REVISED VERSION
HEAVY METALS TOXICITY AFTER ACUTE EXPOSURE OF CULTURED
RENAL CELLS: INTRACELLULAR ACCUMULATION AND REPARTITION
HICHAM KHODJA, MARIE CARRIÈRE, LAURE AVOSCAN, BARBARA GOUGET
Laboratoire Pierre Süe UMR9951, CEA Saclay
91191 Gif sur Yvette, France
hkhodja@cea.fr
ABSTRACT
Lead (Pb), cadmium (Cd) and uranium (U) present no known biological function but are toxic in
various concentration ranges. Pb and Cd lead generally to nephrotoxicity consisting in proximal
renal tubular dysfunction and accumulation while U has been reported to induce chemical kidney
toxicity, functional and histological damages being as well mainly observed in proximal tubule cells.
This work address the question of Cd, Pb, and U cytotoxicity, intracellular accumulation and
repartition after acute intoxication of renal proximal tubule epithelial cells. After cells exposure to
different concentrations of metals for various times, morphological changes were observed and
intracellular concentrations and distributions of toxic metals were specified by PIXE coupled to
RBS. Cell viability, measured by biochemical tests, was used as toxicity indicator. A direct
correlation between cytotoxicity and intracellular accumulation in renal epithelial cells have been
established. Finally, intracellular Pb and U localizations were detected while Cd was found to be
uniformly distributed in renal cells.
Keywords: heavy metals, proximal tubule, nuclear microprobe
1. Introduction
Heavy metals used in various industry sectors may accidentally be absorbed by humans
or animals, either directly or through the food chain. Pb, Cd and U are heavy metals that
are more specifically used in the nuclear industry and share the property of having a
common target organ, the kidney, due to its filtering and excretion functionalities. Pb is
known to produce disruption of the heme synthesis system, nervous system and renal
damages. Cd is considered as the reference nephrotoxic element and its effects are
extremely well documented, both in vivo and in vitro1,2. U use in the industry has raised
concerns regarding the risk associated to a potential contamination. The target at the
kidney level is localized on epithelial cells from proximal tubules3. Mechanisms of
nephrotoxicity are however not well understood. The identification the physico-chemical
state of the heavy metal inside or in the vicinity of the exposed cells will permit to better
understand cellular toxicity after exposure to the toxic.
In this study, we investigated the effect of these metals on two cell lines representative of
the major two parts of the kidney functional unit, namely the nephron. The MDCK cell
line has a distal tubular or cortical collecting duct origin; the NRK52E cell line was used
as a model of the proximal tubule. The aim of the study was focused on establishing
correlations between heavy metals acute exposure, toxicity and intracellular accumulation
and repartition. Parameters were heavy metals concentrations in the culture medium and
exposure times. Various techniques were used to measure the biological effects of the
heavy metals. Among them, PIXE permitted to quantify and localize the heavy metal
accumulation in cellular monolayers.
2. Materials and Methods
2.1. Cell culture and exposure, toxicity tests
MDCK and NRK52E cells were purchased from the American Type Culture Collection
and were routinely grown in 25 cm² flasks in Dulbecco’s Modified Eagle Medium
(DMEM) containing 4.5 g/l glucose and supplemented with 2 mM L-glutamine,
penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) and 10% (v/v) fetal bovine
serum. Cells were maintained at 37°C in a 5% CO2/air incubator. For metal accumulation
measurements, cells were sub-cultured on thin semi permeable polyester membranes
(Transwell Clear from Costar with 0.4 µm pore size). For metal repartition observations
with the NRK52E cell line, cells were seeded onto a thin sterile Pioloform film precoated with poly-L-lysine.
Before metal exposures, cells were systematically rinsed with serum free culture medium.
Cd or Pb was administered as fresh dilutions of cadmium chloride CdCl2 or lead acetate
(CH3CO2)2Pb.3H20 stock solutions (Sigma-Aldrich). Pb precipitation in the culture
medium was controlled by complexing Pb-acetate with glutamic acid in equimolar
amounts. These solutions were directly added to serum, antibiotic, carbonate, phosphate
free DMEM. 10 mM uranyl stock solutions were prepared by dissolution of uranyl nitrate
in 100 mM NaHCO3, stirred for 30 min and filtered through 0.22 µm filter units after pH
adjustment to 7.2. This stock solution was diluted in Minimum Essential Medium
(MEM).
For toxicity assays, cells were sub-cultured in 96-well plates, seeded at 6000 cells per
well. Confluent monolayers, i.e. three days after subculture, were exposed to different
concentrations of metals. Then, the colorimetric methylthiazol tetrazolium (MTT) assay
was used to evaluate metal cytotoxicity4.
2.2. PIXE and RBS measurements
Ion Beam Analyses were carried out with two goals: quantifying intracellular
concentrations of the toxic metals and localizing their intracellular repartition. After
exposure to the toxics, monolayer cell cultures were rinsed, cryofixed in –165 °C chilled
isopentane and freeze-dried as described previously4.
Measurements were all performed at the Nuclear Microprobe at Saclay5. Two run
conditions were used, depending on the aim of the experiment: the first one was
optimized for quantification (3 MeV proton beam, 1 nA beam current, 10 x 10 µm² beam
size and 1 mm² scanning area), while the second one was dedicated to cellular level
observations (3 MeV beam, 50 pA 1 x 1 µm² beam size).
PIXE detection was performed using, for Cd exposures, a Si(Li) x-ray detector collimated
to 38 mm2, in front of which a 150 µm Mylar foil was positioned to remove backscattered
particles. The detector was positioned at the minimal distance (21mm) from the sample in
order to optimize Cd x-ray detection. For Pb and U exposures, a high area and low energy
High Purity Germanium detector has been employed. It was collimated to 50 mm² and
located at 21 mm from the samples.
In all experiments, RBS spectra were simultaneously collected, either for monitoring and
normalization purposes, or for cellular and sub-cellular localization. In that case, large
area charged particles detector (180 mm²) was mounted close to the sample (8 mm
distance) thus offering a large -but not evaluated- solid angle. For quantification
measurements, PIXE spectra normalization was based on the nitrogen contents
evaluation, method described elsewhere6.
Data were acquired in list mode and extracted from useful areas using the RISMIN
software6. Spectra were processed using GUPIX software7 for PIXE and SIMNRA
software8 for RBS.
3. Results and Discussion
3.1. Heavy metals toxicity
Cd, Pb and U toxicities were investigated using the MTT assay. Fig.1 shows the variation
of cellular mortality after 24 hr of exposure to increasing concentrations of the toxic. Cd
induced cytotoxicity even at very low concentration. The inhibitory concentration IC50,
defined as the toxic concentration leading to 50% cell death, was determined to be 60 µM
for the MDCK cells exposed to Cd. Pb cytotoxicity to the NRK52E cells started at 100
µM and presented an IC50 of 350 µM. U cytotoxicity presented an IC50 of 700 µM for the
NRK52E cell line.
100%
Toxicity
80%
60%
Cd (MDCK)
Pb (NRK)
U (NRK)
40%
20%
0%
0
200
400
600
800
1000
[Cd], [Pb], [U] (µM)
Fig.1. Cd, Pb, U cytotoxicity measured by the MTT assay on MDCK and NRK52E cell lines after 24 hr of
exposure.
3.2. Heavy metals intracellular accumulation
After exposure to the toxics, intracellular accumulation was quantified by PIXE as
described upper. Fig.2 presents the results obtained with the MDCK cells exposed to
various Cd concentrations for 6 hr: incorporated Cd remained in the range of several
thousands of ppm. Cd concentrations were determined with the K ray intensity,
notwithstanding the low K-shell ionization cross section compared to the L-shell one.
Indeed, overlaps between Cd L and Ca K lines occured and background levels were
too high high, thus reducing considerably the detection limits.
Cd (ppm)
2000
1600
1200
800
400
0
0
20
40
60
80
100
120
[Cd] (µM)
Fig.2. Cd accumulation in MDCK cell line extracted from PIXE measurements after 6h exposition to toxic.
After Pb and U exposures, heavy metal concentrations were extracted from the L line
intensities. In Fig.3 are given concentrations of Pb and Cd accumulated after 24 hr
exposure of the NRK52E cells. Accumulated concentrations at the IC50 thresholds were
as high as 0.5 % wt. for Pb and 10 % wt. for U.
25000
250000
U
20000
200000
15000
150000
10000
100000
5000
50000
0
0
500
1000
U (ppm)
Pb (ppm)
Pb
0
1500
[Pb] or [U]
Fig.3. Pb and U accumulation in NRK52E cell line extracted from PIXE measurements after 24 hr exposition to
toxics.
3.3. Heavy metals intracellular repartition
Intracellular repartition of heavy metals strongly differed with the considered toxic.
Experiments with MDCK exposed to Cd showed that this element was uniformly
distributed within cells with no apparent concentration inhomogeneity. Fig.4 presents the
elemental maps of MDCK cells exposed for 6 hr to Cd, potassium and calcium x-ray
signals acting as cell indicators.
Fig.4. Elemental maps of MDCK cells exposed for 6 hr to Cd. Left: K and Ca K x-ray map. Right: Cd K xray map. Cd is uniformly present inside cells. Scanning parameters: 60x60 µm², beam spot 1x1 µm², total
charge 2 µC.
10 µm
LEAD
URANIUM
NITROGEN
By contrast, Pb and U repartitions in NRK52E cells presented a different behavior.
Indeed, two physical forms of the toxics were found. Precipitates were visible even at low
toxic concentration and they could be found inside the cells or in their vicinity. For U, it
was already demonstrated in vivo that thin needles of uranium phosphate were
concentrated in proximal tubule cells. The mechanism of their formation was not clearly
specified. Two hypotheses were underlined: direct internalization of these precipitates or
precipitation inside the cytoplasm of the cells9,10. On Fig.5 are shown RBS elemental
images of NRK52E cells exposed to Pb and U. Precipitates are observed close to the
cells. A more detailed analysis of the spectra confirms that both metals are detected also
outside the precipitate region, indicating the presence of a soluble form of the toxics
inside the cells11.
10 µm
Fig.5. Elemental maps of NRK52E exposed 24 hr to 600 µM U (left) and 180 µM (right). Scales are modified
to enhance nitrogen, uranium and lead contents. Scanning parameters: 60x60 µm², beam spot 1x1 µm².
3.4. Discussion
The results obtained in this study establish a clear correlation between Cd, Pb and U
cytotoxicity and heavy metal accumulation in MDCK and NRK52E cells. Cd is more
toxic than Pb, and U is less toxic than Pb. Intracellular accumulation is correlated to
heavy metal sensitivity. Cd is much less accumulated than Pb. The high level of Pb
accumulation has already been established in vivo. It has been shown that kidneys play an
important role in regulating blood Pb levels by accumulating more Pb than other soft
tissues. And Pb has been shown to be more avidly taken up by renal cells than Cd12. U
accumulation reaches extremely high levels in the conditions used in this study.
However, examining the intracellular physical forms of the heavy metals, it should be
noticed that high accumulation levels (Pb, U) are correlated to a partially precipitate form
of the heavy metal, which probably contents an important part of the toxic. The
chemically active soluble form concentration is necessarily lower than the measured one.
By contrast, when Cd is used, it induces toxicity at much lower levels but Cd seems to be
uniformly distributed in the cell.
4. Conclusion
Heavy metal toxicity to cultured renal cells has been investigated by means of PIXE. A
broad beam has been used for heavy metal accumulation measurements and microbeam
has been used for intracellular repartition observations. A correlation between heavy
metal toxicity and cellular accumulation has been established. Cd has been found more
toxic than Pb, itself more toxic than U. Intracellular accumulation is higher with U than
Pb than Cd. When Cd is used, it is uniformly distributed in cells while with Pb and U, an
important part of the toxic is precipitated.
Acknowledgments
This work was supported by the French program ToxNuc-E.
References
1. L. Friberg, C.G. Elinder, I. Kjellström, G. Nordberg, Cadmium and Health: A toxicological and
epidemiological appraisal Vols. 1-2, (CRC Press, Boca Raton, FL, 1986)
2. I. Olabarrieta, B. L'Azou, S. Yuric, J. Cambar, M.P. Cajaraville, Toxicol. In Vitro 15 (2001) 173.
3. T. Mossman, J. Immunol. Methods 65 (1983) 55.
4. H. Khodja, M. Carrière, L. Figard, F. Carrot, B. Gouget, Nucl. Ins. Meth. B210 (2003) 359.
5. H. Khodja, E. Berthoumieux, L. Daudin, J.P. Gallien, Nucl. Ins. Meth. B181 (2001) 83.
6. L. Daudin, H. Khodja, J.P. Gallien, Nucl. Ins. Meth. B210 (2003) 153.
7. J.L. Campbell, T.L. Hopman, J.A. Maxwell, Z. Nejedly, Nucl. Inst. and Meth. B170 (2000) 193.
8. M. Mayer, SIMNRA User's Guide, Technical Report IPP 9/113, Max-Planck-Institut fur
Plasmaphysik, Garching, Germany, 1997.
9. Leggett, R. W. Health Phys. 57 (1989), 365.
10. P. Galle, in Toxiques Nucléaires ed. P. Galle (Masson, France 1982), p. 224.
11. M. Carrière, L. Avoscan, R. Collins, F. Carrot, E. Ansoborlo, B. Gouget, Chem. Res. Toxicol.
17 (2004) 446.
12. M.G. Cherian In vitro Cell Dev. Biol., 9 (1985) 505.