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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

Abstract

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.

Keywords

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

Abbreviations

Apocynin

4-Hydroxy-3-methoxyacetophenone

CAT

Catalase

CuZnSOD

CuZn-superoxide dismutase

DHE

Dihydroethidine

DHR123

Dihydrorhodamine 123

DMEM

Dulbecco’s modified Eagle medium

DPI

Diphenylene-iodonium chloride

DTNB

Dithionitrobenzoic acid

GPx

Glutathione peroxidase

GR

Glutathione reductase

GSH

Glutathione

GSSG

Glutathione disulfide

iNOS

Inducible NOS

nNOS

Neuronal NOS

L-NAME

NG-nitro-l-arginine methyl ester

mClBi

Monochlorobimane

MnSOD

Mn-superoxide dismutase

MTT

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

NBT

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

NOS

Nitric oxide synthase

NOX

NADPH oxidase

PBS

Phosphate buffered saline

PC12adh

PC12 cells, adherent variant

PI

Propidium iodide

Prx

Peroxiredoxin

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

SDS

Sodium dodecyl sulfate

TrxR

Thioredoxin reductase

Notes

Acknowledgements

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)

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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

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