SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 92 ( 20 0 8 ) 5 0–5 8

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

Autophagy as an ultrastructural marker of heavy metal toxicity

in human cord blood hematopoietic stem cells

Mario Di Gioacchino a,b,⁎, Claudia Petrarca a,b , Angela Perrone a,b , Massimo Farina d ,
Enrico Sabbioni d , Thomas Hartung d , Simone Martino e , Diana L. Esposito a,c ,
Lavinia Vittoria Lotti e , Renato Mariani-Costantini a,c
a
Aging Research Center, “G. d'Annunzio” University Foundation, Via Colle dell'Ara, 66100 Chieti, Italy
b
Medicine and Science of Ageing University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti, Italy
c
Oncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti, Italy
d
European Commission, Institute of Health and Consumer Protection, ECVAM Unit, Joint Research Centre, Ispra, Varese, Italy
e
Department of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy

AR TIC LE I N FO ABS TR ACT

Article history: Stem cells are a key target of environmental toxicants, but little is known about their
Received 20 June 2007 toxicological responses. We aimed at developing an in-vitro model based on adult human
Received in revised form stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the
26 October 2007 responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI])
Accepted 5 November 2007 and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were
Available online 31 December 2007 exposed for 48 h to 0.1 μM and 10 μM Cr(VI) or Cd. Cultures treated with 10 μM Cr(VI) or Cd
showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent
Keywords: autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with
Autophagy mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and
Cadmium Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not
Chromium show the morphologic hallmarks of apoptosis. Treatment with 0.1 μM Cr(VI) or Cd did not
CD34+ cells result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum
Stem cells and evidence of mitochondrial damage. We conclude that autophagy is implicated in the
Umbilical cord blood response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd.
In-vitro assay Autophagy, which mediates cell survival and death under stress, deserves further
Toxicity evaluation to be established as biomarker of metal exposure.
© 2007 Elsevier B.V. All rights reserved.

1. Introduction dysfunction, cardiovascular disease and increased risk of cancer

Heavy metals are bio-persistent toxic pollutants that accumulate
in organisms at the top of food chains (Scheifler et al., 2006). Their
widespread environmental diffusion, due to industrial and
consumer waste and leakage from soils, raises concerns about
health hazards (Hirano and Suzuki, 1996). Human exposure occurs
through polluted air, water and/or food and has been linked to
neurodegenerative and developmental disorders, reproductive
(EEA, 2005). However, various factors can confound associations
between exposure to heavy metals and diseases (Goyer, 1986;
Hirano and Suzuki, 1996). Furthermore, as is the case of other
toxicants, risk assessment is hindered by the lack of a toxicity test
that faithfully reproduces human conditions (Combes and Balls,
1999). At present, in vivo tests entail inter-species extrapolation,
while in-vitro assays fail to identify non-genotoxic carcinogens
and, being mostly conducted on immortalized cell lines, may not

⁎ Corresponding author. Aging Research Center, “G. d'Annunzio” University Foundation, Via Colle dell'Ara, 66100 Chieti, Italy. Tel.: +39
0871 541291; fax: +39 0871 541300.
E-mail address: m.digioacchino@unich.it (M. Di Gioacchino).

0048-9697/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2007.11.009
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 92 ( 20 0 8 ) 5 0–5 8
reflect the responses of normal human cells (Combes and Balls,
1999). Accordingly, new in-vitro models for carcinogenicity, based
on human cells, need to be developed in order to better understand
the mechanisms of cell transformation and carcinogenesis
induced by metal compounds.
Increasing evidence indicates that adult stem cells (ASCs),
which are progenitor cells capable of both self-renewal and multi-
potent differentiation (Rando, 2006), have been implicated in
chronic inflammatory diseases and cancer (Reya et al., 2001;
Dalerba et al., 2007; Dietrich and Kempermann 2006). ASCs may
represent a key target of toxicants due to their comparatively
relaxed DNA repair functions, which could facilitate mutagenesis
after toxic exposures (Trosko and Tai, 2006). The toxicological
responses of ASCs are relatively unknown, but might differ from
those of other cells because of ASCs' unique biological properties
linked to their role in tissue renewal and repair (Rando, 2006), and
the early stage of their biochemical processes and intracellular
compartments (Inoue et al., 2002). Therefore, assays based on
human ASC models could improve risk assessment of exposure to
heavy metals. Human hematopoietic stem cells (HHPCs), which
are easily isolated from umbilical cord blood (UCB) without
discomfort or risks to donors, could be a well-characterized ASC
model (Davila et al., 2004) for toxicological and carcinogenicity
studies.
The aim of our study was to investigate whether ex-vivo
expanded UCB HHPCs could be used to evaluate the cytotoxicity
induced by exposure to toxic metals. In particular, we studied the
toxicity of hexavalent chromium (Cr[VI]) and cadmium (Cd), two of
the best known toxic and carcinogenic heavy metal ions (ATSDR
1998, 1999).
Cr[VI] and Cd are powerful oxidizing agents that readily enter
cells, where they are subject to metabolic reduction. Endogenous
oxidants generated by this process may attack the cellular protein
machinery, organelles and nucleic acids (Pritchard et al., 2005;
Rossi and Wetterhahn, 1989; Wetterhahn et al., 1989; Stearns and
Wetterhahn, 1994; Yano and Marcondes, 2005). In turn, damaged
mitochondria, by producing superoxide, may further enhance
oxidative stress, eventually leading to apoptotic cell death
(Blankenship et al., 1994; Evan and Vousden, 2001; Golstein and
Kroemer, 2007).
Transmission electron microscopy (TEM) showed that Cr(VI)-
and Cd-exposed UCB HHPCs respond by activating autophagy, a
specific intralysosomal degradation pathway that controls cell fate
by allowing the turnover of damaged long-lived cellular macro-
molecules and organelles (Cuervo, 2004; Levine and Klionsky, 2004;
Abedin et al., 2007).
2. Materials and methods
2.1. Chemicals
Sodium chromate (Na2CrO4·4H2O, [10034-82-9]) and cadmium
chloride, two highly soluble salts containing Cr(VI) and Cd
respectively, were supplied by Alpha Aesar (Karlsruhe, Germany).
The salts were analysed for elemental impurities and, in the case
of chromate, for oxidation state by HPLC-inductively coupled
plasma mass spectrometry (Perkin-Elmer SCIEX, Ontario, Canada).
These analyses (data not reported) showed that both salts had a
high degree of purity. All other chemicals were of analytical grade.
51
Chromium and cadmium salts (10 mM) were dissolved in MilliQ
water, the freshly-prepared mother solutions were sterilised using
0.2-μm filters (Millipore, Italy) and diluted in complete culture
medium to obtain sub-toxic (0.1 μM) and IC50 (10 μM) concentra-
tions of each metal ion. These concentrations were selected based
on previous studies conducted in human and murine cell systems
(Burastero et al., 2006; Mazzotti et al., 2002; Ceriotti, in press).
2.2. Purification of CD34+ UCB HHPCs
UCB samples (20–60 ml) were collected, with informed consent of
donors, from full-term deliveries at the Department of Gynaecol-
ogy and Obstetrics, SS. Annunziata Hospital, Chieti. UCB was
drained by gravity and gentle squeezing from the maternal end of
the severed cord into sterile flasks filled with 40 ml of endotoxin-
free phosphate-buffered saline (PBS) pH 7.4 (Celbio, Milan, Italy)
containing 1% D-glucose (Invitrogen, Milan) and 10 U/ml heparin
(Bristol-Myers Squibb, Milan). Samples were promptly mixed,
adjusted to 3/1 medium/UCB volumes if necessary and stored at
4 °C up to 12 h.
Mononuclear cells were then isolated by density gradient
centrifugation on lymphocyte separation medium with a density
of 1.077 (Lymphoprep, Sentinelle, Milan), washed twice with
running buffer containing 2 mM EDTA and 0.1% BSA in PBS, pH
7.4 (Miltenyi Biotech, Bologna, Italy), and re-suspended in the same
buffer. CD34+ cells were isolated using the magnetic immunose-
paration MACS system (Miltenyi Biotech), according to the
manufacturer's specifications. Collected cells were counted and
an aliquot of 1.0×105 cells in 200 μl was analysed by flow
cytofluorimetry as described below. Remaining cells were
expanded in culture.
2.3. Ex-vivo expansion and Cr(VI) and Cd treatments
CD34+ HHPCs were cultured at densities between 2.5 and 5.0×105/
ml in 24-well plates (Diagramma, Pescara, Italy) containing 1.5 ml
of StemSpan serum-free medium (StemCell Technologies, Van-
couver, Canada) supplemented with 100 units/ml penicillin G,
0.1 mg/ml streptomycin (Celbio), and Stemspan Cytokine Cocktail
CC110 (StemCell Technologies) containing 100 ng/ml of each
cytokine stem cell factor, of thrombopoietin and of flt-3 ligand.
Cells were expanded at 37 °C in humidified atmosphere with 5%
CO2. Adherent cells (possible non-hematopoietic contaminants)
were discarded. Cultures were observed in phase contrast every
3 days and images were recorded with a digital camera. Cells were
counted (as described below) and diluted 1:2 with complete fresh
medium.
HHPCs grown in 24-well plates (5×105 cells/well in 1.5 ml of
complete culture medium) were exposed in parallel for 48 h to 0.1
and 10 μM Cr(VI) and Cd. Untreated cultures were set-up in parallel
and counted every 24 h. At the end of the exposure period, treated
and control cultures were examined at the phase contrast
microscopy, floating cells were harvested, and cell viability and
number assessed by trypan blue exclusion. To minimize cell
waste, cells were carefully re-suspended, 20 μl of cell suspension
were diluted 1:1 in PBS-0.4% trypan blue and 20 μl of the mix was
used to fill a Neubauer counting chamber. Unstained and stained
cells were counted. Data are reported as mean of triplicate
independent counts. Aliquots from each culture were used for
cytofluorimetry and TEM.
52 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 92 ( 20 0 8 ) 5 0–5 8

2.4. Cytofluorimetry
Analyses were conducted on both freshly isolated and ex-vivo
expanded cells (days 3, 5, 7, 10 of culture). Cr(VI)/Cd-exposed
cultures were analysed before (day 5) and after (day 7) exposure.
Cells (1×105) were incubated with 5 μl of either fluorescein-
isothiocyanate (FITC)-conjugated anti-CD34 MoAb (AC136, Milte-
nyi Biotech) or FITC-conjugated, isotype-matched control MoAb
(MOPC-21, Miltenyi Biotech) in 100 μl of PBS for 20 min at 4 °C in
the dark. Cells were collected by centrifugation (10 min at 200 ×g),
re-suspended in 200 μl PBS and analysed using a FACScalibur
instrument (Becton Dickinson, Milan). Forward and side scatter
parameters were used to exclude dead cells and debris. Data were
analysed with the CELLQuest software (Becton Dickinson). At
least 10,000 events were collected for each analysis.
2.5. TEM
HHPCs were fixed overnight at 25 °C in 2% paraformaldehyde-2%
glutaraldehyde in PBS, post-fixed in 1% osmium tetroxide in
veronal acetate buffer (pH 7.4) for 2 h at 25 °C, stained with uranyl
acetate 2% (5 mg/ml), dehydrated in acetone and embedded in
Epon 812. Thin sections were examined under a Philips CM10
TEM after post-staining with uranyl acetate and lead hydroxide.

Fig. 1 – Ex-vivo expansion of CD34+ UCB HHPCs. Panel A: Phase contrast microscopy of cells from the same well at days 1 and 5 of
culture. Cell proliferation and cluster formation are clearly evident (original magnification ×10). Panel B: growth curves of HHPC
cultures from 5 different donors. Exponential growth rates were observed from day 4 of culture.
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 92 ( 20 0 8 ) 5 0–5 8 53

Fig. 2 – CD34+ FACS profiles of HHPCs from two donors (CB#9, CB#10), after isolation (day 0) and at day 7 of culture. A decrease in
CD34 expression with time of culture is shown by the lower mean FL1-H value of profiles at day 7. Note the significant decrease
in CD34+ cells and the appearance of a CD34− sub-population in CB#10.

Fig. 3 – Conventional electron microscopy of thin sections of ex-vivo expanded HHPCs at day 7 of culture. Cells show round or
slightly indented nuclei and secretory compartments formed by the Golgi apparatus with emerging granules filled by
electron-dense material and vesicular structures. Numerous well-conserved mitochondria are evident. PM: plasma membrane,
N: nucleus; G: Golgi, M: mitochondria, er: endoplasmic reticulum, bar = 1 μm.
54 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 92 ( 20 0 8 ) 5 0–5 8

2.6. Ethics committee
The experimental protocol and the use of cord blood stem
cells were approved by the “G. d'Annunzio” University Ethics
Committee.
3. Results
3.1. Establishment of HHPC cultures
Ten fresh UCB samples were obtained after full-term deliveries.
Total HHPCs retrieved after MACS selection ranged from 0.3×105
to 0.9×105, corresponding to 0.4–1.3% of the isolated mononuclear
cells. Freshly-isolated HHPCs were round, medium to large cells
that tended to aggregate in clusters (Fig. 1A). Exponential growth
started at day 4 of culture and cells were 5–20-fold expanded by
day 10 (Fig. 1B). Phenotypic heterogeneity, inherent to all normal
cells, reflected inter-donor variability and differentiation during
culture. CD34 expression, initially high in terms of both positive
cell percentages (96–98% purity) and fluorescence intensities,
decreased over time, although with relevant inter-sample
differences (Fig. 2). CD34− sub-populations appeared in some
samples. These observations are in accordance with a previous
report (Mayani and Lansdorp, 1998).
3.2. Ultrastructure of ex-vivo-expanded HHPCs
At day 7 of culture, most HHPCs appeared as round villous cells
that had a large nucleus-to-cytoplasm ratio and an immature
morphology (Fig. 3). Cells had an indented euchromatin-rich
nucleus and light cytoplasm with scattered ribosomes, single

Fig. 4 – Phase contrast microscopy of parallel cultures of CB#10 CD34+ HHPCs (day 7) exposed or not to 0.1 or 10 μM Cr(VI) and Cd
for 48 h. The cultures treated with 10 μM Cr(VI) or Cd show cell loss and inhibition of cluster formation. Treatment with 0.1 μM
Cr(VI) or Cd had no appreciable effect (original magnification ×40).
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 92 ( 20 0 8 ) 5 0–5 8 55

profiles of rough endoplasmic reticulum (RER) and numerous
mitochondria with electron-transparent matrix and thin tubular
cristae. Budding of the outer mitochondrial membrane, regarded
as ultrastructural evidence of mitochondrial duplication (Shaw
and Nunnari, 2002; Matarrese et al., 2005; Chan, 2006), was
occasionally observed. Golgi complexes with hypertrophied sacs
and associated multi-vesicular structures, empty or filled with
electron-dense material, were also present, which suggests
secretion vacuoles at various stages of maturation (Fig. 3A, B).
Some cells had more eccentrically polarized nuclei, more
extensive protein synthesis machinery and greater numbers of
multi-vesicular structures, suggestive of more advanced
differentiation.
3.3. Cd and Cr(VI) exposure
Parallel HHPC cultures from 10 UCB samples were exposed for
48 h (days 5–6) to 0.1 μM and 10 μM Cd and Cr(VI). Cell loss after
exposure ranged from 70% to 40% with 10 μM Cr(VI) and from 70%
to 35% with 10 μM Cd. No cell loss was detectable with 0.1 μM Cr
(VI) and Cd. No relationship between CD34+ cell yield and metal
toxicity was evident. Cultures of UCB samples #9 and #10 (Figs. 4
and 5) exemplify typical ranges of cytotoxicities observed in our
experiments. The cultures treated with 10 μM Cd or Cr(VI) showed
inhibition of cluster formation as observed by phase contrast
microscopy (Fig. 4) and significant decreases in live cell numbers
(CB#9: 43–64%; CB#10: 30–48%) were confirmed by trypan blue
exclusion assay (Fig. 5). Sample CB#10, which showed more
marked cell loss after 10 μM Cr(VI) exposure, revealed a higher
frequency of differentiated cells in the control culture by FACS
profile (Figs. 2 and 5). As in the other samples treatment with
0.1 μM Cr(VI) or Cd had no appreciable effect on cell numbers or
cluster formation (Figs. 4 and 5).
3.4. Ultrastructural alterations
HHPCs treated with 10 μM Cr(VI) or Cd manifested marked
alterations of subcellular structures, which tended to be more
severe in Cd-exposed cells (Figs. 6 and 7). Strikingly, cells showed
multiple double-membrane-bound cytoplasmic vacuoles of var-
ious sizes that sequestered portions of the cytosol and mem-
branes, most probably from captured cytoplasmic organelles
(Fig. 6A, C, D and Fig. 7A). These vacuoles frequently showed signs
of fusion with lysosomes (Fig. 6A and Fig. 7A). Double-membrane-
bound cytoplasmic vacuoles (autophagosomes) sequestering
cytoplasmic structures targeted for degradation after fusion
with lysosomes (autophagolysosomes) are diagnostic of autop-
hagy (Cuervo, 2004; Levine and Klionsky, 2004; Eskeline, 2005;
Golstein and Kroemer, 2007). Hypertrophy of the RER, dilatation of
the Golgi complex (Fig. 6A and Fig. 7A), increased electron density

Fig. 5 – Total live cell counts of parallel CD34+ HHPC cultures (day 7) from two donors (CB#9 and CB#10), exposed or not to 0.1 or
10 μM Cr(VI) and Cd for 48 h. The cultures treated with 10 μM Cr(VI) or Cd show marked decreases in live cell numbers.
Treatment with 0.1 μM Cr(VI) or Cd had no appreciable effects.
56 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 92 ( 20 0 8 ) 5 0–5 8

of the mitochondrial matrix, dilatation of the intercristal spaces
(Fig. 6A, F) and occasional lipid droplets were also observed,
mainly in Cd-treated cells (Fig. 6E). Budding of the outer
mitochondrial membranes was more frequent in treated than
in control cells (Fig. 6F), which suggests increased mitochondrial
replication (Matarrese et al., 2005). The nuclei of both Cd- and Cr
(VI)-treated HHPCs tended to manifest convoluted profiles and
different patterns of chromatin condensation at the nuclear
membrane, ranging from thin peripheral margination to forma-
tion of relatively large geometric blocks (Figs. 6A and 7A). Some
nuclei also showed centrally condensed chromatin. Nuclei with
diffuse chromatin condensation, reminiscent of pyknosis,
occurred less frequently. The above-described cytoplasmic and
nuclear alterations tended to vary in degree from cell to cell. In
some cells the cytoplasmic changes were less evident and the
nuclei had normal-appearing granular chromatin. Treated cells
did not show morphologic hallmarks of apoptosis, namely
apoptotic bodies, pyknotic nuclear fragmentation, cell membrane
blebbing and degradation of actin filaments (Leist and Jaattela,
2001). HHPCs exposed to 0.1 μM Cd or Cr(VI) showed only
hypertrophied and dilated RER and/or condensed mitochondria
with swelling of the intercristal spaces (Figs. 6B and 7B).
4. Discussion
We aimed at establishing CD34+ HHPCs from UCB (Davila et al.,
2004) as an ASC model for the assessment of toxicity by heavy
metals. Under the short-term culture conditions used, CD34+
HHPCs maintained a high degree of purity and showed an
immature ultrastructural morphology (Rubinstein and Trobaugh,
1973; Servida et al., 1996). To our knowledge there are no
published data about dose-related toxicity of metal compounds
in HHPCs. The two metal concentrations tested in our study were
chosen based on reported evidence that 0.1 μM Cd and Cr have no
detectable cytotoxic effects on BALBc/3T3 cell cultures, while
10 μM is clearly a toxic dose. In fact, in the BALBc/3T3 model, IC50
values for Cd and Cr(VI) are 3.6 and 3.0 μM respectively (Mazzotti
et al., 2002). Therefore, cultures were exposed for 48 h soon after
the beginning of the exponential growth phase to sub-toxic

Fig. 6 – Conventional electron microscopy of thin sections of HHPCs at day 7 of culture, after 48 h exposure to 10 μM (panels A, C,
D, E, F) or 0.1 μM (panel B) Cd. Cells exposed to 10 μM Cd show hypertrophied endoplasmic reticulum (er), dilated Golgi
complex, convoluted nuclei with heterochromatin margination and partial clumping (panel A). Mitochondria appear electron-
dense; intercristal spaces are dilated and there is frequent budding of the outer mitochondrial membrane, suggesting
mitochondrial duplication (panels A, F). Some cytoplasmic lipid droplets were observed (panel E). Double-membrane-bound
vacuolar structures (“autophagosomes”) that sequester portions of the cytosol and membranes can be seen forming (panel C)
and at various stages of development (panel D, arrows). Cells exposed to 0.1 μM Cd display mitochondrial condensation and
dilatation of intercristal spaces (Panel B and inset). PM: plasma membrane, N: nucleus; G: Golgi, M: mitochondria. Panels A–B,
D–E, bar = 1 μm; panels C and F bar = 0.5 μm.
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 92 ( 20 0 8 ) 5 0–5 8 57

Fig. 7 – Conventional electron microscopy of thin sections of HHPCs at day 7 of culture, after 48 h exposure to 10 μM (panel A) or
0.1 μM (panel B) Cr(VI). Cells exposed to 10 μM Cr(VI) contain many autophagosomes at various stages of maturation and have
convoluted and indented nuclei with geometric heterochromatin clumps (panel A and inset). Cells exposed to 0.1 μM Cr(VI)
show mainly hypertrophied and dilated endoplasmic reticulum and condensed mitochondria. PM: plasma membrane, N:
nucleus; G: Golgi, M: mitochondria, er: endoplasmic reticulum, bar = 1 μm.

(0.1 μM) and toxic (10 μM) concentrations of each metal ion. The
cultures treated with 10 μM Cr(VI) or Cd underwent cell loss. We
found no relationships between CD34+ cell yield from UCB and
metal toxicity. However, we cannot exclude that the more marked
Cr(VI) cytotoxicity observed in CB#10 could be related to a higher
frequency of differentiated cells, as evidenced by the FACS profile.
The open question is whether differentiation influences metal
cytotoxicity in the human hematopoietic cell compartment.
Treated cells did not show the morphologic hallmarks of
apoptosis (Leist and Jaattela, 2001; Ziegler and Groscurth,
2004), whereas autophagosomes and autophagolysosomes,
characteristic of autophagy (Eskeline, 2005) were promi-
nent. The other subcellular alterations we found in treated
cells (i.e., dilatation of the RER and Golgi, mitochondrial
damage and replication, lipid accumulation and chromatin
condensation) coincide with previous reports of an associa-
tion of these alterations with autophagy (Cuervo, 2004;
Levine and Klionsky, 2004; Eskeline, 2005; Golstein and
Kroemer, 2007). Treatment with 0.1 μM Cr(VI) or Cd did not
affect cell growth, but RER dilatation and mitochondrial
damage were detected at ultrastructural level. Such altera-
tions represent evidence of some level of toxicity and
suggest that human hematopoietic progenitor cells could
be sensitive to metal concentrations not regarded as toxic in
other cell systems (Burastero et al., 2006; Mazzotti et al.,
2002; Ceriotti et al., in press). This issue requires further
studies.
Autophagy is a major lysosomal catabolic pathway that, by
recycling damaged cytosolic macromolecules and organelles, is
crucial for cell survival under conditions of environmental stress
(Cuervo, 2004; Edinger and Thompson, 2004; Eskeline, 2005). The
relationships between autophagy and apoptosis remain unclear
(Cuervo, 2004; Gozuacik and Kimchi, 2004; Levine and Klionsky,
2004; Golstein and Kroemer, 2007). While autophagy may prevent
apoptosis, in many situations apoptosis, necrosis and autophagy
all contribute to cell death, with the role of each process
depending on cell type and triggering factor (Abedin et al., 2007;
Golstein and Kroemer, 2007).
Blankenship et al. (1994) found that apoptosis is the major
mode of cell death in rodent ovarian cells after brief exposure
(2 h) to high Cr(VI) concentrations (150–300 μM). However, Ord
et al. (1990) reported that increased efficiency in autophago-
cytosing damaged cell components is associated with the
acquisition of Cd resistance in deer fibroblast cell lines. In our
study, in which HHPCs were exposed to lower Cd and Cr(VI)
concentrations, we found no evidence of apoptosis. While
additional studies are clearly needed also to explore mechan-
istic aspects, our results suggest that reparative autophagy is
characteristic of the response of HHPCs to Cr(VI) and Cd. In
this respect, autophagy, by enhancing the cell's tolerance,
could provide a second tier of defence against oxidative stress
(Abedin et al., 2007; Golstein and Kroemer, 2007). Autophagy
has been implicated in cancer, degenerative disorders, aging,
and modulation of immune functions (Abedin et al., 2007;
Deretic, 2006). Thus, upregulation of autophagy in ASC
compartments could contribute to the pathogenesis of chronic
diseases associated with exposure to heavy metals. It is not
possible, based on the evidence of the present study, to assess
whether the autophagic response to metals in the context
of HHPC cultures is adaptive or death-related. Autophagy
deserves further evaluations, such as quantitative or semi-
quantitative measurements and concentration–effect rela-
tionships, to be established as biomarker of metal exposure.
Acknowledgements
We thank the Department of Gynaecology and Obstetrics, SS.
Annunziata Hospital, for cord blood collection, D. Kempuraj
for help in cell isolation and M. Iezzi for digital images, and
Jean Ann Gilder for text editing. This work was supported by
EC grant n. 2004/S 83-070342.
58 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 92 ( 20 0 8 ) 5 0–5 8
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