In vitro models to study mechanisms of neural cell death induced by toxic agents

Neurotoxicity arises when exposure to toxic agents, either naturally occurring or manmade substances, alters the normal activity and/or structure of the nervous system. This can lead to disruption of vital metabolic processes and eventually to cell death. Certain unique features of the nervous system make it particularly vunerable to toxicants, e.g. its high demand of energy, high content of polyunsaturated fatty acids and low levels of antioxidant enzymes. In this thesis, we have investigated the mechanisms leading to neurotoxic cell death and the role of ion channels in neural apoptosis by using different in vitro experimental models. Such models offer unique advantages in elucidating mechanisms of toxicity, and hence are used to understand the consequences of exposure to toxicants.

Occupational exposure to styrene in industrial workers has been associated with neurobehavioral deficits. Alterations of neurotransmitters and loss of neurons have also been observed in in vivo models. The main metabolite of styrene, styrene 7,8-oxide (SO), is believed to account for most of styrene s toxicity. Carbon monoxide (CO), an endogenous gas, plays important physiological roles, but CO poisoning due to accidental or intentional exposure, occurs frequently. CO has higher affinity for hemoglobin than oxygen, and brain hypoxia due to the binding of CO to hemoglobin is a recognized cause of CO neurotoxicity. However, the direct effect of CO on intracellular targets is still not well understood. We have characterized the cellular damage induced by SO and CO in different in vitro models. Our data show that SO causes apoptosis via activation of caspases and that its neurotoxic effects are related to mitochondrial damage and oxidative stress. CO induces hypoxia-independent apoptotic cell death via parallel activation of both caspases and calpains.

Cell shrinkage is an early morphological feature occurring during apoptosis that is associated with an increased efflux of K+ and Cl- ions. We investigated the role of ion channels in differentiated and non-differentiated neural cells undergoing apoptosis. Our results point to a novel function of the voltage-dependent anion channel in the plasma membrane (pl-VDAC) playing a role in the early phase of neuronal apoptosis. In contrast, pl-VDAC is scarcely seen in apoptotic cortical neural stem cells and instead, an amiloride-sensitive Na+-channel is activated. Thus, it appears that neurons and neural stem cells utilize different apoptotic strategies.

With appropriate in vitro models we have been able to characterize the intracellular pathways affected by SO and CO, and to demonstrate activation of different ion channels during apoptosis in undifferentiated and differentiated neural cells. There is an increasing consensus on in vitro models being useful tools to test neurotoxic agents and dissect their mechanisms of action. In fact, by using multiple cell models it is possible to recognize specific patterns of toxicity of different neurotoxicants and use this information for risk assessement.

List of scientific papers

I. Daré E, Tofighi R, Vettori MV, Momoi T, Poli D, Saido TC, Mutti A, Ceccatelli S. (2002). "Styrene 7,8-oxide induces caspase activation and regular DNA fragmentation in neuronal cells." Brain Res 933(1): 12-22
https://pubmed.ncbi.nlm.nih.gov/11929631

II. Daré E, Tofighi R, Nutt L, Vettori MV, Emgård M, Mutti A, Ceccatelli S. (2004). "Styrene 7,8-oxide induces mitochondrial damage and oxidative stress in neurons." Toxicology 201(1-3): 125-32
https://pubmed.ncbi.nlm.nih.gov/15297027

III. Tofighi R, Tillmark N, Daré E, Aberg AM, Larsson JE, Ceccatelli S. (2006). "Hypoxia-independent apoptosis in neural cells exposed to carbon monoxide in vitro." Brain Res 1098(1): 1-8
https://pubmed.ncbi.nlm.nih.gov/16777078

IV. Elinder F, Akanda N, Tofighi R, Shimizu S, Tsujimoto Y, Orrenius S, Ceccatelli S. (2005). "Opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase activation in neuronal apoptosis induced by toxic stimuli." Cell Death Differ 12(8): 1134-40
https://pubmed.ncbi.nlm.nih.gov/15861186

V. Akanda N, Tofighi R, Brask J, Tamm C, Elinder F, Ceccatelli S. (1970). "Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role in apoptosis in differentiated hippocampal neurons but not in neural stem cells." Journal of Neuroscience (Submitted)