Archives of Toxicology

, Volume 92, Issue 1, pp 195–211 | Cite as

Early response of glutathione- and thioredoxin-dependent antioxidant defense systems to Tl(I)- and Tl(III)-mediated oxidative stress in adherent pheochromocytoma (PC12adh) cells

  • Lis C. Puga Molina
  • Damiana M. Salvatierra Fréchou
  • Sandra V. VerstraetenEmail author
Inorganic Compounds


Thallium (Tl) is a toxic heavy metal that causes oxidative stress both in vitro and in vivo. In this work, we evaluated the production of oxygen (ROS)- and nitrogen (RNS)-reactive species in adherent PC12 (PC12adh) cells exposed for 0.5–6 h to Tl(I) or Tl(III) (10–100 µM). In this system, Tl(I) induced mostly H2O2 generation while Tl(III) induced H2O2 and ONOO·− generation. Both cations enhanced iNOS expression and activity, and decreased CuZnSOD expression but without affecting its activity. Tl(I) increased MnSOD expression and activity but Tl(III) decreased them. NADPH oxidase (NOX) activity remained unaffected throughout the period assessed. Oxidant levels returned to baseline values after 6 h of incubation, suggesting a response of the antioxidant defense system to the oxidative insult imposed by the cations. Tl also affected the glutathione-dependent system: while Tl(III) increased glutathione peroxidase (GPx) expression and activity, Tl(I) and Tl(III) decreased glutathione reductase (GR) expression. However, GR activity was mildly enhanced by Tl(III). Finally, thioredoxin-dependent system was evaluated. Only Tl(I) increased 2-Cys peroxiredoxins (2-Cys Prx) expression, although both cations increased their activity. Tl(I) increased cytosolic thioredoxin reductase (TrxR1) and decreased mitochondrial (TrxR2) expression. Tl(III) had a biphasic effect on TrxR1 expression and slightly increased TrxR2 expression. Despite of this, both cations increased total TrxR activity. Obtained results suggest that in Tl(I)-exposed PC12adh cells, there is an early response to oxidative stress mainly by GSH-dependent system while in Tl(III)-treated cells both GSH- and Trx-dependent systems are involved.


Thallium Oxidative stress Mitochondria Antioxidant enzymes Antioxidant defense system Glutathione Thioredoxin PC12 cells 







CuZn-superoxide dismutase




Dihydrorhodamine 123


Dulbecco’s modified Eagle medium


Diphenylene-iodonium chloride


Dithionitrobenzoic acid


Glutathione peroxidase


Glutathione reductase




Glutathione disulfide


Inducible NOS


Neuronal NOS


NG-nitro-l-arginine methyl ester




Mn-superoxide dismutase


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide


2,2′-bis(4-Nitrophenyl)-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-diphenylene) ditetrazolium chloride


Nitric oxide synthase


NADPH oxidase


Phosphate buffered saline


PC12 cells, adherent variant


Propidium iodide




Reactive nitrogen species


Reactive oxygen species


Sodium dodecyl sulfate


Thioredoxin reductase



This work was supported by grants of the University of Buenos Aires (B086 and 20020100100112) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; PICT2013-1018). SVV is a career investigator of the CONICET (National Research Council, Argentina). LCPM was a recipient of an undergraduate fellowship from the University of Buenos Aires (Argentina).

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

204_2017_2056_MOESM1_ESM.tif (70 kb)
Supp. Figure 1. Tl(I) and Tl(III) did not affect cell viability. PC12adh cells were incubated at 37 °C for 30 min in the absence (○) or presence (●) of 100 µM apocynin (Apo), and further exposed for 6 h to 10-100 µM (A) Tl(I) or (B) Tl(III). Cell viability was evaluated from the reduction of MTT and expressed as the percentage of the value recorded in control cells. Results are shown as the mean ± SEM (n > 3). * denotes a statistical significance respect to the value recorded in cells exposed to the same concentration of Tl but in the absence of apocynin (P < 0.01). (TIFF 69 kb)
204_2017_2056_MOESM2_ESM.tif (44 kb)
Supp. Figure 2. Positive controls used for DHR123 oxidation determination. PC12adh cells were loaded with DHR123, and non-incorporated probe was eliminated by washing. Cells were next incubated at 37 °C for 3 h in the presence of 500 µM H2O2 or 1 mM GSNO. At the end of incubation, DHR123 fluorescence was recorded and normalized by DNA content by reaction with Hoechst 32258. Results are shown as the mean ± SEM (n = 3). * denotes a statistical significance respect to the value recorded in control cells (P < 0.01). (TIFF 44 kb)
204_2017_2056_MOESM3_ESM.tif (69 kb)
Supp. Figure 3. DPI inhibited cell capacity to metabolize MTT. PC12adh cells were incubated at 37 °C for 30 min in the absence (○) or presence (●) of 100 µM DPI, and further exposed for 1 h to 10-100 µM (A) Tl(I) or (B) Tl(III). Cell viability was evaluated from the reduction of MTT and expressed as the percentage of the value recorded in control cells. Results are shown as the mean ± SEM (n = 3). * denotes a statistical significance respect to the value recorded in cells exposed to the same concentration of Tl but in the absence of DPI (P < 0.01). (TIFF 69 kb)
204_2017_2056_MOESM4_ESM.tif (43 kb)
Supp. Figure 4. Positive control used for DHE oxidation determination. PC12adh cells were loaded with DHE, and non-incorporated probe was eliminated by washing. Cells were next incubated at 37 °C for 3 h to 500 µM H2O2 or 100 µM pyrogallol (Pyro). At the end of incubation, DHE fluorescence was recorded and normalized by DNA content by reaction with Hoechst 32258. Results are shown as the mean ± SEM (n = 3). * denotes a statistical significance respect to the value recorded in control cells (P < 0.001). (TIFF 43 kb)
204_2017_2056_MOESM5_ESM.tif (544 kb)
Supp. Figure 5. Tl(I) and Tl(III) did not affect NOX activity. PC12adh cells were loaded with DHE, incubated at 37 °C for 30 min in the absence (●) or the presence (○) of 100 µM of NOX inhibitor apocynin (Apo), and then exposed for (A, B) 0.5 h, (C, D) 3 h, or (E, F) 6 h to Tl(I) or Tl(III) (10-100 µM). Results were normalized by DNA content measured with Hoechst 32258, and are shown as the mean ± SEM (n > 3). (TIFF 544 kb)


  1. ATSDR (1999) Thallium. ATSDR (Agency for Toxic Substances and Disease Registry). Prepared by Clement International Corp., under contract 205-88-0608, Atlanta, GAGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  3. Brismar T, Anderson S, Collins VP (1995) Mechanism of high K+ and Tl+ uptake in cultured human glioma cells. Cell Mol Neurobiol 15(3):351–360CrossRefPubMedGoogle Scholar
  4. Castilho RF, Ward MW, Nicholls DG (1999) Oxidative stress, mitochondrial function, and acute glutamate excitotoxicity in cultured cerebellar granule cells. J Neurochem 72(4):1394–1401CrossRefPubMedGoogle Scholar
  5. Chae HZ, Kim HJ, Kang SW, Rhee SG (1999) Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin. Diabetes Res Clin Pract 45(2–3):101–112CrossRefPubMedGoogle Scholar
  6. Chen HM, Lee YC, Huang CL et al (2007) Methamphetamine downregulates peroxiredoxins in rat pheochromocytoma cells. Biochem Biophys Res Commun 354(1):96–101CrossRefPubMedGoogle Scholar
  7. Ciofani G, Genchi GG, Mazzolai B, Mattoli V (2014) Transcriptional profile of genes involved in oxidative stress and antioxidant defense in PC12 cells following treatment with cerium oxide nanoparticles. Biochim Biophys Acta 1840(1):495–506CrossRefPubMedGoogle Scholar
  8. Crow JP (1997) Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1(2):145–157CrossRefPubMedGoogle Scholar
  9. Dringen R, Kussmaul L, Hamprecht B (1998) Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Res Protoc 2(3):223–228CrossRefGoogle Scholar
  10. Galvan-Arzate S, Rios C (1994) Thallium distribution in organs and brain regions of developing rats. Toxicology 90(1–2):63–69CrossRefPubMedGoogle Scholar
  11. Galván-Arzate S, Martínez A, Medina E, Santamaría A, Ríos C (2000) Subchronic administration of sublethal doses of thallium to rats: effects on distribution and lipid peroxidation in brain regions. Toxicol Lett 116:37–43CrossRefPubMedGoogle Scholar
  12. Grzelak A, Soszynski M, Bartosz G (2000) Inactivation of antioxidant enzymes by peroxynitrite. Scand J Clin Lab Invest 60(4):253–258CrossRefPubMedGoogle Scholar
  13. Guevara I, Iwanejko J, Dembinska-Kiec A et al (1998) Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin Chim Acta 274(2):177–188CrossRefPubMedGoogle Scholar
  14. Halliwell B, Gutteridge J (1999) Free radicals in biology and medicine, 3rd edn. Oxford University Press, New YorkGoogle Scholar
  15. Hanschmann EM, Godoy JR, Berndt C, Hudemann C, Lillig CH (2013) Thioredoxins, glutaredoxins, and peroxiredoxins–molecular mechanisms and health significance: from cofactors to antioxidants to redox signaling. Antioxid Redox Signal 19(13):1539–1605CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hanzel CE, Verstraeten SV (2006) Thallium induces hydrogen peroxide generation by impairing mitochondrial function. Toxicol Appl Pharmacol 216(3):485–492CrossRefPubMedGoogle Scholar
  17. Hanzel CE, Verstraeten SV (2009) Tl(I) and Tl(III) activate both mitochondrial and extrinsic pathways of apoptosis in rat pheochromocytoma (PC12) cells. Toxicol Appl Pharmacol 236(1):59–70CrossRefPubMedGoogle Scholar
  18. Hanzel CE, Almeira Gubiani MF, Verstraeten SV (2012) Endosomes and lysosomes are involved in early steps of Tl(III)-mediated apoptosis in rat pheochromocytoma (PC12) cells. Arch Toxicol 86(11):1667–1680CrossRefPubMedGoogle Scholar
  19. Hasan M, Ali SF (1981) Effects of thallium, nickel, and cobalt administration of the lipid peroxidation in different regions of the rat brain. Toxicol Appl Pharmacol 57(1):8–13CrossRefPubMedGoogle Scholar
  20. Holmgren A, Bjornstedt M (1995) Thioredoxin and thioredoxin reductase. Methods Enzymol 252:199–208CrossRefPubMedGoogle Scholar
  21. Johansson LH, Borg LA (1988) A spectrophotometric method for determination of catalase activity in small tissue samples. Anal Biochem 174(1):331–336CrossRefPubMedGoogle Scholar
  22. Jomova K, Valko M (2011) Advances in metal-induced oxidative stress and human disease. Toxicology 283(2–3):65–87CrossRefPubMedGoogle Scholar
  23. Kim JA, Park S, Kim K, Rhee SG, Kang SW (2005) Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system. Anal Biochem 338(2):216–223CrossRefPubMedGoogle Scholar
  24. Korotkov SM, Brailovskaya LV (2001) Tl+ increases the permeability of the inner membranes of rat liver mitochondria for monovalent cations. Dokl Biochem Biophys 378:145–149CrossRefPubMedGoogle Scholar
  25. Korotkov SM, Lapin AV (2003) Thallium induces opening of the mitochondrial permeability transition pore in the inner membrane of rat liver mitochondria. Dokl Biochem Biophys 392:247–252CrossRefPubMedGoogle Scholar
  26. Lu J, Holmgren A (2014) The thioredoxin antioxidant system. Free Radic Biol Med 66:75–87CrossRefPubMedGoogle Scholar
  27. Manta B, Hugo M, Ortiz C, Ferrer-Sueta G, Trujillo M, Denicola A (2009) The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2. Arch Biochem Biophys 484(2):146–154CrossRefPubMedGoogle Scholar
  28. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63CrossRefPubMedGoogle Scholar
  29. Nakagawa H, Yoshida M, Miyamoto S (2000) Nitric oxide underlies the differentiation of PC12 cells induced by depolarization with high KCl. J Biochem 127(1):113–119CrossRefPubMedGoogle Scholar
  30. Ogusucu R, Rettori D, Munhoz DC, Netto LE, Augusto O (2007) Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics. Free Radic Biol Med 42(3):326–334CrossRefPubMedGoogle Scholar
  31. Peter AL, Viraraghavan T (2005) Thallium: a review of public health and environmental concerns. Environ Int 31(4):493–501CrossRefPubMedGoogle Scholar
  32. Peunova N, Enikolopov G (1995) Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. Nature 375(6526):68–73CrossRefPubMedGoogle Scholar
  33. Pino MT, Marotte C, Verstraeten SV (2017) Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis. Arch Toxicol 91(3):1157–1174CrossRefPubMedGoogle Scholar
  34. Pourahmad J, Eskandari MR, Daraei B (2010) A comparison of hepatocyte cytotoxic mechanisms for thallium (I) and thallium (III). Environ Toxicol 25(5):456–467CrossRefPubMedGoogle Scholar
  35. Rabilloud T, Heller M, Gasnier F et al (2002) Proteomics analysis of cellular response to oxidative stress. Evidence for in vivo overoxidation of peroxiredoxins at their active site. J Biol Chem 277(22):19396–19401CrossRefPubMedGoogle Scholar
  36. Repetto G, Del Peso A, Repetto M (1998) Human thallium toxicity. In: Nriagu J (ed) Thallium in the environment. Advances in environmental science and technology. Wiley, Hoboken, pp 167–199Google Scholar
  37. Rhee SG, Yang KS, Kang SW, Woo HA, Chang TS (2005) Controlled elimination of intracellular H(2)O(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification. Antioxid Redox Signal 7(5–6):619–626CrossRefPubMedGoogle Scholar
  38. Sherstobitov AO, Lapin AV, Glazunov VV, Nikiforov AA (2010) Transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis. Zh Evol Biokhim Fiziol 46(3):198–202PubMedGoogle Scholar
  39. Shimizu N, Kobayashi K, Hayashi K (1984) The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical. J Biol Chem 259(7):4414–4418PubMedGoogle Scholar
  40. Simzar S, Ellyin R, Shau H, Sarafian TA (2000) Contrasting antioxidant and cytotoxic effects of peroxiredoxin I and II in PC12 and NIH3T3 cells. Neurochem Res 25(12):1613–1621CrossRefPubMedGoogle Scholar
  41. Speckmann B, Steinbrenner H, Grune T, Klotz LO (2016) Peroxynitrite: from interception to signaling. Arch Biochem Biophys 595:153–160CrossRefPubMedGoogle Scholar
  42. Sun Y, Oberley LW, Li Y (1988) A simple method for clinical assay of superoxide dismutase. Clin Chem 34(3):497–500PubMedGoogle Scholar
  43. Villaverde MS, Hanzel CE, Verstraeten SV (2004) In vitro interactions of thallium with components of the glutathione-dependent antioxidant defence system. Free Radic Res 38(9):977–984CrossRefPubMedGoogle Scholar
  44. Wagner E, Luche S, Penna L et al (2002) A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress. Biochem J 366(Pt 3):777–785CrossRefPubMedPubMedCentralGoogle Scholar
  45. Wang J, Rahman MF, Duhart HM et al (2009) Expression changes of dopaminergic system-related genes in PC12 cells induced by manganese, silver, or copper nanoparticles. Neurotoxicology 30(6):926–933CrossRefPubMedGoogle Scholar
  46. Wang W-L, Dai R, Yan H-W, Han C-N, Liu L-S, Duan X-H (2015) Current situation of PC12 cell use in neuronal injury study. Int J Biotechnol Well Ind 4(2):61–66CrossRefGoogle Scholar
  47. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND (2004) Glutathione metabolism and its implications for health. J Nutr 134(3):489–492PubMedGoogle Scholar
  48. Yang KS, Kang SW, Woo HA et al (2002) Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid. J Biol Chem 277(41):38029–38036CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Instituto de Biología y Medicina Experimental (IBYME)Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET)Buenos AiresArgentina
  2. 2.Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Química y Fisicoquímica Biológicas (IQUIFIB)Facultad de Farmacia y BioquímicaBuenos AiresArgentina

Personalised recommendations