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