Interception of benzo[a]pyrene-7,8-dione by UDP glucuronosyltransferases (UGTs) in human lung cells

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are environmental and tobacco carcinogens. Proximate carcinogenic PAH trans-dihydrodiols are activated by human aldo-keto reductases (AKRs) to yield electrophilic and redox-active o-quinones. Interconversion among benzo[a]pyrene (B[a]P)-7,8-dione, a representative PAH o-quinone, and its corresponding catechol generates a futile redox-cycle with the concomitant production of reactive oxygen species (ROS). We investigated whether glucuronidation of B[a]P-7,8-catechol by human UDP glucuronosyltransferases (UGTs) could intercept the catechol in three different human lung cells. RT-PCR showed that UGT1A1, 1A3, and 2B7 were only expressed in human lung adenocarcinoma A549 cells. The corresponding recombinant UGTs were examined for their kinetic constants and product profile using B[a]P-7,8-catechol as a substrate. B[a]P-7,8-dione was reduced to B[a]P-7,8-catechol by dithiothreitol under anaerobic conditions and then further glucuronidated by the UGTs in the presence of uridine-5'-diphosphoglucuronic acid as a glucuronic acid group donor. UGT1A1 catalyzed the glucuronidation of B[a]P-7,8-catechol and generated two isomeric O-monoglucuronsyl-B[a]P-7,8-catechol products that were identified by RP-HPLC and by LC-MS/MS. By contrast, UGT1A3 and 2B7 catalyzed the formation of only one monoglucuronide, which was identical to that formed in A549 cells. The kinetic profiles of three UGTs followed Michaelis-Menten kinetics. On the basis of the expression levels of UGT1A3 and UGT2B7 and the observation that a single monoglucuronide was produced in A549 cells, we suggest that the major UGT isoforms in A549 cells that can intercept B[a]P-7,8-catechol are UGT1A3 and 2B7.

Figure 1

Metabolic activation of B[a]P by AKRs and detoxication of B[a]P-7,8-dione by UGTs.

Figure 2

Gene expression of UGTs in various human cell lines. (HepG2, hepatoma cells; A549, human lung adenocarcinoma cells; H358, human bronchoalveolar cells; HBEC-KT, immortalized human bronchial epithelial cells; BEAS-2B, normal human bronchial epithelial cells.)

Figure 3

HPLC/UV/RAM identification of B[a]P-7,8-catechol glucuronide metabolites (M1 and M2). B[a]P-7,8-catechol (20 μM) was generated in situ under anaerobic conditions in an incubation buffer containing 1 mM dithiothreitol. B[a]P-7,8-catechol was converted to the O-glucuronide(s) by UGT Supersomes supplemented with UDPGA (A, UGT1A1; B, UGT1A3; and C, UGT2B7) in the presence of UDPGA. (D) Control incubations were conducted in the absence of UDPGA. (E) HPLC-RAM chromatogram of O-monoglucuronsyl-B[a]P-7,8-catechol formed by UGT1A3 in the presence of [¹⁴C]-UDPGA. The delayed retention time of the M2 metabolite is due to the fact that the radiometeric detector is in-line and downstream from the absorbance (PDA) detector.

Figure 4

UV spectra of O-monoglucuronsyl-B[a]P-7,8-catechol metabolites (M1 and M2). B[a]P-7,8-catechol (20 μM) was generated in situ under anaerobic conditions in an incubation buffer containing 1 mM dithiothreitol. B[a]P-7,8-catechol was converted to O-glucuronide(s) by UGT1A1 plus UDPGA. UV spectra of M1 and M2 were collected by an in-line Waters 996 photodiode array detector.

Figure 5

LC/MS/MS identification of O-monoglucuronsyl-B[a]P-7,8-catechols (M1 and M2). B[a]P-7,8-catechol (20 μM) was generated in situ under anaerobic conditions in the presence of 1 mM dithiothreitol. B[a]P-7,8-catechol was converted to O-glucuronide(s) in the presence of UDPGA and UGTs in the incubation buffer. The reaction was quenched by 1% formic acid and extracted with ethyl acetate. The organic phase was then dried and dissolved in MeOH for LC-MS/MS analysis (positive ion mode, MS² spectrum of isomer M1 and MS² spectrum of isomer M2). The structure of the M2 metabolite is shown.

Figure 6

LC-MS/MS detection of O-monoglucuronsyl-B[a]P-7,8-catechols (M1 and M2) formed by recombinant UGT1A1 (A), 1A3 (B), 2B7 (C), and by A549 (D) at 24 h. B[a]P-7,8-catechol (20 μM) was generated in situ under anaerobic conditions in the presence of 1 mM dithiothreitol. B[a]P-7,8-catechol was converted to the O-glucuronide(s) in the presence of UDPGA and UGTs in the incubation buffer (A, UGT1A1; B, UGT1A3; and C, UGT2B7). (D) B[a]P-7,8-dione (2 μM, 0.2% DMSO) in HBSS medium was incubated with A549 cells and the culture media collected at 0 and 24 h, respectively. The culture media were acidified by formic acid and extracted with ethyl acetate. The organic phases were dried under vacuum and redissolved in methanol. O-Monoglucuronsyl-B[a]P-7,8-catechols were analyzed with LC-MS/MS in a negative ion mode by monitoring the mass transition m/z 459 → 283 ([M – H]⁻ → [M – H-glucuronic acid group]⁻).

Figure 7

Kinetic characterization of glucuronidation of B[a]P-7,8-catechol by UGTs. Kinetic analyses were performed by fitting the Michaelis–Menten equation to the data. Reactions contained 10 mM KPO₄ buffer at pH 7.4, 1.0 mM dithiothreitol, 5.0 mM MgCl₂, 1 mM [¹⁴C]UDPGA, 0–20 μM B[a]P-7,8-dione, and 15–30 μg of human recombinant UGT Supersomes at 25 °C. A, B, and C, velocity versus [S] curve for the glucuronidation of B[a]P-7,8-catechol by UGT1A1, 1A3, and 2B7, respectively.