Effect of Cytochrome P450 Reductase Deficiency on 2-Amino-9H

Nov 19, 2015 - Therefore, hepatic P450 plays an important role in detoxication of AαC, but other hepatic or extrahepatic enzymes contribute to the bi...
2 downloads 8 Views 3MB Size
Download PDF
Article pubs.acs.org/crt

Effect of Cytochrome P450 Reductase Deficiency on 2‑Amino‑9H‑pyrido[2,3‑b]indole Metabolism and DNA Adduct Formation in Liver and Extrahepatic Tissues of Mice Robert J. Turesky,*,† Dmitri Konorev,† Xiaoyu Fan,‡ Yijin Tang,‡ Lihua Yao,† Xinxin Ding,§ Fang Xie,‡ Yi Zhu,‡ and Qing-Yu Zhang*,‡ †

Department of Medicinal Chemistry and Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States ‡ Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, New York 12201, United States § College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, New York 12203, United States ABSTRACT: 2-Amino-9H-pyrido[2,3-b]indole (AαC), a carcinogen formed during the combustion of tobacco and cooking of meat, undergoes cytochrome P450 (P450) metabolism to form the DNA adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dGC8-AαC). We evaluated the roles of P450 expressed in the liver and intestine to bioactivate AαC by employing male B6 wild-type (WT) mice, liver-specific P450 reductase (Cpr)-null (LCN) mice, and intestinal epithelium-specific Cpr-null (IECN) mice. Pharmacokinetic parameters were determined for AαC, 2-amino-9H-pyrido[2,3-b]indol3-yl sulfate (AαC-3-OSO3H), and N2-(β-1-glucosidurony1)-2-amino9H-pyrido[2,3-b]indole (AαC-N2-Glu) with animals dosed by gavage with AαC (13.6 mg/kg). The uptake of AαC was rapid with no difference in the plasma half-lives (t1/2) of AαC, AαC-3-OSO3H, and AαC-N2-Glu among mouse models. The maximal plasma concentrations (Cmax) and the areas under concentration−time curve (AUC0−24h) of AαC and AαC-N2-Glu were 4−24-fold higher in LCN than in WT mice, but they were not different between WT and IECN mice. These findings are consistent with the ablation of hepatic P450 activity in LCN mice. However, the Cmax and AUC0−24h of AαC-3-OSO3H in plasma were not substantially different among the mouse models. Similar pharmacokinetic parameters were obtained with WT and LCN mice treated with a lower AαC dose (1.36 mg kg−1). dG-C8-AαC was detected at similar levels in the livers of all three mouse models at the high AαC dose; levels of dG-C8-AαC in colon, bladder, and lung were greater in LCN than in WT mice and were the same in colon of IECN and WT mice. At the low AαC dose, dG-C8-AαC occurred at ∼40% lower levels in liver of LCN mouse than in WT mouse liver, but adduct levels remained higher in extrahepatic tissues of LCN mice. Therefore, hepatic P450 plays an important role in detoxication of AαC, but other hepatic or extrahepatic enzymes contribute to the bioactivation of AαC. P450s expressed in the intestine do not appreciably contribute to bioactivation of AαC in mice.

■

INTRODUCTION 2-Amino-9H-pyrido[2,3-b]indole (AαC) was originally discovered in a pyrolysate of soybean protein1 and then identified in mainstream cigarette smoke at levels ranging from 60 to 250 ng/cigarette.2,3 These quantities are far greater than those of the aromatic amines 4-aminobiphenyl (4-ABP) and 2-naphthylamine, which are implicated in the pathogenesis of bladder cancer in smokers.4,5 AαC is also formed in well-done charred meats and some vegetables.6 Apart from the endocyclic nitrogen atoms, AαC shares the same structure as 2aminofluorene, one of the most well-studied aromatic amine carcinogens.7 Significant amounts of AαC were detected in the urine of male smokers of the Shanghai cohort in China, providing evidence that tobacco smoke is a major source of AαC exposure.8 AαC is a liver carcinogen in mice, a lacI © 2015 American Chemical Society

transgene colon mutagen, and an inducer of colonic aberrant crypt foci, an early biomarker of colon neoplasia.9−11 Tobacco smoke is now recognized as a risk factor for liver and colorectal cancer in humans.12−15 Given the high levels of AαC that arise in tobacco smoke, AαC may contribute to the DNA damage and cancer risk of liver and colorectum of tobacco smokers.16 AαC undergoes metabolism by N-oxidation of the exocyclic amine group to form 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC) with rodent and human liver microsomes.17,18 HONH-AαC can undergo conjugation by sulfotransferases (SULTs) or N-acetyltransferases (NATs) to form reactive esters.19 These intermediates undergo heterolytic cleavage to Received: September 24, 2015 Published: November 19, 2015 2400

DOI: 10.1021/acs.chemrestox.5b00405 Chem. Res. Toxicol. 2015, 28, 2400−2410

Article

Chemical Research in Toxicology

Figure 1. AαC and its biomarkers.

produce the proposed nitrenium ion of AαC,20 which binds to DNA and can lead to mutations. The major DNA adduct of AαC has been identified as N-(deoxyguanosin-8-yl)-2-amino9H-pyrido[2,3-b]indole (dG-C8-AαC),21,22 which is thought to be responsible for the genotoxicity of AαC.23 While there is extensive exposure to AαC through tobacco smoke and ingestion of charred meat, knowledge about the major metabolic pathways of AαC and the key enzymes involved bioactivation of AαC in liver and extrahepatic tissues in rodent models and humans is not known. In this study, we have employed wild-type male C57BL/6 mice and two tissue-specific NADPH-cytochrome P450 reductase (Cpr) gene knockout mouse models to explore the role of hepatic and intestinal P450s in the bioactivation of AαC. The cytochrome P450 reductase (CPR or POR) is required for the monooxygenase activity of all microsomal P450 enzymes.24 In one mouse model, Cpr was specifically deleted in all hepatocytes of the liver,25 and in the second model, Cpr was selectively deleted in all intestinal epithelial enterocytes.26 Both models have been utilized previously to identify specific contributions of hepatic and intestinal microsomal P450 enzymes to xenobiotic metabolism and bioactivation in vivo.26−30 We have examined the pharmacokinetics of AαC and its metabolism by measurement of the plasma levels of AαC, the oxidative metabolite 2-amino-9H-pyrido[2,3-b]indol3-yl sulfate (AαC-3-OSO3H), and the conjugate N2-(β-1glucosiduronyl)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2Glu). The bioactivation of AαC was assessed through DNA adduct formation in liver and extrahepatic tissues of these mouse models (Figure 1).

■

pyrido[2,3-b]indol-3-yl sulfate (AαC-3-OSO3H) and [13C6] AαC-3OSO3H were prepared by the Boyland−Sims oxidation of AαC with potassium persulfate.32 N2-(β-1-Glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Glu) was prepared with human liver microsomes fortified with UDPGA as previously reported.33 dG-C8-AαC and the isotopically labeled [13C10]-dG-C8-AαC were synthesized as described.34 2-Hydroxamino-3,8-dimethylimidaz[4,5-f ]quinoxaline (HONH-MeIQx) and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP) were prepared from their respective nitro derivatives.35 2-Hydroxyamino-9H-pyrido[2,3-b]indole (HONHAαC was synthesized as described.36 2-Amino-4′-hydroxy-1-methyl-6phenylimidazo[4,5-b]pyridine (4′-HO-PhIP) was prepared with rat liver microsomes and characterized as previously described.37 NMR Characterization of AαC Metabolites. 1H NMR resonance assignments for the metabolites of AαC were conducted at 25 °C with a Bruker Avance III 600 MHz spectrometer equipped with a triple resonance cryoprobe (Bruker BioSpin Corp., Billerica, MA). The 1H chemical shifts were referenced directly from the DMSO-d6 multiplet at 2.50 ppm. Biosynthesis of 2-Amino-3-hydroxy-9H-pyrido[2,3-b]indole (3-HO-AαC) and 2-Amino-6-hydroxy-9H-pyrido[2,3-b]indole (6-HO-AαC). 3-HO-AαC and 6-HO-AαC were prepared enzymatically by incubation of liver microsomes (1 mg mL−1) of rats pretreated with polychlorinated biphenyls with cofactors and AαC (200 μM) for 1 h at 37 °C, followed by HPLC purification as previously described.38 The assignment of the protons of 3-HO-AαC in DMSO-d6 was: δ 10.92 (s, 1H, H−N9) 7.79 (d, 7.67 Hz, 1H, H-5); 7.53 (s, 1H, H-4); 7.34 (d, 7.97 Hz, 1H, H-8), 7.19 (dd, 7.52, 7.97 Hz, 1H, H-7), 7.06 (dd, 7.52, 7.67 Hz, 1H, H-6), 5.75 (s, 2H, NH2). The assignment of the protons of 6-HO-AαC in DMSO-d6 was: δ 10.76 (s, 1H, H−N9), 7.95 (d, 8.33 Hz, 1H, H-4), 7.20 (d, 2.01 Hz, 1H, H-5), 7.14 (d, 8.50 Hz, 1H, H-8), 6.72 (dd, 2.01, 8.50 Hz, 1H, H-7), 6.30 (d, 8.33 Hz, 1H, H-3), 6.01 (s, 2H, NH2). The full scan spectra of 3-HO-AαC and 6HO-AαC under ESI/MS conditions by triple stage quadrupole MS showed the protonated molecules [M + H]+ at m/z 200.1. Animals and Treatments. Male LCN and IECN mice (2−4 months old) and age-matched WT littermates (all on the C57BL/6 background) were obtained from breeding stocks maintained at the Wadsworth Center. Animals were given food and water ad libitum. For AαC administration, WT, LCN, and IECN mice were given a bolus dose of AαC (dissolved in water at 1.36 or 0.136 mg mL−1) via oral gavage (at 13.6 or 1.36 mg kg−1, respectively). All animals were sacrificed 24 h later for tissue collection when dosed at 13.6 mg kg−1 or sacrificed at 6 h for tissue collection when dosed at 1.36 mg kg−1. Plasma, liver, lung, and bladder were frozen on dry ice immediately after collection. Colon was slit open and rinsed with ice-cold PBS before the colon epithelial layer was isolated as described previously.39 All animal studies were approved by the Institutional Animal Care and Use Committee of the Wadsworth Center.

MATERIALS AND METHODS

Caution! AαC is a potential human carcinogen. AαC and its derivatives must be handled in a well-ventilated f ume hood with proper use of gloves and protective clothing. Chemicals. AαC, 2-amino-3,8-dimethylimidaz[4,5-f ]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were purchased from the Toronto Research Chemicals (Toronto, ON, Canada). [4b,5,6,7,8,8a-13C6]-AαC was a gift from Dr. Daniel Doerge, National Center for Toxicological Research (Jefferson, AR). Uridine-5′-diphosphoglucuronic acid (UDPGA), alamethicin, tetrahydrofuran (THF), potassium persulfate, and Pd/C were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). LC/MS grade solvents were from Fisher Scientific. HONH-AαC was prepared by reduction of 2-nitro-9H-pyrido[2,3-b]indole (NO2-AαC) in THF with hydrazine, using Pd/C as a catalyst.31 2-Amino-9H2401

DOI: 10.1021/acs.chemrestox.5b00405 Chem. Res. Toxicol. 2015, 28, 2400−2410

Article

Chemical Research in Toxicology Enzyme Preparation and Assays. Mouse liver microsomes and cytosols were prepared by differential centrifugation in 0.1 M potassium phosphate buffer (pH 7.4), containing 0.125 M potassium chloride, 0.25 M sucrose, and 1.0 mM EDTA. The microsomes and cytosols were stored in 0.1 M potassium phosphate buffer, pH 7.4, 0.1 mM EDTA, containing 20% glycerol.38 Human liver microsomes were obtained from the Tennessee Donor Services, Nashville, TN, and kindly provided by Prof. F. P. Guengerich, Vanderbilt University. The protein content was determined with the bicinchoninic acid (BCA) protein assay using bovine serum albumin as a reference standard. All enzyme assays were performed under linear reaction conditions relative to substrate and protein contents. Liver Microsomal Metabolism Studies. Liver microsomal P450 methoxyresorufin O-demethylation (MROD) activities were determined using the buffer and cofactor conditions previously described employing microsomal protein (0.25 mg mL−1) and methoxyresorufin (10 μM) in a final volume of 0.5 mL.38 Resorufin formation was measured by fluorescence employing a Cary Eclipse fluorescence spectrophotometer (Agilent Technology) with excitation at 530 nm and emission at 585 nm, using a slit width of 5 nm.40,41 Metabolism studies with AαC, MeIQx, or PhIP (100 μM) used microsomal protein (1.0 mg mL−1) in 100 mM potassium phosphate (pH 7.6) containing 0.5 mM EDTA and 1 mM NADPH. Incubations were conducted at 37 °C for up to 30 min and terminated as previously described.38 The supernatant was assayed by HPLC with UV detection monitoring at 274 (MeIQx), 315 (PhIP), and 336 nm (AαC). HPLC was done with an Agilent Technology 1260 Infinity System (Santa Clara, CA) with an Aquasil C18 column (4.6 × 150 mm, 5 μm particle size) from Thermo Scientific. The gradient elution started from 99% of 10 mM NH4CH3CO2 (pH 6.8, containing 5% CH3CN), increased to 40% CH3CN in 16 min, increased to 100% CH3N at 25 min with holding for 2 min, and then returned to 1% CH3CN at 29 min. Microsomal Flavin Monooxygenase (FMO). FMO activities were determined using methimazole (1 mM) as the substrate. The reaction was carried out in 0.1 M Tris-HCl (pH 8.4) containing 1 mM EDTA, 0.025 mM DTT, 0.1 mM NADPH, 0.06 mM 5,5′ dithiobis(2nitrobenzoate), and microsomal protein (0.5 mg mL−1). The rate of oxidation was determined spectrophotometrically at 412 nm, using an extinction coefficient of 28 200 M −1 cm−1 for 5,5′-dithiobis(2nitrobenzoate), which serves as an indirect measure of the sulfenic acid.42 Xanthine Dehydrogenase Assay. The assay employed xanthine (50 μM) as the substrate with NAD+ (500 μM) in 50 mM phosphate buffer (pH 7.8) containing cytosolic protein (1 mg mL−1). Activity was determined by the production of NADH at 340 nm (ε = 6220 M −1 cm−1) at 25 °C.43 Aldehyde Oxidase Assay. The assay employed p-dimethylaminocinnamaldehyde (DMAC) as a substrate (25 μM), monitoring the decrease in its absorbance at 398 nm (ε = 30 500 M −1 cm−1) in 50 mM phosphate buffer, pH 7.8, at 25 °C with cytosolic protein (1 mg mL−1).43 Pharmacokinetic Studies. Male mice (WT, LCN, or IECN, 2−4 months old, 5 in each group) were dosed with AαC as its hydrochloride salt (1.36 or 13.6 mg kg−1 body weight) by oral gavage. Blood samples (20 μL) were collected from the tail vein at time points 0, 1, 2, 4, 6, and 24 h after AαC administration when mice were dosed at 13.6 mg kg−1 and at time points 0, 1, 2, 4, and 6 h after AαC administration for animals dosed at 1.36 mg kg−1. Plasma was obtained by centrifugation at 2000g for 10 min at 4 °C. The plasma was immediately frozen at −80 °C. For the low-dose study, plasma (4 μL) was diluted with CH3OH (26 μL) containing 400 pg of [13C6]-AαC and [13C6]-AαC-3-OSO3H and left on ice for 15 min to precipitate protein, followed by centrifugation. For the high-dose study, plasma samples (4 μL) were diluted with CH3OH (96 μL) containing 2 ng of [13C6]-AαC and [13C6]-AαC-3-OSO3H for time points at 1, 2, and 4 h, and plasma samples (4 μL) were diluted with CH3OH (16 μL) containing 400 pg [13C6]-AαC and [13C6]- AαC-3-OSO3H for the 6 and 24 h time points. The supernatants were diluted with an equal volume of H2O and transferred to LC/MS vials. The pharmacokinetic

parameters were calculated by noncompartmental analysis using WinNonlin software (version 5.1; Pharsight, Mountain View, CA). Clearance was calculated as the hybrid constant CL/F, given that bioavailability (F) is not known. Liquid Chromatography/Mass Spectrometry (UPLC/MS2) of AαC, AαC-3-OSO3H, and AαC-N2-Glu in Plasma. The analysis of AαC and its metabolites was done with the Thermo TSQ Quantiva triple stage quadrupole MS (TSQ/MS) interfaced to a HESI II source (Thermo Scientific, San Jose, CA). Chromatography was performed with a NanoAcquity UPLC system (Waters Corp., Milford, MA) equipped with a Waters BEH130 C18 reversed-phase column (3 × 150 mm, 3 μm particle size). Solvent A was 5 mM NH4HCO3 (pH 9.0), and solvent B was 95% CH3CN containing 5% H2O and 5 mM NH4HCO3, pH 9.0. The flow rate was 5 μL min−1, and a gradient was employed starting at 10%, arriving at 99% B in 7 min, and holding at 99% B for 2 min before returning to starting conditions at 10 min with a post run time of 5 min. AαC and AαC-N2-Glu were assayed in the positive ion mode, employing a spray voltage of 2.9 kV and AαC-3-OSO3H was assayed in the negative ion mode using a spray voltage of 2.4 kV. The sheath gas (N2) was set at 4 units. The collision gas was argon and set at 1.5 mTorr. The ion transfer tube temperature was set at 400 °C, and the capillary temperature was 50 °C. Measurements were done by selected reaction monitoring (SRM) with polarity switching. The transitions employed for AαC and [13C6]-AαC in positive ion mode were, respectively, m/z 184.1 → 167.1 and 140.1, and 190.1 → 173.1 and 146.1 using collision energies of 26 and 36 V. The transition for AαCN2-Glu was m/z 360.1 → 184.1 at 28 V. [13C6]-AαC was employed as an internal standard to measure AαC and AαC-N2-Glu. A correction factor of 10 was used to account for the discrepancy in signal of response between AαC and AαC-N2-Glu because the latter metabolite underwent CID to produce multiple product ions of AαC containing different portions of the glucuronide moiety.33 The transitions for AαC-3-OSO3H and [13C6]-AαC-3-OSO3H were measured in the negative ion mode and were, respectively, m/z 278.1 → 198.1 and 284.1 → 204.1, employing a collision energy of 23 V. Sample Preparation and AαC-DNA Adduct Measurements by UPLC/MS3. Tissues were homogenized in 3 volumes of TE buffer, pH 8.0 (50 mM Tris-HCl, 10 mM EDTA) and incubated with RNase T1, RNase, and proteinase K, followed by isolation of DNA by the phenol/chloroform method.34 DNA (5 or 10 μg) of each sample was spiked with isotopically labeled internal standards at a level of 1 adduct per 107 bases, and the enzymatic digestion of DNA was conducted in 5 mM Bis-Tris-HCl buffer (pH 7.1) with DNase I for 1.5 h, followed by incubation with nuclease P1 for 3 h, and then by digestion with alkaline phosphatase and phosphodiesterase for 18 h.44 The samples were vacuum centrifuged to dryness and resuspended in 1:1 water/ DMSO (30 μL). Following centrifugation (22 000g for 5 min), the supernatant was transferred to capillary LC vials. The DNA adduct analyses were conducted with a Waters NanoAcquity UPLC system (Waters Corp., New Milford, MA) equipped with a Waters Symmetry trap column (180 μm × 20 mm, 5 μm particle size), a Michrom C18 AQ column (0.3 × 150 mm, 3 μm particle size, Michrom Bioresources Inc., Auburn, CA), and a Michrom CaptiveSpray source interfaced to a linear quadrupole ion-trap mass spectrometer (LTQ Velos, Thermo Fisher, San Jose, CA). The adduct measurements were conducted at the MS3 scan stage. For dG-C8AαC, ions at m/z 449.1 (MS) > 333.1 (MS2) > 209.2, 291.4, 316.4 (MS3), and for the internal standard, [13C10]-dG-C8-AαC, ions at m/z 459.1 (MS) > 338.1 (MS2) > 210.2, 295.4, 321.5 (MS3) were monitored. An external calibration curve was employed for quantification.45 The chromatographic conditions and mass spectral parameters have been reported.34,45,46 Statistical Analysis. Statistical significance of differences between groups for pharmacokinetic parameters was examined using Student’s t test or Mann−Whitney rank test. The statistical significance of AαCDNA adduct formation of the high-dose-treated animals was performed by one-way ANOVA, followed by Dunnett’s multiple comparison test with WT mouse serving as the control, and the 2402

DOI: 10.1021/acs.chemrestox.5b00405 Chem. Res. Toxicol. 2015, 28, 2400−2410

Article

Chemical Research in Toxicology

Table 1. Pharmacokinetic Parameters for Plasma AαC and Its Metabolites in LCN, IECN, and WT Mice at a High Dose of AαC (13.6 mg kg−1)a strain

analytes

WT LCN IECN WT LCN IECN WT LCN IECN

AαC AαC AαC AαC-N2-Glu AαC-N2-Glu AαC-N2-Glu AαC-3-OSO3H AαC-3-OSO3H AαC-3-OSO3H

Tmax (h) 1.0 1.0 1.0 1.0 1.0 1.8 1.8 3.5 1.8

± ± ± ± ± ± ± ± ±

0 0 0 0 0 1.2 1.3 2.3 1.2

Cmax (ng mL−1) 26.5 272 25.1 40.2 186 42.4 669 586 913

± ± ± ± ± ± ± ± ±

8.6 85.9b 12.4 9.6 85.0c 15.4 251 128 248

t1/2 (h) 3.0 2.8 3.9 1.5 2.1 2.5 3.4 3.1 2.8

± ± ± ± ± ± ± ± ±

2.4 0.3 2.2 0.7 0.5 2.2 1.2 0.2 0.2

AUC0−24h (h·ng mL−1) 57.6 1360 68.4 128 803 150 3700 6140 5310

± ± ± ± ± ± ± ± ±

14.2 595b 18.2 26.8 241b 30.0 911 1330c 1650

CL/F (mL h−1) 6900 335 5920 3610 543 3180 107 64.3 77.0

± ± ± ± ± ± ± ± ±

1850 146c 1530 566 148b 1031 27.9 13.6b 20.3d

Plasma levels of AαC, AαC-N2-Glu, and AαC-3-OSO3H were determined after a single dose of AαC at 13.6 mg kg−1 via oral gavage. Plasma samples were obtained at various times after dosing. Values represent means ± SD (n = 5). bp < 0.001; cp < 0.01; dp < 0.05 compared to the corresponding WT group (Student’s t-test or Mann−Whitney rank test) a

Table 2. Pharmacokinetic Parameters for Plasma AαC and Its Metabolites in LCN and WT Mice at a Low Dose of AαC (1.36 mg kg−1)a strain

analytes

WT LCN WT LCN WT LCN

AαC AαC AαC-N2-Glu AαC-N2-Glu AαC-3-OSO3H AαC-3-OSO3H

Tmax (h) 1.0 1.0 1.0 1.0 1.0 1.0

± ± ± ± ± ±

0 0 0 0 0 0

Cmax (ng mL−1) 1.4 21.8 19.2 74.3 74.6 177

± ± ± ± ± ±

0.9 13.6b 13.5 48.6b 31.5 73.0b

t1/2 (h) 1.7 1.4 1.4 1.5 5.9 2.5

± ± ± ± ± ±

0.9 0.5 0.7 0.8 6.1 1.7

AUC0−6h (h·ng mL−1) 2.5 45.2 40.5 195 165.6 438

± ± ± ± ± ±

1.3 16.9b 16.6 78.7b 47.6 118b

CL/F (mL h−1) 15 300 884 985 207 180 76.0

± ± ± ± ± ±

5690 378b 372 100b 140 39.2c

Plasma levels of AαC, AαC-N2-Glu, and AαC-3-OSO3H were determined after a single dose of AαC at 1.36 mg kg−1 via oral gavage. Plasma samples were obtained at various times after dosing. Values represent means ± SD (n = 5). bP < 0.001; cP < 0.01 compared to the corresponding WT group (Student’s t-test or Mann−Whitney rank test) a

Figure 2. Representative chromatograms of dG-C8-AaαC adduct formation in extrahepatic tissues of LCN and WT mice dosed with AαC (1.36 mg kg−1). The right panel shows the MS3 product ion spectra of dG-C8-AαC and [13C10]-dG-C8-AαC obtained from LCN bladder DNA. analysis of the low-dose study with AαC was done by the unpaired ttest, employing GraphPad Prism software, v 6 (San Diego, CA).

■

among WT, LCN, and IECN mice at a high dose (13.6 mg kg−1) and between WT and LCN mice at a lower dose (1.36 mg kg−1). The pharmacokinetic parameters are shown in Tables 1 and 2. At a dose of 13.6 mg kg−1 (Table 1), a comparable plasma t1/2 for AαC was observed among WT, LCN, and IECN mice. However, the Cmax value of AαC in

RESULTS

Impact of Hepatic Cpr Deletion on First-Pass Clearance of AαC. Rates of AαC clearance were compared 2403

DOI: 10.1021/acs.chemrestox.5b00405 Chem. Res. Toxicol. 2015, 28, 2400−2410

Article

Chemical Research in Toxicology plasma was 10-fold greater in LCN mice than for WT and IECN mice, and the area under the concentration−time curve (AUC) 0−24 h for unmetabolized AαC in plasma was about 20-fold greater in LCN mice than in WT or IECN mice. The clearance rate for AαC was 20-fold lower in LCN mice than in WT or IECN mice. The Cmax value for AαC-3-OSO3H was not significantly different among the three strains of mice, whereas the (AUC)0−24h for AαC-3-OSO3H in LCN mice was about 1.6-fold greater than WT mice (P < 0.01), and the clearance rate for AαC-3-OSO3H was slightly lower in LCN mice than in WT mice. For AαC-N2-Glu, the AUC0−24h and Cmax values were, respectively, ∼6- and 4.5-fold greater in LCN mice than in WT; and the clearance rate was 6.6-fold lower in LCN mice than in WT mice. There were no significant differences in the pharmacokinetic parameters of AαC-N2-Glu between WT and IECN mice. The increase in the AUC and Cmax values for AαCN2-Glu in LCN mice are consistent with an ablation of P450 activity with higher levels of AαC available to serve as a substrate for uridine 5′-diphospho-glucuronosyltransferases (UGT). The strain-related differences between WT and LCN mice in pharmacokinetic parameters for plasma AαC, AαC-3OSO3H, and AαC-N2-Glu were similar at the lower dose of 1.36 mg kg−1, except for the Cmax value of AαC-3-OSO3H, which was approximately 2-fold higher in LCN mice than in WT mice (Table 2). AαC-DNA Adduct Formation in Liver and Extrahepatic Organs of WT, LCN, and IECN Mice. Representative UPLC/MS3 chromatograms of dG-C8-AαC adduct formation are shown in Figure 2. The levels of dG-C8-AαC formed in liver and extrahepatic tissues of mouse models are summarized in Figure 3. At the high dose of AαC (13.6 mg kg−1), there was no statistically significant difference (one-way ANOVA, p = 0.411) in the levels of dG-C8-AαC adducts formed in liver of WT, LCN, and IECN mice, even though the pharmacokinetics data showed a considerably higher Cmax and AUC0−24h of AαC in plasma of LCN mice, attributed to the ablation of hepatic P450 activity. However, the levels of dG-C8-AαC were 4 times greater in the colon of LCN mice than in the colon of WT and IECN mice, which contained similar levels of adducts. Similarly, adduct levels in lung of LCN mice were also 4-fold higher than adduct levels in lung of WT and IECN mice. These findings signify that at the high dose of AαC (13.6 mg kg−1) administered, dG-C8-AαC formation is not dependent on hepatic microsomal P450, but the latter is important in the detoxification of AαC in WT mice. Moreover, intestinal P450s are not involved in the formation of colonic or hepatic DNA adducts because the same levels of adducts are formed in colon of WT and IECN, the latter strain lacks P450 activity in the intestine. Interestingly, although the variance between levels of dG-C8-AαC adducts formed in the bladder of these three mouse models was considerably lower than the variance in adduct levels observed in colon and lung, the levels of dG-C8AαC were modestly but statistically significantly higher in bladder of IECN mice than bladder of WT (1.5-fold) or LCN (1.2-fold) mice. Further studies are required to determine why the levels of dG-C8-AαC appear to be slightly increased in the bladder of IECN vs WT mice. At the lower dose of AαC (1.36 mg kg−1), dG-C8-AαC adduct formation in liver of WT mice was modestly but significantly higher, by 1.6-fold, in comparison to adduct levels formed in livers of LCN mice. The level of dG-C8-AαC adduct formation in the colon of LCN mice was still greater than adduct levels formed in WT mice. Thus, at this lower dose, it is

Figure 3. (A) AαC DNA adduct formation in liver, colon, lung, and bladder WT, LCN, and IECN mice dosed with AαC at 13.6 mg kg−1 and (B) AαC DNA adduct formation in liver, colon, lung, and bladder of WT and LCN mice dosed with AαC at 1.36 mg kg−1. Data are presented as the mean and standard deviation of 5 or 7 animals per group. High dose performed by one-way ANOVA, and Dunnett’s multiple comparison test with WT DNA serving as the control (*p < 0.01 WT Bladder vs LCN Bladder; **** p < 0.0001 WT Bladder vs IECN Bladder; WT Colon vs LCN Colon; WT Lung vs LCN Lung), and low dose performed by unpaired t-test (** p < 0.05; *** p < 0.001; **** p < 0.0001).

apparent that hepatic P450 contributes to the bioactivation of AαC and DNA adduct formation in liver of WT mice, but the expression of hepatic P450 still strongly contributes to detoxication of AαC, resulting in lower levels of dG-C8-AαC adducts formed in colon, bladder, and lung of WT mice compared to LCN mice. In Vitro Assays with Liver Enzymes. We examined the capacity of AαC to serve as a substrate for several different hepatic enzyme systems that oxidize heterocyclic aromatic compounds (Table 3). Cytochrome P450s, particularly P450 1A2, are generally viewed as the major enzymes involved in the metabolism of AαC and other HAAs in rodents and humans.18,38,47 The levels of MROD activity, a probe for P450, were comparable to those values previously reported in liver microsomes of C57BL/6N mice and humans.38,48 The MROD activity was decreased by greater than 10-fold in LCN liver microsomes compared to WT microsomes, corroborating the ablation of P450 activity in this mouse model. The metabolism of AαC, by hepatic P450s, in WT mouse liver was low and approached the limit of detection by HPLCUV. Only trace levels of the 3-HO-AαC, 6-HO-AαC, and HONH-AαC were detected in reaction mixtures with WT microsomes (Figure 4A). Although reactive HONH-AαC metabolite may have bound to microsomal protein,49 the rate 2404

DOI: 10.1021/acs.chemrestox.5b00405 Chem. Res. Toxicol. 2015, 28, 2400−2410

Article

Chemical Research in Toxicology Table 3. Metabolism of AαC and Other Heterocyclic Amines by Hepatic Enzymesa

FMO activity (nmol min−1 mg−1 protein)

P450 oxidation (pmol min−1 mg−1 protein) AαC WT mouse liver microsomes LCN mouse liver microsomes Human liver microsome (H112)

WT mouse liver cytosol LCN mouse liver cytosol

PhIP

MeIQx

103 ± 33 362 ± 120 404 ± 155 N.D. N.D. 67 ± 44 820 ± 60 1440 ± 280 1010 ± 155 Xanthine dehydrogenase (nmol min−1 mg−1 protein)

MROD

methimazole

AαC

115 ± 23 2.90 ± 0.67 N.D. 3.8 ± 1.0 3.97 ± 0.80 N.D. 90.1 ± 2.5 Aldehyde oxidase (nmol min−1 mg−1 protein)

xanthine

AαC

DMAC

AαC

2.12 ± 0.48 1.72 ± 0.37

N.D. N.D.

783 ± 93 592 ± 148

N.D. N.D.

Enzyme assays were carried out as described in Materials and Methods. Values are reported as the mean and SD of at least 3 mice (n ≥ 3). Substrate concentrations assayed were as follows: 10 μM (MROD), 25 μM (DMAC), 50 μM (xanthine), 100 μM (AαC, PhIP, MelQx), and 1 mM (methimazole). N.D., not detected;