Acetaldehyde cytotoxicity in cultured rat astrocytes

Abstract

The effect of acetaldehyde on astrocytes have been investigated because not only do they play an important role in brain maturation but also recent reports have shown their delayed proliferation following both `in vivo' and `in vitro' ethanol exposure. Biochemical parameters related to apoptotic and necrotic processes were examined in primary cultures of rat astrocytes exposed for 4 days to acetaldehyde generated from ethanol by co-cultured alcohol dehydrogenase-transfected Chinese hamster ovary cells. Acetaldehyde levels in the culture media attained concentrations of approximately 450 μM. To study ethanol effects, alcohol oxidation was inhibited by 4-methylpyrazole (an inhibitor of alcohol dehydrogenase). Acetaldehyde but not ethanol increased intracellular calcium levels by 155%. Moreover, significant DNA fragmentation was detected using a random oligonucleotide primed synthesis assay, by flow cytometry and when using agar gel electrophoresis. Transglutaminase activity was elevated in the cells treated with acetaldehyde but when acetaldehyde formation was inhibited by 4-methylpyrazole the enzyme activity was unaffected. Nitrate levels in the culture media were unchanged. Additionally, microscopic examination of cell nuclei revealed chromatin condensation in astrocytes exposed to acetaldehyde. It can be concluded, that in `in vitro' acetaldehyde exposed rat astrocytes apoptotic pathways are activated.

Introduction

The role of the primary metabolite of ethanol–acetaldehyde (ACH) in alcohol embryotoxicity remains unresolved. It is known that acetaldehyde may reach the human fetus and also those of laboratory animals intoxicated with ethanol, however, its concentration attains only about 50% of maternal blood ACH [47]. On the other hand, acetaldehyde may be produced directly from ethanol in brain tissue and therefore it may play a role in the Fetal Alcohol Syndrome 13, 24, 30, 46.

Astroglial cells form guides for migrating neurons, maintain ion and neurotransmitter balance, produce neurotropic compounds and serve other important roles in the developing central nervous system [31]. In our previous studies we have shown that both ethanol and acetaldehyde disturb astroglial growth and differentiation, however, the mechanism remained elusive 27, 28, 29, 32, 33, 34. These results also evidenced biochemical changes related to oxidative stress, increased lipid peroxidation and necrotic/apoptotic mechanisms of cell death. In recent years, programmed cell death (apoptosis) in the central nervous system has been intensively studied. It has been shown that both neurons and glial cells may die by apoptotic mechanism and that several factors, including reactive oxygen species, influence this process 37, 43. The role of ethanol as one of the factors involved in programmed cell death of thymocytes and hepatocytes has been cited 3, 12. Similar effects were seen in alcohol dehydrogenase (ADH)-transfected Chinese hamster ovary (CHO) cells growing in the presence of ethanol, thus able to produce significant amounts of ACH [50].

In order to address the problem of ACH cytotoxicity in cultured glial cells and thus in ethanol neurotoxicity, we used an in vitro co-culture model comprising ADH-transfected CHO cells and primary cultures of rat astrocytes. ADH-transfected CHO cells are capable of producing ACH from ethanol directly into the culture medium and also both types of cells remain separated, while sharing a common culture medium. Thus, this model effectively eliminates the problem of rapidly declining ACH concentrations when added exogenously to the culture medium.