Diesel exhaust influences carcinogenic PAH-induced genotoxicity and gene expression in human breast epithelial cells in culture

Abstract

The carcinogenic polycyclic aromatic hydrocarbon (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are widespread environmental pollutants, however their toxicological effects within a mixture is not established. We investigated the influence of diesel exhaust (DE) on B[a]P and DB[a,l]P-induced PAH-DNA adduct formation, metabolic activation, gene expression and 8-oxo-dG adduct levels in human breast epithelial cells (MCF-10A) in culture. Following 24 and 48h, cells co-exposed to DE plus B[a]P exhibited a significant decrease in PAH-DNA adduct levels, compared with B[a]P alone, as determined by (33)P-postlabeling combined with reversed-phase high performance liquid chromatography (HPLC). Cytochrome P450 (CYP) enzyme activity, as measured by the ethoxyresorufin O-deethylase (EROD) assay and CYP1B1 expression, significantly increased with co-exposure of DE plus DB[a,l]P, compared with DB[a,l]P alone. Aldo keto-reductase (AKR)1C1, AKR1C2, and AKR1C3 expression also significantly increased in cells exposed to DE plus PAH, compared with PAH exposure alone. Cell populations exhibiting 8-oxo-dG adducts significantly increased in response to exposure to B[a]P or DE plus B[a]P for 24h, compared with vehicle control, as quantified by flow cytometry. These results suggest that complex mixtures may modify the carcinogenic potency of PAH by shifting the metabolic activation pathway from the production of PAH diol-epoxides to AKR pathway-derived metabolites.

Figure 1

The effect of DE exposure on the levels of B[a]P-diolepoxide- or DB[a,l]P-diolepoxide DNA adducts in MCF-10A cells in culture. Isolated DNA was ³³P-postlabled and analyzed by reversed-phase HPLC (as described in Materials and Methods). Data points indicate means with standard error bars over n = 3 or 4 separate experiments. No effect on adduct levels were found between time of exposure (P > 0.05). DE significantly decreased the level of B[a]PDE-DNA adducts, compared with B[a]P treatment alone (P = 0.016). Whereas, DE had no significant influence on the level of DB[a,l]PDE-DNA adducts (P = 0.428).

Figure 2

The influence of DE on B[a]P or DB[a,l]P-induced EROD activity in MCF-10A cells in culture. Data represents three individual experiments averaged over 24 and 48 h, presented as a simple mean ± SEM on log-scale. A statistically significant difference (P = 0.002) was observed in cells co-exposed to DE plus DB[a,l]P, compared with DB[a,l]P alone.

Figure 3

Changes in expression of genes (as measured by quantitative real-time PCR) involved in PAH metabolism following 24 h exposure to PAH or PAH plus DE, compared with dimethyl sulfoxide (DMSO; vehicle control). Data represents mean ± SD (n = 3 replicates per exposure). aGenes that were significantly altered (P ≤ 0.01) compared to B[a]P or DB[a,l]P.

Figure 4

Cell populations exhibiting 8-oxo-dG adducts, following 24 h exposure to vehicle control (tricaprylin), PAH or PAH plus DE. Adduct levels were quantified by fluorescence emitted by FITC-conjugated protein binding agent, measured by flow cytometry using the OxyFLOW® platform (HemoGenix, Inc, Colorado Springs, CO). Data represents mean ± SEM (n = 3 replicates per exposure).

Figure 5

Competing pathways for the metabolic activation of PAH by CYP, cytochrome P450, and AKR, aldo-ketoreductase (adapted from T.M. Penning [56]). Schematic also illustrates the effect of CYP inhibition by mixture components, as suggested previously [44].