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. Author manuscript; available in PMC: 2009 Dec 2.
Published in final edited form as: Toxicol Lett. 2002 Dec 15;136(2):151–158. doi: 10.1016/s0378-4274(02)00332-6

Activation of early signaling transcription factor, NF-κB following low-level manganese exposure

Govindarajan T Ramesh a, Debabrata Ghosh a, Palur G Gunasekar b,*
PMCID: PMC2786211  NIHMSID: NIHMS135245  PMID: 12425965

Abstract

Occupational and environmental exposure to manganese (Mn2+) is an increasing problem. It manifests neuronal degeneration characterized by dyskinesia resembling Parkinson’s disease. The study was performed to test the hypotheses whether exposure to Mn2+ alters cellular physiology and promotes intracellular signaling mechanism in dopaminergic neuronal cell line. Since transcription factors have been shown to play an essential role in the control of cellular proliferation and survival, catecholaminergic rich pheochromocytoma (PC12) cells were used to measure changes in the DNA binding activities of nuclear factor kappa B (NF-κB) by electrophoretic mobility shift assay (EMSA) following Mn2+ (0.1–10 µM) exposure. Cells that were exposed to Mn2+ produced five-fold-activation of transcription factor NF-κB DNA binding activity. This remarkable increase was seen within 30–60 min period of Mn2+ exposure. Activation of NF-κB DNA binding activity by Mn2+ at 1.0 µM correlated with proteolytic degradation of the inhibitory subunit IκBα as evidenced in cytosol. Additional experiments on NF-κB reporter gene assay also showed increased NF-κB gene expression at 1.0 and 5.0 µM Mn2+ and this was completely blocked in the presence of NF-κB translocation inhibitor, IκBα-DN supporting that NF-κB induction occurred during Mn2+ exposure. In addition, Mn2+ exposure to PC 12 cells led to activation of signal responsive mitogen activated protein kinase kinase (MAPKK). These results suggest that Mn2+ at a low dose appears to induce the expression of immediate early gene, NF-κB through MAPKK by a mechanism in which IκBα phosphorylation may be involved.

Keywords: Mn2+, NF-κB, PC12 cells, Oxidative stress, Neurotoxicity

1. Introduction

Manganese (Mn2+) is an essential trace metal required for brain development and functioning of the brain (Prohaska, 1987). On the other hand, Mn2+ intoxication in humans is identified with prolonged occupational exposure to high level of this metal. It alters dopaminergic functions specifically in the basal ganglia and produces Parkinson-like disorder (Daniels and Abarca, 1991). Behavioral deficits and neurodegeneration are suggested to be features noted in patients with liver failure are typical of patients with Mn2+ neurotoxicity (Pomeir-Layragues et al., 1998; Malecki et al., 1999). Mn2+ stimulates DA autoxidation in the dopaminergic neurons, a processes accompanied by an increase in the formation of quinones (Florence and Stauber, 1989; Shen and Dryhurst, 1998) and protein bound cysteinyl DA and cysteinyl dihydroxyphenylacetic acid (DO-PAC; Hastings et al., 1996). Mn2+ uptake, particularly in the globus pallidus, appears to be necessary for the selective vulnerability during Mn2+ exposure. This region (among others) is known to be sensitive to energy deprivation and excitotoxic injury. Administration of Mn2+ into the rat striatum induced a selective vulnerability of globus pallidus associated with decreased DA content (Lista et al., 1986). Mn2+ accumulates within the mitochondria (Gunter et al., 1975) preferentially via the calcium uniporter (Gavin et al., 1990, 1999) and thereby collapsing the mitochondrial membrane potential and producing bio-energetic defect and DNA fragmentation and associated microtubule protein MAP-2 (Malecki, 2001). Apoptosis or necrosis induced by Mn2+ may be related to oxidative stress through oxidizing DNA, lipids and proteins (Roth et al., 2000). Like other chemicals Mn2+ may induce neurotoxicity by modulating signaling molecules via redox-sensitive transcription factors such as activation protein 1 (AP-1) or nuclear factor kappa-B (NF-κB) (Baichwal and Baeuerle, 1997; Schrantz et al., 1999). Studies indicate that NF-κB activation by various chemicals is blocked by antioxidants (Schreck et al., 1991; Schmidt et al., 1995). We hypothesis that activation of these transcription factors would play a significant role in Mn2+ induced neurotoxicity that may contribute to cell death. In the present study, we evaluated and characterized the activation of NF-κB transcription factor and related mitogen activated protein kinase (MAPK kinase) as a target in Mn2+ exposure that will help to understand the neuronal degeneration.

2. Methods and materials

2.1. Chemicals

Antibiotics-antimycotics (contains penicillin, streptomycin and amphotericin B), RPMI 1640 medium, and fetal bovine serum were obtained from GIBCO (Grand Island, NY). Glycine, MnCl2, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). MnCl2 was dissolved at 100 mM in water and then all further dilution made in medium. Antibody against IκBα and double-stranded oligonucleotide having the NF-κB consensus sequence were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The phospho-specific anti-p44/42 MAP kinase (Thr 202/Tyr 204) antibody was obtained from New England Biolabs, Inc.

2.2. Cell lines

PC12 cells were obtained from the American Tissue and Cell Culture Collection (ATCC, Rockville, MD). Cells were cultured as monolayer in RPMI-1640 medium supplemented with 10% FBS, 5% horse serum, 1% l-glutamine and 1 × antibiotic-antimycotics. Cells were free from mycoplasma as detected by Gen-Probe Mycoplasma Rapid Detection Kit (Fisher Scientific, Pittsburgh, PA). Cells were maintained at 37 °C in an air and 5% CO2 atmosphere and were used at 4–6 days after passage.

2.3. NF-κB activation assay

To assay NF-κB DNA binding activity, electrophoretic mobility shift assay (EMSA) was carried out essentially as described previously (Chaturvedi et al., 1994). Briefly, nuclear extracts (28 µg protein) prepared from Mn2+ treated cells (2 × 106 per ml) were incubated with 32P end-labeled 45-mer double-stranded NF-κB oligonucleotide (6 µg protein with 16 fmoles DNA) from the HIV-LTR, 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′ (bold indicates NF-κB binding sites) for 15 min at 37 °C, and the DNA-protein complex formed was resolved from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5′TTGTTACAACT-CACTTTCCGCTGCTCACTTTCCAGG-GAGGCGTGG-3′, was used to examine the specificity of binding of NF-κB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. The dried gels visualized and 32P radioactive NF-κB DNA complex quantitated through Phosphor-Imager (Molecular Dynamics, Sunnyvale, CA) using image quant software.

2.4. Western blot for IκBα

To examine IκBα by Western blot, cytoplasmic extracts were prepared from Mn2+-treated and untreated cells and then resolved on 10% SDS-polyacrylamide gels as per the method described earlier (Reddy et al., 1994). After electrophoresis, the proteins were electrotransferred to nitrocellulose filters, probed with rabbit polyclonal antibodies against IκBα. The filters were detected by chemiluminescence (ECL, Amersham-Pharmacia Biotech, Arlington Heights, IL).

2.5. Transient transfection and NF-κB reporter assay

To examine whether Mn2+ activates NF-κB dependant gene expression, we constructed a synthetic NF-κB-containing promoter element, a PCR-based strategy. The upstream primer (5′-GCGGCCTCGAGGGGACTTTCCCGGG-GACTTTCCGGGGACTTTCCGGGACTTTC-CATCCTGCCATCTCAATTAG-3′) contained four tandem copies of the NF-κB binding site (GGGGACTTTCCC) and 18 base pairs of a sequence complementary to the 5′ end of the SV40 early promoter sequence, and was flanked with an XhoI site. The downstream primer (5′-GCGGCAAGCTTTTTGCAAAGCCTAGGC-3′) was complementary to the 3′ end of the SV40 promoter and was flanked with a HindIII site. PCR was performed using the SV40 promoter template. The resulting PCR fragment was digested with XhoI/HindIII and subcloned into a likewise digested SEAP2-promoter plasmid to replace the SV40 minimal promoter element.

PC12 cells were transiently transfected with NF-κB-SEAP2 (0.5 µg) and dominant negative IκB (1 µM) in addition of empty vector (pCMV) DNA to make up the concentration of DNA 3 µg for each combination. After transfection (12 h), cells were treated with different concentration of Mn2+ for 6 h. Then conditioned medium was removed and assayed for SEAP activity essentially as described by the manufacturer (CLONTECH, Palo Alto, CA). In brief, conditioned medium (25 µl) was mixed with 30 µl of five times buffer (500 mM Tris, pH 9, and 0.5% bovine serum albumin) in a total volume of 100 µl in a 96-well plate and incubated at 65 °C for 30 min. The plate was chilled on ice for 2 min. Then 50 µl of 1 mM 4-methylumbelliferyl phosphate was added to each well and incubated at 37 °C for 2 h. The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan II, Lab Systems, Needham, Heights, MA) with excitation set at 360 nm and emission at 460 nm. The average (±/S.E.M.) number of relative fluorescent light units for each transfection was then determined and reported as fold activation with respect to control vector-transfected cells.

2.6. Western blot analysis of MAP kinase kinase

The change in oxidative species generation, mitochondrial function and associated cell death in response to various chemical stimuli and other neurotoxicants explain the role in part played by activation of redox sensitive transcription factors, NF-κB and AP-1 and other related kinases. Since MAPK kinase (mitogen activated protein kinases) is a regulator of NF-κB transcription factor, the phosphorylated form of MAP Kinase Kinase was assayed in PC 12 cells in response to Mn2+ exposure (0–10 µm) as per the method of Ramesh et al. (1999). Following treatments for 120 min, cell extracts (60 µg aliquot of total protein) was resolved on each lane on 10% SDS-PAGE, electrotransferred onto nitrocellulose membrane, and probed with the phospho-specific anti-p44/42 MAP kinase (Thr 202/Tyr 204) antibody (New England Biolabs, Inc.) raised in rabbit (1:3000 dilution). The membrane was then incubated with peroxidase-conjugated anti-rabbits IgG (1:3000 dilution), and bands were detected by chemiluminescence (ECL, Amersham). Same filter was developed for ERK2 monoclonal antibody for loading control.

2.7. Statistics

Data were expressed as mean ± S.E.M and statistical significance was assessed by using one way analysis of variance followed by Tukeykramer multiple range test. Differences were considered significant at the level of P < 0.05.

3. Results

3.1. Mn2+ activates NF-κB transcription factor

EMSA was performed to analyze NF-κB DNA binding activity in PC12 cells after Mn2+ exposure at different dose (0.01–10 µM) for 120 min. As shown in Fig. 1 Mn2+ enhanced NF-κB DNA binding activity in a concentration dependent manner. The enhancement of NF-κB DNA binding was about five fold with a maximal stimulation at 0.5–10 µM Mn2+. The specificity of NF-κB binding was demonstrated by the disappearance of NF-κB DNA binding signal with inclusion of the excess unlabelled oligonucleotides.

Fig. 1.

Fig. 1

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Dose dependent activation of NF-κB DNA binding by Mn2+. Cells (2 × 106 cells per ml) were incubated with different concentrations of Mn2+ (0–10 µM) for 120 min and then nuclear extracts were prepared and activation was determined by electrophoreticmobility shift assay as described in Section 2. Units at the bottom indicate fold increase in Mn2+-induced NF-κB activation with dose compared with untreated control cells. Similar results were obtained in three independent experiments.

Super shift assay revealed the specificity of NF-κB activated during Mn2+ exposure. Addition of anti-p50 NF-κB antibody resulted in the super shift of two NF-κB binding bands (Fig. 2). Of these, higher band was shifted by the addition of anti-p65 NF-κB antibody. These suggest that the activated complex of NF-κB by Mn2+ consisted of p50 and p65 subunits. Neither pre immune serum (PIS) nor irrelevant antibodies like anti-cRel or anti-cyclin D1 had any effect on the mobility of NF-κB. An excess (100-fold) of unlabelled NF-κB oligo prevented the formation of the band, indicating specificity of NF-κB binding. To investigate the level of kinetics of NF-κB activation in Mn2+ exposed PC12 cells, cells were treated with 1 µM Mn2+ for various time points (5, 10, 15, 30, 60, 120 min) and nuclear extracts were analyzed for NF-κB DNA binding. The results in Fig. 3 indicate that Mn2+ activated NF-κB DNA binding in a time dependent manner with a maximum activation at 30 min.

Fig. 2.

Fig. 2

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Super shift and specificity assay revealed Mn2+-induced activation of NF-κB consists of p50 and p65 sub units complex. Treated and untreated nuclear extracts were incubated with different antibodies and 50 times excess cold probe for 30 min at 37 °C and then assayed by EMSA with labeled wild NF-κB probe as described in Section 2.

Fig. 3.

Fig. 3

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Time dependent activation of NF-κB DNA binding by Mn2+ in PC12 cells. Cells (2 × 106 cells per ml) were exposed to Mn2+ (1 µM) at various time points as indicated and then activation was determined in the nuclear extracts by Electrophoretic mobility shift assay as described in the Section 2. Units at the bottom indicate fold increase in Mn2+-induced NF-κB activation with time compared with untreated control cells. Similar results were obtained in three independent experiments.

3.2. Mn2+ activates degradation of IκBα

The translocation of NF-κB to the nucleus is preceded by the phosphorylation and proteolytic degradation of IκBα in cytosol. To determine the effect of Mn2+ on IκBα degradation, cytosolic extracts were assayed for IκBα by Western blot analysis. Results obtained (Fig. 4) revealed that Mn2+ exposure (1.0 µM) to PC12 cells caused degradation of IκBα, within 5 min and completely degrades by 10 min and then reappeared at maximum by 30 min. This disappearance and reappearance of IκBα corresponds with the kinetics of NF-κB activation (Fig. 3). The alteration of NF-κB activation in respect to concentration of Mn2+ exposure will help in understanding the mechanisms underlying Mn2+-induced toxicity.

Fig. 4.

Fig. 4

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Mn2+-induced IκBα degradation in the cytosolic fraction. Cells (2 × 106 cells per ml) were incubated with Mn2+ (1 µM) at different time periods (0 –120 min) and detected IκBα in the cytosolic fraction by Western blot using polyclonal antibody against IκBα as described in the Section 2. Degradation of IκBα is apparent at 10 min and reappeared at 30 min.

3.3. Mn2+ -induced NF-κB reporter gene expression

Since we have observed that Mn2+ activates NF-κB DNA binding in time and dose dependent manner, further we wanted to examine whether Mn2+ activates NF-κB-dependent gene expression. To determine this, PC12 cells were transfected with a plasmid consisting of gene promotor containing NF-κB binding sites ligated to the SEAP2 reporter gene. After transfection (12 h), the cells were treated with different concentration of Mn2+ for 6 h. and then assayed for SEAP activity. As shown in Fig. 5, there were 2–3-fold increases in SEAP activity in response to 1 and 5 µM Mn2+, respectively. This was completely blocked in the presence of NF-κB translocation inhibitor, IκBα-DN. These results show that Mn2+ activates NF-κB dependent reporter gene expression.

Fig. 5.

Fig. 5

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Mn2+-induced NF-κB reporter gene expression in PC12 cells. Cells were transfected with a plasmid consisting of gene promoter containing NF-κB binding sites ligated to SEAP2 reporter gene and then cells were treated with different concentration of Mn2+ for 6 h. and assayed for SEAP activity as described in the Section 2. After treatment significant increase of SEAP2 expression is observed at (P < 0.001).

3.4. Mn2+-induced activation of MAPKK

To examine the gene regulation pathways leading to cell death induced by manganese, MAPK kinase were analyzed by Western blot in PC12 cells. Cells that were exposed to various concentrations of Mn2+ (0–10 µM) phosphorylated MAPK into 44 and 42 kDa dose dependently as shown in the Fig. 6. In a similar time course, NF-κB was also activated with same dose of Mn2+ exposure as shown in Fig. 3. The dose dependent activation of MAPKK suggests that Mn2+ activates NF-κB may be through signal responsive kinase.

Fig. 6.

Fig. 6

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Mn2+-induced activation of MAPKK in PC-12 cells. Cells were exposed to various concentrations of Mn2+ (0–10 µM) and assayed for MAPKK activation by Western blot using antibody specific to phospho-specific p44/42 MAP kinase as described in the Section 2. Dose dependent activation of MAPKK was observed following Mn2+ exposure. Same filter was developed for ERK2 monoclonal antibody for loading control. Similar results were obtained in three independent experiments.

4. Discussion

Miners exposed to high level of Mn2+ are associated with extrapyramidal symptoms (Barbeau, 1984; Aschner, 2000). Potential risks of Mn2+ exposure to public arise in the form of gasoline additive methylcyclopentadienyl manganese tricarbonyl (MMT). Mn2+ known to induce cell toxicity in which oxidative stress plays an important role (Sun et al., 1993). However, the toxic potential of Mn2+ and the exact mechanism by which Mn2+ leads to cell death remains elusive.

Several lines of evidence indicate that Mn2+ accumulates (~100 nm) within the mitochondria (Maynard and Cotzias, 1955; Gavin et al., 1992; Malecki et al., 1999) and thereby collapses the mitochondrial membrane potential and produces bio-energetic defect impairing oxidative phosphorylation and decreasing ATP synthesis (Brouillet et al., 1993). The decreased cellular protective mechanisms like glutathione and glutathione peroxidase and consequent redox homeostasis loss have been reported in the affected brain regions (Desole et al., 1997; Rabinovich and Hastings, 1998). Our study showed that Mn2+ toxicity at the level of 0.01 –10 µM in PC 12 cells is associated with activation of nuclear transcription factor, NF-κB. Mn2+ is known to disturb the mitochondrial respiration and inhibits the antioxidants system (Maynard and Cotzias, 1955; Brouillet et al., 1993) and consequently straining the cell’s ability to combat oxidative stress mediated effect. NF-κB is activated in many different cell types following challenge with several pathogenic stimuli (Gilmore, 1990). Though most, if not all, inducers of NF-κB seem to relay on the production of reactive oxygen intermediates (Meyer et al., 1993), we observed non-significant generation of oxidative species (data not shown) in Mn2+-induced NF-κB activation. Mn2+ dose for optimal NF-κB activation though, was not the same as that for optimal oxidative species production, it is suggested that oxidative radicals produced in response to Mn2+ might be necessary for NF-κB activation.

NF-κB is present in the cytosol as an inducible multi subunit complex of 50 kDa (p50) and 65 kDa (p65) polypeptide complexed with an inhibitor subunit IκBα. The inhibitory protein, IκBα, tightly controls NF-κB. Upon activation, IκBα dissociates from NF-κB heterodimer by undergoing sequential phosphorylation and degradation, thereby release NF-κB for nuclear translocation and facilitate DNA binding and transcriptional up-regulation of genes down stream of κB motif. Mn2+-induced NF-κB activation in the present study was accompanied by characteristic phosphorylation and degradation of IκBα in a time-dependant way. In Mn2+ exposed PC12 cells, the disappearance and reappearance of IκBα subunit in the cytosolic fraction at the time point in which NF-κB nuclear translocation occurred was well correlated. The studies with monoclonal antibodies directed to p50 and p65 indicated that Mn2+-induced NF-κB complex comprises p50 and p65 heterodimers. To examine whether NF-κB plays a role in signaling, we assayed reported gene expression. Treatment with Mn2+ resulted in 2–3-fold increase of NF-κB -induced SEAP2 expression. This effect was blocked by simultaneous treatment with IκBα-DN (NF-κB translocation inhibitor). This reflects the induction of NF-κB during Mn2+ exposure in the dopaminergic cell death. Though the significant role of NF-κB in Mn2+-induced cell death mechanism is not known, previous reports indicate that (Baichwal and Baeuerle, 1997; Ishikawa et al., 1997) activation of NF-κB transcription factor could lead downstream to excessive transcription of proteins that contribute to cell death. Further, increased oxidative production is also seen in with many other NF-κB stimuli, which was blocked by antioxidants (Schreck et al., 1991; Schmidt et al., 1995). In contrast, in some other cell lines, it was shown that TNF-α, a potent stimulator of NF-κB nuclear translocation prevented cell death (Cheng et al., 1994).

We have previously reported that heavy metals like lead activates NF-κB through MAPKK activation (Ramesh et al., 1999) in PC12 cells. The mechanism by which the NF-κB induction during Mn2+ exposure may be related to MAPKK activation as reported in other studies (Ramesh et al., 1999; Lee et al., 1998). The phosphorylation of IκBα is regulated by a large number of kinases, including IKK-α, IKK-β, and mitogen activated protein/extracellular signal-related kinase kinases (Karin, 1999). The IκBα activation and phosphorylation by MAPKK has been shown in both in vitro and in vivo models (Lee et al., 1997, 1998). In PC 12 cells, the degradation of IκBα during Mn2+ exposure may be due to the activation of either of these kinase regulators. Dose dependent activation of NF-κB regulator MAPKK following Mn2+ in our study further suggesting that MAPKK may influence the activation of NF-κB transcription factor possibly through either IKK-α or IKK-β in the mechanism of Mn2+-induced toxicity.

In summary, although the contribution of oxidative stress to NF-κB activation is speculative, there appears to be ample evidence suggesting that Mn2+ -induced oxidative species may contribute to cell death by multiple mechanisms involving NF-κB activation. Further we suggest that MAPKK activation may initiate NF-κB, which might be involved in the dopaminergic toxicity during Mn2+ exposure. The functions of NF-κB in cell death and the mechanisms of its regulation by oxidative stress in Mn2+ -induced dopaminergic toxicity deserve further study. This finding is important because targeting NF-κB is a potential mechanistic pathway in both environmental and idiopathic forms of PD.

Acknowledgements

This work was funded by grants from The American Parkinson Disease Association, Inc. (P.G.G.) and The National Institutes of Health (Grant No. NIH/RCMI BB03045) (G.T.R).

References

  1. Aschner M. Manganese: brain transport and emerging research needs. Environ. Health Perspect. 2000;108:429–432. doi: 10.1289/ehp.00108s3429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baichwal VR, Baeuerle PA. Apoptosis: activate NF-κB or die. Curr. Biol. 1997;7:R94–R96. doi: 10.1016/s0960-9822(06)00046-7. [DOI] [PubMed] [Google Scholar]
  3. Barbeau A. Manganese and extrapyramidal disorders. Neurotoxicology. 1984;5:13–36. [PubMed] [Google Scholar]
  4. Brouillet EP, Shinobu I, McGarvey U, Hochberg F, Beal MF. Manganese injection into the rat striatum produces excitotoxic lesions by impairing energy metabolism. Exp. Neurol. 1993;120:89–94. doi: 10.1006/exnr.1993.1042. [DOI] [PubMed] [Google Scholar]
  5. Chaturvedi MM, LaPushin R, Aggarwal BB. Tumor necrosis factor and lymphotoxcin. Qualitative and quantitative differences in the mediation of early and late cellular response. J. Biol. Chem. 1994;269:14575–14583. [PubMed] [Google Scholar]
  6. Cheng B, Christakos M, Mattson MP. Tumor necrosis factors protect neurons against metabolic excitotoxic insults and promote maintenance of calcium homeostasis. Neuron. 1994;12:139–153. doi: 10.1016/0896-6273(94)90159-7. [DOI] [PubMed] [Google Scholar]
  7. Daniels AJ, Abarca J. Effect of intranigral Mn2+ on striatal and nigral synthesis and levels of dopamine and cofactor. Neurotoxicol. Teratol. 1991;13:483–487. doi: 10.1016/0892-0362(91)90053-y. [DOI] [PubMed] [Google Scholar]
  8. Desole MS, Esposito G, Migheli R, Sircana S, Delogu MR, Fresu L, Miele M, De Natale G, Miele E. Glutathione deficiency potentiates manganese toxicity in rat striatum and brain stem and PC12 cells. Pharmacol. Res. 1997;36:285–292. doi: 10.1006/phrs.1997.0197. [DOI] [PubMed] [Google Scholar]
  9. Florence TM, Stauber JL. Manganese catalysis of dopamine oxidation. Sci. Total Environ. 1989;78:233–240. doi: 10.1016/0048-9697(89)90036-3. [DOI] [PubMed] [Google Scholar]
  10. Gavin CE, Gunter KK, Gunter TE. Manganese and calcium efflux kinetics in brain mitochondria. Relevance to manganese toxicity. Biochem. J. 1990;266:329–334. doi: 10.1042/bj2660329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gavin CE, Gunter KK, Gunter TE. Mn2+ sequestration by mitochondria and inhibition of oxidative phosphorylation. Toxicol. Appl. Pharmacol. 1992;115:1–5. doi: 10.1016/0041-008x(92)90360-5. [DOI] [PubMed] [Google Scholar]
  12. Gavin CE, Gunter KK, Gunter TE. Manganese and calcium transport in mitochondria: implication for manganese toxicity. Neurotoxicology. 1999;20(2–3):445–454. [PubMed] [Google Scholar]
  13. Gilmore TD. NF-kappa B, KBF1, dorsal, and related matters. Cell. 1990;62(5):841–843. doi: 10.1016/0092-8674(90)90257-f. [DOI] [PubMed] [Google Scholar]
  14. Gunter TE, Puskin JS, Russell PR. Quantitative magnetic resonance studies of manganese uptake by mitochondria. Biophys. J. 1975;15:319–333. doi: 10.1016/S0006-3495(75)85822-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hastings TG, Lewis DA, Zigmond MJ. Role of oxidative stress in the neurotoxic effects of intrastriatal dopamine injections. Proc. Natl. Acad. Sci. USA. 1996;93:1056–1061. doi: 10.1073/pnas.93.5.1956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ishikawa Y, Yokoo T, Kitamura M. c-Jun/AP-1, but not NF-κB is a mediator for oxidant initiated apoptosis in glomerular mesengial cells. Biochem. Biophys. Res. Commun. 1997;240:496–501. doi: 10.1006/bbrc.1997.7665. [DOI] [PubMed] [Google Scholar]
  17. Karin M. How NF-κB is activated: the role of the IB kinase (IKK) complex. Oncogene. 1999;18:6867–6874. doi: 10.1038/sj.onc.1203219. [DOI] [PubMed] [Google Scholar]
  18. Lee FS, Hagler J, Chen ZJ, Maniatis T. Activation of the IkappaB alpha kinase complex by MEKK1, a kinase of the JNK pathway. Cell. 1997;88:213–222. doi: 10.1016/s0092-8674(00)81842-5. [DOI] [PubMed] [Google Scholar]
  19. Lee FS, Peters RT, Dang L, Maniatis T. MEKK1 activates both IκB kinase α and IκB kinase β. Proc. Natl. Acad. Sci. USA. 1998;95:9319–9324. doi: 10.1073/pnas.95.16.9319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lista A, Abarca J, Ramos C, Daniels AJ. Rat striatal dopamine and tetrahydrobiopterin content following an intrastriatal injection of manganese chloride. Life Sci. 1986;38:2121–2127. doi: 10.1016/0024-3205(86)90211-0. [DOI] [PubMed] [Google Scholar]
  21. Maynard LS, Cotzias GC. The partition of manganese among organs and intracellular organelles of the rat. J. Biol. Chem. 1955;214:489–495. [PubMed] [Google Scholar]
  22. Malecki EA. Manganese toxicity is associated with mitochondrial dysfunction and DNA fragmentation in rat primary striatal neurons. Brain Res. Bull. 2001;55:225–228. doi: 10.1016/s0361-9230(01)00456-7. [DOI] [PubMed] [Google Scholar]
  23. Malecki EA, Devenyi AG, Barron TF, Mosher TJ, Eslinger PJ, Flasherty-Craig CV, Rossaro L. Iron and manganese homeostasis in chronic liver disease; relationship to pallidal T1-weighted magnetic resonance signal hyperintensity. Neurotoxicology. 1999;20:647–652. [PubMed] [Google Scholar]
  24. Meyer M, Schreck R, Baeurele PA. H2O2 and antioxidants have opposite effects on activation of NF-κB and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 1993;12:2005–2015. doi: 10.1002/j.1460-2075.1993.tb05850.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pomeir-Layragues G, Rose C, Spahr L, Zayed J, Normandin L, Butterworth RF. Role of manganese in the pathogenesis of portal systemic encepalopathy. Metab. Brain Dis. 1998;13:311–317. doi: 10.1023/a:1020636809063. [DOI] [PubMed] [Google Scholar]
  26. Prohaska Functions of trace elements in brain metabolism. Physiol. Rev. 1987;67(3):858–901. doi: 10.1152/physrev.1987.67.3.858. [DOI] [PubMed] [Google Scholar]
  27. Rabinovich AD, Hastings TG. Role of endogenous glutathione in the oxidation of dopamine. J. Neurochem. 1998;71:2071–2078. doi: 10.1046/j.1471-4159.1998.71052071.x. [DOI] [PubMed] [Google Scholar]
  28. Ramesh GT, Manna SK, Aggarwal BB, Jadhav AL. Lead activates nuclear transcription factor-κB, activator protein-1, and amino-terminal c-Jun kinase in pheochromocytoma cells. Toxicol. Appl. Pharmacol. 1999;155(3):280–286. doi: 10.1006/taap.1999.8624. [DOI] [PubMed] [Google Scholar]
  29. Reddy SA, Chaturvedi MM, Damay BG, Chan H, Higuchi M, Aggarwal BB. Reconstitution of nuclear factor kappa B activation induced by tumor necrosis factor requires membrane associated components, comparison with pathway activated by ceramide. J. Biol. Chem. 1994;269:25369–25372. [PubMed] [Google Scholar]
  30. Roth JA, Jennifer Walowitz LF, Browne RW. Manganese-induced rat pheochromacytoma (PC12) cell death is independent of caspase activation. J. Neurosci. Res. 2000;61:62–171. doi: 10.1002/1097-4547(20000715)61:2<162::AID-JNR7>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  31. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of NF-κB transcription factor and HIV-1. EMBO J. 1991;10:2247–2258. doi: 10.1002/j.1460-2075.1991.tb07761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schmidt KN, Traenckner BM, Meier B, Baeuerle PA. Induction of oxidative stress by okadaic acid is required for activation of transcription factor NF-κB. J. Biol. Chem. 1995;270(45):27136–27142. doi: 10.1074/jbc.270.45.27136. [DOI] [PubMed] [Google Scholar]
  33. Schrantz N, Blanchard DA, Mitenne F, Auffredou MT, Vazquez A, Leca G. Manganese induces apoptosis of human B cells: caspase dependent cell death blocked by Bcl-2. Cell Death Differ. 1999;6:445–453. doi: 10.1038/sj.cdd.4400508. [DOI] [PubMed] [Google Scholar]
  34. Shen XM, Dryhurst G. Iron and manganese-catalyzed autoxidation of dopamine in the presence of l-cysteine: possible insights into iron and manganese-mediated dopaminergic neurotoxicity. Chem.Res. Toxicol. 1998;11:824–837. doi: 10.1021/tx980036t. [DOI] [PubMed] [Google Scholar]
  35. Sun AY, Yang WL, Kim HD. Free radical and lipid peroxidation in manganese-induced neuronal cell injury. Ann. New York Acad. Sci. 1993;679:358–363. doi: 10.1111/j.1749-6632.1993.tb18322.x. [DOI] [PubMed] [Google Scholar]

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