Elsevier

Dental Materials

Volume 24, Issue 6, June 2008, Pages 765-772
Dental Materials

Effect of mercury(II) on Nrf2, thioredoxin reductase-1 and thioredoxin-1 in human monocytes

https://doi.org/10.1016/j.dental.2007.09.002Get rights and content

Abstract

Objectives

Human blood levels of mercury are commonly 10 nM, but may transiently reach 50–75 nM after dental amalgam placement or removal. Controversy persists about the use of mercury because the effects of these ‘trace’ levels of mercury are not clear. Concentrations of mercury ≥5000 nM unequivocally alter redox balance in blood cells including monocytes. In the current study, we tested a hypothesis that concentrations of mercury <100 nM altered levels and activities of key proteins that maintain monocytic redox balance.

Methods

Human THP1 monocytes were exposed to 10–75 nM of Hg(II) for 6–72 h, with or without activation by lipopolysaccharide (LPS). The redox management proteins Nrf2 and thioredoxin-1 (Trx1) were separated by electrophoresis, then quantified by immunoblotting. The activity of the seleno-enzyme thioredoxin reductase (TrxR1), important in maintaining Trx1 redox balance, was measured by cell-free and cell-dependent assays.

Results

Concentrations of Hg(II) between 10–75 nM increased Nrf2 levels (3.5–4.5 fold) and decreased Trx1 levels (2–3 fold), but these changes persisted <24 h. Hg(II) potently inhibited (at concentrations of 5–50 nM) TrxR1 activity in both cell-free and intracellular assays. Furthermore, Hg(II) transiently amplified LPS-induced Nrf2 levels by 2–3 fold and limited LPS-induced decreases in Trx1. All effects of Hg(II) were mitigated by pre-adding N-acetyl-cysteine (NAC) or sodium selenide (Na2SeO3), supplements of cellular thiols and selenols, respectively.

Significance

Our results suggest that nanomolar concentrations of Hg(II) transiently alter cellular redox balance in monocytes that trigger changes in Nrf2 and Trx1 levels. These changes indicate that monocytes have a capacity to adapt to trace concentrations of Hg(II) that are introduced into the bloodstream after dental amalgam procedures or fish consumption. The ability of monocytes to adapt suggests that low levels of mercury exposure from dental amalgam may not overtly compromise monocyte function.

Introduction

Mercury is a major component of dental amalgam and is essential to many manufacturing processes, but its biological and environmental liabilities have been perennially controversial [1], [2]. Human exposure to mercury cannot be completely avoided; the World Health Organization estimates that humans are exposed to as much as 6.6 μg/day of mercury from all sources [1], [2]. The uptake into and distribution of mercury in the body is complex and depends on the route and form of mercury exposure [3]. Elemental (Hg0) and organic (Rx-Hg) forms are efficiently absorbed, usually from the lungs (Hg0) and gut (Rx-Hg), then distributed via the bloodstream [4], [5], [6], [7]. Blood mercury levels of 10 nM are commonplace and may transiently reach 50–75 nM or more after ingestion of certain fish or placement or removal of dental amalgam restorations. A significant problem fueling controversy about the use of mercury is that the ability to detect it has outpaced knowledge of its effects at trace levels (<100 nM) in the bloodstream and other tissues. The effects of mercury are well defined above blood levels of 350 nM [1], [2], [8], [9], [10], [11], but are less clear below this level.

Because the blood is among the earliest tissues exposed to absorbed mercury, the effects of mercury on blood cells, and particularly monocytes, has been one recent research focus [12]. In monocytes, both Hg0 and organomercurials such as methyl mercury (Me-Hg) are rapidly converted by intracellular catalases into Hg(II) [1], [2], [11]. Hg(II) is therefore an important proximate cellular toxin resulting from exposure of monocytes and other cells to Hg0 and Rx-Hg [8], [9]. Monocytes orchestrate inflammatory and immune responses by integrating and amplifying biochemical signals, and may therefore be an important mediator of the effects of trace levels of mercury [13]. Mercury-induced changes in monocyte function may not be manifest until monocytes migrate into tissues and are activated during inflammation by bacterial toxins such as lipopolysaccharide (LPS) or other inflammatory activators.

Hg(II) appears to exert cellular effects at least in part via oxidative stress mechanisms [10], [12], [14]. The cytotoxic effects of Hg(II) are enhanced by known oxidative stressors and mitigated by antioxidants [12]. Hg(II) enhances intracellular glutathione (GSH) levels at near-lethal and sublethal concentrations, perhaps by binding with reduced glutathione to form a mercuric derivative (GS-Hg-SG) that mimics the oxidized form of glutathione (GS-SG, [1], [2], [12], [15]). Intracellular levels of reactive oxygen species (ROS) are elevated by Hg(II) concentrations of 5000 nM in some cells [16]. However, low levels of mercury (<100 nM) do not elevate ROS in monocytes [12], suggesting that other mechanisms play a role in Hg(II)-induced oxidative stress at lower concentrations. Most researchers accept that these other mechanisms involve the high affinity of Hg(II) for cellular thiol (-SH) and selenol (-SeH) groups [1], [2], [11].

Thioredoxin-1 (Trx1) is a 12 kD redox protein that helps maintain cellular redox homeostasis, and plays an important role in repair of oxidized proteins [17]. The reduced di-thiol form of Trx1 (Trxred) also maintains transcription factors such as NFκB in a reduced condition that promotes DNA binding and gene transactivation [18], [19]. Trx1 is secreted, but the full role of the secreted form is not clear [17]. Hg(II) recently has been shown to induce oxidative bias in the Trx1red/Trx1ox redox couple [20], which suggests one mechanism for Hg(II)-induced oxidative stress. Trx1red is highly favored in cells, and is maintained by thioredoxin reductase-1 (TrxR1), which has a selenocysteine residue that is essential to its catalytic activity [21]. The high affinity of Hg(II) for selenols suggests that Hg(II) may bind selenocysteine and inhibit TrxR1 activity, thereby inducing Trx1 oxidative bias in the Trx1red/Trx1ox couple.

The Nrf2 pathway coordinates the cellular response to oxidative stress by inducing the expression of genes for synthesis of over 100 ‘redox rescue’ proteins, including Trx1 and TrxR1 [22], [23]. Nrf2 pathway activation relies on oxidation of key cysteine residues in Keap1, a cysteine-rich, cytosolic protein that binds Nrf2 and directs its degradation [23], [24]. Because of its high affinity for thiols, Hg(II) might alter the regulation of the Nrf2 pathway, either directly by binding cysteines of Keap1 or Nrf2, or indirectly by shifting GSH or Trx1 redox balance to an oxidizing bias. Although, Hg(II)/Nrf2 effects have not been studied directly, several reports support a focus on the Nrf2 pathway as an important component of the intracellular response to trace levels of Hg(II) [16], [25].

In the current study, we investigated a hypothesis that levels of Hg(II) <100 nM alter cellular redox balance. We first show that Hg(II) inhibits TrxR1 activity and that inhibitory concentrations elevate Nrf2 levels and change levels of Trx1 in monocytes. We then demonstrate that some of these Hg(II)-induced changes are mitigated by supplementation with thiol or selenol compounds. We also show that Hg(II) alters the effects of inflammatory activators such as lipopolysaccharide on Nrf2 and Trx1 levels. Our results suggest that trace levels of Hg(II) (<100 nM) alter cellular redox balance in monocytes that trigger adaptive changes in Nrf2 and Trx1 levels to attempt to re-establish redox homeostasis.

Section snippets

Cells and cell-culture

Human THP1 monocytic cells (ATCC TIB 202) were cultured in RPMI 1640 cell-culture medium supplemented with 10% FBS, 2 mM glutamine, 100 μg/mL streptomycin, 100 units/mL of penicillin, and 50 μM β-mercaptoethanol (Invitrogen/Gibco BRL). Because the β-mercaptoethanol is a thiol-based reducing agent, we removed this component from the medium 72 h prior to experiments to avoid masking Hg-induced thiol-mediated effects. This strategy has been successfully used in previous studies [12].

Hg(II) exposure conditions and rationale

Hg(II) (from HgCl2,

TrxR1 Activity

Hg(II) was a potent inhibitor of rTrxR1 activity in both cell-free and cell-dependent assays. When DTNB was used as a rTrxR1 substrate in cell-free assays (Fig. 1, top), concentrations of Hg(II) as low as 10 nM inhibited rTrxR1 activity by 50%, and 50 nM inhibited activity by 80%. When Trx1ox was used as the substrate (Fig. 1, middle), Hg(II) inhibited rTrxR1 completely at 5 nM. After 24 h exposure to THP1 monocytes, Hg(II) concentrations of 100 nM inhibited intracellular TrxR1 activity by 60% (Fig.

Discussion

We observed a potent Hg(II)-induced inhibition of rTrxR1 activity (Fig. 1), which is consistent with the affinity of Hg(II) for selenols [1], [11], the accessibility of the selenocysteine residue at the carboxy terminus of rTrxR1 [37], and critical role selenocysteine plays in the catalytic cycle of this enzyme [30], [37], [38]. Inhibition was less potent in THP1 cells than in cell-free assays, possibly reflecting a reduced access of Hg(II) to TrxR1 in the cell vs. cell-free environment.

Acknowledgement

The authors thank the Dental Research Center at the Medical College of Georgia for financial support of this work.

References (44)

  • J.R. Mackert et al.

    Mercury exposure from dental amalgam fillings: absorbed dose and the potential of adverse health effects

    Crit Rev Oral Biol Med

    (1997)
  • J.B. Hursh et al.

    Clearance of mercury (Hg-197, Hg-203) vapor inhaled by human subjects

    Arch Environ Health

    (1976)
  • A. Berglund

    Estimation by a 24 h study of the daily dose of intra-oral mercury vapor inhaled after released from dental amalgam

    J Dent Res

    (1990)
  • G. Sandborgh-Englund et al.

    The absorption, blood levels, and excretion of mercury after a single dose of mercury vapor in humans

    Toxicol Appl Pharmacol

    (1998)
  • G. Sandborgh-Englund et al.

    Mercury in biological fluids after amalgam removal

    J Dent Res

    (1998)
  • T.W. Clarkson et al.

    Human exposure to mercury: the three modern dilemmas

    J Trace Elem Expt Med

    (2003)
  • T.W. Clarkson et al.

    Current concepts—the toxicology of mercury-current exposures and clinical manifestations

    New Eng J Med

    (2003)
  • R.A. Goyer

    Toxic effects of metals

  • P.D. Whanger

    Selenium and the brain: a review

    Nut Neurobiol

    (2002)
  • R.L.W. Messer et al.

    Mercury (II) alters mitochondrial activity of monocytes at sublethal doses via oxidative stress mechanisms

    J Biomed Mater Res

    (2005)
  • M.J. Auger et al.

    The biology of the macrophage

  • M.B. Wolf et al.

    Cadmium and mercury cause an oxidative stress-induced endothelial cell dysfunction

    Biometals

    (2007)
  • Cited by (30)

    • Thioredoxin reductase as a pharmacological target

      2021, Pharmacological Research
      Citation Excerpt :

      However, it has been reported that methionine sulfoxide reductases (at least one of them) can also use glutaredoxin as an alternative reducing cofactor [85], thus providing a backup when reduced Trx is depleted. TrxR is very sensitive to inhibition by several metals including gold, palladium, and platinum, as well as by silver, zinc, mercury, cadmium and gadolinium (Fig. 3) [86–91]. While TrxR strongly binds particularly toxic metals, its substrate Trx is also a heavy metal-binding protein.

    • The thioredoxin system as a target for mercury compounds

      2019, Biochimica et Biophysica Acta - General Subjects
      Citation Excerpt :

      This effect was subsequently confirmed in HeLa cells where 24 h of exposure to Hg2+ or MeHg inhibited TrxR activity with an IC50 of 5.4 and 1.4 μM, respectively [58]. Additional results supporting these findings were obtained by Wataha and co-workers [59] showing the inhibitory effect of Hg2+ over both purified rat TrxR1 and in cultures of THP1 monocytes. Further data in HepG2 and SH-SY5Y cells also showed that ethylmercury (EtHg) affected TrxR similarly to MeHg [60].

    • Environmental mercury exposure and selenium-associated biomarkers of antioxidant status at molecular and biochemical level. A short-term intervention study

      2019, Food and Chemical Toxicology
      Citation Excerpt :

      Because this Se-dependent enzyme has a redox property, inhibition of TrxR may elevate levels of ROS. Therefore, MeHg may impair the redox state of cells through elevating Trx1 oxidative bias (Wataha et al., 2008). This may explain of another mechanism by which MeHg may induce oxidative stress, i.e. via TrxR inhibition.

    • Chronic Vascular Pathology and Toxicology

      2018, Comprehensive Toxicology: Third Edition
    View all citing articles on Scopus
    View full text