Quantitation of benzo[a]pyrene metabolic profiles in human bronchoalveolar (H358) cells by stable isotope dilution liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and are carcinogenic in multiple organs and species. Benzo[a]pyrene (B[a]P) is a representative PAH and has been studied extensively for its carcinogenicity and toxicity. B[a]P itself is chemically inert and requires metabolic activation to exhibit its toxicity and carcinogenicity. Three major metabolic pathways have been well documented. The signature metabolites generated from the radical cation (peroxidase or monooxygenase mediated) pathway are B[a]P-1,6-dione and B[a]P-3,6-dione, the signature metabolite generated from the diol-epoxide (P450 mediated) pathway is B[a]P-r-7,t-8,t-9,c-10-tetrahydrotetrol (B[a]P-tetrol-1), and the signature metabolite generated from the o-quinone (aldo-keto reductase mediated) pathway is B[a]P-7,8-dione. The contributions of these different metabolic pathways to cancer initiation and the exploitation of this information for cancer prevention are still under debate. With the availability of a library of [(13)C(4)]-labeled B[a]P metabolite internal standards, we developed a sensitive stable isotope dilution atmospheric pressure chemical ionization tandem mass spectrometry method to address this issue by quantitating B[a]P metabolites from each metabolic pathway in human lung cells. This analytical method represents a 500-fold increased sensitivity compared with that of a method using HPLC-radiometric detection. The limit of quantitation (LOQ) was determined to be 6 fmol on column for 3-hydroxybenzo[a]pyrene (3-OH-B[a]P), the generally accepted biomarker for B[a]P exposure. This high level of sensitivity and robustness of the method was demonstrated in a study of B[a]P metabolic profiles in human bronchoalveolar H358 cells induced or uninduced with the AhR ligand, 2,3,7,8-tetrachlorodibenzodioxin (TCDD). All the signature metabolites were detected and successfully quantitated. Our results suggest that all three metabolic pathways contribute equally in the overall metabolism of B[a]P in H358 cells with or without TCDD induction. The sensitivity of the method should permit the identification of cell-type differences in B[a]P activation and detoxication and could also be used for biomonitoring human exposure to PAH.

Figure 1

Proposed B[a]P metabolic pathways and their representative metabolites. The radical cation pathway involves the activity of either P450 peroxidase or P450 monooxygenase; the diol epoxide pathway involves P450 and epoxide hydrolase; and the o-quinone pathway involves P450, epoxide hydrolase and AKR.

Figure 2

HPLC-UV chromatogram of a mixture of B[a]P metabolite standards monitored at 254 nm.

Figure 3

Representative LC-MS chromatograms of a mixture of B[a]P metabolite standards. *low collision energy was used with the loss of one CO was monitored

Figure 4

Two sets of [¹³C₄]-labeled B[a]P metabolite standards showing the position of ¹³C-incorporation.

Figure 5

Representative standard curves performed in MeOH and in the cell matrices. The solid triangle represents the regression line for samples prepared in MeOH, the cross represents the regression lines for samples prepared in matrices and the solid square represents the expected regression lines for samples with 100% recovery. (A) B[a]P-tetrol-1/[¹³C₄]-B[a]P-tetrol-1, (r² = 0.9972 for MeOH and r² = 0.9965 for matrices); (B) 3-OH-B[a]P/[¹³C₄]-3-OH-B[a]P (r² = 0.999 for MeOH and r² = 0.9986 for matrices) and (C) B[a]P-7,8-dione/[¹³C₄]-B[a]P-7,8-dione (r² =0.9977 for MeOH and r² = 0.9946 for matrices).

Figure 6

Distribution of total B[a]P organic metabolites in H358 cells over 24h after pretreatment with TCDD (A) or without TCDD (B). The mean ± SD for n=3 is shown.

Figure 7

Representative 12 h time point of LC/MS/MS chromatograms of B[a]P metabolites from extraction of TCDD treated H358 cells incubated with 4 µM B[a]P. Injection volume was 10 µL. A) B[a]P tetrol-1 (14.6 min); B) B[a]P-9,10-diol (17.2 min), B[a]P-7,8-diol (24.5 min) and 3-OH-B[a]P (35.6 min); C) B[a]P-7,8-dione (27.9 min), B[a]P-1,6-dione (32.2 min) and B[a]P-3,6-dione (33.1 min). CM: cellular metabolite; IS: [¹³C₄]-labeled internal standard.

Figure 8

Appearance of B[a]P major metabolites over 24 h (± TCDD). A). B[a]P-tetrol-1; B). B[a]P-9,10-diol; C). B[a]P-7,8-diol; D). 3-OH-B[a]P; E). B[a]P-7,8-dione; F). B[a]P-1,6-dione; G). B[a]P-3,6-dione. Solid triangle represents metabolites from TCDD treated cells and solid circle represents metabolites from DMSO treated cells. The mean ± SD is shown n=3.

Figure 9

Contribution of the three metabolic pathways of B[a]P metabolism in H358 cells. A). radical cation pathway, (B[a]P-1,6-dione and B[a]P-3,6-dione); B). diol-epoxide pathway, (B[a]P-tetrol-1); C). o-quinone pathway, (B[a]P-7,8-dione). Solid triangle represents metabolites from TCDD treated cells and solid circle represents metabolites from DMSO treated cells. The mean ± SD is shown n=3.