Prenatal dexamethasone, as used in preterm labor, worsens the impact of postnatal chlorpyrifos exposure on serotonergic pathways

Abstract

This study explores how glucocorticoids sensitize the developing brain to the organophosphate pesticide, chlorpyrifos. Pregnant rats received a standard therapeutic dose (0.2mg/kg) of dexamethasone on gestational days 17-19; pups were given subtoxic doses of chlorpyrifos on postnatal days 1-4 (1mg/kg, <10% cholinesterase inhibition). We evaluated serotonin (5HT) synaptic function from postnatal day 30 to day 150, assessing the expression of 5HT receptors and the 5HT transporter, along with 5HT turnover (index of presynaptic impulse activity) in brain regions encompassing all the 5HT projections and cell bodies. These parameters are known targets for neurodevelopmental effects of dexamethasone and chlorpyrifos individually. In males, chlorpyrifos evoked overall elevations in the expression of 5HT synaptic proteins, with a progressive increase from adolescence to adulthood; this effect was attenuated by prenatal dexamethasone treatment. The chlorpyrifos-induced upregulation was preceded by deficits in 5HT turnover, indicating that the receptor upregulation was an adaptive response to deficient presynaptic activity. Turnover deficiencies were magnified by dexamethasone pretreatment, worsening the functional impairment caused by chlorpyrifos. In females, chlorpyrifos-induced receptor changes reflected relative sparing of adverse effects compared to males. Nevertheless, prenatal dexamethasone still worsened the 5HT turnover deficits and reduced 5HT receptor expression in females, demonstrating the same adverse interaction. Glucocorticoids are used in 10% of U.S. pregnancies, and are also elevated in maternal stress; accordingly, our results indicate that this group represents a large subpopulation that may have heightened vulnerability to developmental neurotoxicants such as the organophosphates.

Keywords: 5-hydroxytryptamine, serotonin; 5HT; ANOVA; Chlorpyrifos; Dexamethasone; GD; Glucocorticoids; Organophosphate pesticides; PN; Preterm delivery; Serotonin; analysis of variance; gestational day; postnatal day.

Figure 1

Effects of dexamethasone (A), chlorpyrifos (B), and combined treatment (C) on 5HT_1A receptor binding (mean ± SE, n=6 for each sex in each treatment group), shown as the percent change from control values (see original values in Supplementary Table 1). Because the global ANOVA showed a significant treatment × sex interaction (p < 0.05), values were separated for males and females. Multivariate ANOVA for each treatment appears at the top of the panels. For dexamethasone (A), males showed an interaction of treatment × region and the regions for which differences were significant are indicated; females showed only a main treatment effect, so no lower-order tests were performed. For chlorpyrifos (B), and for combined treatment (C) both sexes showed only main treatment effects, so no lower-order tests were evaluated.. Panel (D) shows the simple main treatment effects for each sex, collapsed across all the other variables. Abbreviations: mb, midbrain; bs, brainstem; f/p, frontal/parietal cortex; t/o, temporal/occipital cortex; hp, hippocampus; st, striatum; Dex, dexamethasone; CPF, chlorpyrifos.

Figure 2

Effects of dexamethasone (A), chlorpyrifos (B), and combined treatment (C) on 5HT₂ receptor binding (mean ± SE, n=6 for each sex in each treatment group), shown as the percent change from control values (see original values in Supplementary Table 2). Because the global ANOVA showed a significant treatment × sex interaction (p < 0.0001), values were separated for males and females. Multivariate ANOVA for each treatment appears at the top of the panels. For males given dexamethasone (A) or chlorpyrifos (B), there was only a main treatment effect, so no lower-order tests were performed. However, females showed treatment interactions with region and age for both the dexamethasone (A) and chlorpyrifos (B) groups, so main treatment effects for each region are indicated at the top, and lower-order tests at each age are at the bottom; for ages that showed a treatment × region interaction, the individual regions that differ from control are indicated with asterisks. For the combined treatment group (C), males showed a main treatment effect as well as a treatment × age interaction, so lower-order tests were performed at each age and are shown at the bottom of the panel; females had only a main treatment effect, so no lower-order tests were performed. Panel (D) shows the simple main treatment effects for each sex, collapsed across all the other variables. Abbreviations: mb, midbrain; bs, brainstem; f/p, frontal/parietal cortex; t/o, temporal/occipital cortex; hp, hippocampus; st, striatum; Dex, dexamethasone; CPF, chlorpyrifos.

Figure 3

Effects of dexamethasone (A), chlorpyrifos (B), and combined treatment (C) on 5HT transporter binding (mean ± SE, n=6 for each sex in each treatment group), shown as the percent change from control values (see original values in Supplementary Table 3). Because the global ANOVA showed a significant treatment × sex interaction (p < 0.0006), values were separated for males and females. Multivariate ANOVA for each treatment appears at the top of the panels. For males or females given dexamethasone (A), there was a main treatment effect as well as a treatment × age interaction, so lower-order tests were performed at each age and are shown at the bottom of the panel. Similarly, for chlorpyrifos (B), males showed a main treatment effect and a treatment × age interaction and females also displayed a treatment × age interaction; accordingly, lower-order tests were performed at each age and are shown at the bottom of the panels. For the combined treatment (C), males had a treatment × age interaction whereas females showed only a main treatment effect, so lower-order tests were performed only for males. Panel (D) shows the simple main treatment effects for each sex, collapsed across all the other variables. Abbreviations: mb, midbrain; bs, brainstem; f/p, frontal/parietal cortex; t/o, temporal/occipital cortex; hp, hippocampus; st, striatum; Dex, dexamethasone; CPF, chlorpyrifos.

Figure 4

Temporal progression of main treatment effects, collapsed across regions and measures. Values were separated for males (A) and females (B) because of the significant interaction of treatment × sex (p < 0.0005) in the repeated-measures ANOVA across all three binding parameters. Percent change was calculated using the geometric means of the original values in Tables 1, 2 and 3. For males, the temporal course of effects for the combined exposure group differs from what would be expected from the individual treatments (dexamethasone × chlorpyrifos × age, p < 0.03), whereas that interaction term is not significant in females. Abbreviations: Dex, dexamethasone; CPF, chlorpyrifos

Figure 5

Effects of dexamethasone (A), chlorpyrifos (B), and combined treatment (C) on 5HT turnover (mean ± SE, n=6 for each sex in each treatment group), shown as the percent change from control values (see original values in Supplementary Table 4). Because the global, repeated-measures ANOVA for 5HT concentration and turnover showed a significant treatment × sex interaction (p < 0.03), values were separated for males and females. Multivariate ANOVA for each treatment appears at the top of the panels. Dexamethasone alone had no significant effect (A). For chlorpyrifos (B), males showed a main treatment effect without interactions with other variables, so no lower-order tests were performed. Similarly, the group receiving combined treatment (C) showed main treatment effects for males and females, without the need for lower-order tests. Panel (D) shows the simple main treatment effects for each sex, collapsed across all the other variables. Abbreviations: f/p, frontal/parietal cortex; t/o, temporal/occipital cortex; Dex, dexamethasone; CPF, chlorpyrifos; NS, not significant.