Effects of hexachlorophene on myelin marker enzymes in rat oligodendrocytes

Toxic. in Vitro Vol. 8, No. 1, pp. I-11, 1994

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Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/94 $6.00 + 0.00

Pergamon

EFFECTS OF HEXACHLOROPHENE ON MYELIN M A R K E R ENZYMES IN RAT OLIGODENDROCYTES

D. E.AMACHER and S.J. SCHOMAKER Cellular Toxicology, Pfizer Central Research, Groton, CT 06340, USA (Received 16 February 1993; revisions received 29 March 1993)

Abstract--The antibacterial substance hexachlorophene (HCP) can affect myelin formation or integrity leading to intramyelinic oedema and vacuolation in the central nervous system through an unknown mechanism. These studies were conducted to investigate the direct, dose-dependent effects of HCP on myelin membrane markers in cultured oligodendrocytes (OLG) isolated from 4-7-day-old rat pups and cultured in vitro for up to 5 wk. 2-wk-old OLG cultures were exposed to 0, 0.24 or 0.74/aM HCP for 48 hr. At 48 hr and again at 5, 12 and 19 days after the end of dosing the myelin markers galactosylceramide (GalC), myelin basic protein (MBP), and 2',Y-cyclic nucleotide Y-phosphohydrolase (CNPase) were quantified by ELISA or biochemical techniques. DNA was measured to estimate total cell mass and astrocyte contamination was determined by an ELISA procedure using anti-glial fibrillary acidic protein (GFAP) as the primary antibody. Because of the use of a selective culture medium, astrocyte contamination was initially low and continued to decrease from wk 2 to 4 as determined by GFAP binding. CNPase, GalC and MBP levelswere similar in control and low-dose(0.24/~MHCP) cultures with a general increase in MBP and CNPase over time. Cultures exposed to 0.74/zM HCP showed a decline in GalC proportional to decreased DNA content with time, but levels of MBP and CNPase increased after dosing and were always greater than the corresponding levels in control or low-dose cultures. These studies suggest a direct, dose-related toxic effect of HCP accompanied by a stimulation of MBP and CNPase but not of GalC production in the membranes of the recovering OLG following removal of HCP.

INTRODUCTION

A number of chemicals are known to damage selectively myelin-forming oligodendroglia or Schwann cells leading to altered function in the central or peripheral nervous systems, respectively. In particular, there is considerable pathological evidence of the neurotoxicity of the antibacterial substance hexachlorophene (HCP) in laboratory animals that involves the disruption and oedema of central nervous system (CNS) myelin (Towfighi, 1980). Pathological studies of HCP toxicity in humans have revealed CNS intramyelinic oedema with vacuolation of myelin in the brain stems of premature infants (Towfighi, 1980). Intramyelinic oedema has also been reported after HCP administration in the rat in which the myelin changes initially are reversible (Towfighi, 1980). In studies in rats fed HCP widespread myelin intraperiod line-splitting, intramyelinic vacuolization, and occasional segmental demyelination of the sciatic nerve have been observed with a Abbreviations: CNPase = 2',Y-cyclicnucleotide 3'-phosphohydrolase; CNS = central nervous system; DMEM = Dulbecco's Modified Eagle's Medium; DPBS= Dulbecco's phosphate buffered saline; GaIC = galactosylceramide (galactocerebroside); GC=anti-galactocerebroside; GFAP=glial fibrillary acidic protein; HCP = hexachlorophene; MES = 2-(N-morpholino)ethanesulfonic acid; MPB = myelin basic protein; OLG = oligodendrocytes; PBS = phosphate buffered saline.

40% reduction of myelin yield reported (Pleasure et al., 1974). When studied in vivo, however, it is difficult to ascertain whether these effects are a consequence of primary damage to myelin, altered myelinogenesis or oligodendrocytes (OLG) death, or whether these phenomena are the secondary effects of axonal damage and atrophy. Some 70-80% of the glial cells in the corpus callosum and centrum semiovale of older animals are oligodendrocytes, the cells that produce myelin in the CNS (Ling and Leblond, 1973). Methods have been developed for the isolation of relatively pure fractions of OLG and for their cultivation for extended periods (Gebicke-Harter et al., 1984; Lisak et al., 1981; Szuchet et al., 1980). Long-term cultures of OLG with high plating efficiencies and good viabilities have been maintained for up to 4 months (Norton et al., 1983). Thus, it should be possible to study the direct acute effects of HCP on the synthesis and the maintenance of mature myelin membrane markers in cultures of OLG (Szuchet et al., 1983) over a time period that is sufficient to allow recovery. The purpose of these studies were two-fold. The primary objective was to investigate the direct, dosedependent effects of HCP on membrane myelin markers in cultured neonatal rat OLG. The secondary goal was to develop a general in vitro system for assessing the direct effect of potential neurotoxicants on myelin-forming cells. Specific markers of myelin were monitored for several weeks after culture initiation

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D.E. AMACHERand S. J. SCHOMAKER

and short-term (48-hr) exposure to HCP. These markers included: galactosylceramide (galactocerebroside or GalC) one of two major galactolipids of myelin; myelin basic protein, a major myelinassociated glycoprotein (Norton, 1977); 2',Y-cyclic nucleotide 3'-phosphohydrolase (CNPase), a marker for the expression of oligodendroglial differentiation (Langan and Volpe, 1987). Using this in vitro system, we were able to monitor primary damage and recovery of myelin-producing cells independently of any secondary effects that might be caused by axonal degeneration in vivo.

medium). O L D M E M allows the long-term survival of mature OLG pure cultures while preventing the growth of contaminating astrocytes (de Los Monteros et al., 1988). Culture medium was replaced every 3 to 4 days with fresh OLDMEM. After 2 wk in culture--a time that coincides with the maximum rate of myelinization that would have occurred (at 20 days) in the brains of the original rat pups (Norton, 1977)--the oligodendroglia were exposed to HCP at 0.24 or 0.74 gM for 48 hr (days 0-2). At the end of the exposure period (day 2) the test medium was removed and replaced with HCP-free medium. A third set of cultures served as untreated controls. Treated and control cultures were mainMATERIALS AND M E T H O D S tained for an additional 0, 1, 2 or 3 wk after the start O L G were isolated from the brains of 4-7-day-old of dosing. The total time in culture therefore ranged rat pups using the procedures described by Koper between 2 and 5 wk. Each of four sets of experimental et al. (1984) and Lisak et al. (1981). For each cultures treated with 0, 0.24 or 0.74#M HCP and preparation, approximately 20 rat pups (Crl: COBS maintained for 0, 1, 2 or 3 wk after the start of CD (SD) BR, Charles River, Wilmington, MA, USA) treatment were derived from a separate group of rat were used. Except during the culture incubation pups. At weekly intervals, levels of DNA, GalC, period, all procedures were carried out at room myelin basic protein (MPB), CNPase and glial fibriltemperature. Brains were collected in Dulbecco's lary acidic protein (GFAP) were measured. Cells in phosphate buffered saline (DPBS) containing peni- two or three independent cultures were used for each cillin and streptomycin. Brains were dissociated with assay at each time point except for some of the assays gentle pipetting. The suspension was filtered through carried out 3 wk after treatment for which only one three consecutive sieves (Tetko, Elmsford, NY, USA) culture was available for each endpoint. with pore sizes of 300, 150 and 70/zm. The resulting CNPase activity was measured by a modification of cell suspension was diluted 3:1 (v/v) with Percoll the procedures of Sogin (1976) and Weissbarth et al. (Pharmacia) to produce a 25% Percoll suspension. (1980). Cells from cultures in six-well plates were 15ml of a 20% Percoll solution was layered over scraped free in 1.5 ml phosphate buffered saline (PBS) 25 ml of the 25% Percoll cell suspension and a step- and centrifuged for 5 min at 700 g in an Eppendorf gradient created by centrifuging for 30 min at 4°C at centrifuge. The resulting pellets were re-suspended in 7030 g in a Sorvall SS-34 rotor. After centrifugation 200~1 1% Triton X-100 (100pl was used in the and removal of the upper 25 ml, the exposed cell layer CNPase assay and for the bicinchoninic acid protein lying just above the red cell layer was removed, assay). The reaction was initiated by adding 100 pl of diluted with DPBS, and centrifuged for 10min at the tissue suspension to 1 ml of the reaction buffer 955 g in the Sorvall. [0.2 M MES (pH 6.0), 30mM MgC12, 1 mM cyclic The cell pellet was resuspended in medium NADP, 5 mM glucose-6-phosphate, 7 U glucose-6(OLG/FCS medium) consisting of a 50% Dulbecco's phosphate dehydrogenase], and the reaction mixture Modified Eagle's Medium (DMEM)-50% Ham's was incubated for 1 hr at room temperature. The F12 mixture to which had been added (per 0.5 litre): N A D P H generated was determined in an LS-5B 10% foetal bovine serum, 10 ml antibiotic mixture spectrofluorimeter using N A D P H standards to containing 50U penicillin/ml and 50#g strepto- quantitate the results. The results were then corrected mycin/ml, 5 ml L-glutamine, 1.1 g sodium bicarbon- for protein and expressed as /~mol N A D P H ate and I ml ITS premix (Collaborative Research, generated/mg protein. Bedford, MA, USA). At a density of 2 x 106 cells/ml, GalC, MBP, G F A P were measured in 24-well suspended cells were plated out in poly-L-lysine plates using antigen detection ELISA procedures. (0.25%, Sigma, St Louis, MO, USA) coated cluster Cells were fixed before the addition of primary well plates (6 or 24 wells/plate). Cultures were main- antibodies. Polyclonal rabbit anti-galactocerebroside tained in an atmosphere of 5% CO2/95% air at 37°C. (anti-GalC), monoclonal mouse anti-myelin basic Medium was changed after the first 24 hr. protein (anti-MBP), and monoclonal mouse anti-glial On day 5 of culture, OLG/FCS medium was fibrillary protein (anti-GFAP) were used as primary replaced with a 50% D M E M - 5 0 % Ham's F I 2 mix- antibodies to quantify the corresponding O L G ture to which had been added (per litre): 20ml membrane antigens using ELISA procedures and antibiotic mixture containing 50 U penicillin/ml and solutions from an Elisamate kit purchased from 50/zg streptomycin/ml, 10ml 200mM L-glutamine Kirkegard and Perry Laboratories, Inc. solution, 2.2 g sodium bicarbonate, 5.0mg insulin (Gaithersburg, MD, USA). Anti-GalC and anti(bovine pancreas), 16mg putrescine, 2.5g D ( + ) G F A P were purchased from Chemicon and antigalactose, and 8/~g sodium selenite/ml (OLDMEM MBP was purchased from Boehringer Mannheim.

HCP and oligodendrocyte myelin markers 0.01 M Tris (pH7.0). D N A was quantified in an LS-5B spectrofluorimeter using the fluorimetric assay of Harris (1987). A 100-#1 sample was combined with 2.4 ml 2 M NaCI containing 50 mM sodium phosphate (pH 7.4), 2 mM EDTA, and 1 pg Hoechst 33258/ml. This mixture was incubated for 5 min at ambient temperature and then the fluorescence at 460 nm was recorded after excitation at 355 nm. Calf thymus D N A (Sigma) was used to prepare standards. Statistical analysis of the data was completed using a two-way ANOVA by SAS Proc GLM, a procedure that could accommodate the unbalanced factorial design of the experiments (i.e. unequal replicate numbers). In each ANOVA, one of the five endpoints was the independent variable and dose and incubation time were the two factors.

Peroxidase-labelled, affinity-purified, goat antimouse IgG (H and L chain) was the secondary antibody used for the detection of bound anti-MBP and anti-GFAP antibodies, and peroxidase-labelled, affinity-purified, goat anti-rabbit IgG (H and L chain) was the secondary antibody for the detection of anti-GalC antibody. Preliminary titration experiments were conducted using both the primary and secondary antibodies to determine the appropriate dilutions of each antibody. A 1:100 dilution was used for all antibody incubations and the results were determined on a Dynatech MR700 at 410 nm. Protein was measured by the bicinchoninic acid method adapted for the Cobas-Bio autoanalyser (Roche) and using Pierce (Rockford, IL, USA) reagents and procedures. The sensitivity of the assay was 10-1200/z g/ml. The total D N A content of the cultures in 24-well plates was determined as follows. Tris-HCl buffer (pH 8.0) containing 1 mM EDTA and 1% SDS was added to individual culture wells. Cells were scraped free and transferred to an Eppendorf centrifuge tube. Proteinase k (10 mg/ml prepared in the same TrisHCI buffer) was added and each tube was incubated at 37°C for 30 min. An equivalent volume of chloroform-isoamyl alcohol (24: 1, v/v) was added, the mixture was shaken for 5 min, then centrifuged at 8160g for 5 min. The upper D N A layer was removed. A 1/10 volume of 2 M NaC1 plus two vols of ethanol were added and the precipitated D N A was removed after centrifugation at 8160g for 5 min. Ethanol was removed and the dried D N A was dissolved in 100 #1

50-

RESULTS

In the 5 days after treatment with HCP, the D N A content of all cultures declined (Fig. 1). This decrease varied from 38% in the controls to 43 and 48% in the cultures exposed to the low and high doses of HCP, respectively. For the next 2 wk, the D N A content followed a similar pattern in the low-dose and control cultures, with small increases noted 12 days after treatment followed by slight decreases 19 days after treatment. In the high-dose cultures, D N A continued to decline throughout the 2 wk after treatment; at 19 days after treatment the D N A content had declined to 27% of the level at the end of the 48-hr treatment period. At later stages of long-term cultures, particu-

• 0.00 ~M HCP • 0.24 ~M HCP • 0.74 ~M HCP

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DAYS POST EXPOSURE Fig. 1. D N A content of OLG cultures after exposure to 0, 0.24 or 0.74 #M HCP for 48 hr (days 0-2).

DNA was isolated from disrupted cells and detected by fluorescent binding with Hoechst 33258. At time zero, cells had been in culture for 14 days. Error bars indicate + 1 SD or + ½the range for independent cultures.

D.E. AMACHERand S. J. SCHOMAKER

4

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Fig. 2. ELISA quantitation of GFAP in OLG cultures after exposure to 0, 0.24 or 0.74/zM HCP for 48 hr (days 0-2). Monoclonal mouse anti-GFAP was the primary antibody and peroxidase-conjugated goat anti-mouse IgG was the secondary antibody. Absorbance was measured at 410 nm. At time zero, cells had been in culture for 14 days. Error bars indicate + ½the range for one to three independent cultures.

lady during the period after treatment, the oligodendrogha appeared to be clustered (Plate 1). GFAP, a marker for astrocytes, declined substantially in all cultures after dosing, with a 70% decrease in the high-dose cultures and a 57-59% decrease in the control and low-dose cultures at day 19 (Fig. 2). O L D M E M medium was used throughout most of the total culture period in order to prevent astrocyte growth. Visual inspection of cultures during the entire study suggested that some astrocytes were present initially, but the predominant cell type throughout the 5-wk culture period was the OLG. The first of the three myelin markers, MBP, was decreased in all three groups (by 11-17%) 5 days after treatment (Fig. 3). Although this initial decline was similar in the controls and both of the HCPtreated groups, the increase that subsequently occurred between 5 and 19 days after dosing was greater in the low-dose HCP cultures (57%, day 19 v. day 5 after dosing) than in either the controls (43%) or the high-dose group (29%). Also at three of the four sampling times, absolute levels of MBP were greater in the cultures treated with the high dose of HCP than in low-dose or control cultures.

The second myelin marker, GalC, had also showed small declines 5 days after treatment, with losses ranging from 15% for controls to 9 and 10% for the high and low-dose cultures, respectively (Fig. 4). However, during the next 2 wk the controls gradually recovered and 19 days after treatment levels of GalC in control cultures were similar to those at the end of the 48-hr treatment period. Levels of GalC in lowdose cultures remained relatively stable, but highdose cultures showed continuing declines with a net loss of 30% between days 5 and 19 after dosing. At all four sampling times, the absolute GalC levels estimated by antibody binding were lowest in the cultures exposed to 0.74 #M HCP. In contrast to the first two myelin markers, CNPase was increased in all three culture groups at 5 days after treatment, with increases of 55, 33 and 36% in the control, low- and high-dose groups, respectively (Fig. 5). During the next 14 days, the control cultures showed a 5% net decline, the lowdose cultures were generally unchanged and the high-dose cultures showed a further increase in CNPase for an overall net increase of 57% during the 19-day period. Absolute levels of CNPase were

Plate 1. Phase-contrast photomicrograph of oligodendrocytes after 20 days in culture. Unstained. × 20.

HCP and oligodendrocyte myelin markers

7

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EXPOSURE

Fig. 3. ELISA quantitation of MBP in OLG cultures after exposure to 0, 0.24 or 0.74 #M HCP for 48 hr (days 0-2). Monoclonal mouse anti-MBP was the primary antibody and peroxidase-conjugated goat anti-mouse IgG was the secondary antibody. Absorbance was measured at 410 nm. At time zero, cells had been in culture for 14 days. Error bars indicate + ½the range for one to three independent cultures.

always higher in the cultures treated with 0.74#M HCP than in control or low-dose cultures, even though independent OLG cultures were used at the various time points. Results of the two-way ANOVA using SAS Proc G L M (SAS Institute Inc., Cary, NC, USA) are presented in Table 1. A pooled estimate of variance was used to compare the controls with either the lowor high-dose group. For all independent variables, the controls were not statistically significantly different (P > 0.05) from the low-dose group, but were significantly different (P < 0.05) from the high-dose group. DISCUSSION Microscopic examination and immunochemical studies with astrocyte-specific antibodies indicate that the studies described here were completed with cultures consisting predominantly of rat oligodendroglia. At later stages of long-term culture, particularly during the period after treatment, the oligodendroglia appeared to be clustered. This suggests either cell division or the possible migration of OLG; however, cellular mobility was not measured. No difference in cell morphology was noted between HCP-treated and control cultures (not

shown). Because there was a net loss of D N A over a 3-wk period, the clustering of OLG could not readily be attributed to mitosis unless some O L G populations died out while others from different brain tissues multiplied. Isolated OLG may or may not proliferate in culture depending on the culture milieu and the developmental state of the brain area--and hence, the age of the animal--used to establish the cultures (Saneto and Vellis, 1985). In these studies, the use of non-proliferating or slowly proliferating OLG cultures was a distinct advantage in that the effects of HCP could be monitored without being obscured by changes in myelin markers that would accompany rapid cell proliferation. Biochemical and immunochemical studies for the quantification of three specific myelin markers in OLG cultures suggested changes in these markers over time in the absence of any chemical treatment. For example, our preliminary experiments with similar, untreated OLG cultures indicated that, when expressed on the basis of total protein, both MBP and GalC declined during the first 18 days of culture (data not shown) but then began to increase to the original levels (DNA was not quantified). This phenomenon was similar to the growth characteristics reported by Norton et al. (1983) for bovine O L G measured by total protein per culture over the first 24 days in vitro.

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D.E. AMACHERand S. J. SCHOMAKER

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Fig. 4. ELISA quantitation of GaIC in OLG cultures after exposure to 0, 0.24 or 0.74/~M HCP for 48 hr (days 0-2). Polyclonal rabbit anti-GFAP was the primary antibody and peroxidase-conjugated goat anti-rabbit IgG was the secondary antibody. Absorbance was measured at 410 nm. At time zero, cells had been in culture for 14 days. Error bars indicate + ½the range for one to three independent cultures.

In particular, o u r preliminary data for GalC were very similar to the levels of GalC estimated by precursor incorporation in a study of ovine OLG from 5 to 35 days in culture by Szuchet et al. (1983). In the present study CNPase, on the other hand, increased in the control cultures between days 16 and 21 after the initiation of culture. The decline in total DNA content, mentioned above, and the quantification of the CNPase marker relative to total cellular protein suggested that the increased activity of this marker was not due simply to an increase in the total number of control cells. The selection of 2-wk-old cultures for HCP exposure was based on the approximate maturation time for myelinization in vivo and on these preliminary GalC and MBP studies which suggested stabilization after preliminary adjustment to culture conditions. Our data indicate that, in primary cultures of OLG derived from 5-7-day-old rat pups and grown in selective OLDMEM medium, there is an initial decline in MBP and GalC as well as a decline in the number of OLG as determined by DNA content. This corresponds to 2-5 days after treatment or 16 to 19 days after culture initiation. The presence of HCP for 48 hr (i.e. 14-16 days after initiation of the

primary culture) results in a dose-dependent effect on all three of these markers (GalC, MBP and CNPase) presumably as a consequence of membrane damage. In contrast to control cultures, the HCP-treated cultures showed a net loss over 21 days in total GalC activity, especially in the cultures exposed to 0.74 gM HCP. It has been suggested that MBP and GalC may be under separate regulatory control and that the diminished expression of GalC in culture may result from the presence of differentiated OLG that are no longer capable of cell division (Saneto and de Vellis, 1985). In vitro studies by Pleasure et al. (1974) suggest that the binding of HCP to neural tissue is not myelin-specific, but is determined by the portion of lipid in the fraction, and GalC is the major glycolipid in myelin (Norton and Autilio, 1966). Thus, a subpopulation of OLG expressing GalC may be selectively damaged by HCP exposure. In this study, the effects of HCP on CNPase activity were two-fold. The presence of HCP increased CNPase activity during long-term culture, and the absolute CNPase levels were greater in the high-dose cultures when HCP exposure was terminated than in low-dose or control cultures. In view of the net loss over time of DNA in the OLG cultures

H C P and oligodendrocyte myelin markers

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DAYS POST EXPOSURE Fig. 5. C N P a s e activity expressed as N A D P H formed per m g proteins in O L G cultures exposed to 0, 0.24 or 0.74 #M H C P for 48 hr (days 0-2). Values are sample means + ½ the range (indicated by error bars) o f one to three independent cultu~-s. At time zero, cells had been in culture for 14 days.

treated with 0.74/~M HCP cultures, the increase in CNPase activity in the high-dose cultures actually represents an increase, after HCP exposure, in enzyme activity relative to total cell mass. Likewise, the

recovery of MBP activity between days 5 and 19 after treatment (19-33 days after culture initiation) suggests a net increase in this marker relative to control cells when changes in D N A content are considered.

Table I. Two-way analysis of variance with contrasts ANOVA table F~ndpoint DNA GalC MBP

GFAP CNPase

Model Error Model Error Model Error Model Error Model Error

P value for dose contrast:

DF

SS

F

P

0.24/~M v. 0 (control)

0.74pM v. 0 (control)

I1

771.84

30.2

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0.44

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15 II

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24.61

0.0001

0.18

0.0001

9

0.00587

0.025

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5.38

0.0087

0.81

9 8 9

0.08055 0.01218 0.00075

18.18

0.0001

0.21

0.024

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78.2377

28.78

0.0001

0.27

0.0001

2.9652

GFAP = glial fibrillary protein CNPase = 2',Y-cyclic nucleotide GaIC = galactosylceramide MBP = myelin basic protein Y-phosphohydrolase In each ANOVA, one of the five endpoints is the dependent variable, and dose and incubation time are the two factors. A pooled estimate of variance was used to compare the controls (0) with either dose (0.24 or 0.74/~M) group. Model refers to the treatment effect or difference between overall mean and the mean at each level; error refers to the replication error or average variance within each cell of the table.

I0

D.E. AMACHERand S. J. SCHOMAKER

Previous workers (Kung et al., 1988) have demonstrated that the in vitro exposure of brain cell marker enzymes, including non-neuronal CNPase, to HCP concentrations up to 100 gM had no direct inhibitory effect. However, whereas in that study (Kung et al., 1988) short-term exposure of rats to HCP (40 mg/kg body weight/day orally for 9 days), resulted in a significant decrease in CNPase in the sensitive optic nerve, longer term exposure of rats to HCP (20 mg/ kg/day orally for 53 days) produced a significant increase in CNPase activities in optic nerve (Kung et al., 1989). Broadly interpreted, our results, which show an increase in cellular CNPase activity with HCP exposure, suggest that the long-term in vivo response reported for CNPase by Kung et al. (1989) may actually represent either a recovery or adaptation response to the primary membrane damage. The effects of HCP on rat oligodendroglia may also indicate a selective effect against a single type of OLG or an OLG at a certain stage of maturation. For example, using adult pig brain and anti-galactocerebroside (GC) antibodies, Gebicke-Harter et al. (1984) have distinguished anti-GC ÷ and anti-GC cells and the latter may represent precursors of mature OLG. Using myelin prepared from 18-day chick embryo sciatic nerves, Pleasure et al. (1984) have suggested that the binding of HCP to neural tissue was determined by the proportion of lipid in the fraction. The expression of other myelin markers, for example MBP, is also time dependent (Monge et al., 1986), and therefore maturation dependent, in vitro. MBP is expressed in 60% of the OLG population after 3-4 wk in culture (Gebicke-Harter et al., 1984; Lisak et al., 1981). However, this mechanism is unlikely to explain the effects seen in the present study because, during myelinogenesis, oligodendroglia have been shown first to express GalC and then to express MBP several days later (Althaus et al., 1984), a sequence that is also observed in vivo; Dubois-Dalcq et al., 1986). In the present study, the high-dose cultures expressed low and decreasing GalC accompanied by high and increasing MBP over time. In conclusion, we have demonstrated for the first time a dose-related effect of HCP on cell viability or proliferation and subsequently altered myelin marker enzymes in OLG cultures prepared from rat neonates and maintained in vitro for a total of 5 wk. This inhibitory effect was manifested as the gradual, dosedependent loss of OLG as estimated by a decreasing DNA content over time and an associated decline in GalC activity. Those cells that remained viable after HCP exposure appeared to have normal general morphology. There was a tendency for the formation of aggregates or clusters in all cultures which increased over time. Comparative increases in MBP content and CNPase activity (relative to DNA content or total cellular protein) over time after HCP exposure and subsequent removal suggest an in vitro recovery phase that is accompanied by stimulation of the synthesis of some myelin markers. The discrep-

ancy between increasing MBP and CNPase production on the one hand and declining GalC content on the other, may indicate: (1) HCP toxicity results in the synthesis of an altered membrane myelin that is specifically deficient in the galactolipid that is recognized by anti-GalC or (2) selective survival of OLG cells with these characteristics in the presence of HCP. The sequence of events at the cellular and molecular levels suggested by these studies are: (1) HCP toxicity which results in cell loss and/or diminished cell division in surviving OLG and (2) a subsequent recovery phase characterized by dose-related increases in MBP and CNPase synthesis but not GalC synthesis. Acknowledgements--The authors thank Dr Annahiti Ghassemi, formerly with the School of Pharmacy, University of Connecticut, Storrs, CT, USA for her preliminary contributions to this study. We also thank Dr Archie Swindell, Clinical Research, Pfizer Central Research, for his advice and statistical evaluation of the data, and Ms Cyndi Schneider for her assistance in the preparation of this manuscript. REFERENCES

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