Exposure to Organophosphates Reduces the Expression of Neurotrophic Factors in Neonatal Rat Brain Regions: Similarities and Differences in the Effects of Chlorpyrifos and Diazinon on the Fibroblast Growth Factor Superfamily

Materials and Methods

Results

Of the FGF and FGFR genes present on the microarray, 19 genes passed the quality control filters, encoding 15 of the FGFs and all 4 FGFRs (Table 1). In control rats, we did not observe any overall pattern of regional preference for expression of these genes: Of the 19 genes evaluated, only 6 showed significant regional differences, with fgf9, fgf22, and fgfr2 more highly expressed in the brain stem, whereas fgf14, fgf20, and fgfr1 were higher in the forebrain. Organophosphate exposures elicited significant, regionally selective changes in gene expression for the FGFs and FGFRs. Multivariate ANOVA (all treatments, all genes, both regions) showed a significant treatment × region × gene interaction (p < 0.0001), enabling separate evaluations for each gene. Out of the 19 genes, 7 displayed significant main treatment effects or an interaction of treatment × region, as compared with an expected false positive rate of only 1 gene (p < 0.02).

For the genes encoding FGFs, chlorpyrifos exposure produced a significant overall decrement (main treatment effect, p < 0.05) and specific reductions in the expression of fgf2, fgf11, fgf20, and fgf22 (Figure 1). By far, the largest effect was on fgf20, which showed a 50% deficit in the forebrain; this region also displayed a significant deficit in fgf2 and fgf11. In contrast, the brain stem showed smaller decreases restricted to fgf2 and fgf22.

The effects of diazinon on the FGF genes displayed similarities to those of chlorpyrifos, but also some differences. The lower dose of diazinon caused a large reduction in forebrain fgf20 expression as did chlorpyrifos, but diazinon failed to decrease forebrain fgf2 or fgf11 significantly, and instead evoked a reduction in fgf14 (Figure 2A). In the brain stem, we again saw a small decrease in fgf2 and fgf22. Increasing the dose of diazinon to 2 mg/kg produced a further divergence from the effects seen with chlorpyrifos (Figure 2B). Although we still saw a significant reduction in fgf20 in the forebrain, no other gene was significantly affected for this region. In the brain stem, the higher dose of diazinon produced an even larger decrease in fgf2 expression than with either chlorpyrifos or the lower diazinon treatment. These regional differences between diazinon and chlorpyrifos were statistically significant (p < 0.02 for the interaction of treatment × region × gene).

Two of the four genes encoding the FGFRs, fgfr1 and fgfr4, showed statistically significant treatment-related changes in expression, but the magnitude of the effect on fgfr1 was quite small, < 10% (Figure 3). In contrast, fgfr4 showed significant increases in expression for all three organophosphate treatment groups, an effect that was restricted to the brain stem. Diazinon produced a larger increase than did chlorpyrifos. Again, the regional differences in the effects of the two organophosphates were statistically distinguishable (p < 0.05 for the interaction of treatment × region × gene).

Earlier work with higher doses of chlorpyrifos administered for longer periods of time—treatments that evoke significant and persistent cholinesterase inhibition and/or growth impairment—identified small (10–20%) decreases in the mRNA encoding nerve growth factor (Betancourt et al. 2006). We also examined expression of the two corresponding genes on our arrays, ngfb (GenBank accession no. XM_227525; GenBank 2007) and ngfg (Genbank NM_031523) but found only a small (6%) decrease in ngfb in the forebrain that did not achieve statistical significance (data not shown). Similarly, we found no significant effects on expression of the gene encoding brain-derived neurotrophic factor (bdnf; Genbank accession no. NM_012513; data not shown).

Discussion

Our results show that neonatal exposure to doses of organophosphates that are below the threshold for any signs of systemic intoxication or growth deficits, and just at the threshold for any detectable inhibition of cholinesterase, nevertheless causes profound suppression of several members of the FGF superfamily of neurotrophic factors. Indeed, the effects for chlorpyrifos or diazinon in the present study are far larger than those reported previously for other neurotrophic factors, even when the earlier work involved chlorpyrifos treatments at higher doses for longer periods, producing much greater cholinesterase inhibition or frank growth impairment (Betancourt and Carr 2004; Jameson et al. 2007). Furthermore, we found a distinct regional hierarchy corresponding to the maturational and anatomical differences between the brain stem and the forebrain (Rodier 1988). The brain stem matures earlier than the forbrain and contains a high proportion of cell bodies for cholinergic, catecholaminergic, and serotonergic neurons; the forebrain develops later and contains the terminal projections of these neurotransmitter systems, all of which are prominent targets for the developmental neurotoxicity of organophosphates (Slotkin 1999, 2004, 2005). In keeping with this regional specificity, fgf20 was suppressed by chlorpyrifos or diazinon in the forebrain, whereas the two organophosphates differentially targeted fgf2 in the brain stem (diazinon > chlorpyrifos). There were also smaller effects on fgf11, fgf14, fgf22, fgfr1, and fgfr4, each of which also displayed either a regionally selective effect or a difference between the two organophosphates. In contrast, no such regional differences were reported for other neurotrophic factors such as nerve growth factor or acetylcholinesterase splice variants associated with neural damage/ repair (Betancourt and Carr 2004; Jameson et al. 2007). Indeed, to obtain any effect on nerve growth factor gene expression, the dose and duration of chlorpyrifos exposure have to be increased to the point where cholinesterase is persistently inhibited and/or growth is impaired; even then, there is only a small (10–20%) decrement (Betancourt et al. 2006). In the present study, we used lower doses and shorter durations of exposure that caused barely detectable cholinesterase inhibition and no growth impairment, and found no significant deficits for either nerve growth factor or brain-derived neurotrophic factor, indicating that selective members of the FGF superfamily are indeed far more sensitive to disruption by the organophosphates.

The regional selectivity suggests that the effects of neonatal exposure to organophosphates on expression of FGFs reflects the targeting of specific processes in brain development rather than a global interference with neurotrophic responses. Below, we will consider each of the FGFs in turn, emphasizing their various roles in neural development and plasticity. However, first it is necessary to consider the important differences between chlorpyrifos and diazinon.

In the developing rat brain, treatment with 1 mg/kg chlorpyrifos produces approximately a 10–20% inhibition of cholinesterase (Song et al. 1997), roughly equivalent to that seen at 2 mg/kg diazinon (Slotkin et al. 2006b); the lower dose of diazinon (1 mg/kg) produces no significant inhibition whatsoever (Slotkin et al. 2006b). If the effects of these agents were the result of cholinesterase inhibition, then the chlorpyrifos treatment should produce the same pattern of effects as the higher dose of diazinon, whereas the lower diazinon dose should have no effect at all. In fact, though, all three treatments shared the same major suppression of fgf20 in the forebrain. Furthermore, the low dose of diazinon inhibited brain stem fgf2 and fgf22 expression, and increased fgfr4, just as did chlorpyrifos. It is therefore apparent that these effects are totally unrelated to cholinesterase inhibition, the mechanism that underlies the systemic toxicity of the organophosphates, thus reinforcing the concept that the developmental neurotoxicity of these agents represents a separable set of mechanisms that operate at lower exposures (Colborn 2006; Slotkin 1999, 2004, 2005; U.S. Environmental Protection Agency 2006).

It is also noteworthy that we saw several important differences in the effects of chlorpyrifos as compared with diazinon: chlorpyrifos decreased fgf2 and fgf11 in the forebrain, whereas diazinon did not; in the brain stem, diazinon reduced fgf2 and induced fgfr4 much more than did chlorpyrifos, and also had effects on forebrain fgf14 and fgfr1 that were not seen with chlorpyrifos. The similarities and disparities suggest that the two organophosphates are likely to produce many parallels in subsequent neurodevelopmental deficits but may also differ in important ways. Although much more information is available for chlorpyrifos than for diazinon, several findings already suggest differential targeting of neural cell replication, neuritic outgrowth, cytotoxic events, and cholinergic and monoaminergic neurotransmitter systems by these two agents (Jameson et al. 2007; Qiao et al. 2001; Slotkin et al. 2006a, 2007).

The two specific members of the FGF superfamily that were most highly affected by neonatal organophosphate exposure were fgf2 and fgf20, both of which have clearly established roles in neurodevelopment, plasticity, damage/repair, and neurodegenerative disorders. The expression of fgf2 shows spatial and temporal relationships to the maturational profile of each brain region (Gomez-Pinilla et al. 1994; Monfils et al. 2006) and up-regulation of this gene is required for the recovery from developmental brain injury (Monfils et al. 2005; Yoshimura et al. 2001). In addition, we recently found that fgf2 is intimately involved in the programming of neural plasticity associated with less injurious perturbations, such as prenatal stress (Fumagalli et al. 2005). Accordingly, the robust down-regulation of this gene caused by neonatal organophosphate treatment is likely to play an important role in the neurodevelopmental outcomes of such exposures; in particular, our finding of regional selectivity (brain stem > forebrain) is in keeping with the targeting of a specific maturational stage and/or anatomical location and similarly, the preferential sensitivity to diazinon predicts a potentially worsened outcome with this agent. For fgf2, our finding of gross suppression by organophosphates during the brain growth spurt (Dobbing and Sands 1979) is highly likely to have long-term, adverse consequences for neural development and behavioral function. Even a brief period of fgf2 down-regulation interferes directly with neurogenesis (Tao et al. 1997), and the period of exposure studied here (first few days after birth) corresponds to the peak proliferation period in a number of neuronal populations, including those of the hippocampal dentate gyrus (Kempermann et al. 1997; Rodier 1988). If similar effects occur with organophosphate exposures in earlier or later developmental periods, this could explain why shifting the exposure window often targets the neural cells and regions that are undergoing the most rapid development (Slotkin 1999, 2004, 2005).

In contrast to the organophosphate-evoked reduction in fgf2, which was more prominent in the brain stem, the suppression of fgf20 was selective for the forebrain. What is particularly notable about the regional difference is that fgf20 is preferentially expressed in a subregion of the forebrain, the striatum (Ohmachi et al. 2000), which contains the majority of dopamine projections, the loss of which results in Parkinson disease. There is growing suspicion that repeated developmental exposures to pesticides that target striatal dopamine projections play a significant role in the later emergence of this neurodegenerative disorder (for review, see Cory-Slechta et al. 2005; Landrigan et al. 2005). Indeed, the relationship of suppressed fgf20 expression to dopaminergic deficits and thence to Parkinson disease is directly supported by human genetic data (Murase and McKay 2006; Takagi et al. 2005; van der Walt et al. 2004) and by the specific role of this neurotrophic factor in promoting survival of the very neurons that are lost in Parkinson disease (Damier et al. 1999; Yamada et al. 1990).

The requirement for fgf20 is similarly found for development of these neurons and for preventing their death from apoptosis secondary to oxidative stress (Murase and McKay 2006); the striatum is especially sensitive to oxidative damage, in part because dopamine itself produces oxidative metabolites (Hirsch 1994; Olanow and Arendash 1994). Organophosphates target striatal dopamine systems by causing release of dopamine while simultaneously evoking oxidative stress through other cellular mechanisms (Bloomquist et al. 2002; Gupta 2004; Jett and Navoa 2000; Karen et al. 2001; Lazarini et al. 2004; Slotkin et al. 2002, 2005; Slotkin and Seidler 2007). Consequently, these neural pathways are among the most vulnerable to long-term damage after developmental exposure to chlorpyrifos (Aldridge et al. 2005b; Dam et al. 1999; Slotkin et al. 2002, 2007; Slotkin and Seidler 2007). Finally, it should be noted that fgf2 is also deficient in dopaminergic neurons in Parkinson disease (Tooyama et al. 1993) and fgf2 and fgf20 actually act in concert to protect dopaminergic neurons from oxidative injury and to promote their repair (Krieglstein et al. 1998; Ohmachi et al. 2000; Otto and Unsicker 1990; Shults et al. 2000), so that the combined deficit in both these neurotrophic factors, superimposed on organophosphate-induced oxidative stress, may render striatal dopamine pathways especially vulnerable. In particular, in Parkinson disease, degeneration begins in the brain stem (Braak et al. 2006), the region in which we found reduced fgf2 expression after neonatal organophosphate exposure. We therefore anticipate that later in life, exposed individuals may show a greater likelihood of neurodegenerative disorders such as Parkinson disease, which is already known to be associated with pesticide exposures in adulthood (Kamel and Hoppin 2004).

In addition to the major changes seen for fgf2 and fgf20 expression, we found significant but smaller effects on other members of the FGF superfamily, including fgf11, fgf14, and fgf22, and also on two of the receptor genes, fgfr1 and fgfr4. Although the roles for these are less well understood, there is substantial evidence for involvement of all of them in neurodevelopment and hence in the developmental neurotoxicity of the organophosphates. Developing neurons show particularly high expression of fgf11 and fgf14 (Luo et al. 2002; Wang et al. 2000), and the latter participates directly in neuronal signaling, axonal trafficking, and development of sodium channels required for neuronal excitability (Lou et al. 2005; Wang et al. 2002). Deficits in fgf14 are associated with the development of movement disorders (Wang et al. 2002) and it is well established that early exposure to organophosphates compromises the subsequent development of motor activity (Carr et al. 2001; Dam et al. 2000). Similarly, fgf22, a recently discovered member of the FGF family, is involved in neural differentiation of granule cells and acts as an organizer of presynaptic activity (Umemori et al. 2004). Again, hippocampal and cerebellar granule cells are known to be targeted by developmental exposure to organophosphates (Abdel-Rahman et al. 2003; Roy et al. 2005) in association with profound alterations in the patterns of pre-synaptic neuronal activity and associated behaviors (Aldridge et al. 2005b; Dam et al. 1999; Icenogle et al. 2004; Levin et al. 2001, 2002; Qiao et al. 2003, 2004; Richardson and Chambers 2004, 2005; Slotkin et al. 2001, 2002; Slotkin and Seidler 2007).

Of the two FGF receptor genes for which we found significant changes, fgfr1 is only weakly expressed in the developing brain but probably serves as an organization factor (Blak et al. 2005); we found only a small effect on this receptor, restricted to diazinon, so that this may ultimately contribute to some differences in outcome between the two organophosphates. We saw a far more robust effect on fgf4, again with a greater action of diazinon as compared to chlorpyrifos. In contrast to fgfr1, fgfr4 is highly expressed in developing brain, especially in the hippocampus (Cool et al. 2002; Limke et al. 2003; Wright et al. 2004) and in cholinergic neurons of the medial habenular nucleus (Miyake and Itoh 1996), and is involved specifically in neurite outgrowth (Hart et al. 2000). Furthermore, this receptor binds fgf2, one of the FGF members highly affected by organophosphate exposure, reinforcing the greater potential contribution of this particular signaling pathway; indeed, the fact that fgf4 was up-regulated suggests that this is a partial, adaptive response to the suppression of fgf2 expression, a conclusion reinforced by the fact that diazinon was more effective than chlorpyrifos for both the down-regulation of brain stem fgf2 and the up-regulation of fgfr4. In keeping with these relationships, developmental exposure to organophosphates especially targets each of the processes associated with fgfr4: neurons of the cholinergic phenotype (Slotkin 1999, 2004, 2005), the hippocampus (Abdel-Rahman et al. 2003; Pung et al. 2006; Roy et al. 2005; Terry et al. 2003), and neuritic outgrowth (Axelrad et al. 2003; Das and Barone 1999; Howard et al. 2005; Slotkin et al. 2006a; Song et al. 1998).

It is important to note a number of limitations of our approach, which relies on measurement of gene expression at the mRNA level assessed in two, broadly defined brain regions. First, the magnitude of changes was small when compared to the fold-change that can be obtained with in vitro studies of pesticide neurotoxicity, where typically one assesses the effects on a single cell type at a fixed stage of differentiation (Mense et al. 2006). That is hardly surprising, given the heterogeneity of the brain stem and the fore-brain, so that even a large change in gene expression in a specific cell population would be “washed out” by mRNA from unaffected areas. In fact, treatment of animals with higher doses of organophosphates that produce outright toxicity or even lethality rarely produces changes in gene expression exceeding 10–30% in vivo (Betancourt et al. 2006; Damodaran et al. 2006a, 2006b).

The second limitation is inherent in any study of mRNA: this measure by itself does not provide a definitive answer about the actual rate of synthesis and degradation of the encoded protein, the factors that actually control the concentrations of the corresponding trophic factors or factors within the cell. Nevertheless, there are two important aspects designed into the current study that render the findings relevant and interpretable. First, we included chlorpyrifos as a test compound whose impact has already been confirmed for the relevant end points of neural cell differentiation, axonogenesis, and other developmental processes known to be regulated by FGFs. Second, our interpretations rely on patterns of changes across multiple regions and FGF/FGFR subtypes rather than on a single change in one region. Nevertheless, it is obvious that a direct mechanistic link needs to be established between the changes seen here at the mRNA level and the known outcomes for chlorpyrifos, or the suspected outcomes for diazinon.

Our results are also limited by the fact that we examined only males, whereas there are numerous studies showing significant sex differences in the outcomes of developmental exposure to organophosphates (Aldridge et al. 2004, 2005a, 2005b; Dam et al. 2000; Levin et al. 2001; Ricceri et al. 2006; Slotkin 2005; Slotkin et al. 2001, 2002; Slotkin and Seidler 2007). Here, we were limited primarily by practical considerations of technical capabilities and cost. We expect, however, that males and females will show important differences in transcriptional profiles in accordance with the sex-selective nature of organophosphate-induced neurodevelopmental anomalies. Despite these limitations, though, our results are likely to be relevant to environmental exposures of fetuses and children to organophosphates. Although our studies were modeled primarily on the upper limits of estimated or measured exposures after home or agricultural application (Gurunathan et al. 1998; Ostrea et al. 2002), recent studies indicate that much lower, long-term exposures of pregnant women result in adverse neurodevelopmental outcomes for their children (Eskenazi et al. 2007; Rauh et al. 2006; Young et al. 2005). Accordingly, modeling the potential mechanisms underlying the adverse effects of these agents at exposures below the threshold for cholinesterase inhibition may provide important insights into the etiology of these orders and thus to potential strategies for amelioration.

In conclusion, our results show that neonatal exposure to low doses of organophosphates, below the threshold for any signs of systemic toxicity and spanning the threshold for any detectable cholinesterase inhibition, evoke profound and regionally selective effects on expression of specific members of the FGF superfamily of neurotrophic factors, with the largest effects seen for fgf2 and fgf20. The fact that there are similarities but also notable disparities in the responses to chlorpyrifos and diazinon, and that robust effects were seen at a dose of diazinon that does not inhibit cholinesterase, supports the idea that organophosphates differ in their propensity to elicit developmental neurotoxicity, unrelated to their anticholinesterase activity. Further, the specific involvement of fgf2 and fgf20 in development of the hippocampus and striatum matches some of the most sensitive regional targets for neurodevelopmental disruption by the organophosphates, reinforcing the potential mechanistic role of suppression of these neurotrophic factors in organophosphate-induced developmental neurotoxicity.

The close relationship between deficiencies in these factors and loss of dopamine neurons in Parkinson disease further indicates the need for long-term studies of the effects of early organophosphate exposure, preferably occupying the entire life span so as to determine whether developmental exposures lead to later emergence of neurodegenerative disorders. Finally, the identification of specific neurotrophic factors targeted by organophosphates may enable the design of targeted, interventional strategies that might prevent or offset neurodevelopmental damage in cases of known exposure. For example, increasing the concentration of FGF2 protein appears to offset the functional outcome of neonatal damage to the motor cortex (Monfils et al. 2005), and the neuroprotective effect of nicotine in animal models of Parkinson disease (Quik and Di Monte 2001) is associated with its ability to up-regulate fgf2 expression (Belluardo et al. 2004). The characterization of neurotrophic factors involved in the developmental neurotoxicity of organophosphates thus establishes a mechanistic link between the initial events in neural cell damage and the eventual outcome, while at the same time providing valuable information to enable discrimination between the effects of different organophosphates as well as potential therapeutic interventions to prevent or offset neural damage.