotoxic activity.