Combustion derived ultrafine particles induce cytochrome P-450 expression in specific lung compartments in the developing neonatal and adult rat

Abstract

Vehicle exhaust is rich in polycyclic aromatic hydrocarbons (PAH) and can be a dominant contributor to ultrafine urban particulate matter (PM). Exposure to ultrafine PM is correlated with respiratory infections and asthmatic symptoms in young children. The lung undergoes substantial growth, alveolarization, and cellular maturation within the first years of life, which may be impacted by environmental pollutants such as PM. PAHs in PM can serve as ligands for the aryl hydrocarbon receptor (AhR) that induces expression of certain isozymes in the cytochrome P-450 superfamily, such as CYP1A1 and CYP1B1, localized in specific lung cell types. Although AhR activation and induction has been widely studied, its context within PM exposure and impact on the developing lung is poorly understood. In response, we have developed a replicable ultrafine premixed flame particle (PFP) generating system and used in vitro and in vivo models to define PM effects on AhR activation in the developing lung. We exposed 7-day neonatal and adult rats to a single 6-h PFP exposure and determined that PFPs cause significant parenchymal toxicity in neonates. PFPs contain weak AhR agonists that upregulate AhR-xenobiotic response element activity and expression and are capable inducers of CYP1A1 and CYP1B1 expression in both ages with different spatial and temporal patterns. Neonatal CYP1A1 expression was muted and delayed compared with adults, possibly because of differences in the enzyme maturation. We conclude that the inability of neonates to sufficiently adapt in response to PFP exposure may, in part, explain their susceptibility to PFP and urban ultrafine PM.

Keywords: aryl hydrocarbon receptor; lung development; particulate matter; polycyclic aromatic hydrocarbons.

Fig. 1.

In vitro xenobiotic response element (XRE) reporter assay. Human U-937 macrophages were transiently transfected with the XRE luciferase reporter construct and subsequently treated with premixed flame particles (PFP), urban dust particles (UDP), or 2,3,7,8-tetrachlorodibenzodioxin (TCDD, positive control). A: a dose-dependent increase in acyl hydrocarbon receptor (AhR)/XRE activity was observed after PFP treatment. Treatment with 25 μg/ml or more PFP yielded a significant response compared with controls. UDP (10 μg/ml) and TCDD (1.0 nM) provide strong AhR agonists and significantly induced XRE luciferase activity. B: PFP treatment increases binding activity of AhR at a consensus XRE-binding element using gel-mobility-shift assay with XRE consensus oligonucleotide after treatment of U-937 macrophages with PFP (25 μg/ml) and TCDD (1.0 nM) for 90 min. C: AhR mRNA was upregulated in a dose-dependent manner at 25 and 50 μg/ml PFP. UDP (10 μg/ml) induced AhR mRNA expression similarly to 25 μg/ml PFP. D: cytochrome P-450 (CYP) 1A1 mRNA level was significantly increased at 25 and 50 μg/ml PFP and strongly activated by UDP or TCDD treatment used as positive control. Data are presented as means + SE (n = 3 separate experiments). *Significantly different compared with controls.

Fig. 2.

Airway cellular toxicity following PFP exposure. In situ ethidium homodimer-1 staining was analyzed using confocal microscopy in 7-day postnatal (A and B) and adult (C and D) rats exposed to either filtered air (FA, A and C) or PFP2 (B and D). Overall, ethidium staining was scarce in either 7-day-old neonates (A) or adults (C) reared in FA. Ethidium-positive cells (white arrows) were observed distributed in both bronchiolar epithelium and parenchyma in neonatal animals (B). Comparatively, the majority of cytotoxic cells was present in bronchiolar airways and was virtually absent in the parenchyma (D). Scale bars are 100 μm. Stereological quantification revealed an increased volume fraction of ethidium homodimer-1-positive cells after exposure. The volume fraction of ethidium-positive cells of neonates increased in both airway and parenchyma after exposure, but this increase was significant only in the parenchyma (E). Adult volume fractions also trended higher in the airways but were unaffected by PFPs in the parenchyma (F). Data are plotted as means ± SE (n = 4–5 rats/group, per compartment). *P < 0.05, significantly different from FA in the same compartment.

Fig. 3.

AhR immunofluorescence. AhR immunofluorescence (red) was overlaid upon DAPI nuclear (blue) staining in neonate (A and B) and adult (C and D) lungs exposed to either FA (A and C) or PFP2 (B and D). AhR staining was virtually absent in FA-treated animals of either age. However, robust AhR staining was observed in both airway and parenchyma compartments in both ages. High-magnification insets are denoted by white squares and separate images showing AhR and DAPI colocalization, presented adjacent to B and D, respectively. Aw, airway; Par, parenchyma.

Fig. 4.

CYP1A1 compartmental mRNA and protein expression. RT-PCR expression in airway and parenchyma compartments in neonatal and adult rats exposed to PFPs. A: basal CYP1A1 mRNA levels were significantly higher in adults. Although no compartmental differences were observed in neonates, there was 4-fold more CYP1A1 in the parenchyma than in airways in adults. B: after PFP exposure, neonate CYP1A1 mRNA was transiently upregulated at PFP2 in the airways and reverted to FA levels by 24 h. C: a time-dependent decrease in CYP1A1 was observed in the adult parenchyma, reaching significance at 24 and 48 h postexposure. Data are presented as means + SE (n = 5–7 rats/group, in each compartment) *Significantly different compared with neonates in the same compartment. †Significantly different compared with airways in the same age. ‡Significantly different compared with FA in the same compartment. CYP1A1 immunohistochemical expression is presented in D. Although basal CYP1A1 was low in both ages across either lung compartment, CYP1A1 protein had different spatial and temporal patterns compared between neonates and adults after exposure. Neonatal CYP1A1 protein trailed mRNA levels, and CYP1A1 airway protein was enhanced at PFP24. CYP1A1 responded more acutely in adult animals, where staining was transiently observed in both lung compartments at PFP2. Scale bar is 50 μm.

Fig. 5.

CYP1B1 compartmental mRNA and protein expression. RT-PCR: basal CYP1B1 mRNA was significantly higher in adults in both compartments, with the highest expression present in the airway (A), no exposure-related effects were observed in neonates (B), and adults had a time-dependent decrease in CYP1B1 expression in the airways at PFP days 24 (PFP24) and 48 (PFP48) (C). Data are presented as means + SE (n = 5–7 rats/group, in each compartment) *Significantly different compared with neonates in the same compartment. †Significantly different compared with airways in the same age. ‡Significantly different compared with FA in the same compartment. CYP1B1 immunohistochemistry showed a different spatial pattern. Whereas adults had more CYP1B1-positive cells basally, CYP1B1 was intensely upregulated in both ages. By PFP24, neonatal CYP1B1 began to fade from the most distal terminal bronchioles, whereas adult CYP1B1 expression remained elevated, as shown in the insets, as denoted by black arrows. At PFP48, neonatal CYP1B1 returned to FA control levels, whereas adults continue to remain slightly upregulated. Scale bar is 50 μm.

Fig. 6.

Cytochrome P-450 oxidoreductase (POR) compartmental mRNA and protein expression. RT-PCR: basal POR expression was higher in adults compared with neonates and POR mRNA levels were lowest in the neonatal parenchyma (A), POR expression was unchanged post-PFP exposure in neonates (B), and, in adults, compartmental differences were observed after exposure (C). Parenchyma mRNA was significantly less than airways in all exposure time points. Data are presented as means + SE (n = 5–7 rats/group, in each compartment) *Significantly different compared with neonates in the same compartment. †Significantly different compared with airways in the same age. POR immunohistochemical protein expression revealed a similar trend. POR expression was highest in the airways. The only differences observed were terminal bronchiole staining, where POR were absent in neonates, vs. heavy staining in adults, as denoted by white arrows and high-magnification insets. Scale bars are 50 μm.