Autophagy , apoptosis and organelle features during cell exposure to cadmium č

Cadmium (Cd) induces several effects in different tissues, but our knowledge of the toxic effects on organelles is insuffi cient. To observe the progression of Cd effects on organelle structure and function, HuH-7 cells (human hepatic carcinoma cell line) were exposed to CdCl2 in increasing concentrations (1 μM – 20 μM) and exposure times (2 h – 24 h). During Cd treatment, the cells exhibited a progressive decrease in viability that was both timeand dose-dependent. Cd treated cells displayed progressive morphological changes that included cytoplasm retraction and nuclear condensation preceding a total loss of cell adhesion. Treatment with 10 μM for 12 h led to irreversible damages. Before these drastic and irreparable damages, treated cells (5 μM for 12 h) presented a progressive loss of mitochondrial function and cytoplasm acidifi cation as well as dysfunction and disorganization of microfi laments and endoplasmic reticulum. These damages led to the induction of apoptotic events and an increase in autophagic bodies in the cytoplasm. These results revealed that Cd affects multiple intra-cellular targets that induce alterations in the mitochondria, cytoskeleton, endoplasmic reticulum and acidic compartments, ultimately culminating in cell death via apoptotic and autophagic pathways. BIOCELL 2013, 37(2): 45-54 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
Cadmium (Cd) is a highly toxic metal that exerts multiple effects on organisms (Filipič, 2012;Waisberg et al., 2003;Bertin and Averbeck, 2006).However, the complexity and diversity of events associated with cell-Cd interactions have resulted in fragmented information mainly related with organelle structure and function (Cannino et al., 2009).
Biochemical studies have shown the involvement of organelles (mitochondria, lysosomes and cytoskeleton) in Cd toxicity in several cell lines (Cannino et al., 2009;Fotakis et al., 2005;Faverney et al., 2004;L'Azou et al., 2002).However, the wide-ranging effects of this metal on organelles and their involvement in induced cell death remain to be fully understood (Fabbri et al., 2012).Therefore, the overall understanding of Cd induced cell damage and toxicity needs the observation of its effects on different intra-cellular targets.
Cd exposure in organisms is followed by injuries in the liver, testes, lungs, kidneys and bones (Ye et al., 2007;Joseph, 2009;Nordberg, 2009;Siu et al., 2009).Cd uptake by hepatocytes makes the liver one of the major sites of Cd accumulation (Fabbri et al., 2012) and reduces its availability to other organs (Souza et al., 1997).Therefore, studies of hepatocyte organelles may help understanding the progression from the direct effects of Cd to its ultimate toxicity.
With this purpose, the structure and function of mitochondria, acidic organelles and vesicles, endoplasmic reticulum elements and microfi laments was assessed in HuH-7 cells (a human hepatic carcinoma cell line) to observe the progression of Cd toxicity.

Cell culture and treatments
HuH-7 cells were maintained in 25 mL cell culture fl asks with Dulbecco's Modifi ed Eagle's Medium (DMEM-1152, Sigma Aldrich®) supplemented with 10% fetal bovine serum (Gibco®) in a humidifi ed atmosphere containing 5% CO 2 at 37ºC.For experimental purposes, the cells were seeded onto 24-well plastic plates.The optimum cell concentration determined from cell line growth profi les was 10 5 cells/mL.Cells were allowed to attach for 24 h before Cd treatments.
For Cd toxicity assays, stock solutions (0.1 M CdCl 2 ) were prepared using ultra-pure quality water, and dilutions were made with culture medium to 1 μM, 5 μM, 10 μM, 15 μM and 20 μM fi nal concentrations.To observe the progression of Cd induced toxic effects, these concentrations were added to cell cultures for 2, 6, 12 and 24 h.

Quantifi cation and morphological analysis of Cd induced toxic effects
Control and Cd exposed cells were fi xed in Bouin's solution and stained with Giemsa (10%) for light mi-croscopy observation.All preparations were examined using a Zeiss Axioplan photomicroscope equipped with 20x and 40x objectives.HuH-7 cell survival was determined by counting the number of living cells in a given area (the cell spread on the substrate and nuclear condensation were considered for discrimination between live and dead cells).For each sample, 6 randomly chosen fi elds were scored at a magnifi cation of 400x, and results were expressed as the mean ± standard deviation.HuH-7 control cell numbers counted at each time point were considered to be 100%.Digital images were obtained using an Axioplan microscope equipped with a Canon Power Shot camera A610/620 employing 20x and 40x objectives.

Scanning and transmission electron microscopy (SEM and TEM)
HuH-7 cells treated with 5 μM CdCl 2 for 12 h were fi xed in 2.5% (v/v) glutaraldehyde and 4% (v/v) formaldehyde in 0.1 M cacodylate buffer (pH 7.2).For  SEM preparations, the samples were washed, dehydrated with a graded series of ethanol, critical-point dried in CO 2 , positioned on a specimen holder and sputtered with gold.All micrographs were recorded using a Zeiss Evo 40 microscope employing secondary electrons.For TEM, the fi xed samples were post-fi xed with (1:1) 1% osmium tetraoxide and 0.8% potassium ferricyanide, dehydrated with acetone and embedded in Epon.Ultra-thin slices (70 nm) were obtained with a Leica Reichert Ultracut S ultramicrotome, contrasted with uranyl acetate (5%) and lead citrate and observed using a Zeiss 900 transmission electron microscope.

Reversibility of Cd induced toxic effects
For reversibility testing, HuH-7 cells were incubated for 6 h with 10 μM or 20 μM CdCl 2 or for 12 h with 5 μM or 10 μM of CdCl 2 .After exposure, the cells were washed and the medium was replaced without Cd addition.After a 24 h recovery period, cells were analysed by light microscopy and quantifi ed as described above.
Given that only apoptotic cells will take up YO-PRO-1 and viable cells exclude the dye, YO-PRO-1 dye was used (Molecular Probes®) for detection of apoptosis (Idziorek et al., 1995;Plantin-Carrenard et al., 2003).YO-PRO-1 (1 μM) was added to HuH-7 cell cultures for 30 min in an incubator with 5% CO 2 at 37ºC.For autophagic vacuole detection, a selective marker monodansylcadaverine (MDC) (Sigma Aldrich®) was used as described by Biederbick et al. (1995).The cell culture was incubated with 0.05 mM MDC in PBS at 37ºC for 10 min.
All the stained cells were observed under a Zeiss Confocal Laser Scan Microscope (CLSM) using a 543 nm argon laser and a 40x objective.

Statistical analyses
All data are expressed as the means ± standard errors.Statistical analyses were made using GraphPad Prism v.4 software (GraphPad Software, Inc. CA, USA).The two-way analysis of variance followed by the Bonferroni test was performed for cell viability data and reversibility test data.Differences were considered signifi cant when p < 0.05.

Results
To determine the threshold of metal damage and its relationship to metal toxicity (induction of cell death), the present study investigated the effects of Cd over the HuH-7 cell machinery after treatments with increasing concentrations and exposure times.
The dose and duration of treatment were critical factors in the induction of cell death (Fig. 1a).These toxic effects were evaluated after each Cd treatment following the observation of reduced cell numbers demonstrated by the attached cell count (Fig. 1a).Cell viability was assessed through the MTT assay, verifying the decrease of cell viability indicated by the failure of mitochondrial function (Fig. 1b).The results obtained by counting the surviving cells or through assessment of mitochondrial function by MTT assay corroborate the Cd toxicity in the culture.
The observation of the Cd induced toxic effects indicated that healthy cells at semi-confl uence, evidenced by adherence and spread cytoplasm on the substrate with prominent nuclei and nucleoli, changed during Cd treatment (Fig. 2a, 2b).Cells experienced different degrees of cytoplasm shrinkage and nuclear condensation (Fig. 2b).This cytoplasmic retraction was more evident at higher doses (20 μM), but occurred asynchronously within the culture (Fig. 2c, inset) and led to the gradual loss of cell viability and subsequent release from the substrate.
Ultrastructural analysis of cell culture indicated that cell morphology (Fig. 3a) changed in the presence of Cd (5 μM for 12 h) as evidenced by cytoplasm retraction (Fig. 3b), severe vacuolization (Fig. 3c, arrows) and alterations in mitochondrial structure (Fig. 3c, inset).The presence of blebs on the membrane cell surface (Fig. 3d, arrows) also indicated apoptosis, and this was also confi rmed by YO-PRO-1 nuclear staining (Fig. 4a-d).No indicative probe (Fig. 4b, arrowhead) was observed in the adherent control cells (Fig. 4a).However, following Cd exposure (5 μM for 12 h), staining was evident in cells with cytoplasmic retraction and nuclear disorganisation (Fig. 4c, d, arrowheads).The cells displayed different stages of cellular retraction (Fig. 4c) with distinct apoptotic staining (Fig. 4d), suggesting that the process occurred asynchronously within the same culture.
To assess the reversibility of Cd induced damage, cells were treated with 5 and 10 μM CdCl 2 for 12 h or with 10 and 20 μM CdCl 2 for 6 h, and then maintained in the absence of Cd for 24 h.After Cd removal, both treatments (10 and 20 μM) for the short period (6 h) and the lower concentration (5 μM) with long-term exposure (12 h) the culture was able to recover (Fig. 5ah).However, treatment with 10 μM for 12 h promoted severe deleterious changes (Fig. 5i, j) that compromised cellular recovery (Fig. 5b).This fi nding is important to understand the kinetics of metal action on the cellular machinery.
The treatment with 5 μM for 12 h was chosen to investigate the Cd induced changes in organelles and severe damages that compromised cell survival were observed.Initially, organelle functionality was analysed using the mitochondrial fl uorescent stain Rhodamine 123 (Fig. 6a-d).The intense and spread fi laments indicative of functional mitochondria (Fig. 6b, arrowheads) present in control cells changed to punctate staining  While mitochondrial function was impaired after Cd exposure, the acidic compartments increased in frequency and size (Fig. 7a-d).This increase in acidic vesicles may correspond with increased abundance of lysosomes in the cytoplasm (Fig. 8a-d).Consequently, the cells changed from a punctate regular fl uorescent staining (Fig. 7b, 8b) to an intense fl uorescence pattern corresponding to acid structures in the cytoplasm (Fig. 7d, 8d arrowheads).
The presence of fl uorescent acidic compartments or lysosomal vacuoles further suggests the possibility of intracellular autophagic digestion.To disclose the presence of autophagosomes during Cd treatment, the cells were incubated with MDC (Fig. 9).Untreated cells (Fig. 9a) exhibited no fl uorescence indicative of autophagic vacuoles (Fig. 9b), while treated cells (Fig. 9c) showed a high frequency of fl uorescent compartments and even the formation of large vacuoles (Fig. 9d).Therefore, these results strongly suggest that the apoptotic pathway and autophagic processes are involved in Cd induced cell death.
Cytoskeleton microfi laments were also analysed for understanding the changes in cell structure after Cd treatment (Fig. 11).The extended microfi lament network (Fig. 11b, arrowheads) changed after Cd treatment, given that the cells lost their microfi lament projections in the cytoplasm (Fig. 11d, arrowheads) and their adhesion points on the substrates (Fig. 11d, arrowheads),    Interestingly, multiple Cd induced damages in organelles were observed in treated cells that remained attached, indicating that the severity of the effect in different targets is important in inducing cell death.Therefore, Cd reached several targets at the same time leading to loss of mitochondrial function, endoplasmic reticulum dysfunction, cytoplasmic acidifi cation and microfi lament disorganisation.These processes are all occurring together in treated cells, and if the exposure is not halted, might lead to cell death by apoptotic and autophagic pathways.

Discussion
The results obtained clearly show that Cd induces a decrease in cell viability and progressive damage to cell morphology in HuH-7 cells through concurrent effects in multiple intracellular targets, including mi-tochondria, cytoskeleton, endoplasmic reticulum and acidic compartments, leading to cell death through the apoptotic and autophagic pathways.
Apoptosis is considered a normal housekeeping event, but it is also necessary to arrest abnormal cell proliferation in development (Pulido and Parrish, 2003).Apoptosis can also be induced by a variety of chemicals, including many toxic metals (Rana, 2008), and is a known pathway of Cd mediated cell death (Wang et al., 2009;Lasfer et al., 2008;Ye et al., 2007;Mao et al., 2007;Pulido and Parrish, 2003;Faverney et al., 2004).However, the present study indicates that apoptosis is not the only process observed in Cd treated cells, given that the autophagic pathway was also observed after sustained Cd exposure.
The autophagic pathway allows the digestion of dysfunctional organelles with resulting recirculation and reuse of their molecular constituents (Templeton and Liu, 2010).Furthermore, when Cd induced cell damage exceeds the repairing capacity of repair, cell death occurred.Dying cells generate increasing amounts of autophagic vacuoles and clear large proportions of their cytoplasm before dying (Bursch et al., 2008).
The induction of Cd toxicity (cell death) in culture was asynchronous, suggesting preferential interference in some stages of the cell cycle.In fact, Cd can lead to cell cycle arrest, which may affect several cellular processes including cell proliferation and differentiation (Hartwig, 2010;Bertin and Averbeck, 2006).G2/M phase arrest was demonstrated after Cd exposure (Bork et al., 2010), preventing damaged cells from entering into mitosis, until DNA damage is repaired.Therefore, some stages of the cell cycle might be more susceptible to Cd damage as suggested by the effect on different cells in the same culture.
Other authors have shown the isolated involvement of mitochondria (Caninno et al., 2009), cytoskeleton (L'Azou et al., 2002), endoplasmic reticulum (Wang et al., 2009) and lysosomes (Lekube et al., 2000) demonstrating the role of separate organelles and structures in Cd induced cell death.The present study shows that these intracellular targets are all being affected concurrently and contribute to cell dysfunction leading to cell death.Moreover, the present study shows that the extent of damage induced by Cd treatment is eventually so severe that cells cannot reverse the toxic effects as shown by the 10 μM treatment for 12 h in the reversibility test.
The present study has increased our understanding of the cellular mechanisms of Cd toxicity on HuH-7 cells, by showing the progression of Cd induced damage in cells, and the involvement of mitochondria,

DIC image
Cd treatment RP control lysosomes, acidic compartments, cytoskeleton and endoplasmic reticulum as targets of Cd toxicity.Further investigations should be addressed to show the effects of this metal on each of these organelles.

FIGURE 1 .
FIGURE 1. CdCl 2 toxic effects on viability of HuH-7 cells.(a) Quantifi cation of Giemsa stained HuH-7 cells after CdCl 2 treatment.All concentrations tested were compared to a control group that was defi ned as 100%.(b) Decrease in cell viability by MTT assay in a dose/time dependent manner.*Signifi cantly different from control (p < 0.001).

FIGURE 2 .
FIGURE 2. Light microscopy of HuH-7 cells showing morphological alterations induced by CdCl 2 .(a) Control cells in monolayer.(b) Changes in the cell monolayer following incubation with 5 μM for 12 h.(c) Complete cell detachment after incubation with 20 μM for 12 h.(b) and (c) also show treated cells with retraction and nuclear condensation (arrows).Cells displaying normal morphology are also seen (arrowheads).n = nucleus.Scale bars: A and C: 200 μm; B: 100 μm.

FIGURE 4 .FIGURE 5 .
FIGURE 4. Differential interference contrast microscopy (DIC) (a and c) and confocal laser scanning microscopy of HuH-7 cells stained with YO-PRO-1 (YP-1) (1 μM) (b an d) before (a and b) and after Cd treatment with 5 μM for 12 h (c and d).(a) Control cells.(b) No fl uorescence signal in untreated cell.(c) Differential levels of cytoplasm retraction and nuclear disorganisation, both characteristics of apoptotic processes observed in Cd treated cells.(d) Cellular staining indicative of cell death via apoptotic processes following CdCl 2 exposure.n= nucleus.Scale bar: 10 μm.

FIGURE 10 .
FIGURE 10.Differential interference contrast microscopy (DIC) (a, c) and confocal laser scanning microscopy of HuH-7 cells stained with DiOC 6 (2.5 μg/mL) (b, d).(a) Control cells.(b) Morphological aspect of the reticular network of control cells with thinner peripheral regions (arrow) and regions with high fl uorescence close to the nucleus, mainly because of the concentration of reticulum and other membranes, such as mitochondria (arrowheads) (c) Cd treated cells (5 μM for 12 h).(d) Weaker fl uorescence signal in treated cells (5 μM for 12 h); with evidence of the disorganisation in reticular arrangement close to nucleus (arrowheads) and in cell periphery (arrow).n = nucleus.Scale bar: 10 μm.