Cobalt distribution in keratinocyte cells indicates nuclear and perinuclear accumulation and interaction with magnesium and zinc homeostasis
Introduction
Cobalt is an essential trace element for humans, present in vitamin B12 cobalamins, but become toxic at high concentrations, thus leading to adverse health effects (Barceloux, 1999, Lison et al., 2001, De Boeck et al., 2003, Kim et al., 2006). Some compounds of the stable isotope 59Co are classified as possible human carcinogens by the International Agency in Cancer Research (IARC, 2003). Sources of environmental cobalt are both natural and anthropogenic (Barceloux, 1999). Occupational exposure to cobalt occurs in several industries including hard metal manufacturing, welding, and grinding. On the other hand, workers in the nuclear industry can be exposed to cobalt radioisotopes, particularly 60Co (Le Guen and Ansoborlo, 2005, Davis et al., 2007), a neutron activation product. During occupational exposure internal and/or external contamination might occur. Dermal exposure to stable cobalt can induce allergic contact dermatitis in workers exposed to specific occupational conditions (Barceloux, 1999, Lison et al., 2001, Athavale et al., 2007), and cutaneous absorption of radiocobalt is the principal risk of for workers in the nuclear industry (Le Guen and Ansoborlo, 2005).
As the mechanisms of cobalt-induced toxicity on skin have not been fully elucidated, in vitro toxicological studies have been carried out in HaCaT human keratinocyte cell line as a cellular model (Brosin et al., 1997, Ermolli et al., 2001, Bresson et al., 2006). Co(II) compounds can induce DNA damage, DNA cross links, gene mutations, sister chromatid exchanges and aneuploidy in in vitro studies on animal and human cells (for review: Beyersmann and Hartwig, 1992, Hartwig, 1995, Lison et al., 2001). In a previous study (Bresson et al., 2006), effects of cobalt on HaCaT cells were evaluated in terms of cellular viability and impact on the DNA integrity. Cobalt cytotoxicity was assessed through WST-1 and Neutral Red assays, giving EC50 values at 620 and 860 μM, respectively. The aim of the present study was to investigate the intracellular distribution of cobalt in HaCaT cells exposed to CoCl2 at two model concentrations, 40 μM, a non-cytotoxic dose, and 400 μM, a low-toxic dose representative of skin exposures that can be encountered in occupational setting. The specific aims were first to determine how cobalt is distributed in sub-cellular compartments, in order to assess if cobalt could interact directly with nuclear proteins and DNA, and second, to evaluate the effect of cobalt on essential trace elements homeostasis. Indeed, it has been proposed that cobalt could impact on DNA repair mechanisms through its interaction with essential trace metals such as Mg or Zn involved in DNA repair enzymes (Lison et al., 2001).
Three analytical procedures were carried out and compared to determine the sub-cellular distribution of cobalt in HaCaT cells: (i) direct 2D quantitative chemical imaging based upon ion beam microprobe analysis, (ii) direct 3D chemical imaging using synchrotron radiation X-ray fluorescence computed tomography (SR-XFCT), and (iii) indirect analysis coupling cellular fractionation by differential ultracentrifugation followed by elemental analysis by inductively coupled plasma atomic emission spectrometry (ICP-AES). SR-XFCT was applied to single cell analysis for the first time in this study. Through these techniques, quantitative intracellular distribution of cobalt could be assessed, as well as effects induced by this metal on other trace elements within human keratinocytes.
Section snippets
Cell culture
HaCaT cells were a kind gift of Dr Gauthier (GlaxoWellcome). Dulbecco's Modified Eagle Medium (DMEM), supplements and trypsin were purchased from Cambrex (France). Fetal calf serum was from Dutscher (France), l-glutamine, CoCl2, 6H2O and Hanks balanced salt solution (HBSS) buffer were from Sigma (France). Nitric and hydrochloric acid of high purity grade (HNO3 65% normatom and HCl 37% normatom) were purchased from VWR Prolabo (France). HaCaT cells were grown at 37 °C in complete DMEM at pH 7.4
2D sub-cellular imaging of cobalt distribution
At subcytotoxic concentration of CoCl2 (40 μM), the intracellular cobalt content was below the detection limit of the micro-PIXE imaging technique (<5 μg g−1). At 400 μM CoCl2, cobalt is located mainly within the cell nucleus, but also in the cytosol, as presented in Fig. 1. In some cases, cobalt also accumulates within perinuclear structures in the cytosol. The precise location of the cytosol and nucleus was determined by two independent methods. First, by using optical microscopy, cells were
Discussion
Cobalt is known to induce toxic effects on skin cells (Brosin et al., 1997, Ermolli et al., 2001, Bresson et al., 2006). In occupational exposures cobalt can induce contact dermatosis (Athavale et al., 2007), and radiocobalt exposure in the nuclear industry can induce external contamination by skin contact (Le Guen and Ansoborlo, 2005). DNA damage and interaction with DNA repair mechanisms have been proposed to be involved in cobalt toxicity directly or indirectly (Lison et al., 2001, De Boeck
Conclusion
This study has shown that in HaCaT human keratinocyte cells exposed in vitro to cobalt at subcytotoxic concentration (40 μM), the cobalt intracellular content was very low (<5 μg g−1), whereas at a low cytotoxic concentration (400 μM), cobalt intracellular content increased up to 680 μg g−1. More than half of this intracellular cobalt was present in the cell nucleus indicating the possible direct interaction with genomic DNA and nuclear proteins. The remaining cobalt was distributed in the cytosol
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This study was supported by a grant from the national program Nuclear and Environmental Toxicology (TOXNUC-E), by the CNRS and the CEA/DEN/DDIN/MR. We are also grateful to TOXNUC-E program for the postdoctoral fellowships of Caroline Sandre, Ph.D. fellowship of Aurélien Fraysse, and engineering fellowship of Priscillia Soudant. The authors are sincerely grateful to the technical staff at AIFIRA facility (Application Interdisciplinaires des Faisceaux d’Ions en Région Aquitaine), and ESRF
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Present address: CEA, DAM, DIF, F-91297 Arpajon Cedex, France.