1. Anne Heller, Jannette Wober
Cytotoxicity of rare earth elements (REE) towards mammalian renal epithelial cells
Anne Heller, Jannette Wober

2. Motivation Rare Earth Elements = family of 16 elements
naturally occuring elements (0.02 % of earth crust)
widely used in high-tech industry (fuel cells, catalysts, lasers), metallurgy, medicine (MRI contrast agents) & agriculture (fertilizers)
extensive exploitation and release into the environment
REE concentrations in natural and drinking waters
∑ [REE] = 0.05 – 10 µg/L (Germany, France, China)[1-3]
single [REE] = 0.01 – 8 µg/L[1-3]
∑ [REE] = 30 – 110 µg/L (China, mining areas)[1]
( )
( )
∑ [REE] = 0.05 – 10 µg/L ~ 10-9 – 10-7 M (nanomolar range)
∑ [REE] = 0.03 – 0.11 mg/L ~ 10-7 – 10-6 M (micromolar range)
[1] Zhang et al. (1999) Biolog. Trace Elem [2] Négrel et al. (2000) Appl. Geochem [3] Kulksiz et al. (2013) Earth Plan. Sci. Lett. 362

3. Motivation entrance of REE into the food chain & uptake
possible health risk to humans due to
heavy metal toxicity
Calcium analogy
enrichment in body fluids and tissues (∑[REE] = 5 – 644 µg/L in blood)[1,2]
excretion very low (5 – 10 %) but mainly with urine[3]
[1] Li et al. (2014) Environ. Earth Sci [2] Yuan et al. (2003) Chin. Publ. Health 19 [3] Bingham and Dobrota (1994) Biometals 7

4. Objectives studying the interaction of REE with mammalian kidney cells
determination of the cellular tolerance towards REE and cytotoxicity of REE as a function of
REE concentration
incubation time
REE speciation
comparison within the REE family
improvement of risk assessment and knowledge about the biological behavior of REE on a cellular level

5. Cell line epithelial-like normal rat kidney cells (NRK-52E)[1]
purchased from DSMZ (ACC No. 199)
cultured in DMEM + 10 % FBS, 4 mM Gln and 1 % Pen/Strep at 37 °C, 95 % rH & 5 % CO2
adherent mono-layer
doubling time ~14 h
flat polyhedral cells with distinct nuclei
typical cobblestone shape
[1] de Larco et al. (1978) J. Cell. Physiol. 94 (3)

6. solubility of REE in medium[1]
Cell-free experiments
10-7 M
24 / 48 h
37 °C, 95 % rH & 5 % CO2
solubility of REE in medium[1]
filtration through 0.45 µm sterile filters
measurement of REE concentration with ICP-MS (after acidification)
10-3 M
12-well plates with
cell culture medium + REE
[1] Sachs et al. (2015) Toxicol. In Vitro 29 (7)

7. Solubility of REE in medium
unpublished results, distribution prohibited
no precipitation observed after incubation for 24 and 48 h
all REE 100 % soluble in cell culture medium up to 10-3 M
applied REE concentration is fully bioavailable for the cells
no difference within REE family
unpublished results, distribution prohibited

8. cell viability / cytotoxicity
Cell culture experiments
morphology
immunocytochemistry +
fluorescence microscopy
nuclei staining with DAPI
actin staining (cytoskeleton) with phalloidin conjugate
REE exposure
8-chamber slides, 24 h,
37 °C, 95 % rH & 5 % CO2
REE
exposure
96-well plates,
37 °C, 95 % rH & 5 % CO2
all studies were performed
as independent experiments
with each control/sample in quadruplicate
statistical evaluation
outlier detection by Grubbs‘ test
check for significances by one-way ANOVA with Tukey post hoc test[2]
cell viability / cytotoxicity
XTT test[1] (mitochondrial activity)
dose response curves (10-9 – 10-3 M)
time dependent studies (8 – 48 h)
[1] Scudiero et al. (1988) Cancer Res. 48 (17) [2] Tukey (1949) Biometrics 5 (2)

9. Dose response curves (24 h exposure)
unpublished results, distribution prohibited
for all REE, loss of cell viability at concentrations > 10-4 M
REE cytotoxicity is concentration-dependend
onset of significant cytotoxic effects* varies from 4 ∙ 10-4 M (Ce) to 10-3 M (most REE)
* one-Way-ANOVA with Tukey post hoc test; p < 0.05 (95 % confidence)
unpublished results, distribution prohibited

10. Comparison within REE family
unpublished results, distribution prohibited
cell viability after 24 h exposure with 10-3 M REE
EC50 values (µM)
Ce is significantly more cytotoxic then other REE La Ce Nd Eu Tb Er Yb
967 ± 10
528 ± 28
1240 ± 116
1480 ± 456
946 ± 20
952 ± 34
902 ± 24
1800 ± 180[1]
* one-Way-ANOVA with Tukey post hoc test; p < 0.05 (95 % confidence) [1] Sachs et al. (2015) Toxicol. In Vitro 29 (FaDu cells)
unpublished results, distribution prohibited

11. Time-dependent studies
unpublished results, distribution prohibited
dose response curves for Ce
cell viability at 10-3 M REE
dose response curves exhibit a time-independent manner
cell viability at 10-3 M REE and EC50 values are also very similar
cell viability and REE cytotoxicity show no significant dependence on exposure time
unpublished results, distribution prohibited

12. Cell morphology (light microscopy)
unpublished results, distribution prohibited
untreated cells M Ce/Eu
reduced cell density and loss of cell-cell contact
shrinking and rounding of cells
morphological changes start to occur at 10-5 M and are significantly observable at 10-3 M REE
unpublished results, distribution prohibited

13. Cell morphology (ICC)
unpublished results, distribution prohibited
untreated cells M Ce/Eu
staining with
DAPI  nuclei
Phalloidin  actin/cytoskeleton
reduced cell density and loss of cell-cell contact
shrinking and rounding of cells
morphological changes start to occur at 10-5 M and are significantly observable at 10-3 M REE
generally, analog morphological alterations upon Ce and Eu exposition but to a higher extent for Ce
unpublished results, distribution prohibited

14. Summary / Take home messages
unpublished results, distribution prohibited
REE are able to induce morphological changes in NRK-52E cells and a loss of cell viability
cytotoxicity of REE is concentration-dependent but independent from exposure time
cytotoxic effect is caused by soluble REE species
out of all REE, Ce is particularly cytotoxic
at environmental concentrations (nanomolar range), REE exhibit no cytotoxic effect onto NRK-52E cells
at elevated concentrations in mining or industrial areas and REE levels in blood serum of exposed workers (micromolar range), especially Ce starts to be cytotoxic to NRK-52E cells
blood level: 600 µg/l = 4.3 µM [Ce]
unpublished results, distribution prohibited

15. Outlook / Open questions
unpublished results, distribution prohibited
What is the pathway/mechanism of REE cytotoxicity?
apoptosis vs. necrosis vs. alteration of proliferation
detection by selective staining + fluorescence microscopy
quantification by assays & flow cytometry
determination of the intracellular uptake of REE
Is there a correlation between chemical and in-vitro properties to explain the significantly higher cytotoxicity of Ce?
determination of the speciation of selected REE in cell culture medium by spectroscopy
quantification of the protein-bound REE fraction
Is the actinide-lanthanide analogy valid for renal cells?
random analog experiments with radioactive Am
apoptosis = programmed cell death; necrosis = traumatic cell death; proliferation = cell cycle
Staining: Ethidium homodimer III (necrosis), Annexin V or fluoerescin (apoptosis), Bromdesoxyuridin (proliferation)
Assays: dead-cell-protease (necrosis), caspase 3/7 (apoptosis), Bromdesoxyuridin (proliferation)
unpublished results, distribution prohibited

16. Acknowledgement Founded by the DFG under contract number HE 7054/2-1.
Thanks to S. Gurlit (HZDR/IRE) for ICP-MS measurements.
Special thanks go to all collegues from „Molecular Cell Physiology and Endocrinology“.
Oliver Zierau, Georg Kretzschmar, Nicole Dressel, Anne-Kathrin Keiler, Prof. Günter Vollmer,
Miriam Stürze, Sebastian Müller
Carolina Blum, Katrin Richter,
Antje Beyer, Renate Linke,
Lotta Dittmann, Jannette Wober,
Susanne Broschk, Matthias Langner,
Pia Bräuer