Metabolism of an Alkylated Polycyclic Aromatic Hydrocarbon 5-Methylchrysene in Human Hepatoma (HepG2) Cells

Abstract

Exposure to polycyclic aromatic hydrocarbons (PAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. C1-chrysenes are representative PAHs present in the crude oil and have been detected in contaminated sea food in amounts that exceed their permissible safety thresholds. We describe the metabolism of the most carcinogenic C1-chrysene regioisomer, 5-methylchrysene (5-MC), in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS. 5-MC-tetraol, a signature metabolite of the diol-epoxide pathway, was identified as reported previously. Novel O-monosulfonated-5-MC-catechol isomers and O-monomethyl-O-monosulfonated-5-MC-catechol were discovered, and evidence for their precursor ortho-quinones was obtained. The identities of O-monosulfonated-5-MC-1,2-catechol, O-monomethyl-O-monosulfonated-5-MC-1,2-catechol, and 5-MC-1,2-dione were validated by comparison to authentic synthesized standards. Dual metabolic activation of 5-MC involving the formation of bis-electrophiles, i.e., a mono-diol-epoxide and a mono-ortho-quinone within the same structure, bis-diol-epoxides, and bis-ortho-quinones is reported for the first time. Evidence was also obtained for minor metabolic conversion of 5-MC to form monohydroxylated-quinones and bis-phenols. The identification of 5-MC-tetraol, O-monosulfonated-5-MC-1,2-catechol, O-monomethyl-O-monosulfonated-5-MC-1,2-catechol, and 5-MC-1,2-dione supports metabolic activation of 5-MC by P450 and AKR isozymes followed by metabolic detoxification of the ortho-quinone through interception of redox cycling by COMT and SULT isozymes. The major metabolites, O-monosulfonated-catechols and tetraols, could be used as biomarkers of human exposure to 5-MC resulting from oil spills.

Figure 1

HPLC detection of 5-MC metabolites in human HepG2 cells. (A) UV chromatogram at λ_max 268 nm at 0 h. (B) UV chromatogram at λ_max 268 nm at 24 h. (C) FLR chromatogram at λ_ex 273 nm and λ_em 391 nm at 0 h. (D) FLR chromatogram at λ_ex 273 nm and λ_em 391 nm at 24 h. Human HepG2 cells (~5 × 10⁶) were treated with 5-MC (1 μM, 0.2% (v/v) DMSO) in MEM (without phenol red) containing 10 mM glucose. The cell media were collected at 0 and 24 h and subsequently acidified with 0.1% formic acid before extraction with ethyl acetate. The extracts were analyzed on a HPLC-UV-FLR. 5-MeC = 5-methylchrysene.

Figure 2

Detection of O-monosulfonated-5-MC-catechol isomers in human HepG2 cells. (A) MS³ chromatogram at 0 h. (B) MS³ chromatogram at 24 h. (C) MS² spectrum of the peak at 22.88 min. (D) MS³ spectrum of the peak at 22.88 min. The samples were prepared as described in the caption to Figure 1 and were subsequently analyzed on an ion-trap LC-MS/MS.

Figure 3

Characterization of synthetic O-monosulfonated-5-MC-1,2-catechol. (A) UV chromatogram at λ_max 268 nm. (B) FLR chromatogram at λ_ex 273 nm and λ_em 391 nm. (C) Extracted ion chromatogram of pseudo SRM transition. (D) MS² spectrum. (E) MS³ spectrum. The product profiles were obtained after 1 h incubation of 5-MC-1,2-catechol with SULT1A1 and PAPS.

Figure 4

Detection of O-monomethyl-O-monosulfonated-5-MC-1,2-catechol in human HepG2 cells. (A) Extracted ion chromatogram of pseudo SRM transition at 0 h. (B) Extracted ion chromatogram of pseudo SRM transition at 24 h. (C) MS³ spectrum of the peak at 22.54 min. The samples were prepared as described in the caption to Figure 1 and were subsequently analyzed on an ion-trap LC-MS/MS.

Figure 5

Characterization of synthetic O-monomethyl-O-monosulfonated-5-MC-1,2-catechol. (A) UV chromatogram at λ_max 268 nm. (B) FLR chromatogram at λ_ex 273 nm and λ_em 391 nm. (C) Extracted ion chromatogram of pseudo SRM transition. (D) MS² spectrum. (E) MS³ spectrum. The product profiles were obtained after 1 h incubation of 5-MC-1,2-catechol with COMT and AdoMet followed by an additional 1 h incubation with SULT1A1 and PAPS.

Figure 6

Detection of 5-MC-1,2-dione in human HepG2 cells. (A) Extracted ion chromatogram of pseudo SRM transition at 0 h. (B) Extracted ion chromatogram of pseudo SRM transition at 24 h. (C) MS² spectrum of the peak at 31.98 min. (D) MS³ spectrum of the peak at 31.98 min. The samples were prepared as described in the caption to Figure 1 and were subsequently analyzed on an ion-trap LC-MS/MS. Another peak with a retention time of 23.05 min and with relatively high polarity could be an isomer of O-monosulfonated-5-MC-catechol, which could undergo cleavage of the sulfate conjugate in the mass spectrometer followed by auto-oxidation and thus result in the detection of quinone instead.

Figure 7

Detection of mono-hydroxy-5-MC-dione in human HepG2 cells. (A) Extracted ion chromatogram of pseudo SRM transition at 0 h. (B) Extracted ion chromatogram of pseudo SRM transition at 24 h. (C) MS² spectrum of the peak at 31.38 min. (D) MS³ spectrum of the peak at 31.38 min. The samples were prepared as described in the caption to Figure 1 and were subsequently analyzed on an ion-trap LC-MS/MS.

Figure 8

Detection of tetrahydroxy-O-monomethyl-5-MC-catechols in human HepG2 cells. (A) Extracted ion chromatogram of the Orbitrap full scan at 0 h. (B) Extracted ion chromatogram of the Orbitrap full scan at 24 h. (C, D) MS spectra of the peaks eluting at 14.66 and 15.65 min. The samples were prepared as described in the caption to Figure 1 and were subsequently analyzed on an Orbitrap LC-MS/MS.

Figure 9

Detection of monodehydrated O-monosulfonated-5-MC-dihydrodiol in human HepG2 cells. (A) Extracted ion chromatogram of pseudo SRM transition at 0 h. (B) Extracted ion chromatogram of pseudo SRM transition at 24 h. (C) MS² spectrum of the peak at 22.01 min. (D) MS³ spectrum of the peak at 22.01 min. The samples were prepared as described in the caption to Figure 1 and were subsequently analyzed on an ion-trap LC-MS/MS.

Scheme 1. Synthetic Routes of Four 5-MC-1,2-Catechol Conjugates

Scheme 2. Proposed Metabolic Pathway of 5-MC in Human HepG2 Cells^a

^aThe numbers for each metabolite correspond to the metabolites labeled in the UV and fluorescence chromatograms in Figure 1.