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Autophagy as an ultrastructural marker of heavy metal toxicityin human cord blood hematopoietic stem cells

Mario Di Gioacchinoa,b,⁎, Claudia Petrarcaa,b, Angela Perronea,b, Massimo Farinad,Enrico Sabbionid, Thomas Hartungd, Simone Martinoe, Diana L. Espositoa,c,Lavinia Vittoria Lottie, Renato Mariani-Costantinia,c

aAging Research Center, “G. d'Annunzio” University Foundation, Via Colle dell'Ara, 66100 Chieti, ItalybMedicine and Science of Ageing University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti, ItalycOncology and Neurosciences University of Chieti-Pescara, Via dei Vestini 1, 66100 Chieti, ItalydEuropean Commission, Institute of Health and Consumer Protection, ECVAM Unit, Joint Research Centre, Ispra, Varese, ItalyeDepartment of Experimental Medicine, University La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy

A R T I C L E I N F O

⁎ Corresponding author. Aging Research Cen0871 541291; fax: +39 0871 541300.

E-mail address: m.digioacchino@unich.it (M

0048-9697/$ – see front matter © 2007 Elsevidoi:10.1016/j.scitotenv.2007.11.009

A B S T R A C T

Article history:Received 20 June 2007Received in revised form26 October 2007Accepted 5 November 2007Available online 31 December 2007

Stem cells are a key target of environmental toxicants, but little is known about theirtoxicological responses. We aimed at developing an in-vitro model based on adult humanstem cells to identify biomarkers of heavy metal exposure. To this end we investigated theresponses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI])and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood wereexposed for 48 h to 0.1 μM and 10 μM Cr(VI) or Cd. Cultures treated with 10 μM Cr(VI) or Cdshowed marked cell loss. Ultrastructural analysis of surviving cells revealed prominentautophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated withmitochondrial damage and replication, dilatation of the rough endoplasmic reticulum andGolgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did notshow the morphologic hallmarks of apoptosis. Treatment with 0.1 μM Cr(VI) or Cd did notresult in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulumand evidence of mitochondrial damage. We conclude that autophagy is implicated in theresponse of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd.Autophagy, which mediates cell survival and death under stress, deserves furtherevaluation to be established as biomarker of metal exposure.

© 2007 Elsevier B.V. All rights reserved.

Keywords:AutophagyCadmiumChromiumCD34+ cellsStem cellsUmbilical cord bloodIn-vitro assayToxicity

1. Introduction

Heavy metals are bio-persistent toxic pollutants that accumulatein organisms at the top of food chains (Scheifler et al., 2006). Theirwidespread environmental diffusion, due to industrial andconsumer waste and leakage from soils, raises concerns abouthealthhazards (HiranoandSuzuki, 1996).Humanexposureoccursthrough polluted air, water and/or food and has been linked toneurodegenerative and developmental disorders, reproductive

ter, “G. d'Annunzio” Univ

. Di Gioacchino).

er B.V. All rights reserved

dysfunction, cardiovascular disease and increased risk of cancer(EEA, 2005). However, various factors can confound associationsbetween exposure to heavy metals and diseases (Goyer, 1986;Hirano and Suzuki, 1996). Furthermore, as is the case of othertoxicants, risk assessment is hindered by the lack of a toxicity testthat 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 carcinogensand, being mostly conducted on immortalized cell lines, may not

ersity Foundation, Via Colle dell'Ara, 66100 Chieti, Italy. Tel.: +39

.

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reflect the responses of normal human cells (Combes and Balls,1999). Accordingly, new in-vitro models for carcinogenicity, basedonhumancells,needtobedeveloped inorder tobetterunderstandthe mechanisms of cell transformation and carcinogenesisinduced bymetal compounds.

Increasing evidence indicates that adult stem cells (ASCs),which are progenitor cells capable of both self-renewal andmulti-potent differentiation (Rando, 2006), have been implicated inchronic inflammatory diseases and cancer (Reya et al., 2001;Dalerba et al., 2007; Dietrich and Kempermann 2006). ASCs mayrepresent a key target of toxicants due to their comparativelyrelaxed DNA repair functions, which could facilitate mutagenesisafter toxic exposures (Trosko and Tai, 2006). The toxicologicalresponses of ASCs are relatively unknown, but might differ fromthose of other cells because of ASCs' unique biological propertieslinked to their role in tissue renewal and repair (Rando, 2006), andthe early stage of their biochemical processes and intracellularcompartments (Inoue et al., 2002). Therefore, assays based onhumanASCmodels could improve risk assessment of exposure toheavy metals. Human hematopoietic stem cells (HHPCs), whichare easily isolated from umbilical cord blood (UCB) withoutdiscomfort or risks to donors, could be a well-characterized ASCmodel (Davila et al., 2004) for toxicological and carcinogenicitystudies.

The aim of our study was to investigate whether ex-vivoexpanded UCB HHPCs could be used to evaluate the cytotoxicityinduced by exposure to toxic metals. In particular, we studied thetoxicityofhexavalent chromium(Cr[VI]) andcadmium(Cd), twoofthe best known toxic and carcinogenic heavy metal ions (ATSDR1998, 1999).

Cr[VI] and Cd are powerful oxidizing agents that readily entercells, where they are subject tometabolic reduction. Endogenousoxidants generatedby thisprocessmayattack thecellular proteinmachinery, organelles and nucleic acids (Pritchard et al., 2005;Rossi andWetterhahn, 1989;Wetterhahn et al., 1989; Stearns andWetterhahn, 1994; Yano andMarcondes, 2005). In turn, damagedmitochondria, by producing superoxide, may further enhanceoxidative stress, eventually leading to apoptotic cell death(Blankenship et al., 1994; Evan and Vousden, 2001; Golstein andKroemer, 2007).

Transmission electron microscopy (TEM) showed that Cr(VI)-and Cd-exposed UCB HHPCs respond by activating autophagy, aspecific intralysosomaldegradationpathwaythatcontrols cell fateby allowing the turnover of damaged long-lived cellular macro-moleculesandorganelles (Cuervo,2004; LevineandKlionsky,2004;Abedin et al., 2007).

2. Materials and methods

2.1. Chemicals

Sodium chromate (Na2CrO4·4H2O, [10034-82-9]) and cadmiumchloride, two highly soluble salts containing Cr(VI) and Cdrespectively, were supplied by Alpha Aesar (Karlsruhe, Germany).The salts were analysed for elemental impurities and, in the caseof chromate, for oxidation state by HPLC-inductively coupledplasmamassspectrometry (Perkin-ElmerSCIEX,Ontario,Canada).These analyses (data not reported) showed that both salts had ahigh degree of purity. All other chemicalswere of analytical grade.

Chromium and cadmium salts (10 mM) were dissolved in MilliQwater, the freshly-preparedmother solutionswere sterilisedusing0.2-μm filters (Millipore, Italy) and diluted in complete culturemedium to obtain sub-toxic (0.1 μM) and IC50 (10 μM) concentra-tions of eachmetal ion. These concentrationswere selected basedonprevious studies conducted inhumanandmurine cell systems(Burastero et al., 2006; Mazzotti et al., 2002; Ceriotti, in press).

2.2. Purification of CD34+ UCB HHPCs

UCB samples (20–60ml) were collected, with informed consent ofdonors, from full-term deliveries at the Department of Gynaecol-ogy and Obstetrics, SS. Annunziata Hospital, Chieti. UCB wasdrainedby gravity and gentle squeezing from thematernal endofthe severed cord into sterile flasks filled with 40ml 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 at4 °C up to 12 h.

Mononuclear cells were then isolated by density gradientcentrifugation on lymphocyte separation medium with a densityof 1.077 (Lymphoprep, Sentinelle, Milan), washed twice withrunning buffer containing 2 mM EDTA and 0.1% BSA in PBS, pH7.4 (MiltenyiBiotech,Bologna, Italy), andre-suspended in thesamebuffer. CD34+ cells were isolated using the magnetic immunose-paration MACS system (Miltenyi Biotech), according to themanufacturer's specifications. Collected cells were counted andan aliquot of 1.0×105 cells in 200 μl was analysed by flowcytofluorimetry as described below. Remaining cells wereexpanded in culture.

2.3. Ex-vivo expansion and Cr(VI) and Cd treatments

CD34+HHPCswere culturedatdensities between2.5and5.0×105/ml in 24-well plates (Diagramma, Pescara, Italy) containing 1.5mlof StemSpan serum-free medium (StemCell Technologies, Van-couver, Canada) supplemented with 100 units/ml penicillin G,0.1mg/ml streptomycin (Celbio), andStemspanCytokineCocktailCC110 (StemCell Technologies) containing 100 ng/ml of eachcytokine 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 every3daysand imageswere recordedwithadigital camera. Cellswerecounted (as described below) and diluted 1:2 with complete freshmedium.

HHPCs grown in 24-well plates (5×105 cells/well in 1.5 ml ofcomplete culturemedium) were exposed in parallel for 48 h to 0.1and10μMCr(VI) andCd.Untreatedcultureswere set-up inparalleland counted every 24 h. At the end of the exposure period, treatedand control cultures were examined at the phase contrastmicroscopy, floating cells were harvested, and cell viability andnumber assessed by trypan blue exclusion. To minimize cellwaste, cells were carefully re-suspended, 20 μl of cell suspensionwere diluted 1:1 in PBS-0.4% trypan blue and 20 μl of the mix wasused to fill a Neubauer counting chamber. Unstained and stainedcells were counted. Data are reported as mean of triplicateindependent counts. Aliquots from each culture were used forcytofluorimetry and TEM.

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2.4. Cytofluorimetry

Analyses were conducted on both freshly isolated and ex-vivoexpanded cells (days 3, 5, 7, 10 of culture). Cr(VI)/Cd-exposedcultures 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 inthe dark. Cells were collected by centrifugation (10min at 200 ×g),re-suspended in 200 μl PBS and analysed using a FACScaliburinstrument (Becton Dickinson, Milan). Forward and side scatter

Fig. 1 –Ex-vivo expansion of CD34+UCBHHPCs. Panel A: Phase conculture. Cell proliferation and cluster formation are clearly evidencultures from 5 different donors. Exponential growth rates were

parameterswereused to excludedead cells anddebris. Datawereanalysed with the CELLQuest software (Becton Dickinson). Atleast 10,000 events were collected for each analysis.

2.5. TEM

HHPCswere fixedovernight at 25 °C in2%paraformaldehyde-2%glutaraldehyde in PBS, post-fixed in 1% osmium tetroxide inveronal acetatebuffer (pH7.4) for 2hat 25 °C, stainedwithuranylacetate 2% (5 mg/ml), dehydrated in acetone and embedded inEpon 812. Thin sections were examined under a Philips CM10TEM after post-stainingwith uranyl acetate and lead hydroxide.

trastmicroscopy of cells from the samewell at days 1 and 5 oft (original magnification ×10). Panel B: growth curves of HHPCobserved from day 4 of culture.

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 inCD34 expressionwith time of culture is shown by the lowermean FL1-H value of profiles at day 7. Note the significant decreasein 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 orslightly indented nuclei and secretory compartments formed by the Golgi apparatus with emerging granules filled byelectron-densematerial and vesicular structures. Numerouswell-conservedmitochondria are evident. PM: plasmamembrane,N: nucleus; G: Golgi, M: mitochondria, er: endoplasmic reticulum, bar=1 μm.

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2.6. Ethics committee

The experimental protocol and the use of cord blood stemcells were approved by the “G. d'Annunzio” University EthicsCommittee.

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 to0.4–1.3%of the isolatedmononuclearcells. Freshly-isolated HHPCs were round, medium to large cellsthat tended to aggregate in clusters (Fig. 1A). Exponential growth

Fig. 4 –Phase contrastmicroscopy of parallel cultures of CB#10 CDfor 48 h. The cultures treated with 10 μMCr(VI) or Cd show cell loCr(VI) or Cd had no appreciable effect (original magnification ×40

started at day 4 of culture and cells were 5–20-fold expanded byday 10 (Fig. 1B). Phenotypic heterogeneity, inherent to all normalcells, reflected inter-donor variability and differentiation duringculture. CD34 expression, initially high in terms of both positivecell percentages (96–98% purity) and fluorescence intensities,decreased over time, although with relevant inter-sampledifferences (Fig. 2). CD34− sub-populations appeared in somesamples. These observations are in accordance with a previousreport (Mayani and Lansdorp, 1998).

3.2. Ultrastructure of ex-vivo-expanded HHPCs

At day 7 of culture, most HHPCs appeared as round villous cellsthat had a large nucleus-to-cytoplasm ratio and an immaturemorphology (Fig. 3). Cells had an indented euchromatin-richnucleus and light cytoplasm with scattered ribosomes, single

34+ HHPCs (day 7) exposed or not to 0.1 or 10μMCr(VI) and Cdss and inhibition of cluster formation. Treatment with 0.1 μM).

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profiles of rough endoplasmic reticulum (RER) and numerousmitochondria with electron-transparent matrix and thin tubularcristae. Budding of the outer mitochondrial membrane, regardedas ultrastructural evidence of mitochondrial duplication (Shawand Nunnari, 2002; Matarrese et al., 2005; Chan, 2006), wasoccasionally observed. Golgi complexes with hypertrophied sacsand associated multi-vesicular structures, empty or filled withelectron-dense material, were also present, which suggestssecretion vacuoles at various stages of maturation (Fig. 3A, B).Some cells had more eccentrically polarized nuclei, moreextensive protein synthesis machinery and greater numbers ofmulti-vesicular structures, suggestive of more advanceddifferentiation.

3.3. Cd and Cr(VI) exposure

Parallel HHPC cultures from 10 UCB samples were exposed for48 h (days 5–6) to 0.1 μM and 10 μM Cd and Cr(VI). Cell loss afterexposure ranged from70% to 40%with 10 μMCr(VI) and from70%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 metaltoxicity was evident. Cultures of UCB samples #9 and #10 (Figs. 4and 5) exemplify typical ranges of cytotoxicities observed in ourexperiments. The cultures treatedwith 10μMCdorCr(VI) showedinhibition of cluster formation as observed by phase contrast

Fig. 5 –Total live cell counts of parallel CD34+ HHPC cultures (day10 μM Cr(VI) and Cd for 48 h. The cultures treated with 10 μM CrTreatment with 0.1 μM Cr(VI) or Cd had no appreciable effects.

microscopy (Fig. 4) and significant decreases in live cell numbers(CB#9: 43–64%; CB#10: 30–48%) were confirmed by trypan blueexclusion assay (Fig. 5). Sample CB#10, which showed moremarked cell loss after 10 μM Cr(VI) exposure, revealed a higherfrequency of differentiated cells in the control culture by FACSprofile (Figs. 2 and 5). As in the other samples treatment with0.1 μM Cr(VI) or Cd had no appreciable effect on cell numbers orcluster formation (Figs. 4 and 5).

3.4. Ultrastructural alterations

HHPCs treated with 10 μM Cr(VI) or Cd manifested markedalterations of subcellular structures, which tended to be moresevere in Cd-exposed cells (Figs. 6 and 7). Strikingly, cells showedmultiple 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,DandFig. 7A). Thesevacuoles frequently showedsignsof fusionwith lysosomes (Fig. 6AandFig. 7A).Double-membrane-bound cytoplasmic vacuoles (autophagosomes) sequesteringcytoplasmic structures targeted for degradation after fusionwith lysosomes (autophagolysosomes) are diagnostic of autop-hagy (Cuervo, 2004; Levine and Klionsky, 2004; Eskeline, 2005;Golstein andKroemer, 2007).Hypertrophyof theRER, dilatationofthe Golgi complex (Fig. 6A and Fig. 7A), increased electron density

7) from two donors (CB#9 and CB#10), exposed or not to 0.1 or(VI) or Cd show marked decreases in live cell numbers.

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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 outermitochondrial membranes was more frequent in treated thanin control cells (Fig. 6F), which suggests increased mitochondrialreplication (Matarrese et al., 2005). The nuclei of both Cd- and Cr(VI)-treated HHPCs tended to manifest convoluted profiles anddifferent patterns of chromatin condensation at the nuclearmembrane, ranging from thin peripheral margination to forma-tion of relatively large geometric blocks (Figs. 6A and 7A). Somenuclei also showed centrally condensed chromatin. Nuclei withdiffuse chromatin condensation, reminiscent of pyknosis,occurred less frequently. The above-described cytoplasmic andnuclear alterations tended to vary in degree from cell to cell. Insome cells the cytoplasmic changes were less evident and thenuclei had normal-appearing granular chromatin. Treated cellsdid not show morphologic hallmarks of apoptosis, namelyapoptotic bodies, pyknoticnuclear fragmentation, cellmembraneblebbing and degradation of actin filaments (Leist and Jaattela,2001). HHPCs exposed to 0.1 μM Cd or Cr(VI) showed only

Fig. 6 –Conventional electronmicroscopy of thin sections of HHPCD, E, F) or 0.1 μM (panel B) Cd. Cells exposed to 10 μM Cd showcomplex, convoluted nuclei with heterochromatin margination adense; intercristal spaces are dilated and there is frequent buddmitochondrial duplication (panels A, F). Some cytoplasmic lipidvacuolar structures (“autophagosomes”) that sequester portionsand at various stages of development (panel D, arrows). Cells exdilatation of intercristal spaces (Panel B and inset). PM: plasma mD–E, bar=1 μm; panels C and F bar=0.5 μm.

hypertrophied and dilated RER and/or condensed mitochondriawith 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 heavymetals. Under the short-term culture conditions used, CD34+HHPCs maintained a high degree of purity and showed animmature ultrastructuralmorphology (Rubinstein and Trobaugh,1973; Servida et al., 1996). To our knowledge there are nopublished data about dose-related toxicity of metal compoundsinHHPCs. The twometal concentrations tested in our studywerechosen based on reported evidence that 0.1 μMCdandCrhavenodetectable cytotoxic effects on BALBc/3T3 cell cultures, while10 μM is clearly a toxic dose. In fact, in the BALBc/3T3model, IC50

values for Cd and Cr(VI) are 3.6 and 3.0 μM respectively (Mazzottiet al., 2002). Therefore, cultures were exposed for 48 h soon afterthe beginning of the exponential growth phase to sub-toxic

s at day 7 of culture, after 48 h exposure to 10μM (panels A, C,hypertrophied endoplasmic reticulum (er), dilated Golgind partial clumping (panel A). Mitochondria appear electron-ing of the outer mitochondrial membrane, suggestingdroplets were observed (panel E). Double-membrane-boundof the cytosol and membranes can be seen forming (panel C)posed to 0.1 μM Cd display mitochondrial condensation andembrane, N: nucleus; G: Golgi, M: mitochondria. Panels A–B,

Fig. 7 –Conventional electronmicroscopy of thin sections of HHPCs at day 7 of culture, after 48 h exposure to 10 μM (panel A) or0.1 μM (panel B) Cr(VI). Cells exposed to 10 μMCr(VI) contain many autophagosomes at various stages of maturation and haveconvoluted 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.

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(0.1 μM) and toxic (10 μM) concentrations of each metal ion. Thecultures treated with 10 μM Cr(VI) or Cd underwent cell loss. Wefound no relationships between CD34+ cell yield from UCB andmetal toxicity.However,wecannotexclude that themoremarkedCr(VI) cytotoxicity observed in CB#10 could be related to a higherfrequencyofdifferentiated cells, as evidencedby theFACSprofile.The open question is whether differentiation influences metalcytotoxicity in the human hematopoietic cell compartment.

Treated cells did not show the morphologic hallmarks ofapoptosis (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 treatedcells (i.e., dilatation of the RER and Golgi, mitochondrialdamage and replication, lipid accumulation and chromatincondensation) coincide with previous reports of an associa-tion of these alterations with autophagy (Cuervo, 2004;Levine and Klionsky, 2004; Eskeline, 2005; Golstein andKroemer, 2007). Treatment with 0.1 μM Cr(VI) or Cd did notaffect cell growth, but RER dilatation and mitochondrialdamage were detected at ultrastructural level. Such altera-tions represent evidence of some level of toxicity andsuggest that human hematopoietic progenitor cells couldbe sensitive tometal concentrations not regarded as toxic inother cell systems (Burastero et al., 2006; Mazzotti et al.,2002; Ceriotti et al., in press). This issue requires furtherstudies.

Autophagy is a major lysosomal catabolic pathway that, byrecycling damaged cytosolic macromolecules and organelles, iscrucial for cell survival under conditions of environmental stress(Cuervo, 2004; Edinger and Thompson, 2004; Eskeline, 2005). Therelationships between autophagy and apoptosis remain unclear(Cuervo, 2004; Gozuacik and Kimchi, 2004; Levine and Klionsky,2004; Golstein andKroemer, 2007).While autophagymay preventapoptosis, inmany situations apoptosis, necrosis and autophagyall 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 majormode of cell death in rodent ovarian cells after brief exposure(2 h) to high Cr(VI) concentrations (150–300 μM). However, Ordet al. (1990) reported that increased efficiency in autophago-cytosing damaged cell components is associated with theacquisition of Cd resistance in deer fibroblast cell lines. In ourstudy, in which HHPCs were exposed to lower Cd and Cr(VI)concentrations, we found no evidence of apoptosis. Whileadditional studies are clearly needed also to explore mechan-istic aspects, our results suggest that reparative autophagy ischaracteristic of the response of HHPCs to Cr(VI) and Cd. Inthis 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). Autophagyhas been implicated in cancer, degenerative disorders, aging,and modulation of immune functions (Abedin et al., 2007;Deretic, 2006). Thus, upregulation of autophagy in ASCcompartments could contribute to the pathogenesis of chronicdiseases associated with exposure to heavy metals. It is notpossible, based on the evidence of the present study, to assesswhether the autophagic response to metals in the contextof HHPC cultures is adaptive or death-related. Autophagydeserves 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. Kempurajfor help in cell isolation and M. Iezzi for digital images, andJean Ann Gilder for text editing. This work was supported byEC grant n. 2004/S 83-070342.

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