Nicholas School of the Environment and Earth Sciences, Duke University, Durham, North Carolina 27708-0328
Submitted 10 July 2003 ; accepted in final form 14 October 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
metallothionein; signal transduction; MAPK
Copper is a redox-active metal that is able to catalyze the formation of hydroxyl radicals via a Haber-Weiss or Fenton-like reaction (19, 44, 50). In addition, copper can be toxic by directly binding to sulfhydryl groups in proteins, which results in enzyme inactivation or altered protein conformation (26). The wide variety of adverse effects attributed to excess copper is likely to be due to the various mechanisms by which this metal can induce intracellular damage.
Exposure to toxic concentrations of copper will produce an intracellular stress response. The copper-induced stress response involves altered transcription of multiple genes, which are responsible for maintaining metal homeostasis and protecting cellular components from damage (55). To help maintain copper homeostasis and scavenge toxic by-products of copper exposure, cells express metallothioneins (MTs). Metallothioneins are small, cysteine-rich proteins that are ubiquitous among eukaryotes (21, 27). Proposed functions of MTs include maintaining homeostasis for essential metals such as zinc and copper, cellular detoxification, and scavenging free radicals. Elevated concentrations of many transition metals have been shown to elicit rapid induction of MT mRNAs and proteins (21, 23, 27, 35, 42). In addition, a variety of nonmetal stressors such as heat shock, alkylating agents, oxidative stress, and UV radiation induce MT transcription (9, 38, 40, 43, 48). This suggests that MT may function as a general stress-response protein.
Metal-inducible MT transcription is regulated primarily through the interaction between metal response elements (MREs) and metal transcription factor (MTF)-1 (22, 37). MREs are 13- to 15-bp upstream regulatory elements with the core consensus sequence CTNTGCRCNCGG that are found in the promoter region of most metal-inducible MT genes (47). MTF-1 specifically binds to the MRE and has been shown to be essential for the metal-inducible transcription of MT (22, 37). Although MTF-1 is essential for copper-inducible MT transcription, the interaction among MREs, MTF-1, and copper has not been defined.
Additional cis regulatory elements that also regulate inducible MT transcription are the antioxidant response elements (AREs), which have a core consensus sequence of TGACNNNGC (7). AREs regulate oxidative stress-inducible transcription of many genes, including glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase (NQO1), and several MTs (7, 24). The ARE of the GST Ya gene is activated in response to a variety of chemical agents, including metals, -naphthoflavone, t-butylhydroquinone (tBHQ), butylhydroxyanisol, and hydrogen peroxide (11, 3133, 49, 52). The ARE-mediated induction of detoxification genes is thought to be a critical mechanism involved in protecting cells from challenges by electrophiles and reactive oxygen species (ROS). The ability of redox cycling transition metals to activate MT transcription through the ARE has not been fully addressed.
One mechanism by which the transcription of detoxification genes are activated is via mitogen-activated protein kinase (MAPK) pathways, which include extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38. Many forms of cellular stress preferentially activate the JNK/SAPK and p38 pathways (16, 29). In human bronchial epithelial cell line BEAS 2B cells, exposure to copper, vanadium, arsenic, zinc, or chromium results in the phosphorylation and subsequent activation of JNK, p38, and ERK (39). Once activated, ERK, JNK, and p38 can phosphorylate a number of proteins and transcription factors. This results in the enhancement of the transcriptional activity of a multitude of genes (28).
Copper has the potential to mediate several important biological processes through multiple signal transduction pathways. However, the cellular and molecular responses underlying copper-regulated gene expression and toxicity are poorly understood. In this study, we report that copper increases the transcription of MRE- and ARE-containing genes. Transcriptional activation by copper involves MAPK pathways and changes in cellular glutathione status. Results from this study suggest that copper is capable of activating transcription through both metal- and oxidative stress-mediated mechanisms.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reporter gene constructs. The level of MT transcription was established by measuring the amount of chloramphenicol acetyltransferase (CAT) produced in cells transfected with 42CAT, which contains the minimal mouse MT-I promoter (42 to +62); 153CAT, which contains the region of the mouse MT-I gene from 153 to +62; MREd'5CAT, which contains five tandem copies of MRE-d' inserted upstream of TATA box in 42CAT; and ARE4CAT, which contains four tandem copies of the upstream stimulatory factor (USF)/ARE from the mouse MT-I gene inserted into 42CAT. The 42CAT plasmid contains a TATA box but lacks metal- or stress-responsive regulatory elements (i.e., negative control). In contrast, 153CAT contains 153 bp of the mouse MT-I regulatory region and contains four functional MREs, as well as an ARE. Complete descriptions of the MT-CAT reporter genes can be found in Dalton et al. (7).
The level of oxidative stress-responsive transcription was measured by using a CAT reporter gene that contains the ARE from the rat NQO1 gene (pARE). pARE consists of a single copy of the ARE from NQO1 fused to a rat GST minimal promoter. As a negative control, a two-nucleotide mutant form of the ARE, which is unresponsive to oxidative stress, was used (pM1) (10, 11). The pM1 plasmid is identical to pARE with the exception of a mutation of the GC box in the consensus ARE sequence (GTGACNNNGC).
Transient transfection and reporter gene assays. For transient transfection studies, cells were grown to 60% confluence in complete DMEM in 24-well dishes. The medium was removed, and cells were washed with Opti-MEM and then transfected using Lipofectin (COS-7) or LipofectAMINE (dko7), according to the manufacturer's instructions (Life Technologies). Cells were transfected for 36 h with 650 ng/well of one of the CAT reporter plasmids and 160 ng/well of the control plasmid pSV-
gal (Promega). To inhibit JNK activity, cells were cotransfected with pcDNA3-Flag-MKK4 (Ala), which expresses a dominant negative form of the JNK-activating kinase MEK-4/SEK-1, thereby inhibiting JNK phosphorylation and activation (51). In experiments utilizing the dominant negative SEK plasmid, cells were transfected with 650 ng/well of CAT reporter plasmid, 160 ng/well of pSV-
gal, and 300 ng/well of pcDNA3-Flag-MKK4 (Ala).
After transfection, the medium was replaced with complete DMEM, and the cells were allowed to recover for 12 h. Copper (as CuSO4), ROS modulators, or signal transduction inhibitors were then added as described above. CAT concentrations and
-galactosidase activities were determined by sandwich ELISA, using a CAT-ELISA kit (Roche Biotechnologies) and the
-Galactosidase Enzyme Assay System (Promega), respectively. All assays were performed in triplicate, and CAT protein levels were normalized to
-galactosidase activity.
Cytotoxicity. Cytotoxicity assays were performed as described by Shokri et al. (45). Briefly, cells were seeded in a 48-well tissue culture dish in complete DMEM and incubated for 24 h. Copper sulfate was then added, and incubation continued for an additional 24 h. In experiments in which glutathione was depleted, cells were exposed to 1 mM buthionine sulfoximine (BSO) for 8 h before the addition of copper. After copper exposure, the medium was removed and the cells were then incubated with neutral red solution (40 µg/ml) in DMEM for 3 h at 37°C. The cells were subsequently washed and then fixed with calcium chloride (1%, wt/vol) in formaldehyde (0.5%, vol/vol). To extract the dye from viable cells, cells were lysed with acetic acid (1%, vol/vol) in 50% (vol/vol) ethanol. Optical density was then measured at 540 nm (Abs540). Positive controls, representing 100% cytotoxicity, consisted of cells exposed to distilled water. Cells incubated with complete DMEM in the absence of copper represented 100% viability and were included as a negative control. The level of cytotoxicity was calculated by using the following calculation:
![]() |
Glutathione measurements. Cells were grown for 72 h and then incubated in the presence of copper for an additional 24 h. Extracts were prepared from phosphate-buffered saline-washed cells by sonicating cell pellets suspended in 0.5 ml of 20 mM sulfosalicyclic acid. The lysate was centrifuged at 10,000 g for 2 min, and the supernatants were collected. Total glutathione (GSH + GSSG) and GSSG concentrations were determined by using the enzymatic recycling assay for plate readers (1, 2).
Statistical analysis. All statistical analysis was performed using StatView software (SAS Institute). Results are presented as means ± SE. The significance of mean differences was detected by analysis of variance (ANOVA) followed by Fisher's protected least-squares difference post hoc test for individual comparisons. The criterion for statistical significance was set at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Effect of copper on glutathione levels. Exposure of COS-7 cells to 400 µM copper for 24 h caused a significant decrease (35%) in total glutathione (oxidized plus reduced) (Table 1). Treatment of cells with BSO resulted in a 93% decrease in total glutathione. In addition, treatment with BSO and 400 µM copper caused a 97% decrease in total glutathione. This level of depletion was significantly greater than that observed with BSO alone.
|
The ratio of reduced to oxidized glutathione is commonly used as an indicator of intracellular oxidative stress. In untreated cells, oxidized glutathione accounted for 1.5% of the total glutathione. This value significantly changed when cells were treated with 400 µM copper for 24 h or when they were exposed to BSO for 8 h (Table 1). Likewise, cells exposed to 400 µM copper following GSH depletion showed a significant, synergistic increase of the level of oxidized glutathione (Table 1). These results suggest that exposure to copper alters the intracellular redox potential. It should be noted, however, that this observation is confounded by the high degree of toxicity observed in cells exposed to both copper and BSO (Fig. 1).
Effect of copper on metal- and oxidative stress-responsive transcription. The ability of copper to induce MT transcription (i.e., MT transcriptional activity) was investigated by using CAT reporter genes whose expression is regulated by portions of the mouse MT-I promoter, or concatenated copies of MT upstream regulatory elements (7). COS-7 cells, transfected with MT-I-based reporter plasmids, were exposed to 0600 µM copper for 24 h. Copper at any concentration did not significantly induce transcription when assayed using the 42CAT reporter gene (i.e., negative control; Fig. 2A). In contrast, significant increases in transcriptional activity were observed at copper concentrations of 400 and 600 µM in cells that contained the 153CAT, MREd'5CAT, and ARE4CAT reporters. The maximum response (3.5-fold increase) was observed at 400 µM in cells transfected with ARE4CAT. The decrease in reporter gene activity that was observed at higher concentrations may be attributed to the cytotoxicity observed at these metal concentrations (Fig. 1).
|
The increase in ARE4CAT transcriptional activity reported above suggests that copper may activate transcription by an oxidative stress-responsive mechanism. The ARE in the mouse MT-I promoter, however, overlaps with a USF sequence. The USF element may influence the responsiveness to copper exposure. To further define the role of oxidative stress in copper-inducible transcription, plasmids containing an ARE from the rat quinone reductase gene were used. A 24-h exposure to 400 µM copper caused a 5.5-fold increase in reporter gene expression in cells transfected with pARE (Fig. 2B). The addition of copper did not cause an increase in the level of reporter gene expression in cells transfected with the negative control plasmid pM1 (Fig. 2B).
MTF-I involvement in copper-induced MT transcription. To further investigate the role of metal-responsive gene regulation in copper-induced transcription, the effect of copper exposure on reporter gene activity was examined in the MTF-1 knockout cell line dko7 (18). Cells were transfected with 42CAT, 153CAT, MREd'5CAT, or ARE4CAT plasmids and then exposed to 600 µM copper for 24 h. There was no significant induction of 153CAT and MREd'5CAT expression in dko7 cells, which indicates an essential role for MTF-1 in copper-inducible transcription, via the MRE (Fig. 3). In contrast, there was a significant (5.3-fold) increase in the level of expression when ARE4CAT was used. This finding suggests that copper also activates transcription by an oxidative stress-response mechanism. To further investigate the roles of AREs and oxidative stress in copper-inducible transcription, dko7 cells were transfected with pARE and pM1. Consistent with the earlier results, copper exposure resulted in a significant (4.6-fold) increase in pARE reporter gene expression. These results indicate that both metal- and oxidative stress-responsive mechanisms contribute to copper-inducible transcription. Furthermore, these pathways can function independently.
|
Effects of GSH depletion on copper-inducible transcription. Treatment of COS-7 cells with BSO caused a >90% depletion of total glutathione (Table 1). A significant increase in the level of copper-inducible expression was observed when glutathione levels were reduced before copper addition, compared with copper-exposed cells that were not pretreated with BSO (Fig. 4A). The depletion of glutathione alone caused a significant increase in the level of reporter gene expression (Fig. 4A). Thus glutathione depletion did not significantly increase the level of-153CAT and MREd'5CAT reporter gene expression. Statistical analysis shows that there was not a synergistic interaction between copper and BSO for any of the reporter genes.
|
In cells transfected with ARE4CAT (5.26.8 fold), a significant increase in transcription was observed, which suggests that an ROS-mediated mechanism may be involved in copper-activated transcription. To confirm the involvement of ROS, cells were transfected with pARE and pM1 plasmids. Consistent with the above results, glutathione depletion increased copper-inducible pARE expression compared with cells exposed to copper alone (Fig. 4B). However, the degree of induction for the pARE plasmid (1.5-fold) was much less than was seen for the ARE4CAT plasmid. In addition, reporter gene expression in the absence of added copper was higher in BSO-treated cells for all four MT plasmids compared with cells not treated with BSO. These results suggest that the levels of ROS generated in the absence of glutathione are sufficient to activate MT transcription via the ARE-mediated pathway.
In cells transfected with the 42CAT reporter, gene expression was significantly higher than in cells exposed to BSO and copper compared with cells treated with copper alone (4.1- to 4.4-fold). The same effect, however, was not observed in cells transfected with the pM1 plasmid. This finding indicates that GSH depletion increases the basal level of transcription.
Effects of ROS scavengers and antioxidants on copper-induced transcription. Previous experiments demonstrated that pretreatment of cells with the ROS scavengers aspirin and vitamin E partially protects cells from the cytotoxic effects of transition metals (34). Aspirin and/or vitamin E had no effect on levels of expression observed using the 42CAT reporter (Table 2). Aspirin or vitamin E treatment had no significant effect on the level of 153CAT expression, relative to the level of expression observed with copper alone. In combination, however, aspirin and vitamin E caused a 10.6% increase that was significantly higher than treatment with copper alone.
|
Pretreatment of cells with aspirin, vitamin E, or the combination of the two before copper addition significantly decreased MREd5 'CAT expression (12.5% for each). Similar results were obtained in cells transfected with ARE4CAT. Aspirin caused a 36.3% decrease in copper-inducible transcription, vitamin E caused a 26.5% decrease, and in combination, aspirin and vitamin E caused a 26.5% decrease. The combination of aspirin and vitamin E was not additive.
Effects of signal transduction inhibitors on copper-induced transcription. COS-7 cells were treated with kinase inhibitors to identify potential signal transduction pathways that may regulate copper-inducible transcription. The protein kinase C (PKC) inhibitor H-7 was used to determine the role of PKC in the transcriptional response to copper. Treatment with 100 µM H-7 completely blocked copper-induced 153CAT, MREd5 'CAT, and pARE expression (Fig. 5). Copper (400 µM) did not significantly change the level of transcription of ARE4CAT during the 4-h incubation used in this and subsequent inhibitor studies. Time-course studies (results not shown) have shown that a 4-h exposure to 400 µM copper is insufficient to induce ARE4CAT reporter gene expression, whereas a 24-h exposure as used above induces significant levels of expression.
|
MAPK inhibitors were used to investigate the role of ERK1/2, p38, and JNK signaling pathways on copper-inducible gene expression. ERK1/2 and p38 were inhibited by the chemicals PD-98059 and SB-203580, respectively. JNK inhibition was achieved by transfecting the cells with a plasmid expressing a dominant negative form of SEK-1 that prevents the phosphorylation and activation of JNK (51). Kinase inhibitors were added individually (Fig. 6) or in combinations (Fig. 7). Inhibition of p38 kinase caused a significant increase in copper-inducible expression of 153CAT and MREd5'CAT. Similarly, inhibition of JNK activity caused an increase in 153CAT. The inhibition of ERK1/2 activity did not significantly affect copper-inducible transcription of any of the reporter genes.
|
|
Exposure to the combination of ERK and p38 inhibitors caused a significant decrease in the level of copper-inducible expression of 153CAT and MREd'5CAT. Similarly, inhibition of ERK in combination with either p38 or JNK inhibition caused a significant decrease in copper-inducible expression of 153CAT and MREd5'CAT. In contrast, the combination of JNK and p38 inhibitors caused a small but significant increase in 153CAT and MREd5'CAT. Simultaneous inhibition of the three MAPKs caused a significant decrease in copper-inducible expression of 153CAT and MREd5'CAT. In all of the inhibitor studies, treatment with inhibitors alone did not significantly affect reporter gene expression (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Copper activation of transcription via a metal-responsive pathway was demonstrated using reporter genes in wild-type and MTF-1 null cells. Copper activates transcription via the MRE in a concentration-dependent manner (Fig. 2). In addition, copper did not induce MRE-mediated transcription in the absence of MTF-1 (Fig. 3.). These results are consistent with those previously observed in BHK cells containing a MRE-Geo reporter gene (36). Copper has also been shown to increase transcription factor binding to the MRE in the human MT-2A gene (20).
Several experiments indicate that copper activates transcription by inducing oxidative stress and that the oxidative stress response is independent of the metal response. First, exposure to copper induces transcription of reporter genes containing AREs from MT-I and NQO1 (Fig. 2). The magnitude of the response is attenuated when cells are exposed to chemicals that reduce ROS (Table 2) and increases following glutathione depletion (Fig. 4). The observation that ROS-mediated gene expression occurs in the absence of MTF-1 suggests that the metal-responsive transcription machinery is not required for ROS-activated transcription.
Copper can induce oxidative stress by two mechanisms. First, it can directly catalyze the formation of ROS via a Fenton-like reaction (19, 44, 50). Second, exposure to elevated levels of copper significantly decreases glutathione levels (Table 1). An underlying mechanism of copper- and cadmium-induced oxidative damage is the depletion of glutathione. Depletion of intracellular GSH has also been shown to increase the cytotoxic effects of other classes of xenobiotics (5, 15, 46). A decrease in glutathione would allow the endogenous levels of ROS to become cytotoxic. The large increase in copper toxicity due to GSH depletion (Fig. 1) demonstrates that GSH contributes to the cellular defense against copper toxicity.
Depletion of glutathione by BSO treatment causes an increase in oxidative stress responsive MT transcription (Fig. 4). Glutathione has multiple functions in intracellular copper metabolism and detoxification. It has been proposed that glutathione quickly binds the metal following uptake. Once copper is bound, the metal can then be transferred to other proteins (13, 14). Glutathione also has several roles in copper detoxification. It can ameliorate copper toxicity by directly chelating the metal. Binding by the thiol group can protect cells by maintaining copper in a reduced state, making it unavailable for redox cycling. Glutathione is also a substrate for several enzymes that remove ROS as well as a hydroxyl radical scavenger. The depletion of glutathione may allow the metal to remain in a state that is 1) a stronger inducer of metalresponsive transcription and 2) more catalytically active, thus producing higher levels of ROS, which activates oxidative stress-responsive transcription.
Additional evidence that copper activates transcription via ROS was demonstrated in studies in which cells were treated with aspirin or vitamin E. Treatment with aspirin or vitamin E led to decreases in copper-induced transcription, using MREd'5CAT or ARE4CAT reporters (Table 2). The decrease was greater for ARE4CAT, which is consistent with reduction in oxidative stress. In addition, the level of reporter gene expression did not change when aspirin or vitamin E was added simultaneously with the metal, suggesting that the effects of copper were primarily intracellular (results not shown). The mechanisms by which aspirin and vitamin E treatment reduce copper-inducible transcription are currently unclear. Vitamin E is a lipid soluble vitamin that acts as an antioxidant in cells to interrupt the propagation of lipid peroxidation in the plasma membrane (6). Treatment with vitamin E may reduce the formation of secondary products of lipid peroxidation that can potentially trigger signal transduction pathways. The effects of aspirin are likely due to a different mechanism. Salicylic acid is commonly used as a trap for hydroxyl radicals, which suggests that aspirin may act as a radical scavenger. Aspirin also possesses some degree of metal-binding ability and may thus withdraw copper ions from the site of ROS formation.
To identify potential signal transduction pathways that may mediate copper-activated transcription, the effects of kinase inhibitors on metal-activated transcription were examined. Inhibition of PKC activity prevents both ARE- and MRE-mediated transcription (Fig. 5). This observation suggests that copper activates both metal- and oxidative stress-responsive cascades through signaling pathways that are regulated by PKC. This is similar to previous results that demonstrated the involvement of PKC in metal-inducible transcription, which is activated by zinc and cadmium (30, 41). In addition, it has been shown that inhibition of PKC in Chinese hamster ovary cells blocks MT mRNA transcription, suggesting that PKC is essential for MT expression (53). The inhibition of ARE-mediated, copper-inducible transcription is consistent with PKC regulation of the oxidative stress response (17).
MAPK inhibitors were used individually and in combinations to identify the MAPK pathways that may be involved in regulating copper-inducible transcription. Copper is capable of activating the three MAPK pathways (ERK, p38, and JNK), often in a cell type-specific manner (25, 39). Inhibition of ERK1/2 did not have a significant effect on copper activation of 153CAT and MREd5'CAT reporter genes (Fig. 6). Inhibition of JNK/SAPK or p38 activity led to an increase in copper-inducible transcription, which suggests that JNK/SAPK and p38 may be negative regulators of the response to copper. Whereas ERK1/2 inhibition alone had little effect, in combination with p38 inhibition there was a significant decrease in reporter gene activity (Fig. 7). A similar response was observed when ERK1/2 and JNK/SAPK were both inhibited. Inhibition of both JNK/SAPK and p38 led to a significant increase in copper activation, consistent with inhibition of either pathway alone. Inhibition of all three kinases led to a strong reduction in copper-inducible transcription, which suggests that ERK1/2 has a dominant effect over both p38 and JNK (Fig. 7). Comparable results were obtained when the three MAPK pathways and zinc- and cadmium-inducible transcription were examined (unpublished data). It has been suggested that ERK is capable of attenuating both p38 and JNK activity through its activation of MAP kinase phosphatase-1 (MKP-1). ERK phosphorylation of MKP-1 thereby activates it to dephosphorylate p38 and JNK, resulting in an attenuation of their activity (12). These findings may explain the decreases in copper-induced MT transcription in response to the inhibitor combinations (Fig. 7).
The kinase inhibitor studies are consistent with a model in which copper activates a MAPK signal transduction cascade to ultimately affect the level of MTF-1 phosphorylation to induce transcription. This model is similar to that previously reported for cadmium and zinc (41). It is unlikely that copper is directly binding to MTF-1 to activate transcription; however, an alternative model suggests that copper may displace zinc from MT (54). The "free" zinc would then bind to a zinc finger in MTF-1 that would allow the transcription factor to bind to the MRE and activate transcription.
The results presented in this report support a model in which copper activates transcription via both metal- and oxidative stress-responsive mechanisms. Copper is able to activate transcription through MREs and AREs. In addition, glutathione modulates the level of metal- and oxidative stress-inducible transcription. In COS-7 cells, the ERK1/2 pathway exerts a dominant effect relative to p38 and JNK in regulating metal-inducible MT transcription. Both metals and ROS can independently affect the activity of members of the three MAPK signaling pathways. Thus the roles of specific kinases in regulating transcription in response to these activators may be overlapping. Furthermore, there are additional signaling pathways (casein kinase II, calcium-activated kinase) that participate in the regulation of metal-responsive MT activation (41). Additional studies will be required to more adequately assess copper activation of signal transduction and transcription.
![]() |
ACKNOWLEDGMENTS |
---|
GRANTS
This work was supported in part by National Institute of Environmental Health Sciences Grants ES-10356 and 5T32-ES-07031-22.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Baker MA, Cerniglia GG, and Zaman A. Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal Biochem 190: 360365, 1990.[ISI][Medline]
3. Barceloux DG. Copper. J Toxicol Clin Toxicol 37: 217230, 1999.[CrossRef][ISI][Medline]
4. Bauman JW, Liu J, Liu YP, and Klaassen CD. Increase in metallothionein produced by chemicals that induce oxidative stress. Toxicol Appl Pharmacol 110: 347354, 1991.[ISI][Medline]
5. Chan HM and Cherian MG. Protective roles of metallothionein and glutathione in copper toxicity in rat hepatoma tissue culture cells. Toxicology 72: 281290, 1992.[CrossRef][ISI][Medline]
6. Chow CK. Vitamin E and oxidative stress. Free Radic Biol Med 11: 215232, 1991.[CrossRef][ISI][Medline]
7. Dalton T, Palmiter RD, and Andrews GK. Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elements. Nucleic Acids Res 22: 50165023, 1994.[Abstract]
8. Dalton TP, Li Q, Bittel D, Liang L, and Andrews GK. Oxidative stress activates metal-responsive transcription factor-1 binding activity. Occupancy in vivo of metal response elements in the metallothionein-I gene promoter. J Biol Chem 271: 2623326241, 1996.
9. Durnam DM and Palmiter RD. Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers. Mol Cell Biol 4: 484491, 1984.[ISI][Medline]
10. Favreau LV and Pickett CB. The rat quinone reductase antioxidant response element. Identification of the nucleotide sequence required for basal and inducible activity and detection of antioxidant response element-binding proteins in hepatoma and non-hepatoma cell lines. J Biol Chem 279: 2446824474, 1995.[CrossRef]
11. Favreau LV and Pickett CB. Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression of planar aromatic compounds and phenolic antioxidants. J Biol Chem 266: 45564561, 1991.
12. Franklin CC and Kraft AS. Conditional expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress activated protein kinase in U937 cells. J Biol Chem 272: 1691716923, 1997.
13. Freedman JH, Ciriolo MR, and Peisach J. The role of glutathione in copper metabolism and toxicity. J Biol Chem 264: 55985605, 1989.
14. Freedman JH and Peisach J. Intracellular copper transport in cultured hepatoma cells. Biochem Biophys Res Commun 164: 134140, 1989.[ISI][Medline]
15. Fukino H, Hirai M, Hsueh YM, Moriyasu SB, and Yamane YJ. Mechanism of protection by zinc against mercuric chloride toxicity in rats: effects of zinc and mercury on glutathione metabolism. J Toxicol Environ Health 19: 7589, 1986.[ISI][Medline]
16. Galcheva-Garzova Z, Derijard B, Wu JH, and Davis RJ. An osmosensing signal transduction pathway in mammalian cells. Science 265: 806808, 1994.[ISI][Medline]
17. Gopalakrishna R and Jaken S. Protein kinase C signaling and oxidative stress. Free Radic Biol Med 28: 13491361, 2000.[CrossRef][ISI][Medline]
18. Gunes C, Heuchel R, Georgiev O, Muller KH, Lichtlen P, Bluthmann H, Marino S, Aguzzi A, and Schaffner W. Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. EMBO J 17: 28462854, 1998.
19. Gutteridge JMC. Superoxide dismutase inhibits the superoxide-driven Fenton reaction at two different levels. FEBS Lett 185: 1923, 1985.[CrossRef][ISI][Medline]
20. Hahn SH and Gahl WA. Copper effects on metal regulatory factors of cultured human fibroblasts. Biochem Med Metab Biol 50: 346357, 1993.[CrossRef][ISI][Medline]
21. Hamer DH. Metallothionein. Annu Rev Biochem 55: 913951, 1986.[CrossRef][ISI][Medline]
22. Heuchel R, Radtke F, Georgiev O, Stark G, Aguet M, and Schaffner W. The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression. EMBO J 13: 28702875, 1994.[Abstract]
23. Jahroudi N, Foster R, Price-Haughey J, Beitel G, and Gedamu L. Cell-type specific and differential regulation of the human metallothionein genes. Correlation with DNA methylation and chromatin structure. J Biol Chem 265: 65066511, 1990.
24. Jaiswal AK. Antioxidant response element. Biochem Pharmacol 48: 439444, 1994.[CrossRef][ISI][Medline]
25. Jenkins AJ, Velarde V, Klein RL, Joyce KC, Phillips KD, Mayfield RK, Lyons TJ, and Jaffa AA. Native and modified LDL activate extracellular signal-related kinases in mesangial cells. Diabetes 49: 21602169, 2000.[Abstract]
26. Jeon KI, Jeong JY, and Jue DM. Thiol-reactive metal compounds inhibit NF-B activation by blocking I
B kinase. J Immunol 164: 59815989, 2000.
27. Kagi JHR and Schaffer A. Biochemistry of metallothionein. Biochemistry 27: 85098515, 1988.[ISI][Medline]
28. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem 270: 1648316486, 1995.
29. Kyriakis JM and Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 18: 567577, 1996.[ISI][Medline]
30. LaRochelle OV, Gagne V, Charron J, Soh J, and Seguin C. Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions. J Biol Chem 276: 4187941888, 2001.
31. Li Q, Hu N, Dagget MAF, Chu WA, Bittel D, Johnson JA, and Andrews GK. Participation of upstream stimulator factor (USF) in cadmium-induction of the mouse metallothionein-I gene. Nucleic Acids Res 26: 51825189, 1998.
32. Li Y and Jaiswal AK. Human antioxidant response element-mediated regulation of type 1 NAD(P)H:quinone oxidoreductase gene expression. Eur J Biochem 226: 3139, 1994.[Abstract]
33. Li Y and Jaiswal AK. Regulation of human NADPH:quinone oxidoreductase gene: Role of AP1 binding site contained within human antioxidant response element. J Biol Chem 267: 1509715104, 1992.
34. Mattie MD and Freedman JH. Protective effects of aspirin and vitamin E (-tocopherol) on copper- and cadmium-induced toxicity. Biochem Biophys Res Commun 285: 921925, 2001.[CrossRef][ISI][Medline]
35. Nemer M, Thornton RD, Stuebing EW, and Harlow P. Structure, spatial, and temporal expression of two sea urchin metallothionein genes, SpMTB1 and SpMTA. J Biol Chem 266: 65866593, 1991.
36. Palmiter RD. Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc Natl Acad Sci USA 91: 12191223, 1994.[Abstract]
37. Radtke F, Heuchel R, Georgiev O, Hergersberg M, Gariglio M, Dembic Z, and Schaffner W. Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J 12: 13551362, 1993.[Abstract]
38. Rimoldi D, Flessate DM, and Samid D. Changes in gene expression by 193- and 248-nm excimer laser radiation in cultured human fibroblasts. Radiat Res 131: 325331, 1992.[ISI][Medline]
39. Samet JM, Graves LM, Quay J, Dailey LA, Devlin RB, Ghio AJ, Wu W, Bromberg PA, and Reed W. Activation of MAPKs in human bronchial epithelial cells exposed to metals. Am J Physiol Lung Cell Mol Physiol 275: L551L558, 1998.
40. Sato M and Bremner I. Oxygen free radicals and metallothionein. Free Radic Biol Med 14: 325337, 1993.[CrossRef][ISI][Medline]
41. Saydam N, Adams TK, Steiner F, Schaffner W, and Freedman JH. Regulation of metallothionein transcription by the metal-responsive transcription factor MTF-1: identification of signal transduction cascades that control metal-inducible transcription. J Biol Chem 277: 2043820445, 2002.
42. Schmidt CJ and Hamer DH. Cell specificity and an effect of ras on human metallothionein gene expression. Proc Natl Acad Sci USA 83: 33463350, 1986.[Abstract]
43. Schroder JJ and Cousins RJ. Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures. Proc Natl Acad Sci USA 87: 31373141, 1990.[Abstract]
44. Shi S and Dalal NS. The role of superoxide radical in chromium (VI)-generated hydroxyl radical: the Cr (VI) Haber-Weiss cycle. Arch Biochem Biophys 292: 323327, 1992.[ISI][Medline]
45. Shokri F, Heidari, M, Gharagozloo, S, and Ghazi-Khansari, M. In vitro inhibitory effects of antioxidants on cytotoxicity of T-2 toxin. Toxicology 146: 171176, 2000.[CrossRef][ISI][Medline]
46. Steinebach OM and Wolterbeek HT. Role of cytosolic copper, metallothionein and glutathione in copper toxicity in rat hepatoma tissue culture cells. Toxicology 92: 7590, 1994.[CrossRef][ISI][Medline]
47. Stuart GW, Searle PF, and Palmiter RD. Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature 317: 828831, 1985.[ISI][Medline]
48. Tamai KT, Liu X, Silar P, Sosinowski T, and Thiele DJ. Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signaling pathways. Mol Cell Biol 14: 81558165, 1994.[Abstract]
49. Venugopal R and Jaiswal AK. Coordinated induction of the c-jun gene with genes encoding quinone oxidoreductases in response to xenobiotics and antioxidants. Biochem Pharmacol 58: 597603, 1999.[CrossRef][ISI][Medline]
50. Walling C. Fenton's reagent revisited. Acc Chem Res 8: 125131, 1975.[ISI]
51. Whitmarsh AJ, Shore P, Sharrocks AD, and Davis RJ. Integration of MAP kinase signal transduction pathways at the serum response element. Science 269: 17601763, 1995.
52. Xie T, Belinsky T, Xu Y, and Jaiswal AK. ARE and TRE-mediated regulation of gene expression. J Biol Chem 270: 68946900, 1995.
53. Yu CW, Chen JH, and Lin LY. Metal-induced metallothionein gene expression can be inactivated by protein kinase C inhibitor. FEBS Lett 420: 6973, 1997.[CrossRef][ISI][Medline]
54. Zhang B, Georgiev O, Hagmann M, Gnes Ç, Cramer M, Faller P, Vasák M, and Schaffner W. Activity of metal responsive transcription factor-1 (MTF-1) by toxic heavy metals and H2O2 in vitro is modulated by metallothionein. Mol Cell Biol 23: 84718485, 2003.
55. Zhu J and Thiele DJ. Toxic metal-responsive gene transcription. In: Stress-Inducible Cellular Response, edited by Feige U, Morimoto RI, Yahara I, and Polla BS. Basel, Switzerland: Springer, 1996, p. 306320.