Our goals were to identify protein adducts generated in lungs of mice treated with BHT and to determine which of these modifications are most likely to contribute to tumor promotion. Eight of the proteins detected on 2-DE immunoblots () were present in cytosols from all or most of six treatment groups and three proteins—Prx6, SOD1, and CR—protect cells from ROS and/or reactive products of lipid peroxidation. In addition, a fourth adducted protein (SBP1) may have antioxidant functions. Prx6 is of particular interest as it is highly expressed in lung and has a role in maintaining the appropriate intracellular levels of H2
). This enzyme catalyses both peroxidase and phospholipase A2
reactions at different active sites and is termed a 1-Cys Prx because of a single conserved Cys residue in the peroxidatic site (Cys 47 in the human enzyme) (22
). The human and murine forms of Prx6 also contain a second Cys residue near the protein surface (i.e., Cys 91 in the human form). Cys 47 is directly involved in the reduction of peroxy bonds of H2
and lipid-derived hydroperoxides forming, respectively, water and an alcohol. The present results demonstrate that human Prx6 is highly sensitive to inhibition by QMs; approximately 50% of the activity of the human enzyme was destroyed by treatment with 50 μM BHT-QM and 80% was lost after treatment with 50 μM BHTOH-QM (). The levels of H2
were dramatically increased in S9 fractions from mouse lung exposed to BHT-QM (), consistent with Prx6 inhibition, although there may be additional QM targets in the S9 fraction that contributed to this effect. Clearly, elevated H2
may lead to increased lipid peroxidation in lung S9 fractions treated with BHT-QM.
Attempts to identify specific alkylated peptides in digests of Prx6 isolated from lung cytosols were not successful; some of the difficulties normally associated with mass spectrometric detection of protein adducts from cells and tissues have been discussed previously (13
). QM alkylation sites were characterized, however, by LC-MS/MS analysis of QM-treated purified human Prx6. Peptides containing a BHT group and also containing either Cys 47 or Cys 91 were identified in tryptic digests. In each case, CID resulted in a facile neutral loss of BHT-QM, not unexpected because of the labile nature of BHT-thioether adducts (12
). Other examples of Prx6 adduction are known, for example in lung by epoxides formed during the metabolism of naphthalene (28
). To our knowledge, however, the present study is the first to demonstrate alkylation of the catalytic Cys 47 residue by an electrophilic metabolite accompanied by enzyme inhibition. This residue is located in the interior of the protein and it has been suggested that protein unfolding occurs to enable substrate access (29
). Given that lipid hydroperoxides also are Prx6 substrates, it is possible that BHT-QM gains access to the catalytic Cys 47 because it is also strongly hydrophobic.
Another factor that may contribute to Prx6 inhibition in vivo
is alkylation of GSTP1. It was reported that the latter is involved in regenerating the reduced (i.e., active) peroxidase during the Prx6 catalytic cycle (29
). In earlier work, we demonstrated adduction of GSTP1 by BHT-QM in a transformed cell line derived from mouse lung epithelium, and confirmed that alkylation of Cys residues destroyed conjugation activity (12
). The most abundant form of GSH S-transferases in mouse lung, GSTM1 (31
), was detected in immunoreactive 2-DE spots from cytosols of two treatment groups (data not shown), but no GSTP1 adduct was found. However, GSTP1 is expressed at higher levels in tumors than in non-tumor tissue (30
) so adducts may have been present at undetectable levels in the normal lungs examined here. Taken together, these data indicate that alkylation and inhibition of pulmonary Prx6 (and possibly GSTP1) by BHT-derived QMs resulted in increased levels of H2
leading to oxidative damage and, presumably, to alterations in cell signaling that play key roles in tumor development (15
Adducts of SOD1 also were detected in lungs of BHT-treated mice and were evaluated in vitro
after treatment of purified bovine protein with BHT-QM. Inhibition occurred to a lesser degree than for Prx6. SOD1 activity decreased by about 10% at 50 μM BHT-QM and about 60% at 200 μM BHT-QM, compared to control values (). The rate of formation of O2−
in BHT-QM-treated lung S9 fractions was higher than in untreated S9, consistent with impaired SOD1 activity. MALDI-TOF analysis of the intact protein treated with a 10-fold excess of BHT-QM demonstrated approximately 50% conversion to a mixture of mono- and di-adducts with the former predominating (). Analysis of an Asp-N digest revealed a single adducted peptide consisting of residues 74−87 with a mass increment of 218 Da over its theoretical mass corresponding to the addition of BHT-QM. The most likely site of attachment is the imidazole ring of His 78 (11
Residues His 61, 69, and 78, and Asp 81 of bovine SOD1 are involved in zinc ligation (23
). We initially speculated that alkylation of the imidazole group of His 78 inhibited enzyme activity by disrupting zinc binding. Several attempts to detect release of the metal from QM-treated protein by atomic absorption spectroscopy demonstrated that the metal remained bound to the protein despite alkylation. It may be that alkylation of His 78 is sufficient to affect enzyme activity but not to dislodge zinc from the protein, or alternatively that His 78 is actually a minor alkylation site and another, more important site was not detected. Interestingly, the proteolytic fragment containing His 41, the only metal-free His (33
), produced a very intense MALDI-TOF ion at m/z
1180 Da demonstrating that this residue was not appreciably alkylated. In summary, the alkylation of SOD1 in lungs of BHT-treated mice is strongly supported by immunochemical and MS detection of the adduct in 4 out of 6 treatment groups and by immunochemical and MS studies of the closely related bovine enzyme treated with BHT-QM. Inhibition of SOD1 by treatment with BHT-QM was clearly demonstrated with the bovine enzyme and further supported by increased rates of O2−
formation in NADPH-fortified lung S9 treated with BHT-QM. In contrast to strong evidence implicating Prx6 alkylation in the actions of BHT, however, the contribution of SOD1 inhibition to oxidative damage and the specific mechanism of inhibition remain to be clarified.
Two additional proteins listed in , CR and SBP1, may also contribute to the pulmonary effects of BHT. Carbonyl reductases are highly expressed in lungs and activity is substantially higher in tumors than in normal tissue (34
). These enzymes catalyze the reduction of endogenous and exogenous aldehydes and ketones, including products of lipid peroxidation (34
), and a role for CR in antioxidant protection has been demonstrated (36
). Although some forms of CR contain critical Cys residues in the substrate or cofactor binding sites, the mouse protein contains only two Cys residues located near the solvent surface (refer to PDB entry 1CYD) (37
). If alkylation occurs on these residues, formation of the active homotetrameric form of the enzyme may be hindered causing a loss of activity. Inhibition of CR could also occur if a critical amine-containing residue is alkylated, for example Lys 17 or Lys 153 involved in NADPH binding (37
). Regardless of the specific mechanism involved, QM-induced inhibition of CR could exacerbate cellular damage resulting from oxidative stress and lipid peroxidation.
The functions of selenium binding protein are not fully understood. The closely related forms SBP1 and SBP2 are highly expressed in many tissues, contain several Cys residues, and are commonly targeted by electrophiles including metabolites of acetaminophen and naphthalene (28
). SBP1 expression is lower in lung adenocarcinomas than in normal tissue and downregulation has been correlated with low survival rates for cancer patients (39
). Expression of SBP2 decreases in response to cell proliferation induced by clofibrate and other peroxisome proliferators (40
). The apparent growth-inhibitory characteristics of SBPs indicate an anticarcinogenesis role for these proteins. SBPs are believed to participate in the intracellular transport of selenium and downregulation is expected to disrupt selenium metabolism. In addition, direct antioxidant activity has been postulated for SBP in analogy to other selenocysteine-containing enzymes such as thioredoxin reductase and GSH peroxidase (42
). Adduction of SBP1 may interfere with its normal functions, effectively mimicking downregulation and the associated loss of growth inhibitory and other protective functions.
Several protein targets of BHT-derived QMs discussed above have roles in protecting cells from oxidative stress, and their inactivation provides a mechanistic basis for understanding pulmonary inflammation and tumorigenesis in mice treated with BHT. The most compelling data implicating protein adduction in the underlying mechanisms concern Prx6 inhibition. We currently have no evidence that QMs alkylate other peroxidases (i.e., catalase and glutathione peroxidase); however, the important role of peroxiredoxins in metabolizing H2
has become clear in recent years (27
). In addition, the role of H2
in cell signaling is well established (15
). This oxidant influences the redox states of Cys residues in signaling molecules, including transcription factors, kinases, and phosphatases, with consequences on cell growth, survival, and apoptosis. The finding that Prx6 is rapidly adducted following the first dose of BHT is consistent with the rapid onset of pulmonary inflammation (5
). Adducted Prx6 was detected in lung cytosols 24 hours after treatment with BHT and throughout the 4-week treatment period necessary for tumor promotion, indicating that a period of sustained oxidative stress is required to induce cell proliferation and the resulting accumulation of preneoplastic lesions.
The abundant cytoskeletal proteins tropomyosin, annexin A3, and β-actin were also alkylated in most or all of the treatment groups (); however, none of these proteins are likely to play a role in the effects of BHT on tumorigenesis. Only protein adducts present in relatively high amounts were detected in this work, but less abundant proteins related to signal transduction, including kinases and transcription factors, have Cys residues that may also be alkylated providing a direct route for the modification of cell signaling pathways (44
). The data presently in hand, however, support a state of persistent oxidative stress over several weeks as the major cause of cell proliferation. Other than QMs derived from BHT or structurally-similar alkylphenols (8
) and an electrophilic metabolite of safrole (45
), there is a paucity of specific examples of tumor promotion by electrophiles. Nevertheless, it is probable that other electrophiles of exogenous or endogenous origin influence tumor development by epigenetic mechanisms, especially if exposures occur over a prolonged period of time.