The extended version of restriction analysis approach for the examination of the ability of low-molecular-weight compounds to modify DNA in a cell-free system

Abstract

One of the primary requirements in toxicology is the assessment of ability of chemicals to induce DNA covalent modification. There are several well-established methods used for this purpose such as (32)P-Postlabeling or HPLC-MS. However, all of these approaches have difficult to overcome limitations, which prevents their use in genotoxin screening. Here, we describe the simple protocol exploiting specificity of restriction enzymes for the detection of DNA modification. It uses a specifically designed DNA amplicon, which contains two restriction sites recognized by Tru1I or MspI/HapII endonucleases. Modification of a restriction site abolishes its recognition and thus cleavage by the corresponding enzyme. The inhibition of cleavage indicates the occurrence of DNA modification of the restriction site(s), simultaneously pointing at the kind of base pairs (AT or GC) involved in DNA adduct formation. Previously, the application of this method was demonstrated for two antitumor compounds. Current study shows the extended version, that includes different ways of activation of tested compounds. Moreover, we propose an array of applications being of interest in toxicological research such as monitoring the kinetics of DNA adduct formation, detection of oxidative DNA damage, as well as assessment of the ability of antioxidative phytochemicals to prevent the latter DNA lesions.

Keywords: DNA covalent modification; Genotoxic compounds; Oxidative damage; Phytochemicals; Restriction analysis; Screening.