Oxidative Damage in Human Periodontal Ligament Fibroblast (hPLF) after Methylmercury Exposure

Abstract

Human exposure to mercury (Hg) is primary associated with its organic form, methylmercury (MeHg), through the ingestion of contaminated seafood. However, Hg contamination is also positively correlated with the number of dental restorations, total surface of amalgam, and organic mercury concentration in the saliva. Among the cells existing in the oral cavity, human periodontal ligament fibroblast (hPLF) cells are important cells responsible for the production of matrix and extracellular collagen, besides sustentation, renewal, repair, and tissue regeneration. In this way, the present study is aimed at investigating the potential oxidative effects caused by MeHg on hPLF. Firstly, we analyzed the cytotoxic effects of MeHg (general metabolism status, cell viability, and mercury accumulation) followed by the parameters related to oxidative stress (total antioxidant capacity, GSH levels, and DNA damage). Our results demonstrated that MeHg toxicity increased in accordance with the rise of MeHg concentration in the exposure solutions (1-7 μM) causing 100% of cell death at 7 μM MeHg exposure. The general metabolism status was firstly affected by 2 μM MeHg exposure (43.8 ± 1.7%), while a significant decrease of cell viability has arisen significantly only at 3 μM MeHg exposure (68.7 ± 1.4%). The ratio among these two analyses (named fold change) demonstrated viable hPLF with compromised cellular machinery along with the range of MeHg exposure. Subsequently, two distinct MeHg concentrations (0.3 and 3 μM) were chosen based on LC50 value (4.2 μM). hPLF exposed to these two MeHg concentrations showed an intracellular Hg accumulation as a linear-type saturation curve indicating that metal accumulated diffusively in the cells, typical for metal organic forms such as methyl. The levels of total GSH decreased 50% at exposure to 3 μM MeHg when compared to control. Finally, no alteration in the DNA integrity was observed at 0.3 μM MeHg exposure, but 3 μM MeHg caused significant damage. In conclusion, it was observed that MeHg exposure affected the general metabolism status of hPLF with no necessary decrease on the cell death. Additionally, although the oxidative imbalance in the hPLF was confirmed only at 3 μM MeHg through the increase of total GSH level and DNA damage, the lower concentration of MeHg used (0.3 μM) requires attention since the intracellular mercury accumulation may be toxic at chronic exposures.

Methylmercury-induced cytotoxicity and oxidative biochemistry impairment in dental pulp stem cells: the first toxicological findings

“…The slight decrease on cell viability with no changes on cell metabolism at 2.5 µM of MeHg seems to be related to the induction of cell survival responses by the activation of protective signaling pathways and consequently high metabolic demand at low concentrations of MeHg (Levonen et al, 2004). Similar results for cell viability were found by Nogueira et al (2019) in a study using human periodontal ligament fibroblasts (hPLF) cells and different MeHg concentrations. But in the referred study, the metabolic viability decreased with a lower dose of MeHg, 2 µM.…”

“…In order to bring cell exposure closer to what occurs in human exposure, the lowest MeHg concentration chosen in our study is related to the Hg levels detected in chronically exposed riverine population in the Amazon river (Castilhos et al, 2015). The 2.5 and 5 µM MeHg were based on previous in vitro studies with oral cavity cells with the human periodontal ligament fibroblast (hPLF) (Nogueira et al, 2019). The 5 µM MeHg exerted significant toxic effects on viability and cell metabolism, while the 2.5 µM MeHg is within the range of concentration that significantly affected cell metabolism but minor changes on viability.…”

“…However, only few studies have investigated the effects of MeHg on oral structures. Some of these focused on the amount of organic and inorganic mercury in human saliva (Leistevuo et al, 2001) and recently our group have shown the impact of mercury accumulation and toxicity on rat salivary glands (Lima et al, 2018;Farias-Junior et al, 2017; and human periodontal ligament fibroblast (Nogueira et al, 2019). Nevertheless, no studies have been specifically evaluated the effect of MeHg on human dental pulp cells.…”

Occurrence of mercury in various types of honey

“…Dochodząca do tego postępująca dysfunkcja mitochondriów i zmiany przepuszczalności struktur komórkowych (m.in. białek, lipidów, DNA) wywoływane ekspozycją na rtęć, mogą prowadzić do powstawania stresu oksydacyjnego w organizmie [8,9]. Metylortęć bardzo łatwo przenika przez barierę krew-mózg poprzez duże transportery obojętnych aminokwasów typu L oraz dzięki dużemu powinowactwu do struktur lipidowych, co prowadzi do gromadzenia się jej w mózgu i neurotoksyczności [10,11].…”

Gene Expression Profile in Immortalized Human Periodontal Ligament Fibroblasts Through hTERT Ectopic Expression: Transcriptome and Bioinformatic Analysis

“…Both original hPLFs and immortalized hPLF-hTERT were cultured in Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 nutrient medium (1:1), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin, incubated at 37°C in 5% CO 2 (Nogueira et al, 2019). The medium was changed every 48 h. When cells became fully confluent, they were passaged using 0.25% trypsin solutions and seeded in new flasks.…”