The roles of cytochrome P450s in the toxicity of polycyclic aromatic hydrocarbons (PAHs)

Polycyclic aromatic hydrocarbons (PAHs) are one of the most important and ubiquitous groups of environmental pollutants. Cytochrome P450 enzymes are involved in PAH metabolism, activation and carcinogenesis; and are also used as biomarkers of PAH exposure. Normal operation of oil well platforms results in the discharge of "produced formation water" (PFW), which contains PAHs. As a part of a project conducted with the Australian Institute of Marine Science, to investigate the potential impact of PFW to the marine environment, the expression of CYP1A, CYP2M1-- and 2K1--like proteins was examined for use as possible biomarkers of PFW exposure. A pilot study on the Northwest Shelf of Australia had indicated that PFW contamination possibly contributes to induction of CYP1A-like proteins in Gold--Spotted Trevally ( Carangoides fulvoguttatus). The pilot study samples were re-examined for CYP1A- and in addition, CYP2K1/2M1-like proteins. In a subsequent caged fish study using Stripey seaperch (Lutjanus carponotatus) caught at a clean site, fish were distributed to three caging sites in a gradient from the Harriet A production platform: A (near-field), B (far-field) and C (a non-impacted reference site). Fish were sampled at time (T) T=0, T=3 and T=10 days. Significant increases of CYP1A, one CYP2K1- and two CYP2M1-like proteins were noted at Site A at T=10 d. For another CYP2K1-like protein, a significant increase was observed at Site A only at T=3 d. These results support a previous study indicating that CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1- and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and CYP2 family induction may represent possible new biomarkers (other than CYP1A) associated with PFW. In addition, the novel response of one CYP2K--like protein requires further verification but offers promise for improved monitoring of sub-lethal responses in marine organisms.;To further understand the function of cytochrome P450 in activation of PAH, the metabolism of benzo[alpha]pyrene (BaP), a representative carcinogenic PAH, was studied in the fish Fundulus heteroclitus. A sensitive and fast method was developed to quantitate BaP and eight of its oxidized metabolites by ultra-performance liquid chromatography (UPLC) coupling with mass spectrometry (MS). The new UPLC-MS-based method allows for separation and analysis of BaP metabolites with increased resolution, rapid analysis time, and high detection sensitivity compared to conventional HPLC methods. With this method, it was demonstrated that BaP metabolites existed mainly as glucuronic acid conjugates in exposed Fundulus bile, and 3-hydroxyBaP, BaP diones, BaP-7,8--dihydrodiol were predominate metabolites. Fundulus CYP1A and CYP 1 C 1 were successfully expressed in Escherichia coli. The partially purified proteins showed low EROD activities. Preliminary studies of in vitro BaP metabolism by recombinant proteins suggested differences in metabolite profiles between Fundulus and human, and between CYP1A and CYP1C1. Together these results provided additional methods and results to further understand the role of individual CYP1s in the mechanisms of PAH toxicity.