Abstrakt

The bioactive phytochemicals found in Brassica vegetables belonging to glucosinolates (GLS) and especially the products of their degradation isothiocyanates (ITC) and indoles are regarded as the most promising cancer chemopreventive compounds. These secondary metabolites constitute defence system repelling or preventing the development of agrophages attacking brassica plants. The antibiological properties of these compounds suggest that they may also pose risk for humans when ingested in higher amounts in a form of e.g. dietary supplements. These presumptions have been confirmed by showing mutagenic activity and DNA adduct formation in vitro and in vivo for indoles found in brassica vegetables. Our research concentrated on the ability of degradation products of major aliphatic and indole GLS (AITC, PEITC, sulforaphane, indole-3-carbinol (I3C), indole-3-acetic acid (IAA), indole-3-acetonitril (IAN) and 3,3'-diindolomethan (DIM) a product of I3C dimerization) to induce covalent DNA modification. These mechanistic studies involved both metabolic and kinetic aspects. For this purpose, the restriction analysis method developed by our group was used. This method employs a 695 bp long DNA amplicon that contains two restriction sites: one consisting of only GC base pairs recognized by restriction endonuclease MspI or HpaII and the other consisting of only AT base pairs recognized by the enzyme Tru1I. The modification of the restriction sites abolishes their recognition and thus cleavage by the restriction enzymes. By incubating the DNA amplicon with increasing concentrations of the studied compound(s) in the appropriate activating system (for example sulfotransferases (SULTs) which activate benzylic alcohols), the formation of DNA adducts may be detected and its kinetics monitored. The modified amplicon is submitted to digestion by the mentioned endonucleases and DNA fragments are separated by polyacrylamide gel electrophoresis. The inhibition of cleavage indicates the occurrence of covalent modification of the restriction site(s) simultaneously pointing at the kind of base pairs involved in DNA adduct formation. The restriction analysis demonstrated that some ITC as well as some indole derivatives exhibit the capability of covalent DNA modification.

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