Thyme oil mitigates cadmium-induced oxidative and genotoxic stress in Vicia faba root meristem cells under in vitro conditions

Abstract

Cadmium is considered one of the most toxic and dangerous environmental agents among heavy metals. It induces oxidative stress state, which is a source of disruption to a wide range of key cellular processes in plants. The increasing problem of heavy metal accumulation in the environment necessitates the search for strategies that enhance plant resistance to the toxic effects of these elements. Therefore, the purpose of this study was to evaluate the protective potential of thyme oil (TO) against 175 µM cadmium chloride (CdCl2)-induced toxicity in meristematic cells of Vicia faba var. minor primary roots under in vitro conditions. To assess the impact of TO, we analyzed reactive oxygen species (ROS) production, as cadmium-induced oxidative stress can trigger cellular damage. Hydrogen peroxide (H₂O₂) was detected using 3,3′-diaminobenzidine (DAB) staining, while superoxide anion (O₂•−) was visualized via Nitro Blue Tetrazolium (NBT) reduction. Since excessive ROS can lead to DNA damage, impair replication, and alter the proportion of interphase cells, we performed immunofluorescence labeling to examine replicating cells using a DNA synthesis marker (IdU). Additionally, we quantified disruptions in DNA replication and assessed the level of DNA damage using γ-phosphorylation of histone H2A.X at serine 139, a marker of DNA double-strand breaks (DSBs). Our results demonstrated that TO significantly mitigated cadmium-induced oxidative and genotoxic stress. Notably, TO treatment reduced the percentage of γ-H2A.X-positive cells by approximately 25% compared to cadmium-only treatment. Moreover, a 41,5% decrease in in the intensity of NBT staining (indicative of superoxide accumulation) and a 72,45% reduction in DAB staining intensity (reflecting hydrogen peroxide levels) were observed. These findings indicate that TO application may serve as a promising strategy for protecting plant tissues exposed to cadmium contamination in the future.