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 therefore apply to other human cell types as well. Acrolein also causes redox changes to Trx1 in bovine aortic endothelial cells (BAECs) (Go et al., 2007
) and to purified Trx1 in vitro (Go et al., 2007
). In BAECs, Trx1-acrolein adducts were noted with just 1 μM acrolein (Go et al., 2007
), but acrolein exposure was twice as long as in our studies. Further redox shifts in Trx1, similar to those observed here, were seen in BAECs following 5 and 10 μM acrolein (Go et al., 2007
Trx1 and Trx2 are not in redox equilibrium with each other, so they have been used to differentially assess the impacts of other oxidants on the thiol redox status of the cytosolic and mitochondrial compartments (Halvey et al., 2005
; Hansen et al., 2006a
). For the results reported here, Trx1 was more sensitive to oxidation by acrolein than is Trx2. At 1 and 2.5 μM acrolein, 19 and 50% of the Trx1 was oxidized, whereas Trx2 maintained a reduced state until 5 μM acrolein. Similar differential effects were reported in human microvascular endothelial cells treated with acrolein (Szadkowski and Myers, 2007
). The relative susceptibility is in contrast to that for t
-butyl hydroperoxide or diamide which preferentially oxidize Trx2 relative to Trx1 (Chen et al., 2006
; Hansen et al., 2006a
When compared to another human lung cell line (A549 lung adenocarcinoma cells), the effects on Trxs in BEAS-2B cells occurred at much lower concentrations. Whereas essentially all of the Trx1 and Trx2 was oxidized in BEAS-2B exposed to 5 μM acrolein, ≥25 μM acrolein was required to inhibit >90% of total Trx activity in A549 cells (Yang et al., 2004
). Only ca. 50% inhibition was observed with 10 μM acrolein (Yang et al., 2004
TrxR activity in BEAS-2B cells was also about 10 times more sensitive to acrolein than that in A549 cells. In A549, 50–75 μM acrolein for 30 min caused ca. 65% inhibition (Yang et al., 2004
), whereas in BEAS-2B cells just 2.5 and 5 μM caused 60% and >85% inhibition, respectively (). TrxR in BEAS-2B cells was also more sensitive to acrolein than that in human umbilical vein endothelial cells (HUVECs), which required 25 μM acrolein for 30 min to inhibit 88% of TrxR (Park et al., 2005
). While TrxR protein levels were not examined in the HUVECs or A549 cells, we noted that the amount of TrxR protein remained constant ().
The declines in activity do not therefore result from declines in TrxR protein. We also noted that TrxR activity remained suppressed in BEAS-2B cells even 4 hr after the 2.5 to 12.5 μM acrolein treatment is removed (). The effects on TrxR in BEAS-2B cells are therefore prolonged and TrxR activity is not easily restored in these cells. Even intermittent exposure to acrolein may therefore have prolonged effects in normal bronchial epithelial cells. This is in contrast to A549 cells, in which TrxR activity recovered to 70% and 100% of normal 2 hrs and 4 hrs after removal of the 50–75 μM acrolein treatment (Yang et al., 2004
). It was not determined if this recovery was due to new protein synthesis or reactivation of existing TrxR (Yang et al., 2004
). The differences between the A549 and BEAS-2B cells could reflect their different origins and are consistent with their relative resistance to oxidant stress. A549 cells have proven considerably more resistant to other oxidants than other cell types (Watjen and Beyersmann, 2004
). The A549s consist of four different cell types which can be distinguished both morphologically and biochemically (Croce et al., 1999
), and one or more of these subpopulations could account for the enhanced oxidant tolerance. It is important to note that differences between BEAS-2B cells and other cell types may not necessarily reflect inherent differences in acrolein susceptibility. Differences in growth medium, stage of growth, and other specifics of culture conditions could contribute to differential susceptibility to acrolein. The findings with BEAS-2B cells do, however, indicate that acrolein can cause significant effects at low micromolar concentrations.
Since the BEAS-2B cells originate from normal human bronchial epithelium, they should better reflect normal cells. BEAS-2B cells are a recognized model of normal human bronchial epithelium, and they have been used as a model for cytotoxicity associated with other airborne toxins (Frampton et al., 1999
; Ghio et al., 1999
; Van Vleet et al., 2002
; Gurr et al., 2005
). Whereas A549s are tumorigenic, BEAS-2B cells are not (Reddel et al., 1988
). Overall, the Trxs and TrxR in BEAS-2B cells were considerably more sensitive to the effects of acrolein than would have been predicted by the A549 cells. TrxR also proved more sensitive to acrolein than another unsaturated aldehyde 4-hydroxynonenal (4-HNE). After 6 hr, 10 μM 4-HNE caused no inhibition of TrxR in HeLa cells, whereas 50 μM caused ca. 60% inhibition (Fang and Holmgren, 2006
). These 4-HNE treatments are higher and longer than the 5 μM acrolein which causes >85% inhibition after just 30 min in BEAS-2B cells. These differences between acrolein and 4-HNE directly reflect their differential reactivity with other species, i.e. acrolein is 100–150 times more reactive than 4-HNE (Esterbauer et al., 1991
The detailed mechanism by which acrolein mediates inhibition/oxidation of TrxR and Trxs remains to be determined in these cells. However, given that acrolein can react with some thiols, it is plausible that acrolein is reacting with one or more of the sulfhydryls in the Trxs. Human Trx1 has 5 thiols (C32/C35, C62/C69, and C73), and the detection of at least 3 redox states () implies effects on more than one of the thiols. One possibility is that acrolein reacts directly with one or more of the thiols to form Trx-S-acrolein adducts. While different from conventional oxidation in which disulfides (–S–S–) are formed, Trx-acrolein adducts in essence represent Trx oxidation because they block its sulfhydryl groups, which is reflected in altered mobility in the redox blots. Since disulfide reductants could not reverse the redox changes to Trx1 or Trx2, Trx oxidation following acrolein exposure is not disulfide in nature. Whatever the nature of the changes to Trx, they are not readily reversed. Trx-S-acrolein adducts are one possibility that could explain the results. Adducts of acrolein to Cys73 of Trx1 have been reported following in vitro incubation of equal amounts of Trx1 and acrolein, while adducts to dithiols were not observed (Go et al., 2007
). Adducts to the disulfides may occur in cells, however, and would explain the inability of disulfide reductants to shift either of the oxidized states to a more reduced form (). Similarly, in BAECs exposed to acrolein, dithiothreitol could not reverse the putative acrolein-Trx adducts (Go et al., 2007
). Since thiol-acrolein adducts are generally quite stable (Esterbauer et al., 1991
), Trx-S-acrolein adducts could be difficult to reverse. The Trx1 redox changes may also be due to other effects of acrolein, however. It has been suggested that some of the changes in BAECs may result from oxidation by ROS or potential inhibition of TrxR (Go et al., 2007
). Other as yet undescribed effects of acrolein may be responsible for the Trx oxidation. Regardless of the mechanism, the inability to readily reverse Trx oxidation suggests long-term effects that could impact the redox status and functions of proteins that are dependent on Trx for keeping their thiols reduced, and could alter Trx-mediated signaling.
Acrolein did inhibit TrxR activity in these cells (). Redox-active sites within TrxR include the flavin (FAD), the active site Cys-SeCys (C-terminal) and the N-terminal domain dithiol. NADPH donates electrons to the FAD which then reduces the N-terminal dithiol which then reduces the active site Cys-SeCys. Disruption or inhibition of one or more of these sites should inhibit TrxR activity. This disruption was not reversible. Unreacted acrolein was removed by prolonged dialysis of the cell lysates, and activity was not reversed by NADPH, the native electron donor for TrxR. TrxR inhibition is therefore not likely due to a simple oxidation of the redox-active thiols or selenol as would occur when TrxR reduces endogenous substrates such as Trx. The lack of recovery in cells after a 4-hr acrolein-free period also shows that the changes to TrxR are not easily reversed, and implies that new protein synthesis does not significantly restore TrxR activity during this 4-hr recovery. One plausible mechanism for prolonged TrxR inhibition would be covalent adduction of acrolein to active site Cys-SeCys or to the N-terminal dithiol. Of these, the active site is likely more reactive. It is exposed on the surface of the enzyme (Sandalova et al., 2001
) and the selenol of the SeCys residue (pKa
ca. 5.2) (Jacob et al., 2003
) should be ionized to selenolate at physiological pH making it more reactive. 2,4-dinitrochlorobenzene covalently binds the Cys-SeCys, irreversibly inhibiting the enzyme (Arner et al., 1995
), but it does not modify the N-terminal dithiol (Nordberg et al., 1998
). Both curcumin and 4-HNE covalently bind the active site Cys and SeCys residues of TrxR, causing irreversible inactivation (Fang et al., 2005
; Cassidy et al., 2006
; Fang and Holmgren, 2006
). However, the mechanism by which acrolein inhibits TrxR remains to be determined, and non-adduct mechanisms of inhibition must also be considered at this point.
In these studies, the examination of TrxR, Trxs, and Prxs within the same cells allowed for direct examination of the relationship of the effects of acrolein on these proteins. Given the dependence of Prxs on Trxs, the oxidation of Trxs could influence the redox status and function of the Prxs. Similarly, inhibition of TrxR could influence the redox status and function of the Trxs. There was a strong relationship between the inhibition of TrxR and the oxidation of Trx1 (). This might imply that Trx1 oxidation results from the inability of TrxR to maintain Trx1 in a reduced state. However, this type of Trx1 oxidation should be reversible by disulfide reductants, which proved not to be true (). As discussed above, the data are more consistent with some other type of modification that is not reversed by disulfide reductants, of which Trx1-S-acrolein adducts are one possibility.
Relationship between the inhibition/oxidation of various proteins in acrolein-exposed cells: TrxR and Trx1 (A), TrxR and Trx2 (B), Trx1 and Prx1 (C), and Trx2 and Prx3 (D).
Unlike Trx1 which exhibited progressive oxidation with increasing acrolein concentrations, Trx2 remained reduced until 5 μM acrolein at which point there was an abrupt shift to the oxidized form. This abrupt shift is apparent in the relationship between TrxR inhibition and Trx2 oxidation (). At the point of this shift, 90% of total TrxR was inhibited. There is not therefore a linear relationship between TrxR inhibition and Trx2 oxidation. However, the assay measured total TrxR activity; it did not distinguish between TrxR1 and TrxR2. It is possible that Trx2 oxidation might result from TrxR2 inhibition. However, the inability to reverse Trx2 oxidation by disulfide reductants argues that Trx2 oxidation results from other effects of acrolein and not from loss of reducing equivalents from TrxR2.
The cytosolic Prxs such as Prx1 should be dependent on Trx1 to maintain their redox state. At ≤2.5 μM acrolein, Prx1 redox state resembled that of untreated cells, but at 5 μM acrolein it abruptly shifted to approximately 75% oxidized (). This is the point at which >90% of Trx1 is oxidized (). In contrast, the normal Prx1 redox state is maintained when 50% of Trx1 is oxidized (). Thus, these cells can maintain Prx1 redox status until almost all of the Trx1 is oxidized.
Mitochondrial Prxs such as Prx3 should be dependent on Trx2 to maintain their redox state. At 5 μM acrolein, Trx2 shifted to the oxidized form and at this point a significant increase in Prx3 oxidation was noted (). Prx3 oxidation increased to >90% at the next acrolein concentration (12.5 μM) (). further illustrates the graphical relationship between Trx2 and Prx3 redox state. Thus, for both Prx1 and Prx3, increased Prx oxidation is not seen until the respective Trxs are almost completely oxidized. The data suggest that Prx oxidation occurs because the Trxs can no longer maintain the redox status of their Prxs. For Prx1 and Prx3, this type of Prx oxidation should yield intermolecular disulfide homodimers in non-reducing gels, which is exactly what was observed (). Such intermolecular disulfides should be reduced by disulfide reductants, which proved to be the case (). An alternative mechanism of Prx oxidation would be the direct reaction of acrolein with Prx to form Prx-S-acrolein adducts. Such adducts should prevent dimer formation and should not be readily reversed by disulfide reductants. The formation of dimers and their reversal in reducing gels (, ) are therefore inconsistent with Prx-S-acrolein adducts. While it is possible that a small percentage of Prx sulfhydryls react with acrolein, the data are consistent with the formation of intermolecular disulfides as the prominent form of oxidized Prx. The conformation of the Prxs may protect their –SH groups from direct reaction with acrolein. While Prx2 shows a high rate of reaction with peroxide substrates, its sulfhydryls are not readily alkylated with iodoacetamide or various amino acid chloroamines, despite the high reactivity of these agents with other cellular –SH groups (Peskin et al., 2007
). A third possible form of Prx oxidation is overoxidation of the sulfhydryls to sulfinic (Prx-SO2
) and sulfonic (Prx-SO3
) forms. For example, for Prx oxidation mediated by lipid hydroperoxide metabolites of arachidonic acid, about 65% is reversible (disulfide) whereas the remainder represents overoxidation to Prx-SO2
(Cordray et al., 2007
). However, using an antibody that recognizes the Prx-SO2
forms of Prx1, Prx2, Prx3, and Prx4, we found no evidence for overoxidation in acrolein-exposed cells. Furthermore, overoxidation should diminish Prx dimerization. Together, the data imply that Prx oxidation in acrolein-exposed cells largely generates intermolecular disulfides which are reversible by disulfide reduction. Since Prx oxidation only occurs once the Trxs are completely or almost completely oxidized, the underlying cause is likely the inability of the Trx system to maintain the redox state of the Prxs. Consistent with this, inhibition of TrxR in erythrocytes causes Prx oxidation (Low et al., 2008
). In acrolein-exposed cells, therefore, the redox status of Prx3 and Prx1 may largely reflect the functional state of the respective Trx systems. The reversibility of the Prx redox changes also underscores the fundamental differences in the effects on Prxs (reversible) vs. Trxs and TrxR (irreversible).
Acrolein-mediated oxidation of Trxs and Prxs, and inhibition of TrxR, could conceivably be mediated by increased reactive oxygen species (ROS). Since Prxs function as peroxidases, increases in peroxides could result following Prx oxidation. Acrolein-mediated increases in ROS have usually been reported at much higher concentrations of acrolein, however. 30 to 100 μM acrolein has been reported to significantly increase superoxide (O2−·
) and H2
in cells (Jaimes et al., 2004
; Wu et al., 2006
), but this is well above the 5 μM acrolein that caused prominent effects on the Trx system and Prxs reported here. However, a 52% increase in dichlorofluorescein fluorescence was seen in BAECs following 5 μM acrolein for 1 hr (Go et al., 2007
), although the responsible oxidants were not determined. However, millimolar levels of H2
have not resulted in significant Trx oxidation in some other cells (Fernando et al., 1992
; Szadkowski and Myers, 2007
), and Trx2 was not significantly oxidized by epidermal growth factor-induced reactive oxygen species (ROS) (Halvey et al., 2005
; Hansen et al., 2006a
). Furthermore, oxidation mediated by H2
would be expected to be readily reversible, which was not the case for Trx and TrxR. Therefore, acrolein-mediated increases in ROS may not account for the bulk of the effects on TrxR and Trx, but the inhibition of these enzymes may result in Prx oxidation and contribute to increases in ROS. Since the Prx oxidation was reversible, it is plausible that endogenous peroxide substrates contributed to, or were largely responsible for, shifting the Prxs to a more oxidized state following acrolein exposure. The ability of various aldehydes to increase peroxide production in cells (Uchida, 2000
) may in part be due to the oxidation of the Prxs following Trx oxidation.
The acrolein-induced changes to the redox state/activity could have important implications for cell growth and survival. Inhibition of TrxR increases the susceptibility to oxidants and favors apoptosis (Nordberg and Arner, 2001
). Genetic suppression or inhibition of Trx results in increased oxidant stress and apoptosis (Hansen et al., 2006a
) and increased sensitivity to oxidants (Chen et al., 2006
). Conversely, overexpression of Trx enhances resistance to oxidant-induced apoptosis (Chen et al., 2006
; Hansen et al., 2006a
). The overoxidation of Prx1 following 6-hydroxydopamine exposure enhances apoptosis, whereas overexpression of Prx1 protects cells from 6-hydroxydopamine (Lee et al., 2008
). Prx3 may form a primary defense against peroxides in mitochondria and depletion of Prx3 renders HeLa cells more susceptible to apoptosis caused by tumor necrosis factor-α and staurosporine (Chang et al., 2004
). Loss of protection from oxidant stress is just one of the potential consequences of the effects of acrolein on the Trx/Prx system. Other more direct effects are also possible. For example, deletion of, or adduction to, the SeCys residue of TrxR can have negative effects that extend beyond those of just blocking TrxR activity (Anestål et al., 2008
). Further studies on the consequences of the effects of acrolein on the TrxR/Trx/Prx system are warranted.
In summary, this report describes the effects of acrolein on the thioredoxins, peroxiredoxins, and TrxR in human bronchial epithelial cells. Low micromolar concentrations of acrolein caused oxidation of cytosolic and mitochondrial Trxs and Prxs and inhibited TrxR. The effects on Trxs and TrxR were not reversible in vitro and TrxR activity did not recover following acrolein removal. Redox changes to Prxs were not observed until 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. These effects of acrolein on the Trx system and Prxs could have important implications for the normal function and survival of the bronchial epithelium, and the ability of these cells to tolerate other oxidant insults.