Studies on the mutagenic properties of alkenylbenzenes

Alkenylbenzenes are secondary metabolites produced in plants, primarily serving plants as a defensive mechanism against herbivores. But they are also used by humans in foods or cosmetics for their aromatic properties. Some of the alkenylbenzenes, such as safrole, are already regulated by the European Commission (EC1334/2008) due to their genotoxic properties. There is a growing awareness that other alkenylbenzenes may also exhibit genotoxic potential and need further evaluation considering the high structural similarity. In the last couple years, the German federal institute for risk assessment (BfR) raises awareness of this missing data gaps and attempts to expand its knowledge base by evaluating additional alkenylbenzenes. To examine further alkenylbenzenes and expand knowledge, this master’s thesis aims to investigate mutagenic properties of parsley apiole dillapiole, honokiol and magnolol. Safrole was also included in experiments to provide a comparison with known genotoxicity. It was decided to perform a hypoxanthine-guanine phosphoribosyl transferase transgene (HPRT) as it already has been used by Groh et al. (2012) to assess mutation frequency of the alkenylbenzene methyl eugenol. First, an establishment of the assay within the laboratories of the BfR was attempted. Due to methodological issues, a micronucleus assay was performed later. Alkenylbenzenes were tested at concentrations between 1 μM and 500 μM based on previous tests conducted with other alkenylbenzenes within the same group. V79 cells were treated for 4 h, harvested 24 h later and micronuclei generation measured in a fluorescence-activated cell sorting (FACS) device. Cytotoxicity and ethidium monoazido bromide positive (EMA +) cells were determined as well. The micronucleus assay showed micronuclei generation in all tested alkenylbenzenes. However only in parsley apiole increased micronuclei generation was found without very high cytotoxic effects and EMA + cells. To gain further information on cytotoxicity a 7-amino-actinomycin D/annexin V phosphatidylethanolamine (7-AAD/annexin V PE) assay was performed as well. A very high cytotoxicity especially for honokiol and magnolol was determined at higher concentrations. To be able to make robust statements about mutagenic properties, other tests should be conducted. For further assessment lower doses with a smaller range should be tested to avoid exceeding cytotoxicity limits and allow clear statements.