Elsevier

NeuroToxicology

Volume 30, Issue 4, July 2009, Pages 544-554
NeuroToxicology

Cadmium exposure induces mitochondria-dependent apoptosis in oligodendrocytes

https://doi.org/10.1016/j.neuro.2009.06.001Get rights and content

Abstract

Cadmium toxicity has been associated with learning disabilities and Parkinsonian symptoms in humans. We have previously shown that cultured oligodendrocytes are directly damaged by cadmium exposure. Here, we characterized the molecular mechanisms underlying cadmium-induced cell death in oligodendrocyte progenitors (OLP). Cadmium caused a concentration-dependent decrease in cell viability as assessed by mitochondrial dehydrogenase activity and by the cellular release of lactate dehydrogenase (LDH). A short exposure (1 h) to cadmium (25–100 μM), followed by several hours of recovery, produced a predominant apoptotic mechanism of cell death, involving the mitochondrial intrinsic pathway, as evidenced by nuclear condensation, DNA fragmentation, bax integration into the outer mitochondrial membrane, cytochrome c release, and activation of caspases-9 and -3. Pretreatment of OLPs with the pan-caspase inhibitor, zVAD-fmk, prevented caspase-3 activation but only slightly reduced cell death 11 h after cadmium exposure and failed to prevent cadmium-induced bax insertion into the mitochondrial membrane. In contrast, the anti-oxidant N-acetyl cysteine blocked caspase-3 activation and significantly protected OLPs from cadmium-induced cell death. Continuous exposure (18–48 h) of OLPs to low micromolar concentrations (0.001–25 μM) of cadmium significantly decreased mitochondrial metabolic activity, increased LDH leakage starting at 5 μM and maximally activated caspase-3. These results suggest that cadmium induces OLP cell death mainly by apoptosis, and at higher concentrations or with prolonged exposure to the heavy metal there is an increase in cytoplasmic membrane damage, an index of necrosis. More importantly, transient exposure to cadmium is sufficient to damage OLPs and could in principle impair myelination in the neonate.

Introduction

Cadmium, a transition metal, is a pollutant and carcinogen. Human exposure occurs by inhalation (ex. cigarette smoke) and by ingestion of cadmium-contaminated food or water (Bernard and Lauwerys, 1984, Jarup et al., 1998). It is toxic even at low doses since the metal accumulates and has a long biological half-life in humans (10–30 years) (Wu et al., 2008). Acute exposure can also occur in large doses as an occupational hazard or by accidental leakage of cadmium-containing batteries. In addition to peripheral organs (Bernard and Lauwerys, 1984, Fassett, 1975, Jarup et al., 1998), the central nervous system (CNS) is also subjected to cadmium toxicity (Gabbiani et al., 1967, Lafuente and Esquifino, 1999). Cadmium increases permeability of the blood–brain barrier in rats (Shukla et al., 1987) and may enter the CNS by the olfactory system to induce injury (Clark et al., 1985, Evans and Hastings, 1992, Kumar et al., 1996). Behavioral defects, neurochemical changes and brain lesions were reported in experimental animals (Fern et al., 1996), while in humans acute cadmium poisoning produced Parkinsonism symptoms (Okuda et al., 1997). In addition, high levels of cadmium and lead in children's hair were associated with learning disabilities (Bonithon-Kopp et al., 1986, Thatcher et al., 1984). The CNS is especially vulnerable to damage during early neonatal development; cadmium was shown to readily pass to the fetus via the placenta and was detected in milk during lactation (Korpela et al., 1986). Motor and perceptual abilities of children exposed to cadmium in uteri were significantly affected (Bonithon-Kopp et al., 1986). Cadmium has been shown to produce free radicals in the brain (Shukla et al., 1987), which may potentially damage both neurons and oligodendrocytes (OLG).

OLGs are the glial cells which myelinate axons in the CNS. An early study reported that cadmium toxicity affected CNS white matter (Fern et al., 1996), and our laboratory demonstrated that OLGs are direct targets of this insult (Almazan et al., 2000). Thus, we found that cadmium reduced cell viability of OLGs at various stages of development and oligodendrocyte progenitors (OLPs) were more vulnerable than mature cells. As in other cellular systems, cadmium toxicity was related to its ability to generate reactive oxygen species (ROS). Thus, cadmium was found to reduce intracellular glutathione levels and to increase ROS. N-acetylcysteine, a thiocompound with antioxidant activity and a precursor of glutathione, prevented cadmium-evoked cell death. In contrast, buthionine sulfoximine, an inhibitor of γ-glutamyl-cysteine synthetase, depleted glutathione and potentiated the toxic effects of cadmium on cultured OLGs.

In other cellular systems, cadmium produces either apoptotic or necrotic cell death depending on culture conditions as well as on the concentration and the duration of exposure. Apoptosis and necrosis are two morphologically and biochemically distinct processes of cell death, discernable based on cellular and organellar membrane integrity, nuclear fragmentation, enzymatic activation and release of cellular content (Wyllie et al., 1980). Cadmium-induced apoptosis in vivo (Chuang et al., 2000, Lag et al., 2002, Shih et al., 2003) and in vitro (Lasfer et al., 2008, Mao et al., 2007, Shih et al., 2003) was found to primarily involve the intrinsic mitochondrial-dependent pathway. In this pathway, toxic insults cause the disruption of the mitochondrial inner membrane releasing cytochrome c (cyt c), which complexes with apoptosis initiation factor (AIF), the latter forming a hexameric complex that binds procaspase-9 (pro-cas9) and promotes its activation. The balance between pro-apoptotic (bax, bak and bid) and anti-apoptotic (bcl-2, bcl-XL, mcl-1) factors helps regulate the intrinsic pathway by maintaining mitochondrial membrane integrity (reviewed in Borner, 2003). Pro-apoptotic factors either alone or in combination with other mitochondrial proteins such as voltage directed anion channel (VDAC) form large conductance channels in the outer mitochondrial membrane, thereby permitting pro-apoptotic protein efflux. Following chronic cadmium exposure, there was an upregulation in gene expression of bax (Fernandez et al., 2003, Shin et al., 2003), p53, p21 and a downregulation of bcl-2 (Fernandez et al., 2003). Caspase-3 (cas3), an effector caspase (Nicholson, 1999), has been shown to be critical in cadmium-induced apoptosis in the above mentioned studies.

The objective of our study was to examine the molecular mechanisms of and mode of cell death involved in cadmium-induced toxicity in OLPs. To mimic the acute and chronic cadmium exposures which may take place in humans, OLPs were either exposed for a short period of time (1 h) followed by recovery or a long (18 h) period of time. Using this experimental design, we have addressed the question of whether there is a correlation between cas3 activation and mitochondrial dehydrogenase activity as a marker of cell viability. The involvement of Bax translocation, cytochrome c release and cas9 activation were also explored. Finally, an inhibitor of caspases and the anti-oxidant N-acetylcysteine were evaluated for their ability to reduce cadmium-induced cytotoxicity and cas3 activation.

Section snippets

Materials

Dulbecco's modified Eagle medium (DMEM), Ham's F12 medium, phosphate buffered saline (PBS), Hank's balanced salt solution, 7.5% bovine serum albumin fraction V, fetal calf serum (FCS), penicillin/streptomycin were from Invitrogen (Burlington, ON, Canada). The protein assay kit was from BIO-RAD (Mississauga, ON, Canada). Poly-d-lysine, poly-l-ornithine, Triton X-100, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), human transferrin, insulin, and HEPES were from

Cadmium causes a dose-dependent decrease in cell viability

The toxic effects of cadmium on OLP viability were assessed using the MTT reduction assay under two different experimental conditions. For acute experiments (short-term exposure), OLPs were exposed to CdCl2 (5, 25, 50, 75 and 100 μM) for 1 h and mitochondrial respiration was assessed either immediately or following an 18-h recovery period (Fig. 1C). No significant differences were found in MTT reduction 1 h after acute exposure. On the other hand, cadmium caused a dose-dependent decrease in cell

Discussion

We previously showed that OLGs are a direct target of cadmium-induced toxicity with a higher vulnerability of progenitors as compared to mature cells (Almazan et al., 2000). In the present study we further explored the mechanisms of toxicity elicited by cadmium in OLPs after short (1 h, followed by several hours of recovery) and long (18 h continuous) exposures to either 0–100 μM or 0–25 μM CdCl2, respectively. Both short and long-term exposure caused cell death of OLPs mainly by apoptosis, which

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was funded by operating grants from the Multiple Sclerosis Society of Canada (MSSC) and the Canadian Institutes of Health Research (CIHR) to Dr. Guillermina Almazan. Shireen Hossain was supported by a studentship from the MSSC. We thank Dr. Walter E. Mushynski for editorial help with this manuscript.

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