Marina Guizzetti, Michelle Catlin, and Lucio G. Costa

Department of Environmental Health, University of Washington, Seattle, WA

Received 9/23/97 Accepted 10/2/97

5. INHIBITION OF GLIAL CELL PROLIFERATION AN DETHANOL'S DEVELOPMENTAL NEUROTOXICITY

As mentioned in the introduction, one of the main findings in individuals diagnosed with the Fetal Alcohol Syndrome, is the presence of microencephaly (1-4, 14). Animal models of FAS have shown that microencephaly is observed when ethanol is given during the first two postnatal weeks in the rat, a period corresponding to the third trimester of pregnancy in humans (12, 13, 41). This period of brain development corresponds to the so called "brain growth spurt," which is characterized by proliferation of glial cells and synaptogenesis (11). Thus, it is plausible to hypothesize that the effect of ethanol on glial cell proliferation may play a relevant role in its developmental neurotoxicity, most notably microencephaly.

There is evidence that developmental exposure to ethanol in vivo may affect the development and migration of glial cells (18, 42), and cause a reduction in cell number (43, 44). Microencephaly has been produced in rats following in vivo exposure from postnatal day 4 to 10 to ethanol doses which produced blood alcohol concentrations (BAC) of about 50 mM (45). Other investigators also found microencephaly following similar ethanol exposures and BACs of about 40 mM (41, 46). At these concentrations, ethanol has limited or no effect on the in vitro proliferation of glial cells cultured in the presence of serum. However, as discussed above, at concentrations of about 50 mM, ethanol has been shown to profoundly inhibit the proliferation induced by several mitogens. In case of acetylcholine- and IGF-I-induced proliferation there is initial evidence that the inhibitory effect of ethanol, observed so far only in vitro, may be relevant to in vivo situations.

There is growing evidence that acetylcholine may influence various aspects of brain development (47). Of particular interest is that acetylcholine levels are particularly high in the neonatal rat brain (80-90% of adult values), and that muscarinic receptor-activated signal transduction systems (notably phospholipases C and D) are greatly enhanced in the neonatal rat brain compared to adults (40, 48), despite a lower receptor density. A good correlation has been found between the ability of ethanol to induce microencephaly, and its ability to inhibit muscarinic receptor-stimulated phosphoinositide metabolism (45, 49). Additionally, as discussed earlier, muscarinic agonists can cause proliferation of rat cortical astrocytes and their action is significantly inhibited (about 80%) by 50 mM ethanol. Thus, during the brain growth spurt, acetylcholine, by activating muscarinic receptors coupled to phospholipase C (e.g. the m₃ subtype, which is expressed in astrocytes; 50) may contribute to the proliferation of astrocytes which occurs during this period (51). This process may be a target for ethanol, and may underlie, at least in part, the microencephaly observed following developmental ethanol exposure.

A similar set of considerations may also apply to IGF-I, though less evidence is so far available. This growth factor and its receptor appears to play a significant role in development, including the development of the nervous system (52). In addition to the aforementioned in vitro inhibition by ethanol of IGF-I-induced proliferation of C6 glioma cells, there is also evidence that in vivo administration of alcohol to pregnant rats (yielding BAC of about 13 mM), causes a decrease in circulating levels of IGF-I (53), by still unknown mechanisms. Thus, in this case, ethanol would alter the levels of IGF-I, in addition to directly inhibiting its mitogenic action in astrocytes.

In summary, several in vitro studies have shown that alcohol can inhibit the proliferation of glial cells (either astrocytes or transformed cell lines). The effect of ethanol is more pronounced toward proliferation induced by mitogens, and occurs at concentrations (20-50 mM) which correspond to moderate to high drinking. The action of ethanol also appears to have some degree of specificity, as only the effect of certain mitogens is affected. The ability of ethanol to interfere with the mitogenic action of certain neurotransmitters and growth factors (e.g., acetylcholine or IGF-I) in glial cells, may explain some aspects of the developmental neurotoxicity of alcohol, most notably microencephaly. When exposure to ethanol occurs at different stages of brain development (e.g. during the first or second trimester equivalent in human), inhibition of neuronal proliferation may also occur. Indeed, ethanol has been shown to inhibit proliferation of cells other than glia, including neurons, hepatocytes, lymphocytes and osteoblasts (54-57).

These combined effects on CNS cell proliferation, together with possible direct or indirect toxic effect of ethanol on developing neurons, may provide a comprehensive picture of the events responsible for CNS dysfunction in FAS patients.