Cadmium Toxicity on Developing Oligodendrocytes: Effects on Signal Transduction and Gene Expression

Guillermina Almazan
McGill University
Department of Pharmacology and Therapeutics
3655 Drummond St., Room 1321,
Montreal, Quebec H3G-1Y6, Canada
Tel: (514)-398-6222; Fax: (514) 398-6690
E-mail: galmazan@pharma.mcgill.ca

Many drugs and substances can affect brain function and development. In the central nervous system, the function of oligodendrocytes is the production of the myelin membrane that wraps nerve fibers and insulates them facilitating the conduction of nerve impulses. Myelin destruction causes neurological impairments such as those observed after trauma and in multiple sclerosis patients. In order to better understand the process of demyelination and myelin repair, it is essential to identify the toxic substances that affect oligodendrocytes growth and development. Because of the inherent cellular complexity of the nervous system, it is often difficult to determine whether oligodendrocytes are a direct target of a toxic insult in vivo. We believe that in vitro alternatives to the use of animals to test potential neurotoxins should include oligodendrocytes, in addition to neurons and astrocytes.

The long term goal of our research is to establish the ideal conditions for the use of low density cell cultures of oligodendrocytes as a test system for the screening of substances for potential neurotoxicity, and to elucidate their toxic cellular mechanism.

We have used oligodendrocyte progenitor primary cultures to test the toxic effects of heavy metals with emphasis on cadmium (cadmium chloride). Heavy metals are serious environmental pollutants which cause functional, morphological and biochemical deficits in the CNS of humans and animals who are exposed to them. Exposure to cadmium has been associated with behavioral defects in animal experiments and a toxic effect has been reported on glioma cells (1). There are, however, no studies describing the direct action of this metal on oligodendrocytes.

Our specific objectives were to determine whether selective heavy metals interfere with the following processes in oligodendrocytes: a) cell survival, b) signal transduction mechanisms and c) specific gene expression.

Culture system: The cultures were purified from the brain of newborn rats according to the procedure previously described (2). The system allows large and homogeneous populations of oligodendrocyte precursors to proliferate selectively in the presence of platelet derived growth factor and basic fibroblast growth factor, and to differentiate in serum-free medium. The advantage of this system is that progenitors in culture can recapitulate many of their differentiation processes, so that, they constitute an ideal system to study the differential susceptibility of various developmental stages to the same toxin.

Results

  1. Cadmium toxicity was evaluated with the MTT assay which is based on the tetrazolium ring cleavage by active mitochondria into a visible dark blue formazan product and measures only living cells. Cultures were exposed to increasing CdCl2 concentrations (0.1-1000 µM) for 30 or 180 min. Cadmium caused toxicity in both progenitors and mature oligodendrocytes. A significant effect was observed with 10 µM in progenitor cultures treated for 30 min (65% of control values). The 3 h exposure was more toxic, with 10 µM the values decreased to 35% of control and with 50 µM the values approached background. Interestingly, 9-days differentiated oligodendrocytes were less sensitive to the toxic effect of cadmium. A concentration of 10 µM had only a significant effect when cells were treated for 3h (65% of control values) and reached background levels with 100 µM for 3h and 1mM for 30 min. Our results suggest that oligodendrocyte progenitors are more susceptible to cadmium toxicity than mature oligodendrocytes.
  2. Interference with cellular signaling molecules. High concentrations of CdCl2 (100-1000 µM) blocked the increases in inositol phosphates accumulation evoked by several neurotransmitter, including the action of acetylcholine on muscarinic receptors, noradrenaline on a1A receptors and glutamate on AMPA/KA receptors in oligodendrocyte progenitors. CdCl2 was also found to reduce the kainate-stimulated 45Ca2+ uptake suggesting its interference with voltage-gated or receptor-operated Ca2+ channels. The effect of cadmium on mitogen-activated protein kinases (MAPKs, p42 and p44mapk) was also examined. MAPKs are part of a family of protein Ser/Thr-kinases that are activated rapidly in response to various growth factors and neurotransmitters to control cellular growth. We found that both p42 and p44mapk were activated several hundred-fold by cadmium treatment suggesting their involvement in the mechanism of toxicity.
  3. Effects on gene regulation. We have examined the effect of cadmium on the expression of c-fos, the prototype of immediate early genes, and heat shock protein 72 (HSP72), a protein induced under conditions of cellular stress. The expression of c-fos mRNA was induced by 25 µM and was less pronounced with higher concentrations of CdCl2. Similarly, HSP72 was induced with a cadmium concentration as low as 2.5 µM, reaching a maximum with 25 µM.

References

  1. Stark, M., et al. (1992). Neurotoxicol. Teratol. 14: 247-252.
  2. Almazan, G. et al. (1993). J. Neurosci. Res. 36:163-172.

In conclusion, our results suggest that cadmium induces a cascade of biochemical changes, which may be related to oligodendrocyte injury. Further work is required to identify the molecular targets of cadmium toxicity and the link among the various components affected in the system.

Acknowledgments. This research was supported by a grant from the Johns Hopkins Center for Alternatives to Animal Testing.