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

3. EFFECT OF ETHANOL ON GLIAL CELL PROLIFERATION

More than a decade ago, Davies and Vernadakis (21) examined the effect of exposure of glial cells, prepared from 15-day-old chick embryos, to ethanol (22, 109, 217, and 434 mM) for four days (day 6 to 10 in culture). The two higher concentrations of ethanol caused a 20-40% reduction in DNA content, while at the lower concentrations cell growth did not differ from control. These observations were confirmed in a subsequent study, where exposure or rat mixed glial primary cultures to ethanol (17, 43, and 86 mM) for four days had no effect on DNA synthesis (22). On the other hand, Kennedy and Mukerji (23) reported that astrocytes from newborn mice cultured from postnatal day 6 to 17 in the presence of ethanol (11, 22, and 45 mM) displayed a decrease content of DNA compared to untreated controls; the effect was quite small and was seen only at the highest alcohol concentration (45 mM). Yet, in another study, no effects of ethanol (10-60 mM) were observed following a four day exposure of fetal rat astrocytes (24).

In two additional studies (25,26) ethanol (44, 109, and 217 mM) was found to inhibit the growth of rat cerebral glial cultures following an exposure for seven days or longer. Inhibition of [³H]-thymidine incorporation by a 28 day exposure of rat cortical astrocytes to 100 mM ethanol was also reported (27). Similarly, 100 or 200 mM ethanol were found to inhibit [3H]-thymidine incorporation of rat astrocytes following a 18-24 hr exposure (28,29), though no effects were seen in another study (30). The proliferation of astroglia from adult human cerebrum, measured by 5-bromo-2'-deoxyuridine-5'-monophosphate incorporation into DNA, was also inhibited by ethanol (22, 45, and 109 mM) (31).

Two studies also examined the effect of ethanol on proliferation of C6 glioma cells (measured by cell counting; 32,33) and found contrasting results. In one case, inhibition of proliferation was found, at ethanol concentrations of 30-120 mM, while in the other no effect of ethanol at 80 mM was found (33).

Altogether, these findings consistently indicate that ethanol can inhibit the proliferation of glial cells in vitro. Though in some studies ethanol exerted an effect at concentrations below 100 mM, most studies found that high concentrations of ethanol (>100 mM) were necessary for significant inhibitory effect on proliferation to be manifest. The reason for these quantitative differences are not clear, but may be related to the different systems utilized; for example, Kane et al. (31) observed a small (10%) inhibition of proliferation by 22 mM ethanol in astrocytes derived from human adult temporal lobe, while all other studies made use of fetal or neonatal astrocytes from rodent brain. In all these studies, cells were cultured in medium containing serum and concomitantly exposed to ethanol. Under these conditions, cells are expected to be divided among the G0/G1, S and G2/M phases of the cycle, and the inhibitory effect of ethanol may be related to its ability to produce a block in the G0/G1 phase (34). Indeed, when human astrocytoma cells were serum deprived, so that >98% were in G0/G1, no effect of ethanol were measured by flow cytometry (35). In this case, the observed inhibition of [³H]-thymidine incorporation by 100-200 mM ethanol could be attributed no nonspecific effects on thymidine uptake, or to other interferences with cellular metabolism (e.g. DNA repair) (35).

A major metabolite of ethanol is acetaldehyde, which has been shown to inhibit cell growth and induce apoptosis in other cell types (e.g., the Chinese hamster ovary cell line A-10; 36). However, evidence suggests that ethanol itself, and not acetaldehyde, may be responsible for the observed inhibitory effect on cell proliferation. Astrocytes lack alcohol dehydrogenase, which catalyzes the conversion to ethanol to this metabolite (37), and the lack of this enzymatic pathways was also suggested in human astrocytoma cells (35). Furthermore, co-incubation of ethanol with 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, failed to affect the inhibitory action of ethanol (32, 35). Thus, ethanol, at high concentrations, appears to be able to inhibit proliferation of astrocytes or glioma cells, without the need for metabolic activation.