Beiträge zur Bestimmung von lokalen Verteilungen ausgewählter toxischer Elemente

[no abstract

Platinum corrosion products from electrode contacts of human cochlear implants induce cell death in cell culture models

Despite the technological progress made with cochlear implants (CI), impedances and their diagnosis remain a focus of interest. Increases in impedance have been related to technical defects of the electrode as well as inflammatory and/or fibrosis along the electrode. Recent studies have demonstrated highly increased impedances as the result of corroded platinum (Pt) electrode contacts. This in vitro study examined the effects of Pt ions and compounds generated by corrosion of the electrode contacts of a human CI on cell metabolism. Since traces of solid Pt in surrounding cochlear tissues have been reported, the impact of commercially available Pt nanoparticles (Pt-NP, size 3 nm) on the cell culture model was also determined. For this purpose, the electrode contacts were electrically stimulated in a 0.5% aqueous NaCl solution for four weeks and the mass fraction of the platinum dissolute (Pt-Diss) was determined by mass spectrometry (ICP-MS). Metabolic activity of the murine fibroblasts (NIH 3T3) and the human neuroblastoma (SH-SY5Y) cells was determined using the WST-1 assay following exposure to Pt-Diss and Pt-NP. It was found that 5-50 μg/ml of the Pt-NP did not affect the viability of both cell types. In contrast, 100 μg/ml of the nanoparticles caused significant loss in metabolic activity. Furthermore, transmission electron microscopy (TEM) revealed mitochondrial swelling in both cell types indicating cytotoxicity. Additionally, TEM demonstrated internalized Pt-NP in NIH 3T3 cells in a concentration dependent manner, whereas endocytosis in SH-SY5Y cells was virtually absent. In comparison with the Pt-NP, the corrosion products (Pt-Diss) with concentrations between 1.64 μg/ml and 8.2 μg/ml induced cell death in both cell lines in a concentration dependent manner. TEM imaging revealed both mitochondrial disintegration and swelling of the endoplasmic reticulum, suggesting that Pt ions trigger cytotoxicity in both NIH 3T3 and SH-SY5Y cell lines by interacting with the respiratory chain

Ultrastructural morphology of NIH 3T3 cells following exposure to Pt-Diss.

<p>After cultivation either without Pt as reference (<b>A</b>) or in culture medium containing 6.0 μg/ml (<b>B</b>), 0.11 μg/ml (<b>C</b>) or 0.02 μg/ml Pt (<b>D</b>) the ultrastructure of 3T3 fibroblasts was compared. They demonstrated mitochondrial swelling (arrow in <b>B</b>) only at the highest tested Pt concentration. At 0.11 μg/ml Pt the cells showed greater phagocytic activity (arrowhead in <b>C</b>). Lower amount of Pt in the culture medium induced no morphological changes in comparison with the control. Size of bars: 2 μm.</p

Microscopic characterization of the morphology of NIH 3T3 cells following exposure to Pt-NP.

<p>NIH 3T3 cells were cultivated either without any additional Pt particles as reference (<b>A</b>) or in culture medium containing 25 μg/ml (<b>B</b>), 50 μg/ml (<b>C</b>) and 100 μg/ml (<b>D</b>) of the Pt-NP. The images demonstrated highly uniform cell adhesion without any morphological impairment throughout the cell cultures assays with varying Pt-NP concentrations. Size of bars: 200 μm.</p

Determination of the effects of Pt-Diss with varying Pt-Diss concentrations in cell cultivation.

<p>Metabolic activity of both NIH 3T3 (<b>A</b>) and SH-SY5Y cells (<b>B</b>) grown in culture medium supplied with 0.82 μg/ml– 8.2 μg/ml Pt-Diss concentration was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–16; SH-SY5Y, n = 10). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SE_M. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).</p

Determination of the effects of Pt-NP with varying concentrations in cell cultivation.

<p>Metabolic activity of both NIH 3T3 (<b>A</b>) and SH-SY5Y cells (<b>B</b>) grown in culture medium supplied with 5 μg/ml– 100 μg/ml Pt-NP was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–14; SH-SY5Y, n = 11–15). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SE_M. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).</p

Microscopic characterization of the morphology of NIH 3T3 cells following exposure to Pt-Diss.

<p>NIH 3T3 cells were cultivated either without any additional Pt-Diss as reference (<b>A</b>) or in culture medium containing 8.2 μg/ml (<b>B</b>), 4.1 μg/ml (<b>C</b>), 1.64 μg/ml (<b>D</b>) and 0.82 μg/ml (<b>E</b>) of the Pt components. Microscopic images demonstrated emerging cytotoxic effects of Pt-Diss in a concentration-dependent manner between 1.64 μg/ml and 8.2 μg/ml Pt-Diss concentration. Beyond Pt-Diss concentration of 1.64 μg/ml cell adhesion and morphology appeared normal, whereas a Pt-Diss concentration of 8.2 μg/ml strongly reduced NIH 3T3 cell growth and probably induced cell death. Size of bars: 200 μm.</p

Ultrastructure of the SH-SY5Y cell line cultivated in absence and presence of Pt-Diss.

<p>After cultivation either without any Pt as reference (<b>A</b>) or in culture medium containing 6.0 μg/ml (<b>B</b>), 0.11 μg/ml (<b>C</b>) or 0.02 μg/ml Pt (<b>D</b>) SH-SY5Y were characterized by large euchromatic nucleus, abundant endoplasmic reticulum and a few synaptic granules (arrowheads). At the highest tested Pt concentration these cells were adversely affected, as proven by mitochondrial swelling (arrow in <b>B</b>). A smaller amount of Pt in the culture medium induced no morphological changes in comparison with the control. Size of bars: 2 μm.</p

Microscopic characterization of the morphology of SH-SY5Y cells following exposure to Pt-NP.

<p>SH-SY5Y cells were cultivated either without any additional Pt particles as reference (<b>A</b>) or in culture medium containing 25 μg/ml (<b>B</b>), 50 μg/ml (<b>C</b>) and 100 μg/ml (<b>D</b>) of the Pt-NP. Morphology and adhesion behavior of the SH-SY5Y cells did not change throughout the cultivation assays at varying Pt-NP concentrations. Size of bars: 200 μm.</p

Microscopic characterization of the morphology of SH-SY5Y cells following exposure to varying NaCl concentrations.

<p>SH-SY5Y cells were cultivated either in normal cell culture medium containing 6.4 mg/ml NaCl as reference (<b>A</b>) or in culture medium containing 6.26 mg/ml (1:10) (<b>B</b>), 6.12 mg/ml (1:5) (<b>C</b>) and 5.93 mg/ml (1:3) NaCl (<b>D</b>). Microscopic images demonstrated reduced cell attachment and growth in a concentration dependent manner without any signs of cytotoxicity.</p