Comparative mechanisms of PAH toxicity by benzo[a]pyrene and dibenzo[def,p]chrysene in primary human bronchial epithelial cells cultured at air-liquid interface

Abstract

Current assumption for assessing carcinogenic risk of polycyclic aromatic hydrocarbons (PAHs) is that they function through a common mechanism of action; however, recent studies demonstrate that PAHs can act through unique mechanisms potentially contributing to cancer outcomes in a non-additive manner. Using a primary human 3D bronchial epithelial culture (HBEC) model, we assessed potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from transcriptional profiling. Differentiated bronchial epithelial cells were treated with BAP (100-500 μg/ml), DBC (10 μg/ml), and coal tar extract (CTE 500-1500 μg/ml, SRM1597a) for 48 h and gene expression was measured by RNA sequencing or quantitative PCR. Comparison of BAP and DBC gene signatures showed that the majority of genes (~60%) were uniquely regulated by treatment, including signaling pathways for inflammation and DNA damage by DBC and processes for cell cycle, hypoxia and oxidative stress by BAP. Specifically, BAP upregulated targets of AhR, NRF2, and KLF4, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. Other processes were regulated in common by all PAH treatments, BAP, DBC and CTE, including downregulation of genes involved in cell adhesion and reduced functional measurements of barrier integrity. This work supports prior in vivo studies and demonstrates the utility of profiling short-term biosignatures in an organotypic 3D model to identify mechanisms linked to carcinogenic risk of PAHs in humans.

Keywords: Benzo[a]pyrene; Bronchial epithelial cells; Mixtures; Organotypic culture; Polycyclic aromatic hydrocarbons; Toxicogenomics.

Figure 1.. Morphological characterization of primary 3D human bronchial epithelial cells (HBEC) in culture.

HBEC cultures were fixed in 10% formalin and tissue sections were stained with H&E (A) to observe ciliated cells, Ki67 (B) for actively proliferating cells, p63 (C) for basal cells, and PAS (D) for goblet cells. These features are noted by arrows showing positively stained cells in panels A-D. (E) Immunofluorescence of MUC5B and β-tubulin (red with DAPI counterstain in blue) as markers of glandular mucous cells and ciliated respiratory cells, respectively.

Figure 2.. Cell viability measured by lactate dehydrogenase (LDH) leakage in cell media 48 hrs after exposure to PAHs.

Values are presented as LDH leakage in media for treated cells compared to acetone vehicle control (% vehicle control ± standard error). LDH leakage from HBEC treated with benzo[a]pyrene (BAP), dibenzo[def,p]chrysene (DBC) and coal tar extract (CTE) for 48 hr were not significantly different (p>0.05) from vehicle control at any concentration (one-way ANOVA).

Figure 3.. HBEC transcriptional response to BAP and DBC.

Global gene expression was measured in HBEC 48 hrs after treatment with 500 ug/ml (19.8 nmol) benzo[a]pyrene (BAP) and 10 ug/ml (0.35 nmol) dibenzo[def,p]chrysene (DBC) by RNA sequencing. (A) Bidirectional hierarchical clustering by Euclidean distance of genes differentially expressed (q<0.05) by BAP and DBC compared to vehicle control. Values are log2 fold change for all treatments compared with control; red, green, and black represent increased, decreased and unchanged gene expression, respectively. (B) Venn diagram showing overlap of significantly regulated (q <0.05) genes by BAP and DBC in HBEC. (C) Functional enrichment of gene processes in HBEC using MetaCore network processes (GeneGo) for genes commonly regulated by BAP and DBC (q<0.05). Black bars represent functions for genes up-regulated and gray bars represent functions for genes down-regulated in common by BAP and DBC (q<0.05). The dashed line indicates the threshold for significance (p<0.05).

Figure 4.. Decrease in barrier integrity of HBEC after treatment with BAP and DBC.

(A) Bidirectional hierarchical clustering by Euclidean distance of differentially expressed genes associated with gap junction and tight junction signaling (q<0.05) after 48 hr treatment with benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC) compared to vehicle control. Values are log2 fold change for all treatments compared with control; red, green, and black represent up-regulated, down-regulated and unchanged genes, respectively. (B) Transepithelial electrical resistance (TEER) measured in HBEC 48 hr after treatment with BAP, DBC and coal tar extract (CTE). Values are TEER (Ω-cm2) normalized to vehicle control (scaled to 100%). *Indicates significant reduction in TEER (p<0.05; one-way ANOVA with Tukey’s pairwise comparison). Expression of genes associated with barrier integrity were measured by quantitative PCR in HBEC after 48 hr treatment with BAP and DBC (C) and CTE (D), respectively. Values are expressed as fold change (Log2; mean ± SE) compared to vehicle control. Asterisks indicate significance compared to vehicle control (*p<0.05; **p<0.001; one-way ANOVA with Tukey’s pairwise comparison).

Figure 5.. Genes uniquely regulated by BAP and DBC in HBEC.

Global gene expression was measured in HBEC 48 hrs after treatment with 500 ug/ml (19.8 nmol) benzo[a]pyrene (BAP) and 10 ug/ml (0.35 nmol) dibenzo[def,p]chrysene (DBC) by RNA sequencing. Venn diagram shows genes uniquely regulated (q <0.05) by BAP and DBC in HBEC. Statistical enrichment of biological network processes (MetaCore) is shown for genes uniquely up-regulated (right-panel) and down-regulated (left-panel) by BAP (light gray bars) and DBC (dark gray bars). The dashed line indicates the threshold for significance (p<0.05).

Figure 6.. Unique regulation of AHR- and NRF2-mediated genes by BAP and DBC in HBEC.

(A) Expression of CYP1A1 and CYP1B1 was measured by quantitative PCR in HBEC 6-48 hrs after treatment with BAP, DBC and CTE (48 hr only). (B) Expression of NQO1, ALDH3A1 and GSTA was measured by quantitative PCR in HBEC 48 hrs after treatment with BAP, DBC and CTE. (C) Transcription factor signaling networks for AHR and NRF2 in HBEC after treatment with 500ug/ml (19.8 nmol) BAP (right, red) and 10 ug/ml (0.35 nmol) DBC (left, green). Target nodes are up-regulated by BAP (red) and down-regulated by DBC (green). For panesl A and B, values are expressed as fold change (Log2; mean ± SE) compared to vehicle control. Asterisks indicate significance compared to vehicle control (*p<0.05; **p<0.001; oneway ANOVA with Tukey’s pairwise comparison). For panel C, as indicated in the legends, the size of nodes is associated with significance (larger is more significant) and the color intensity represents magnitude of response (darker is larger response) for BAP and DBC compared to vehicle control.