Elsevier

Toxicology

Volume 257, Issues 1–2, 4 March 2009, Pages 95-104
Toxicology

The effects of acrolein on peroxiredoxins, thioredoxins, and thioredoxin reductase in human bronchial epithelial cells

https://doi.org/10.1016/j.tox.2008.12.013Get rights and content

Abstract

Inhalation is a common form of exposure to acrolein, a toxic reactive volatile aldehyde that is a ubiquitous environmental pollutant. Bronchial epithelial cells would be directly exposed to inhaled acrolein. The thioredoxin (Trx) system is essential for the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, thioredoxin reductase (TrxR) maintains the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins in the reduced state, and the thioredoxins keep the peroxiredoxins (Prx) reduced, thereby supporting their peroxidase function. The effects of acrolein on TrxR, Trx and Prx in human bronchial epithelial (BEAS-2B) cells were determined. A 30-min exposure to 5 μM acrolein oxidized both Trx1 and Trx2, although significant effects were noted for Trx1 at even lower acrolein concentrations. The effects on Trx1 and Trx2 could not be reversed by treatment with disulfide reductants. TrxR activity was inhibited 60% and >85% by 2.5 and 5 μM acrolein, respectively. The endogenous electron donor for TrxR, NADPH, could not restore its activity, and activity did not recover in cells during a 4-h acrolein-free period in complete medium. The effects of acrolein on TrxR and Trx therefore extend beyond the duration of exposure. While there was a strong correlation between TrxR inhibition and Trx1 oxidation, the irreversible effects on Trx1 suggest direct effects of acrolein rather than loss of reducing equivalents from TrxR. Trx2 did not become oxidized until ≥90% of TrxR was inhibited, but irreversible effects on Trx2 also suggest direct effects of acrolein. Prx1 (cytosolic) and Prx3 (mitochondrial) shifted to a largely oxidized state only when >90 and 100% of their respective Trxs were oxidized. Prx oxidation was readily reversed with a disulfide reductant, suggesting that Prx oxidation resulted from lack of reducing equivalents from Trx and not direct reaction with acrolein. The effects of acrolein on the thioredoxin system and peroxiredoxins could have important implications for cell survival, redox-sensitive cell signaling, and tolerance to other oxidant insults.

Introduction

Acrolein is a ubiquitous environmental pollutant, and inhalation is a prominent form of exposure. Major environmental sources include exhaust from internal combustion engines (Beauchamp et al., 1985) and wood combustion. Smoke from burning buildings and vegetative fires can have acrolein levels that are considered acutely dangerous (Beauchamp et al., 1985, Ghilarducci and Tjeerdema, 1995). Photochemical oxidation of airborne hydrocarbons also generates acrolein (Ghilarducci and Tjeerdema, 1995). Large amounts of acrolein are used as an aquatic pesticide and herbicide, and occupations with significant potential exposure include the manufacture of acrylate polymers, foundry operations, welding coated metals, coffee roasting, printing, linoleum production, tin plating, and rubber vulcanization (Beauchamp et al., 1985, Ghilarducci and Tjeerdema, 1995). Cigarette smoke is a major contributor to various lung diseases, and acrolein is a prominent component of the gas phase of cigarette smoke (Beauchamp et al., 1985), with 85–228 μg acrolein per cigarette (Esterbauer et al., 1991, Ghilarducci and Tjeerdema, 1995, Carnevali et al., 1998). Some of the inhaled acrolein can also be absorbed and distributed, causing systemic effects (Uchida et al., 1998a, Uchida et al., 1998b, Uchida, 2000, Barnoya and Glantz, 2005). Bronchial epithelial cells are directly exposed to inhaled acrolein and would therefore receive the highest doses. It is important to understand the effects of acrolein in these cells.

Acrolein is highly toxic to many cells including human bronchial epithelial cells and fibroblasts (Esterbauer et al., 1991), neurons (Lovell et al., 2001), vascular smooth muscle cells (Conklin et al., 1998), and endothelial cells (Patel and Block, 1993, Kachel and Martin, 1994, Szadkowski and Myers, 2007). Depending on the cell type, acrolein is 10–200 times more toxic than formaldehyde (Esterbauer et al., 1991, Conklin et al., 1998). Acrolein is more toxic than hydroxynonenal (4-HNE) (Lovell et al., 2001), and 100–150 times more reactive than 4-HNE (Esterbauer et al., 1991). Because it is a strong electrophile, acrolein can react nonenzymatically with nucleophiles including sulfhydryl (–SH) groups (Uchida et al., 1998b). This could have significant consequences for sulfhydryl-containing proteins and could disrupt the intracellular thiol redox equilibrium.

The maintenance of cellular thiol redox balance is critical for normal function and cell survival. Glutathione and the thioredoxins are major players in this maintenance, although these systems are not in redox equilibrium with each other (Nkabyo et al., 2002, Hansen et al., 2006a). The redox status of the thioredoxin (Trx) system may be more critical to cell survival than is glutathione (Hansen et al., 2006b). All mammalian cells have cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins (Powis and Montfort, 2001). Trx1 and Trx2 are encoded by distinct genes, but they share a conserved active site (Trp-Cys-Gly-Pro-Cys-Lys) that is cycled between the reduced (dithiol) and oxidized (disulfide) forms (Watson et al., 2003). Trx2 has two Cys residues, both in the active site. Trx1 has five Cys residues, two in the active site (C32/C35), and another two in a second dithiol motif (C62/C69) (Watson et al., 2003). Of the two Trx1 dithiols, the C32/C35 active site is more readily oxidized (Watson et al., 2003). Cells normally maintain Trx1 and Trx2 largely in the reduced state (Nordberg and Arnér, 2001, Powis and Montfort, 2001, Watson et al., 2003).

A major role of thioredoxins is to maintain the cysteine residues of intracellular proteins in a reduced state (Arnér and Holmgren, 2000). In this way, they support the function of a variety of proteins including ribonucleotide reductase, protein disulfide isomerase, peroxiredoxins, and many others (Nordberg and Arnér, 2001, Powis and Montfort, 2001). Peroxiredoxins (Prxs) are ubiquitous peroxidases that contain Cys at their active site. Peroxide substrates such as H2O2 and organic hydroperoxides oxidize the Prx active site Cys to sulfenic acid (–SOH). The 2-Cys Prxs (e.g. cytosolic Prx1 and Prx2, and mitochondrial Prx3) are homodimers in which this sulfenic acid reacts with the resolving Cys on the other subunit to form a disulfide-linked dimer (Wood et al., 2003a, Wood et al., 2003b, Peskin et al., 2007). The Trxs reduce these disulfides to regenerate the active Prx (Kim et al., 2005). The Trxs are therefore critical to supporting the peroxidase function of Prxs (Nordberg and Arnér, 2001, Powis and Montfort, 2001).

In cells, the Trxs are kept reduced (active) by Trx reductases (TrxRs) (Powis and Montfort, 2001). Trxs are therefore directly dependent on TrxRs. Through Trxs, Prxs are also dependent on TrxRs. TrxR1 (cytosolic) and TrxR2 (mitochondrial) are NADPH-dependent homodimers (Nordberg and Arnér, 2001, Powis and Montfort, 2001). They contain selenium (Se) as SeCys within the C-terminal Gly-Cys-SeCys-Gly active site (Nordberg and Arnér, 2001).

TrxRs, Trxs, and Prxs are all important for cellular growth and survival. Knockout mice lacking either Trx1 or Trx2 do not survive (Powis and Montfort, 2001, Nonn et al., 2003). Inhibition or genetic suppression of Trx results in increased oxidant stress and apoptosis (Hansen et al., 2006a) and increased sensitivity to oxidants (Chen et al., 2006). Conversely, Trx overexpression enhances resistance to oxidant-induced apoptosis (Chen et al., 2006, Hansen et al., 2006a). TrxR inhibition increases oxidant susceptibility and favors apoptosis (Nordberg and Arnér, 2001). Similarly, depletion of Prx3 enhances susceptibility to apoptotic insults (Chang et al., 2004). Overall, factors which oxidize or inhibit one or more of these proteins could decrease cell survival.

Initial studies noted that acrolein inhibited the activity of Trx1 in vitro (Yang et al., 2004), and caused a dose-dependent decline in Trx activity in A549 cancer cells, with maximal inhibition after 30 min with ≥25 μM (Yang et al., 2004). However, neither the specific Trx(s) affected or the redox status of the Trxs were determined (Yang et al., 2004). Subsequent studies with human endothelial cells showed a much greater sensitivity to acrolein with 5 μM sufficient to oxidize both Trx1 and Trx2 (Szadkowski and Myers, 2007). Trx1 was sensitive to even lower concentrations (Szadkowski and Myers, 2007). Go et al. (2007) noted that adduction of acrolein to Cys73 of Trx1 can inhibit Trx1 activity and change the redox state of Trx1 in bovine endothelial cells.

Relatively high doses of acrolein have been shown to inhibit TrxR activity in cells, e.g. 50–75 μM for 30 min caused ∼65% inhibition in A549 cells (Yang et al., 2004), whereas 25 μM for 30 min inhibited 88% of TrxR activity in human umbilical vein endothelial cells (HUVECs) (Park et al., 2005). No studies to date have examined the effects of acrolein on Prxs.

The effects of acrolein on one or more of these proteins in cells may be direct, whereas effects on others may be indirect, e.g. inhibition of TrxR could result in an inability to keep the Trxs and Prxs reduced. While previous papers have looked at TrxR and Trxs in different systems, none have examined the coordinated effects on TrxR, Trxs, and Prxs within the same system. Furthermore, none have examined the effects of acrolein on these proteins in normal bronchial epithelium, which would receive the highest dose of inhaled acrolein in vivo.

This paper examines the effects of low micromolar concentrations of acrolein on the activity of TrxR and on the redox status of Trxs and Prxs in human bronchial epithelial cells. All three protein types are oxidized/inhibited by ≥5 μM acrolein, with significant effects on TrxR and Trx1 at even lower concentrations. The effects on TrxR and Trx were consistent with acrolein–protein adducts: they were not reversible in vitro, and TrxR activity did not recover in cells after acrolein removal. In contrast, the oxidation of Prxs did not occur until nearly all of the respective Trx was oxidized, and Prx oxidation was reversible in vitro by disulfide reduction. The effects of acrolein on TrxR and Trx may therefore result from direct effects of acrolein, whereas Prx oxidation likely results from loss of function of the upstream Trxs.

Section snippets

Reagents

The following were purchased from Invitrogen Corp. (Carlsbad, CA): Hank's balanced salt solution (HBSS), 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate (AMS), and pre-cast gels (12% Bis–Tris, 16% Tris–Glycine) and matching electrophoresis and loading buffers. Phenylmethylsulfonyl fluoride (PMSF) and Tris were from Research Organics (Cleveland, OH). EDTA, guanidine–HCl, and trichloroacetic acid (TCA) were obtained from Fisher Scientific (Hampton, NH). Primary antibodies specific for Trx1,

Acrolein oxidizes Trx1 and Trx2

The effects of acrolein on the redox status of Trx1 (cytosolic) were analyzed by redox western blot. Iodoacetate covalently labels the –SH groups of reduced Trx1; the resulting negative charge increases migration in native PAGE relative to oxidized Trx1 which does not react with iodoacetate. Reduced Trx1 refers to the form in which both dithiols are reduced and represents the active form. The active site dithiol is the more easily oxidized, so partially oxidized Trx1 likely represents oxidation

Discussion

The acrolein exposures that oxidized/inhibited these proteins in BEAS-2B cells are consistent with those that cause cytotoxicity in human endothelial cells (Szadkowski and Myers, 2007), porcine pulmonary cells (Patel and Block, 1993), and murine lymphocytes (Kern and Kehrer, 2002). The effects on the redox state of Trx1 and Trx2 in BEAS-2B cells are similar both quantitatively and qualitatively to those reported for human endothelial cells (Szadkowski and Myers, 2007). These effects may

Conflict of interest

There are no conflict of interests.

Acknowledgments

This research was supported by grant ES012707 from the National Institute of Environmental Health Sciences (NIEHS), and by the Dept. of Pharmacology & Toxicology of the Medical College of Wisconsin.

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