Lead Exposure Delays the Differentiation of Oligodendroglial Progenitors in Vitro

Toxicology and Applied Pharmacology 174, 235–244 (2001) doi:10.1006/taap.2001.9219, available online at http://www.idealibrary.com on

Lead Exposure Delays the Differentiation of Oligodendroglial Progenitors in Vitro Wenbin Deng,* Randall D. McKinnon,† and Ronald D. Poretz* ,1 *Department of Biochemistry and Microbiology, Rutgers University, 76 Lipman Drive, New Brunswick, New Jersey 08901; and †Department of Surgery (Neurosurgery), Department of Molecular Genetics and Microbiology, and The Cancer Institute of New Jersey, UMDNJ–Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Received January 11, 2001; accepted May 21, 2001

maintenance of myelin in the CNS. The myelin sheath facilitates transmission of nerve impulse by saltatory conduction (Pfeiffer et al., 1993; Vos et al., 1994). Recent studies have demonstrated that OLs produce factors that induce neuronal sodium channel clustering (Kaplan et al., 1997), enhance axon growth (Sa´nchez et al., 1996), and support neuron survival (Sortwell et al., 2000). These are important aspects for optimal synapse formation and maintenance and brain development. The mechanism by which Pb causes CNS deficits is unclear. The metal, however, may affect the development of OLs. In vitro experiments revealed that OLs are the most Pb-sensitive cell type in the brain (Tiffany-Castaglioni, 1993). Mature myelin-competent OLs are exceptionally rich in galactolipids (Coetzee et al., 1998; Pfeiffer et al., 1993), and Pb causes reduced cellular levels of myelin-specific galactolipids and enzymes in the developing rat brain (Deng and Poretz, 2001). Furthermore, exposure of cultured human fibroblasts to Pb-containing medium causes reduced amounts of arylsulfatase A (ARSA), the catabolic enzyme for sulfatide, a major OL galactolipid (Poretz et al., 2000). OLs are generated from oligodendroglial progenitor (OP) cells that, when isolated and grown in vitro, are able to generate either OLs or type-2 astrocytes under different culture conditions (Raff et al., 1983). Primary cultured OP cells are well characterized and serve as a model to study the factors and agents influencing OL survival and differentiation during development (Pfeiffer et al., 1993). OL lineage cells are morphologically and functionally distinct at each stage of maturation and display a temporal expression of stage-specific markers that serve to define OL lineage progression. A large array of reagents capable of detecting these markers is available to monitor the in vitro differentiation process as these cultures develop into mature OLs (Bansal et al., 1999). Significant advances in the understanding of OL biology have resulted from studies of both primary cultures of OP cells and propagating OP cell lines such as central glia-4 (CG-4), established

Lead Exposure Delays the Differentiation of Oligodendroglial Progenitors in vitro. Deng, W., McKinnon, R. D., and Poretz, R. D. (2001). Toxicol. Appl. Pharmacol. 174, 235–244. Lead (Pb) is an environmental neurotoxicant that can cause hypoand demyelination. Oligodendrocytes (OLs), the myelin-forming cells in the central nervous system, may be a possible target for Pb toxicity. The present study describes the effect of Pb on the maturation of rat OL progenitor (OP) cells and the developmental expression of myelinspecific galactolipids. Dose–response studies showed that OP cultures were more sensitive to Pb than mature OLs. Pb delayed the differentiation of OL progenitors, as demonstrated by cell morphology and immunostaining with a panel of stage-specific differentiation markers. Pb given prior to and during differentiation caused a decrease in the biosynthesis of galactolipids in both undifferentiated and differentiated OLs, as detected by metabolic radiolabeling with 3H-D-galactose. While the ratios of galacto/gluco-cerebrosides, hydroxy fatty acid/nonhydroxy fatty acid galactolipids, and galactocerebrosides/ sulfatides increased in control cultures during cell differentiation, Pb treatment prevented these changes. The results suggest that chronic Pb exposure may impact brain development by interfering with the timely developmental maturation of OL progenitors. © 2001 Academic Press

Key Words: lead toxicity; oligodendrocyte; differentiation; myelination; galactolipid.

Lead (Pb) poisoning is a major environmental problem of public concern. Chronic low-level exposure of the pediatric population can result in subtle cognitive and neurobehavioral deficits (Banks et al., 1997; Cory-Slechta, 1995; Goyer, 1993; Bellinger and Needleman, 1992), while at higher levels Pb causes obvious myelin defects (Morell, 1984; Duncan, 1985). One potential target of Pb toxicity in the CNS is oligodendrocytes (OLs). 2 These cells are responsible for the synthesis and 1

To whom correspondence should be addressed. Fax: (732) 932-8965; E-mail: [email protected]. 2 Abbreviations used: ARSA, arylsulfatase A; bFGF, basic fibroblast growth factor; CG-4, central glia-4; CM, conditioned medium; CNPase, 2⬘,3⬘-cyclic nucleotide 3⬘-phosphohydrolase; GC, galactocerebroside; GFAP, glial fibrillary acidic protein; HFA, hydroxy fatty acid; MAb, monoclonal antibody; 235

MBP, myelin basic protein; NFA, non-hydroxy fatty acid; OL, oligodendrocyte; OP, oligodendroglial progenitor; PDGF, platelet-derived growth factor. 0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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from a primary OP culture of neonatal rat cerebral cortex (Louis et al., 1992). Such cell lines have been used as a model to study the biology of OL development (Espinosa de los Monteros et al., 1997; Fatatis and Miller, 1997; Yim et al., 1995; Franklin and Blakemore, 1997; Osterhout et al., 1997). The present study demonstrates that a significant target of Pb-induced OL toxicity is OP cells, as evidenced by analysis of the emergence of OL maturation markers. Chronic Pb exposure may thus impact brain development by impairing the timely OL development and expression of specific OL elements at defined developmental stages. MATERIALS AND METHODS Cell culture. OP cells were isolated from mixed glial cultures established from neonatal rat cortex (McCarthy and de Vellis, 1980) as described by McKinnon et al. (1990). Cerebral cortexes were dissected from 2-day-old Sprague–Dawley rats, the meninges were removed, and the brains were dissociated by gentle trituration in minimum essential medium (MEM) containing Hepes buffer (Gibco–BRL, Bethesda, MD) with a 10-ml pipette and, sequentially, 19-, 22-, and 25-gauge syringe needles. The cell suspension was filtered through a 70-␮m Nylon cell strainer (Falcon), centrifuged at 1000 rpm for 5 min, and the cells were resuspended and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco) and sodium pyruvate (1 mM), penicillin (50 IU/ml), and streptomycin (50 ␮g/ml). The mixed glial cells from two brains were plated in a 75-cm 2 flask precoated by incubation with poly-L-ornithine (100 ␮g/ml) and were allowed to grow for 7 days at 37°C in a humidified atmosphere of 90% air and 10% CO 2. The medium was changed every 3 days and, after confluency, loosely attached microglia were depleted by shaking (80 rpm for 2 h) on a horizontal rotary platform. The cells, collected by further vigorous shaking (120 rpm for 16 h), were mainly OP cells. Contaminating cells (principally astrocytes and microglia) were further depleted by placing the cells on uncoated dishes for 20 min. The OP cells that did not adhere were recovered from the medium by centrifugation at 1000 rpm for 5 min and plated in a 25-cm 2 poly-L-ornithinecoated flask with Silberberg’s medium, a chemically defined medium that contains 0.5% FBS (McKinnon et al., 1990), supplemented with basic fibroblast growth factor (bFGF, 5 ng/ml) and platelet-derived growth factor (PDGF, 10 ng/ml, Sigma). The OP cells were grown in the presence of growth factors by feeding with fresh medium every 2 days. OL differentiation was initiated by replacing this medium with Silberberg’s medium lacking supplemental mitogens. A CG-4-like OP cell line was developed from the primary OP cultures by the procedure described by Louis et al. (1992). It was originated by repeated passage of expanded populations of wild-type progenitors in a mixture of 70% Silberberg’s medium and 30% neuroblastoma B104 conditioned medium (B104-CM). B104-CM is Silberberg’s medium conditioned by the presence of a confluent monolayer of neuroblastoma cells for 48 h as described previously (Louis et al., 1992). The CG-4-like OP cells were maintained and amplified in poly-L-ornithine-coated flasks in 80% Silberberg’s medium supplemented with 20% B104-CM and passaged by trypsinization and replating at a 1:5 split every 3 or 5 days as described (Espinosa de los Monteros et al., 1997). Passages 5 to 25 were employed for the experiments.

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Effect of Pb on the survival and growth of oligodendroglial cells. Dose– response relationships were established with OP cells, the CG-4-like OP cell line, and mature OLs derived from primary OP cultures. OP cells were maintained in Silberberg’s medium supplemented with bFGF and PDGF, and the CG-4-like OP cell line remained in Silberberg’s medium supplemented with 20% B104-CM. Mature OLs were developed from OP cells maintained for 6 days in Silberberg’s medium without the supplemental growth factors. These target cells were exposed to Pb acetate for 0 –7 days and enumerated daily. Each coverslip was analyzed by counting a total of at least 200 cells in a minimum of six randomly chosen fields. Immunocytochemistry. Cells grown on poly-L-ornithine-coated 12-mmdiameter glass coverslips were washed with phosphate-buffered saline (PBS), fixed for 20 min with 2% paraformylaldehyde in PBS, blocked with 2% BSA in PBS, and sequentially exposed to primary and secondary antibodies as described previously (McKinnon et al., 1990). Primary antibody dilutions in PBS containing 0.1% BSA were monoclonal antibody (MAb) A2B5, 1:20; MAb O4 and anti-galactocerebroside (GC, Ranscht-MAb) (Ranscht et al., 1982), 1:10; anti-2⬘,3⬘-cyclic nucleotide 3⬘-phosphohydrolase (CNPase, Sigma), anti-myelin basic protein (MBP, Boehringer Mannhein), 1:250; and anti-glial fibrillary acidic protein (GFAP, Roche), 1:100. Cells stained with anti-CNPase, anti-MBP, and anti-GFAP were first permeabilized with 0.1% Triton X-100 for 10 min. Fluorescent-conjugated secondary antibodies (Pierce) were used at a 1:50 dilution. A2B5 (murine IgM) and anti-GC (murine IgG) were used as pairs for double staining. Fluorescent secondary antibodies were also employed as a pair of anti-mouse IgM TRITC and anti-mouse IgG FITC-conjugated immunoglobulins. The mounted coverslips were viewed with a Leitz microscope, and fluorescence and phase contrast images were captured with a Nikon digital camera. Each coverslip was analyzed by counting a total of at least 200 cells in a minimum of six randomly chosen fields. Lipid extraction and separation of glycolipids. Cells grown in the presence of D-[4,5- 3H(N)]galactose (10 ␮Ci/ml, 58.7 Ci/mmol, DuPont–New England Nuclear) for 20 h were harvested, and lipids were extracted as described by Yim et al. (1995). Separate aliquots were taken from the total cell suspension and the crude mixture of lipids in the lower organic phase to determine the total cellular uptake and extractable lipids associated radioactivity, respectively. Total lipids were subjected to mild alkali-catalyzed methanolysis to partially destroy alkali-labile glycerophospholipids. A solution of lipids in 1 ml of 0.6 N methanolic KOH and 1 ml of chloroform was allowed to stand at 37°C. After 1 h, 1.2 ml of 0.5 N methanolic HCl, 1.7 ml of H 2O, and 3.4 ml of chloroform were added and mixed vigorously. The biphasic system was centrifuged to recover glycolipids in the lower layer. After this extract was washed twice with equal volumes of C/M/0.74% KCl (3:48:47, v/v), the solvents were removed under a stream of nitrogen. Glycolipids were then separated by chromatography on silica gel 60 high-performance thin-layer chromatography (HPTLC) plates developed twice in C/M/H 2O (144:25:2.8, v/v). Radiolabeled lipids were determined by autoradiography using En 3Hancer (New England Nuclear) and X-OMAT AR5 film (Kodak) and quantified by densitometry using the NIH Image computer program (version 1.62). Individual lipids were identified by their comigration with authentic glycolipid standards (Sigma), which were chromatographed with experimental samples and visualized with Orcinol spray. Statistical analysis. Data were presented as mean ⫾ SD (n ⫽ 3, each the average of duplicates). Statistical comparisons on the data of all experiments were performed using one-way analysis of variance with Tukey post-hoc analysis. A p value ⬍0.05 was used to determine statistical significance.

FIG. 1. Effect of Pb on cell growth and/or survival. The OL progenitors, mature OLs, and CG-4-like cells were cultured in the presence of Pb acetate, at the concentrations indicated, for 0 –7 days and enumerated daily. All cultures were maintained in Silberberg’s medium as described under Materials and Methods. OP cells were supplemented with bFGF (5 ng/ml) and PDGF (10 ng/ml), and the CG-4-like OP cell line was supplemented with 20% B104-CM. Mature OLs were developed from OP cells cultured for 6 days in Silberberg’s medium without the supplemental growth factors. Each data point represents the mean ⫾ SD (n ⫽ 3, each an average of duplicates). *Lowest concentration showing significant difference ( p ⬍ 0.05) compared to its respective Pb-free control.

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FIG. 2. Pb treatment protocols of OL lineage cells. Pb concentration, 1 ␮M; GF, growth factors (PDGF and bFGF). The cultures employed for protocols A and C were exposed to 1 ␮M Pb at only one time point and those used for protocol B were exposed a second time to fresh Pb containing medium to ensure continuous Pb exposure before and after removal of growth factors. These cultures were washed three times with PBS between Pb dosing.

RESULTS

OPs and mature OLs exhibit differential sensitivity to Pb. In order to characterize Pb toxicity in OLs and determine the appropriate Pb concentrations to employ, dose–response relationships with Pb treatment for 0 –7 days were established with the CG-4-like OP cell line, OP cells, and mature OLs. The cell populations were characterized immunochemically as described under Materials and Methods and previously (McKinnon et al., 1990; Osterhout et al., 1997). More than 90% of the cells in the CG-4-like OP cell line cultures were A2B5-positive. The OP cell population was ⬎85% A2B5-positive, and the mature OL cultures were ⬎80% MBP-positive. All cultures contained less than 5% GFAP-immunoreactive astrocytes. As shown in Fig. 1A, the CG-4-like OP cell line maintained in the presence of 80% Silberberg’s/20% B104-CM continued to proliferate when exposed to 0 –20 ␮M Pb acetate. Pb treatment, however, decreased the extent of population expansion of these cells in a dose-dependent manner. Treatment of OP cells with a low concentration of Pb (1 ␮M) for 48 h in the presence of PDGF and bFGF had no significant effect on cell number (Fig. 1B). Longer exposure or higher concentration of the metal was, however, either cytostatic or cytotoxic to these cells (Fig. 1B). Mature OLs, developed from primary progenitor cells over the course of 6 days following removal of growth factors, were exposed to medium containing Pb acetate (Fig. 1C). These mature OLs were much more resistant to the cytostatic or cytotoxic effect of Pb than progenitor cells (Fig. 1C). As shown in Fig. 1, the minimum Pb concentration range affecting cell growth and/or survival after a 3-day exposure was 0.1–1.0, 5–10, and ⬎100 ␮M for OP cells, the CG-4-like OP cell line, and mature OLs, respectively. Though the CG4-like and OP cells are proliferative and mature OLs are not, the data clearly indicate that OP cells are more sensitive than either the CG-4-like OP cell line or the mature OLs to Pb.

Pb delays the differentiation of OP cells. We next examined the effects of low levels of Pb on OP maturation in vitro following the protocols illustrated in Fig. 2. OP cells maintained in the presence of growth factors were pretreated with 1 ␮M Pb acetate for 24 h and subsequently allowed to differentiate in the absence of mitogens for 1–5 days, either in the absence or continuous presence of 1 ␮M Pb acetate (Figs. 2A and 2B, respectively). Alternately, a postmitotic Pb exposure protocol was employed in which OP cells were maintained in the absence of growth factors for 24 h prior to exposure to medium lacking mitogens but containing 1 ␮M Pb acetate (Fig. 2C). These Pb treatments appeared not to be cytocidal under these conditions since no significant change in total cell number was found in all three groups of the Pb-treated cells compared to the Pb-free control cells. Cells were stained with a panel of stage-specific antibodies to monitor cell differentiation. The progenitor cells were defined by expression of A2B5-immunoreactive surface gangliosides, late progenitors which acquired sulfated glycans were detected by O4-immunoreactivity, and more mature OLs were identified with the

FIG. 3. Effect of Pb on OL progenitor cells. Phase– contrast images of cells cultured in 1 ␮M Pb acetate as described for protocol A in Fig. 2 (B, D, F) or respective Pb-free controls (A, C, E). (A and B) Cells cultured continuously in the presence of bFGF and PGGF for 48 h. (C and D) Forty-eight hours after withdrawal of growth factors. (E and F) Ninety-six hours after withdrawal of growth factors. Most of the control (A) and Pb-treated (24 h) cells (B) exhibited a bipolar morphology in the presence of growth factors. Two days following withdrawal of mitogens, control cells showed a multibranched morphology (C) while Pb-treated cells gained fewer processes (D). By day 4, both control (E) and Pb-treated (F) cells showed a complex network of cellular processes indicative of mature OLs. Bar represents 30 ␮m.

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(groups A and B) appeared to be more immature with fewer processes emanating from the cell body (Fig. 3D). The Pb pretreated cultures also had a lower percentage of O4 ⫹, GC ⫹,

FIG. 4. Representative immunofluorescence (indicated by uppercase letters) and corresponding phase– contrast (indicated by lowercase letters) images of OL lineage cells in the absence (A, a, C, c, E, e, G, g, I, i) or presence (B, b, D, d, F, f, H, h, J, j) of 1 ␮M Pb acetate as described for protocol A in Fig. 2. Two days after initiating differentiation, control cells developed multibranched processes with loss of A2B5 immunoreactivity (A), while Pb-treated cells (exposed for 24 h prior to removal of growth factors) remained mostly A2B5-positive (B). After 3 days of differentiation, control cells were strongly immunoreactive to O4 (C), GC (E), CNPase (G), and MBP (I), while Pbtreated cells were much more immature and stained weakly for these markers (D, F, H, J). Bar represents 30 ␮m.

Ranscht (anti-GC) antibody as well as the appearance of the OL protein markers, CNPase and MBP. Representative phase-contrast and fluorescence images of control and Pb-treated cells at various developmental stages are shown in Figs. 3 and 4, respectively, and the results of quantitative analysis are shown in Fig. 5. In the presence of mitogens, the majority of the OP cells exhibited a bipolar morphology (Figs. 3A and 3B), and approximately 80% of the total population were immunoreactive with A2B5 (Fig. 5A). The few cells that acquired O4 and GC immunoreactivity under these culture conditions were larger, with multiple branches characteristic of more mature OLs that had escaped mitotic regulation by bFGF and PDGF. In contrast, after 24 h in culture medium lacking growth factors, an increased number of cells in the Pb-free control cultures acquired an O4-immunoreactive multibranched morphology, while most of the Pb pretreated cells (both experimental groups A and B, see Fig. 2) remained bipolar and showed a significantly lower percentage of O4 ⫹/ GC ⫹ cells (see Figs. 5B and 5C). After 2 days of differentiation, the control cells gained more complex processes (Fig. 3C), while the Pb pretreated cells

FIG. 5. Percentage of immunoreactive cells, identified using a panel of stage-specific markers, in the absence or presence of Pb during OP cell development. Three protocols of Pb treatments were employed (see Fig. 2): Pb (1 ␮M) treated 24 h prior to differentiation only; Pb (1 ␮M) treated 24 h prior to and during differentiation; and postmitotic exposure to Pb (1 ␮M) after 1 day of differentiation. No significant change in total cell number was found in the Pb-treated populations compared to the Pb-free control cultures. The cells were stained with a panel of stage-specific antibodies to monitor cell differentiation, and the results represent mean ⫾ SD (n ⫽ 3, each an average of duplicates) with at least 200 cells counted in a minimum of six randomly chosen fields per condition. *p ⬍ 0.05 vs control; ** p ⬍ 0.01 vs control; ***p ⬍ 0.001 vs control.

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FIG. 6. HPTLC chromatogram of glycolipids from 3H-D-galactose-labeled CG-4-like cells at different times during differentiation. Glycolipids were extracted from control and Pb-treated cells representing equal amounts of total protein and separated by chromatography on silica gel 60 HPTLC plates developed twice in C/M/H 2O (144:25:2.8, v/v).

and CNPase ⫹ cells but a higher proportion in A2B5 ⫹ cells compared to the controls (Fig. 5). After 3 days in the absence of growth factors, control cells were highly branched with broad areas of membranes that were strongly reactive with antibodies against O4, GC, CNPase, and MBP but not MAb A2B5 (Figs. 4C, 4E, 4G, and 4I). In contrast, cultures exposed to Pb (both groups A and B) appeared to be less branched and contained a much lower percentage of cells immunoreactive to reagents for O4, GC, CNPase, and MBP (Figs. 4D, 4F, 4H, and 4J; Fig. 5). These cultures also contained a lower percentage of A2B5 immunoreactive cells than the initial OP cultures (Fig. 5A). After 4 days of differentiation, both control and Pb-treated cells had a complex network of cellular processes (Figs. 3E and 3F). Immunostaining showed that the Pb-treated cultures contained cells exhibiting O4, GC, and CNPase epitopes with a percentage similar to the control cultures after 4 days of differentiation (Fig. 5). The Pb-treated cells, however, appeared like the control cells with regard to percentage of anti-MBP reactivity only after 5 days following withdrawal of growth factors (Fig. 5E). In control cultures, the number of A2B5-immunoreactive cells decreased from approximately 80 to 40% within 3 days following removal of growth factors, while the number of O4-, GC-, CNPase-, and MBP-positive cells increased from 17 to 80, 18 to 55, 0 to 25, and 0 to 20%, respectively (Fig. 5), consistent with the progress of OL differentiation in culture. OP cells treated with Pb prior to differentiation (groups A and B) displayed a very different reactivity profile indicative of a

retarded differentiation. A2B5-positive cells remained at approximately 75% of the total cells after 2 days in growth factor-free medium, while the percentages of O4 ⫹, GC ⫹, and CNPase ⫹ cells were as much as 20 – 60% lower than those of the control cultures. The stage-specific markers demonstrated a significant difference in reactivity between group A or B and control cultures. The continuous presence of Pb, even following removal of growth factors, did not alter the outcome, since there was no significant difference between the two Pb-pretreated groups ( p ⬎ 0.05). The cultures exposed to Pb postmitotically (group C) progressed in a manner indistinguishable from control cultures (Fig. 5), demonstrating that exposure of young OLs to Pb had no observable effect on the subsequent maturation of these cells. This observation is consistent with the greater vulnerability to Pb of OP cells relative to differentiating or fully differentiated OL lineage cells. Pb alters the developmental expression of galactolipids. The immunocytochemical analysis indicated that Pb affected OL maturation at an early stage of cell differentiation. To examine this further, we determined the effect of Pb on the developmental expression profile of myelin-specific galactolipids during differentiation. Since this analysis required a large starting cell population, the CG-4-like OP cell line was used for this study. Unlike primary OP cells, the initiation of differentiation of the CG-4-like OP cell line was found to be density dependent. Cells plated at initial densities of 50 and 100 cells/mm 2 continued to proliferate in mitogen-free medium

FIG. 7. Effect of Pb on the developmental expression profile of glycolipids in CG-4-like OP cells. The data represent mean ⫾ SD (n ⫽ 3, each an average of duplicates). *p ⬍ 0.05 vs control; **p ⬍ 0.01 vs control. Pb (5 ␮M) treatment caused a decrease in the biosynthesis of total glycolipids in both undifferentiated and differentiated cells (A). While the ratios of NFA-galacto/glucocerebrosides (B), HFA/NFA-galactolipids (C), and galactocerebrosides/ sulfatides (D) increased during cell differentiation, Pb treatment was found to prevent this increase.

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and failed to express the morphological characteristics of mature OLs. Cells plated initially at 5 and 25 cells/mm 2, however, showed a high percentage of cells that exhibited the morphological changes characteristic of OL development as described by Louis et al. (1992). Cells were, therefore, plated at a low (25 cells/mm 2) initial density. Based upon the dose–response relationship of Pb, the cells were cultured in the presence of 5 ␮M Pb acetate 24 h prior to replacement of B104-conditioned Silberberg’s medium with unsupplemented Silberberg’s medium containing 5 ␮M Pb acetate in order to initiate cell differentiation. The biosynthesis of galactolipids was studied using metabolic radiolabeling with 3H-D-galactose. The uptake of 3H-Dgalactose, measured by either total cell- or extractable lipidassociated radioactivity, was not significantly different in the absence or presence of Pb both before and during cell differentiation. The total cell- or extractable lipid-associated radioactivities were 214.2 ⫾ 69.1 and 42.3 ⫾ 12.6 (mean ⫾ SD, n ⫽ 6) dpm/␮g of cell protein, respectively. As shown in Fig. 6, hydroxy fatty acid (HFA) and non-hydroxy fatty acid (NFA) forms of glucocerebrosides, galactocerebrosides, and sulfatides were separated using HPTLC, although no HFA-glucocerebroside was detected. Continuous Pb treatment caused a decrease in the cellular biosynthesis of total glycolipids in both undifferentiated and differentiated cells (Figs. 6 and 7A). Consistent with the results of Yim et al. (1995), the ratios of NFA-galacto/ glucocerebrosides (Fig. 7B), HFA/NFA-galactolipids (Fig. 7C), and galactocerebrosides/sulfatides (Fig. 7D) increased during differentiation. We found that, in the presence of Pb, this increase in the values of the ratios was significantly less (Fig. 7). DISCUSSION

OLs are more sensitive to Pb toxicity than neurons or astrocytes (Tiffany-Castiglioni et al., 1989). Oligodendroglia exhibit differential sensitivity to potentially damaging factors at different stages of maturation (Pfeiffer et al., 1993). In this study, we report that OP cells are significantly more sensitive than mature OLs to the effects of Pb. Concentrations of Pb as low as 1 ␮M interfere with the normal pathway for OL differentiation, as detected by the biosynthesis of galactolipids and the emergence of the OL lineage markers A2B5, O4, GC, CNPase, and MBP. This concentration of Pb appears to be subcytocidal or -cytostatic, since there was no difference in total cell number between the Pb-treated and control cells during the period of time of each experiment. The Pb exposure did not affect the normal order of appearance or loss of the various markers, consistent with the interpretation that the metal alters the rate, but not the pathway, for maturation of A2B5 ⫹ cells into myelin-capable OLs. Pb also appeared not to direct OP cells to other phenotypes. The Pb-treated cultures exhibited strong MBP immunoreactivity to an extent similar to control cultures following 4 –5 days of differentiation, and the

cell populations were always less than 5% positive to the astrocytic marker GFAP regardless the presence of Pb. Interestingly, both the cultures exposed to 1 ␮M Pb for 24 h prior to removal of medium and replenishment with fresh medium lacking growth factors that either contained or lacked 1 ␮M Pb exhibited delayed developmental progression to a similar extent. Apparently, Pb affects the progenitors during this proliferative period. Cells treated by either of the two protocols eventually expressed the late-stage markers by 4 –5 days following removal of the growth factors. In contrast, Pb exposure of cells following commitment to OL differentiation (1 day after removal of growth factors) did not delay developmental progression to mature OLs. Therefore, it appears that passage of the OP cell from cell cycle into the terminal differentiation pathway represents a transition from relative sensitivity to insensitivity toward Pb. The observed difference in vulnerability to Pb of OP cells and mature OLs is consistent with this conclusion. A consequence of environmental Pb toxicity may be an alteration of the cellular process regulating the normal developmental emergence of OL-lineage cells in the CNS. Pb exposure is known to cause hypo- and demyelination (Morell, 1984; Duncan, 1985; Coria et al., 1984). We have found that rat pups on a protocol resulting in blood Pb levels of approximately 3 ␮M exhibited significant loss of mature OLs as measured by decreased brain CNPase activity and OL specific galactolipids (Deng and Poretz, 2001). These observations in animals are consistent with the notion that OL development may be a target of Pb-induced neurotoxicity. OP cells are present in developing and adult animals throughout life (Shi et al., 1998; Rogister et al., 1999) and continuously contribute to normal brain functioning by active involvement in the dynamics of glia–axon interactions (Rogister et al., 1999). Successful brain development depends upon appropriate intercellular communication involving signaling and timing factors. In addition to myelin deficits, altered OL development can ultimately affect axonal growth and development (Sa´nchez et al., 1996) and neuronal survival (Sortwell et al., 2000) and function (Kaplan et al., 1997). Chronic Pb exposure may impact brain development, in part, by impairing the timely developmental commitment of OL progenitors, thereby affecting optimal neuron survival, axon growth, and synapse formation. Analysis of the biochemical parameters related to galactolipid metabolism during lineage progression of oligodendroglia was accomplished with a CG-4-like OP cell line, since primary OP cell cultures provided too few cells for quantification of these glycolipids. The unique characteristics of CG-4 cells make this cell line a useful model for exploring questions regarding myelin and OL development (Louis et al., 1992). These cells behave in a manner similar to that of OP cells following transplantation into focal areas of CNS demyelination or into myelin-deficient rats (Franklin et al., 1996; Esponosa de los Monteros et al., 1997). There are, however,

LEAD DELAYS OLIGODENDROCYTE DEVELOPMENT

differences between OP and CG-4 cells, including levels of a variety of myelin-specific components. Yim et al. (1995) demonstrated that, during differentiation of CG-4 cells to mature OLs, specific galactocerebrosides and sulfatides were expressed similar to, though quantitatively different from, those of primary OP cells. Nevertheless, the capacity to differentiate into myelin-competent OLs allows such OP cell lines to serve as a convenient model to explore the impact of Pb on OL development. In the present study, we have utilized a CG-4like cell line developed under conditions identical to the original CG-4 isolate. We find that galactolipid biosynthesis in the CG-4-like OP cell line was similar to that reported by Yim et al. (1995), with an increase in the ratios of galacto/glucocerebrosides, HFA/NFA-galactolipids, and galactocerebrosides/sulfatides during differentiation. Pb treatment caused a decrease in galactolipid biosynthesis and prevented the increase of the above ratios, which were used as differentiation parameters. These results are consistent with those expected for a delay in terminal differentiation, as noted for primary OP cells with the immunochemical markers for OL maturation. The synthesis and catabolism of galactocerebrosides and sulfatides affect OP cell differentiation (Bansal et al., 1999) and the ability of OLs to compose and maintain a normal myelin sheath (Coetzee et al., 1996; Bosio et al., 1996). Though the molecular mechanisms by which these galactolipids influence OL development and myelination are unknown, they appear to be critical elements in the process. The CG-4like OP cells (data not shown) and human fibroblasts cultured in Pb-containing medium have greatly reduced levels of ARSA (Poretz et al., 2000). Pb treatment causes inadequate sulfatide metabolism in fibroblasts from individuals homozygous for the pseudodeficiency polymorphisms of the ARSA gene (which results in reduced levels of the enzyme) but not those with the normal alleles (Poretz et al., 2000). It is envisioned that OL development in Pb-exposed children homozygous for the pseudodeficiency polymorphisms of the ARSA gene may be impacted to a greater degree than in children possessing the normal alleles. This would be due to the synergistic effects of the Pb-induced reduction of ARSA resulting in sulfatide accumulation and other detrimental effects of Pb on OL development. ACKNOWLEDGMENTS We thank Sylvie Ebner, Victoria Wu, and Sharon Chen for assistance with the preparation of OP cultures and Dr. Kenneth Reuhl for his advice and review of the manuscript. This research was supported in part by grants to R.D.P. from NIEHS (R01-ES08373) and the March of Dimes Foundation, and to R.D. McK. from NINDS and NIMH.

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