The developmental neurotoxicity of the organophosphate pesticide chlorpyrifos (CPF) is thought to involve both neurons and glia, thus producing a prolonged window of vulnerability. To characterize the cell types and brain regions involved in these effects, we administered CPF to developing rats and examined neuroprotein markers for oligodendrocytes (myelin basic protein, MBP), for neuronal cell bodies (neurofilament 68 kDa, NF6 cool , and for developing axons (neurofilament 200 kDa, NF200). Prenatal CPF administration on gestational days (GDs) 17-20 elicited an immediate (GD21) enhancement of MBP and NF68; by postnatal day (PN) 30, however, there were deficits in all three biomarkers, with the effect restricted to females. Exposure in the early postnatal period, PN1-4, did not evoke significant short-term or long-term changes in the neuroproteins. However, with treatment on PN11-14, we found reductions in MBP in the immediate posttreatment period (PN15, PN20) throughout the brain, and deficiencies across all three proteins emerged by PN30. With this regimen, males were targeted preferentially. The sex-selective effects seen here for the GD17-20 and PN11-14 regimens match those reported earlier for subsequent behavioral performance. These results indicate a shift in the populations of neural cells targeted by CPF, dependent upon the period of exposure. Similarly, developmental differences in the sex selectivity of the biochemical mechanisms underlying neurotoxicant actions are likely to contribute to discrete behavioral outcomes. Key words: brain development, chlorpyrifos, glia, myelin basic protein, neurofilament protein, oligodendrocytes.
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The neurobehavioral consequences of fetal or childhood pesticide exposure are a major biomedical and societal concern (Eriksson 1997; Eriksson and Talts 2000; Landrigan 2001; Landrigan et al. 1999; May 2000; Physicians for Social Responsibility 1995). Chlorpyrifos (CPF), one of the most widely used organophosphate pesticides, is a developmental neurotoxicant specifically targeting the immature brain (Barone et al. 2000; Pope 1999; Rice and Barone 2000; Slotkin 1999). Recent U.S. regulatory provisions have thus curtailed the domestic use of CPF (U.S. Environmental Protection Agency 2000), although worldwide use, particularly in agriculture, will continue for the foreseeable future. The actual mechanisms by which CPF perturbs neural development remain elusive and complicated. A variety of in vitro and in vivo model systems indicate impairment of neural cell replication and differentiation, as well as disruption of axonogenesis and synaptic function, all culminating in disruption of behavioral performance (for reviews, see Barone et al. 2000; Pope 1999; Slotkin 1999). Superimposed on neurospecific effects, CPF may exert more generalized cytotoxicity from oxidative stress that affects the developing brain because of its lower reserve of antioxidants (Bagchi et al. 1995, 1996; Crumpton et al. 2000).
One unusual property of CPF is the apparently wide window of maturational vulnerability, with adverse neurodevelopmental effects noted for exposures ranging from embryonic stages through the postweaning period (Barone et al. 2000; Buznikov et al. 2001; Dam et al. 2000; Garcia et al. 2001, 2002; Levin et al. 2001; Pope 1999; Qiao et al. 2002; Rice and Barone 2000; Roy et al. 1998; Slotkin 1999; Slotkin et al. 2001, 2002). Recent findings from our laboratory (Garcia et al. 2001, 2002; Qiao et al. 2001) and others (Barone et al. 2000; Monnet-Tschudi et al. 2000) suggest that CPF has a shifting cellular target, initially impairing development of neurons and subsequently affecting glia, which develop much later (Aschner 2000; Garcia et al. 2001, 2002; Monnet-Tschudi et at. 2000; Qiao et al. 2001). All three major classes of glia, astrocytes, oligodendrocytes, and microglia, are critical to brain development (Aschner et al. 1999; Barone et al. 2000; Compston et al. 1997; Guerri and Renau-Piqueras 1997). Astrocytes provide nutrition, structural support, and protection from oxidative stress, and additionally guide migrating neurons; oligodendrocytes ensheath axons with myelin; and microglia serve as macrophages (Aschner et al. 1999; Barone et al. 2000; Compston et al. 1997; Guerri and Renau-Piqueras 1997). In most brain regions, neurons exit the cell cycle and undergo terminal differentiation relatively early (i.e., prenatally in the rat), whereas gliogenesis and glial cell differentiation continue well into postnatal development (Aschner et al. 1999; Barone et al. 2000; Cameron and Rakic 1991; Compston et al. 1997; Guerri and Renau-Piqueras 1997; Wiggins 1986). The cerebellum, the brain region that develops last, is an exception, with a peak of neurogenesis in the second postnatal week (Rodier 198 cool . The finding that CPF targets glia as well as (or perhaps more than) neurons (Barone et al. 2000; Garcia et al. 2001, 2002; Monnet-Tschudi et al. 2000; Qiao et al. 2001) thus provides a partial explanation for the exceptionally long maturational period in which brain development is sensitive to this agent. Interference with the numerous roles of glia in synapse formation (Ullian et al. 2001), axon migration, and myelination (Compston et al. 1997; Riederer et al. 1992) may all contribute to eventual adverse outcomes.
In a recent study (Garcia et al. 2002), we evaluated the effects of CPF exposure on glial fibrillary acidic protein (GFAP), an astrocyte-associated protein (Garcia et al. 2002). In keeping with the concept that CPF targets glial development, postnatal treatment initially decreased GFAP levels. However, several weeks later we found elevations of GFAP, a pattern typically associated with gliosis in response to injury to other neural cells (Norton et al. 1992; O'Callaghan 1993). This implied that astroglia were not the only target for CPF. Furthermore, with http://www.cdc.gov/pregnancy/during.html prenatal CPF treatment, unless the dose was raised above the threshold for systemic toxicity, we did not find any changes in GFAP, despite the fact that fetal CPF exposure evokes changes in brain morphology (Lassiter et al. 2002; White et al. 2002) and subsequent behavioral anomalies (Levin et al. 2002). Accordingly, in the current study we examined the potential for CPF to perturb the development of other neural cell populations, using a design similar to our GFAP study (Garcia et al. 2002). We compared the effects of prenatal versus postnatal CPF exposure on the development and regional targeting of myelin basic protein (MBP) and the small (68 kDa) and large (200 kDa) neurofilament proteins (NF68 and NF200). MBP, an oligodendrocyte marker, is a major component of myelin and increases with oligodendrocyte differentiation and myelination, primarily during the second and third postnatal weeks in the rat (Wiggins 1986). The neurofilament proteins are found in neurons and assemble to form intermediate filaments that regulate axon growth, axoplasmic transport, and axon caliber (Capano et al. 2001; Escurat et al. 1990; Lee and Cleveland 1996; Schlaepfer and Bruce 1990). NF68 is expressed early in the postmitotic development of neuronal cell bodies, whereas NF200 expression is associated primarily with the later growth of axons (Capano et al. 2001; Carden et al. 1987; Escurat et al. 1990; Lee and Cleveland 1996; Schlaepfer and Bruce 1990; Yang et al. 1996). By examining these neuroprotein markers, we can now elucidate how the cellular target and regional specificity for CPF-induced alterations of brain development shift with development.
Materials and Methods
Animal treatments. All experiments were conducted in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Timed-pregnant Sprague-Dawley rats (Zivic Laboratories, Pittsburgh, PA) were housed in breeding cages, with a 12-hr light-dark cycle and free access to food and water. CPF (Chem Service Inc., West Chester, PA) was dissolved in dimethylsulfoxide to provide rapid and complete absorption (Whitney et al. 1995) and was injected subcutaneously in a volume of 1 mL/kg body weight. For prenatal CPF exposure, dams were injected daily with CPF in doses of 1-40 mg/kg of body weight, or with vehicle. Twenty-four hours after the last injection [gestational day (GD) 21], fetuses were removed and brains were dissected. Additional dams in the control, 1 mg/kg, and 5 mg/kg treatment groups were allowed to reach term, at which point their pups were randomized within treatment groups and redistributed to the nursing dams with a litter size of 10 to maintain a standard nutritional status. Randomization was repeated at intervals of several days; in addition, dams were rotated among litters to distribute any maternal caretaking differences randomly across litters and treatment groups. Animals were weaned on PN21, and all determinations used no more than one male and one female from each litter.
For measurements on GD21, fetal brains were separated into forebrain and the rest of the brain by making a cut rostral to the thalamus. Because the cerebellum represents an inappreciable proportion of brain weight on GD21, the remainder was designated "midbrain + brainstem." For studies on PN5, PN10, PN15, and PN21, brains were dissected into three regions: blunt cuts were made through the cerebellar peduncles, whereupon the cerebellum (including flocculi) was lifted from the underlying tissue. Then, as for the fetal brain, a cut was made rostral to the thalamus to separate the forebrain from the midbrain + brainstem. This dissection, which follows the planes of the fetal and neonatal rat brain, includes the corpus striatum, hippocampal formation, and neo-cortex within the area designated "forebrain." The region designated "midbrain + brainstem" includes the midbrain, colliculi, pons, and medulla oblongata (but not cervical spinal cord), as well as the thalamus. On PN30, brains were dissected into the same three major regions, and the midbrain + brainstem and forebrain were further subdivided into midbrain, brainstem, cerebral cortex, hippocampus, and striatum.
For postnatal CPF treatments, all pups were randomized the day after birth and redistributed to the dams as already described. For studies of CPF effects in the first few days after birth, animals were given 1 mg/kg daily on PN1-4. For studies in older animals, which tolerate higher doses (Campbell et al. 1997; Pope and Chakraborti 1992; Pope et al. 1991; Whitney et al. 1995), daily treatment with 5 mg/kg was given on PN11-14. These doses have been shown previously to alter neural function without eliciting overt systemic toxicity (Campbell et al. 1997; Song et al. 1997; Whitney et al. 1995). Behavioral differences remain apparent, or may first emerge, after weaning, despite the rapid recovery of cholinesterase activity (Dam et al. 2000; Song et al. 1997). Neither regimen evokes weight loss or mortality (Campbell et al. 1997; Dam et al. 1998; Johnson et al. 1998; Song et al. 1997), and in the current study we did not observe any changes in suckling or maternal caretaking. Animals were weaned and selected from each litter as detailed above.
Tissues were frozen in liquid nitrogen and stored at -45[degrees]C.
Assays. Neurospecific proteins were assayed by a modified (Garcia et al. 2002) dot-immunobinding technique (O'Callaghan 1991; O'Callaghan et al. 1999). Briefly, tissues were homogenized with a sonic probe (Heat Systems-Ultrasonics, Inc., Plainview, NY) in nine volumes of hot 1% sodium dodecyl sulfate (Bio-Rad, Hercules, CA) and were diluted in 120 mM KCl, 20 mM NaCl, 2 mM Mg[Cl.sub.2], 2 mM NaHC[O.sub.3], 0.7% Triton X-100, 0.2% Na[N.sub.3], and 5 mM HEPES (pH 7.4). Ten-microliter aliquots containing 2-15 [micro]g protein were blotted onto prewashed nitrocellulose membranes (0.2 [micro]m; Bio-Rad). Blots were dried and fixed in 25% isopropanol, 10% acetic acid, and 65% water, incubated for 5 min in Tris-buffered saline (200 mM NaCl, 50 mM Tris, 0.002% Na[N.sub.3], pH 7.4) and treated for 1 hr with a blocking solution of 0.5% gelatin (EIA grade; Bio-Rad) in Tris-buffered saline. Blots were then incubated in blocking solution containing 0.1% Triton X-100 with addition of the appropriate antibodies (Chemicon International Inc, Temecula, CA): rabbit polyclonal anti-NF68 (diluted 1:2,000), rabbit polyclonal anti-NF200 (diluted 1:2,000), or mouse monoclonal anti-MBP (diluted 1:300). Because the MBP antibody was monoclonal, blots also were incubated with rabbit anti-mouse IgG (1:500; Dako Corporation, Carpentaria, CA) in blocking solution with Triton. To assess antibody binding, blots were incubated with 20 [micro]g [[sup.125]I]Protein-A (specific activity, 382 Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA) in blocking solution with Triton X-100, washed repeatedly, dried overnight, and counted for radioactivity.
Each blot included serial dilutions of a single preparation of adult midbrain, which was then used to construct a standard curve to normalize the values across blots. Thus, although values are reported in relative units, quantitative comparisons across treatments, regions, and ages could be carried out. Because the development of NF200 is delayed relative to MBP and NF68 (Capano et al. 2001; Escurat et al. 1990; Lee and Cleveland 1996; Schlaepfer and Bruce 1990), determinations for this neurospecific protein were limited to PN30.
Study design and data analysis. Experiments were conducted on four different cohorts of animals. Two cohorts were used for CPF exposure on GD17-20, with each cohort comprising at least 12 dams per treatment group. One cohort received 0, 1, 2, or 5 mg/kg daily, whereas the second received 0, 10, 20, or 40 mg/kg per day. The other two cohorts, again with at least 12 litters per treatment group, were used for CPF exposures on PN1-4 and PN11-14. For presentation purposes, control values were combined across cohorts, as they did not differ significantly from each other. However, treatment differences were established using only the control values for each matched cohort.
Data were compiled as means and standard errors. Differences between treatment groups were assessed first by a multivariate ANOVA (data log-transformed because of heterogeneous variance) incorporating all relevant variables: treatment (control, CPF), treatment period (regimen), age, region, sex, and neuroprotein (MBP, NF68, NF200). Whenever the initial ANOVA indicated an interaction of CPF treatment with other variables, data were separated according to the interactive variable(s) and lower-order ANOVAs were conducted. Individual differences between control and CPF groups were then evaluated post hoc by Fisher's protected least significant difference. However, in the absence of interaction terms, only main treatment effects were compiled, without subdivision into individual determinations. For convenience, some data are presented as the percentage change from the corresponding controls, but statistical significance was always assessed on the original data. Control data were combined across the different treatment regimens (GD17-20, PN1-4, PN11-14) for presentation purposes, but in all cases, CPF effects were established using only the matched control groups. For experiments involving dose-effect determinations, data were analyzed by multiple regression, incorporating all variables (dose, brain region, neuroprotein). Significance was assumed at the level of p 0.05 for main effects; for interactions at p 0.1, we examined lower-order main effects after subdivision of the interactive variables (Snedecor and Cochran 1967).
Results
Development of neuroproteins in controls. Both MBP and NF68 were measurable at all ages from GD21 through PN30, whereas accurate determinations of NF200 were limited to PN30. In control brain, MBP (Figure 1A) was low in the fetus, without significant distinctions between the midbrain + brainstem and the forebrain. Regional differences emerged postnatally, with the greatest increases evident in postnatal weeks 2-4, in agreement with earlier findings (Wiggins 1986). By PN30, MBP in the midbrain + brainstem was 5 times that in the forebrain or cerebellum. Further division of the brain into subregions on PN30 revealed even larger distributional differences: values were highest in the brainstem, followed by the midbrain and striatum, whereas low values were seen in the cerebral cortex and hippocampus. In keeping with the fact that neurogenesis precedes myelination (Rodier 198 cool , NF68 showed an earlier ontogenetic increase than MBP (Figure 1B), with significant regional differences present as early as GD21. Again, by PN30, there were large disparities between the midbrain + brainstem and the forebrain or cerebellum; with regional subdivision, the rank order was brainstem midbrain striatum cerebral cortex hippocampus. On PN30, NF200 (Figure 1C) showed a similar hierarchy. These patterns for ontogeny and regional specialization of the neurofilament proteins are in agreement with earlier results (Capano et al. 2001; Schlaepfer and Bruce 1990). None of the regions showed significant overall sex differences or interactions of sex x other variables, so control values were combined across males and females. However, as shown below, sex differences did emerge in the effects of CPF on these neuroproteins.
[FIGURE 1 OMITTED]
Global statistical analyses of chlorpyrifos effects. We evaluated several data groupings for main treatment effects and interactions prior to subdividing data into separate treatment regimens, sexes, and brain regions. We first compared effects across all three regimens (GD17-20, PN1-4, and PN11-14), limiting the determinations to two brain regions (midbrain + brainstem and forebrain), one time point (24 hr posttreatment), and without regard to sex, as all three regimens shared only these variables. The result (CPF x regimen, p 0.1) indicated the need to examine each regimen separately. Next, on PN30, we compared the effects of CPF on the striatum, a region for which all three regimens and all three neuroproteins (MBP, NF68, and NF200) were evaluated, this time including sex as a factor. The outcome indicated the need to subdivide the data by regimen and sex (CPF x regimen x sex, p 0.01). The next grouping evaluated effects on PN30 across all six subregions (midbrain, brainstem, cerebral cortex, hippocampus, striatum, and cerebellum) for all three neuroproteins, determinations that were shared only by the two postnatal treatment regimens (PN1-4, PN11-14), again including the sex variable. This also indicated the need to look for treatment-related differences after separating the data by regimen and sex (CPF x sex, p 0.06; CPF x regimen x sex, p 0.06). Finally, because MBP and NF68 were evaluated across three time points (24 hr after the last injection, 5 days later, PN30) for both of the postnatal treatment regimens, we conducted analyses for those two regimens, weighting the values for subregions on PN30 to obtain estimates for forebrain (cerebral cortex + hippocampus + striatum) and midbrain + brainstem. For the PN11-14 treatment group, we obtained interaction terms indicative of treatment effects separable by sex and by specific neuroprotein (CPF x sex, p 0.04; CPF x sex x neuroprotein, p 0.06).
Chlorpyrifos treatment on GD17-20. CPF exposure from GD17-20 had a significant effect on MBP and NF68 on GD21, 24 hr after the last injection, characterized by a significant increase (main effect of CPF), assessed across both of the neuroproteins and both regions (Figure 2A). Because there was no interaction of treatment x other variables, we did not conduct lower-order statistical analyses for separate regions or neuroproteins, but post hoc analysis across those factors indicated significant increments at 5, 20 and 40 mg/kg/day of CPF. Multiple regression analysis, including the factors of dose, neuroprotein type, and brain region, confirmed the significant relationship between dose and effect (p 0.0001). Earlier studies defined 10 mg/kg/day as the threshold for fetal weight deficits (Garcia et al. 2002; Qiao et al. 2002), so it was evident that the immediate effects of CPF on MBP and NF68 in the fetus involve doses spanning that threshold. Accordingly, we next assessed whether there might be delayed effects of CPF after gestational administration, concentrating on the striatum, a region that we previously found to be particularly susceptible to subsequent emergence of CPF-induced damage (Garcia et al. 2002; Slotkin et al. 2002). Studies were limited to doses below the threshold for fetal growth impairment (1 or 5 mg/kg/day). On PN30, measurements across all three neuroproteins (MBP, NF68, NF200) indicated sex-dependent effects of CPF (Figure 2B). Females exposed to 5 mg/kg/day during fetal development showed a significant decrease in striatal neuroproteins on PN30. Although effects were not significant for 1 mg/kg/day when compared with control values, the lower-dose group also could not be distinguished statistically from the high-dose group, so an effect at the lower dose could not be ruled out. In contrast to females, gestational CPF administration failed to cause significant deficits in males and in fact tended to elevate the values. Again, these results were confirmed by multiple regression analysis (factors of dose and neuroprotein type), which indicated a significant dose-effect relationship in females (p 0.00 cool but not in males.
[FIGURE 2 OMITTED]
Postnatal chlorpyrifos treatment. CPF administration on PN1-4 did not evoke statistically significant changes in MBP or NF68, assessed 24 hr after the last injection (PN5) or 5 days later on PN10 (Figure 3A). Although there was an increase in the average MBP value in the cerebellum on PN5, the results for this region were highly variable at this age, a likely consequence of its small size and especially rapid rate of growth. Similarly, there were no changes in MBP, NF68, or NF200 in any of the subregions evaluated on PN30 (Figure 3B). The absence of statistically significant effects for this treatment regimen was itself distinguishable from the significant differences seen for gestational CPF treatment: comparing the significant values for GD21 with those for PN5 (24 hr after the last injection for each regimen), treatment x regimen, p 0.02. In the striatum on PN30, values for the sex-dependent effect of gestational CPF were similarly distinguishable from the lack of effect of the PN1-4 regimen in the same region, as shown by a significant interaction of treatment x regimen x sex, p 0.05.
[FIGURE 3 OMITTED]
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