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

4. EFFECT OF ETHANOL ON MITOGEN-STIMULATED GLIAL CELL PROLIFERATION

Only a limited number of studies have examined the effect of ethanol on the proliferation of glial cells stimulated by mitogens. Overall, ethanol appears to be a potent, but selective, inhibitor of stimulated proliferation, as it is capable of completely blocking the action of certain mitogens at concentrations of 50-100 mM, while it is ineffective toward other mitogens.

In a study by Luo and Miller (33) the proliferation of C6 rat glioma cells induced by basic fibroblast growth factor (bFGF) was measured by cell counting and by [³H]-thymidine and bromodeoxyuridine incorporation. Incubation with 80 mM ethanol caused a total block of cell proliferation and the IC₅₀ was about 20 mM (33). These results differ from those of de Vito et al. (29) who reported a lack of effect of ethanol (50 mM) on [³H]-thymidine incorporation in rat cortical astrocytes induced by FGF. These authors, however, reported that ethanol inhibited the proliferation of astrocytes induced by prolactin, with an IC₅₀ of about 25 mM (29). Interestingly, inhibition of prolactin-induced [³H]-thymidine incorporation was seen not only when ethanol and the mitogen were co-incubated, but also when cells had been exposed to ethanol for at least 6-18 hours prior to the addition of prolactin. A decrease in prolactin-induced astrocyte proliferation was also found in astrocytes prepared from rats that had been prenatally exposed to ethanol (5% ethanol in drinking water for the last five days of gestation). Thus, it would appear that the presence of ethanol together with this mitogen is not necessary for its inhibitory effect to be manifest, and that prolonged exposure to ethanol may cause changes in the cells that prevent the action of this mitogen.

In another study, ethanol was found to be a potent inhibitor of proliferation of rat cortical astrocytes and human astrocytoma cells induced by muscarinic agonists (carbachol, methacholine, acetylcholine, bethanecol), with an IC₅₀ of about 10 mM (35). Inhibition of carbachol-stimulated proliferation of glial cells was of the noncompetitive type, and was observed by measuring [³H]-thymidine incorporation into DNA, as well as by flow cytometry. Using this latter technique (which utilizes 5-bromodeoxyuridine and Hoechst 3325), carbachol (0.1 mM) was found to increase the percent of cells in S/G2 from 0.83 to 23.3. Ethanol (25 mM) had no effect when present alone, but significantly decreased the effect of carbachol (to 12.3% of cell in S/G2 phase) (35).

Resnicoff et al, have examined the effect of ethanol on proliferation of rat C6 glioblastoma cells induced by insulin-like-growth-factor I (IGF-I); alcohol completely inhibited proliferation (determined by cell counting with an hemocytometer) with an IC₅₀ of 10-25 mM (38,39).

Though these four mitogens have been investigated more in detail with regard to their sensitivity to ethanol, a few others have also been examined (Table 1). Of notice is that ethanol was ineffective in inhibiting proliferation of glial cells induced by insulin and platelet-derived-growth factor (PGDF), while it was a potent inhibitor of the mitogenic action of the phorbol ester 12-O- tetradecanoylphorbol 13-acetate, TPA), with an IC₅₀ of 15.6 mM (35).

Little is known, so far, on the mechanism(s) involved in ethanol's inhibition of mitogen-stimulated proliferation. In case of IGF-I, it has been shown that the receptor of this growth factor undergoes tyrosine-autophosphorylation of the beta-subunit upon stimulation with the agonist, and this process was completely inhibited by 100 mM ethanol (38). This mechanism may also account for ethanol's inhibition of EGF (epidermal growth factor)-induced proliferation; the latter has indeed been shown to be due to induction of IGF-I receptors by EGF, and a subsequent mitogenic action mediated by an IGF-I/IGF-I receptor autocrine pathway (39).

Ethanol has been shown to inhibit muscarinic receptor-coupled second messenger systems in glial cells, including formation of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium, and activation of phospholipase D (40; Catlin and Costa, unpublished). However, the relative roles of these intracellular pathways in the inhibition of carbachol-stimulated proliferation by ethanol, is still not clear. The finding that proliferation induced by TPA is inhibited by ethanol with high potency has led to the hypothesis that protein kinase C (PKC) may play a role in the inhibitory effect of alcohol (35). However, the reported effects of ethanol on PKC isozymes seem to differ (i.e., stimulation vs. inhibition) depending on the alcohol concentration, the cell type and the mode of exposure (i.e., acute vs. chronic). Thus, substantial additional research is needed to test this hypothesis. The ability of ethanol to potently inhibit mitogen-induced proliferation of glial cells is in agreement with its reported ability to block cells in the G0/G1 phase (34). Still, to be understood, however, is why some mitogens are not sensitive (or less sensitive) to the inhibitory action of ethanol.