Prenatal polycyclic aromatic hydrocarbon exposure leads to behavioral deficits and downregulation of receptor tyrosine kinase, MET

Abstract

Gene by environment interactions (G × E) are thought to underlie neurodevelopmental disorder, etiology, neurodegenerative disorders, including the multiple forms of autism spectrum disorder. However, there is limited biological information, indicating an interaction between specific genes and environmental components. The present study focuses on a major component of airborne pollutants, polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene [B(a)P], which negatively impacts cognitive development in children who have been exposed in utero. In our study, prenatal exposure of Cpr(lox/lox) timed-pregnant dams to B(a)P (0, 150, 300, and 600 μg/kg body weight via oral gavage) on embryonic day (E14-E17) consistent with our susceptibility-exposure paradigm was combined with the analysis of a replicated autism risk gene, the receptor tyrosine kinase, Met. The results demonstrate a dose-dependent increase in B(a)P metabolite generation in B(a)P-exposed Cpr(lox/lox) offspring. Additionally, a sustained persistence of hydroxy metabolites during the onset of synapse formation was noted, corresponding to the peak of Met expression. Prenatal B(a)P exposure also downregulated Met RNA and protein levels and dysregulated normal temporal patterns of expression during synaptogenesis. Consistent with these data, transcriptional cell-based assays demonstrated that B(a)P exposure directly reduces human MET promoter activity. Furthermore, a functional readout of in utero B(a)P exposure showed a robust reduction in novel object discrimination in B(a)P-exposed Cpr(lox/lox) offspring. These results confirm the notion that common pollutants, such as the PAH B(a)P, can have a direct negative impact on the regulated developmental expression of an autism risk gene with associated negative behavioral learning and memory outcomes.

FIG. 1.

B(a)P metabolite disposition during postnatal brain/cortical development. (A) Distribution of metabolite types in B(a)P-exposed C57BL/6 timed-pregnant dams. C57BL timed-pregnant dams received 600 μg B(a)P/kg BW via oral gavage on E14–E17. Neocortical tissue was retrieved from B(a)P-exposed C57BL dam on PND 3 and the distribution of the individual B(a)P metabolites was determined by high-performance liquid chromatography (HPLC) as outlined in Ramesh et al. (2001). Values represent mean ± SEM. The asterisk denotes statistical significance (p < 0.001) for different metabolite concentrations as compared with the 4,5-diol metabolite. (B) Distribution of metabolite types in B(a)P-exposed Cpr^lox/lox offspring demonstrating comparable POR activity in brain tissue. Timed-pregnant Cpr^lox/lox dams received 600 μg B(a)P/kg BW via oral gavage on E14–E17. Neocortical tissue was retrieved from B(a)P-exposed Cpr^lox/lox offspring on PND 3, and the distribution of the individual B(a)P metabolites was determined by HPLC as outlined in Ramesh et al. (2001). Values represent mean ± SEM. The asterisk denotes statistical significance (p < 0.001) for different metabolite concentrations as compared with the 4,5-diol metabolite. (C) Dose-dependent accumulation of total neocortical B(a)P metabolites during the critical postnatal period when synapses are first formed in B(a)P-exposed Cpr^lox/lox offspring. Cpr^lox/lox offspring dosed in utero with either 150, 300, or 600 μg/kg BW were sacrificed on the indicated postnatal day, and brain tissue was frozen in liquid nitrogen subsequent to genotyping on PND 20. Offspring pups that were identified as carrying the allele Cpr^lox/lox were pooled, extracted, and the B(a)P metabolite concentrations was determined by HPLC as outlined in Ramesh et al. (2001). Shown are total metabolite concentrations measured in B(a)P-exposed Cpr^lox/lox offspring for the three B(a)P doses. Values represent mean ± SEM. Due to the limited volume of neocortical tissue present on PND 3 and 5, whole-brain tissue was used for these time points. The asterisk denotes statistical significance at p < 0.05.

FIG. 2.

Analysis of Met mRNA in the cerebral cortex of B(a)P-exposed Cpr^lox/lox offspring. The top panel shows the results of electrophoresis of PCR products from PND 0, 1, 3, 5, and 7. The upper band is MET, and the lower band is 18sRNA (internal standard). The bottom panel shows the densitometric quantitation of the bands. There is a statistically significant dose-dependent reduction in the levels of cortical Met mRNA expression, depicted by the relative ratio between the intensity of the MET band and that of the 18sRNA band. The data are shown as mean ± SEM from three separate experiments; p < 0.05. M is the marker; a = Control Cpr^lox/lox offspring (white bar); b = 150 (black bar); c = 300 (gray bar); d = 600 (white-gray bar) μg/kg BW B(a)P-exposed Cpr^lox/lox offspring.

FIG. 3.

Analysis of B(a)P exposure on human MET promoter activity in HEK cells. Twenty-four hours of exposure to different concentrations of B(a)P results in a statistically significant reduction in transcriptional activity from the human MET promoter. Transcriptional efficiency was measured by luciferase assay following transfection of a construct that drives a luciferase reporter with a 762-bp fragment of the MET promoter. *p < 0.05 by ANOVA followed by the Dunnett’s t-test. Shown is the mean ± SEM. of four independent determinations.

FIG. 4.

Analysis of Met protein expression in the cerebral cortex following in utero B(a)P exposure. The upper panel shows a representative experiment. The upper band is MET, and the lower band is the β-actin internal control (lanes 1–4). Control Cpr^lox/lox offspring; MET expression on PND 0, lane1; PND 5, lane 2; PND 10, lane 3; and PND 15, lane 4 (lanes 5–8) 150 μg/kg BW B(a)P-exposed Cpr^lox/lox offspring; PND 0, lane 5, PND 5, lane 6; PND 10, lane 7; and PND 15, lane 8 (lanes 9–12) 300 μg/kg BW B(a)P-exposed Cpr^lox/lox offspring; PND 0, lane 9; PND 5, lane 10; PND 10, lane 11; and PND 15 lane 12. There is an apparent dose-dependent decrease in Met protein detection, characterized by a premature peak at P5 in B(a)P-exposed Cpr^lox/lox offspring as compared with control Cpr^lox/lox offspring (P10). This was confirmed using semiquantitative analysis. The lower panel displays the histogram of the densitometric quantitation of the relative expression of Met to an internal β-actin internal control. *p < 0.05 relative to control in the 150 and 300 μg/kg BW B(a)P-exposed Cpr^lox/lox offspring.

FIG. 5.

Analysis of the response to novelty. Prenatal B(a)P exposure induces a robust behavioral deficit in novel object discrimination compared with control Cpr^lox/lox offspring. Control and B(a)P-exposed Cpr^lox/lox offspring mice (n = 5 per group) were acclimated and tested as described in the “Materials and Methods” section. Zones of two sizes (novel and familiar objects) were quantitated around the novel and familiar objects in order to compare the observational data. Control Cpr^lox/lox offspring mice spent more time with the novel than the familiar object, which produced a positive mean novelty discrimination index of 0.54 ± 0.13 (SEM) for control mice. Testing of B(a)P-exposed Cpr^lox/lox offspring revealed that significantly less time was spent with the novel than the familiar object as compared with controls, producing a negative mean novelty discrimination index score of −0.26 ± 0.07, p < 0.05 for the 150 μg/kg BW group. For the B(a)P-exposed Cpr^lox/lox offspring in the 300 μg/kg BW group, the statistical significance of the deficit in response to novelty was even greater, −0.230 ± 0.10. Post hoc analysis with the Bonferroni test revealed the significance at p < 0.01.