ScopeGlucosinolates are secondary metabolites present in Brassica vegetables. Alkenyl glucosinolates are enzymatically degraded forming nitriles or isothiocyanates, but in the presence of epithiospecifier protein, epithionitriles are released. However, studies on the occurrence of epithionitriles in Brassica food and knowledge about their biological effects are scarce.Methods and resultsEpithionitrile formation from glucosinolates of seven Brassica vegetables was analyzed using GC‐MS and HPLC‐DAD. Bioactivity of synthetic and plant‐derived 1‐cyano‐2,3‐epithiopropane (CETP) – the predominant epithionitrile in Brassica vegetables – in three human hepatocellular carcinoma (HCC) cell lines and primary murine hepatocytes was also evaluated. The majority of the Brassica vegetables were producers of nitriles or epithionitriles as hydrolysis products and not of isothiocyanates. For example, Brussels sprouts and savoy cabbage contained up to 0.8 μmol CETP/g vegetable. Using formazan dye assays, concentrations of 380–1500 nM CETP were observed to inhibit the mitochondrial dehydrogenase activity of human HCC cells without impairment of cell growth. At 100‐fold higher CETP concentrations, cell death was observed. Presence of plant matrix increased CETP‐based toxicity.ConclusionThese in vitro data provide no indication that epithionitriles will severely affect human health by Brassica consumption. In contrast to isothiocyanates, no evidence of selective toxicity against HCC cells was found.