Inorganic Hg toxicity in plants: A comparison of different genotoxic parameters

Abstract

Inorganic Mercury (Hg) contamination persists an environmental problem, but its cyto- and genotoxicity in plants remains yet unquantified. To determine the extent of Hg-induced cyto- and genotoxicity, and assess most sensitive endpoints in plants, Pisum sativum L. seedlings were exposed for 14 days to different HgCl₂ concentrations up to 100 μM. Shoots and roots from hydroponic exposure presented growth impairment and/or morphological disorders for doses >1 μM, being the roots more sensitive. Plant growth, ploidy changes, clastogenicity (HPCV), cell cycle dynamics (G₁-S-G₂), Comet-tail moment (TM), Comet-TD, Mitotic-index (MI) and cell proliferation index (CPI) were used to evaluate Hg-induced cyto/genotoxicity. Both leaf and root DNA-ploidy levels, assessed by flow cytometry (FCM), remained unaltered after exposure. Root cell cycle impairment occurred at lower doses (≥1 μM) than structural DNA damages (≥10 μM). Cytostatic effects depended on the Hg concentration, with delays during S-phase at lower doses, and arrests at G₁ at higher ones. This arrest was paralleled with decreases of both mitotic index (MI) and cell proliferation index (CPI). DNA fragmentation, assessed by the Comet assay parameters of TD and TM, could be visualized for conditions ≥10 μM, while FCM-clastogenic parameter (FPCV) and micronuclei (MNC) were only altered in roots exposed to 100 μM. We demonstrate that inorganic-Hg induced cytostaticity is detectable even at 1 μM (a value found in contaminated sites), while structural DNA breaks/damage are only visualized in plants at concentrations ≥10 μM. We also demonstrate that among the different techniques tested for cyto- and genotoxicity, TD and TM Comet endpoints were more sensitive than FPCV or MNC. Regarding cytostatic effects, cell cycle analysis by FCM, including the difference in % cell cycle phases and CPI were more sensitive than MI or MNC frequency. Our data contribute to better understand Hg cyto- and genotoxicity in plants and to understand the information and sensitivity provided by each of the genotoxic techniques used.

Keywords: Cell cycle; Comet assay; Flow cytometry; Mercury; Micronuclei; Pisum sativum.