Response of DNA, proteins and membrane bilayer in the digestive gland cells of freshwater mussel Unio tumidus to tannins exposure

Abstract

Exposure of digestive gland cells ( Unio tumidus) in in vitro study to tannins (tannic, ellagic and gallic acid) caused changes in three types of cellular macromolecules, i.e. lipids, proteins and DNA. Two fluorescence probes: TMA-DPH and 12-AS were used to assess the membrane fluidity after incubating cells with different concentrations of tannins (1–60 μM). It was noticed that tannins increased the fluidity in the internal region of the lipid bilayer, but no changes at the surface of the plasma membrane were observed. The number of protein-bound carbonyl groups was quantitated spectrophotometrically using 2,4-dinitrophenylhydrazine (DNPH) assay. Oxidative modification of proteins increased in the cells exposed to tannins in a concentration-dependent manner. Results also showed an increase in carbonyl groups formation after exposing the cells to complex: tannin–Cu 2+ ions. We also investigated the effect of tannins on the level of nuclear DNA damage using the comet assay. We showed that these compounds at 15, 30 and 60 μM induced DNA damage. At these concentrations cell viability measured by trypan blue exclusion assay was not reduced by more than 30%. We also added Cu 2+ ions at 50 μM to assess the influence of the complex tannin–Cu 2+ on DNA lesions. The same method was used to study the possibility to repair this damage in DNA. Experimental data showed that tested polyphenolic acids do not contribute to the induction of strand breaks in nucleic acid at the smallest dose––1 μM and in this range they have a protective effect against the activity of 50 μM Cu 2+ ions.