Pivotal Role for Two Electron Reduction in 2,3-Dimethoxy-1,4

Mar 27, 2009 - Imperial College London. , #. All of these authors made substantial contributions to the practical data. J.D.P., A.V.P., and J.K.C. und...
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Chem. Res. Toxicol. 2009, 22, 717–725

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Pivotal Role for Two Electron Reduction in 2,3-Dimethoxy-1,4-naphthoquinone and 2-Methyl-1,4-naphthoquinone Metabolism and Kinetics in Vivo That Prevents Liver Redox Stress Joel D. Parry,‡,# Amy V. Pointon,†,# Ursula Lutz,§,# Friederike Teichert,|,# Joanne K. Charlwood,‡,# Pui Hei Chan,⊥,# Toby J. Athersuch,⊥,# Emma L. Taylor,† Rajinder Singh,| JinLi Luo,† Kate M. Phillips,† Angelique Vetillard,† Jonathan J. Lyon,‡ Hector C. Keun,⊥ Werner K. Lutz,§ and Timothy W. Gant*,† Medical Research Council Toxicology Unit, UniVersity of Leicester, Lancaster Road, Leicester LE1 9HN, U.K., Department of InVestigatiVe Preclinical Toxicology, Safety Assessment, GSK R&D Ltd., Ware, U.K., UniVersity of Wu¨rzburg, Department of Toxicology, DE-97078 Wu¨rzburg, Germany, Cancer Biomarkers and PreVention Group, Biocentre, UniVersity of Leicester, UniVersity Road, Leicester LE1 7RH, U.K., and Department of Biomolecular Medicine, DiVision of Surgery, Oncology, ReproductiVe Biology and Anaesthetics, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, U.K. ReceiVed December 11, 2008

2,3-Dimethoxy-1,4-naphthoquinone (CAS-RN 6959-96-3) (DMNQ) and 2-methyl-1,4-naphthoquinone (CAS-RN 58-27-5) (MNQ:menadione) are effective one electron redox cycling chemicals in vitro. In addition, in vitro MNQ forms a thioether conjugate with glutathione by nucleophilic attack at the third carbon. In contrast, here we demonstrate that in vivo the major metabolic route is directly to the dihydronaphthoquinone for both DMNQ and MNQ followed by conjugation to mono- and di-glucuronides and sulfate. Analysis of urine and bile showed that glutathione conjugation of MNQ was only a very minor route of metabolism. DMNQ was distributed to all tissues including the brain, and MNQ was much less widely distributed. For DMNQ tissue half-life, in particular for the heart, was considerably longer than the plasma half-life. For both DMNQ and MNQ, urine 8-oxo-7,8-dihydro-2′-deoxyguanosine and liver transcriptomic analysis failed to show any evidence of redox stress. Oxidized glutathione (GSSG) in liver increased significantly at the 10 min postdosing time point only. Metabonomic analysis 96 h after DMNQ administration indicated decreased liver glucose and increased lactate and creatine suggesting an impairment of oxidative metabolism. We conclude that in vivo DMNQ and MNQ are primarily two electron reduced to the dihydronaphthoquinones and undergo little one electron redox cycling. For DMNQ, disruption of cellular oxidative metabolism may be a primary mechanism of toxicity rather than redox stress. Introduction Reactive oxygen species (ROS) have been implicated as primary components in the mechanism of toxicity of a considerable number of chemicals and drugs, disease processes, and aging (1-6). ROS can also modulate the expression of certain genes, thereby affecting cellular biochemistry (7). Redox action is associated with many of the quinone species that are found extensively in nature and physiology (8-11) and utilized in electron transport processes such as in the electron transport chain (12). Quinones may undergo redox cycling following either one electron reduction, which in the cell may be mediated by microsomal NADPH P450 reductase, NADH cytochrome b5 * Corresponding author. † Medical Research Council Toxicology Unit, University of Leicester. ‡ GSK R&D Ltd. § University of Wu¨rzburg. | Cancer Biomarkers and Prevention Group, University of Leicester. ⊥ Imperial College London. # All of these authors made substantial contributions to the practical data. J.D.P., A.V.P., and J.K.C. undertook the experiments, toxicokinetic analysis, and GSH and GSSG measurements; J.D.P. the genomics analysis; U.L. the metabolite analysis; F.T. the creatinine and 8-oxo-dG measurements; and P.H.C. and T.A. the metabonomics.

reductase, mitochondrial NADH-oxidoreductase (13), or alternatively via a two electron reduction mediated by NAD(P)H: quinone oxidoreductase (nqo1) (DT-diaphorase) followed by a disproportionation reaction (14). Loss of nqo1 in mice results in increased 2-methyl-1,4-naphthoquinone (MNQ: menadione) toxicity suggesting that the two electron reduction pathway in this system is more important for detoxification than for activation (15). In contrast, in A549 cells with 2,3-dimethoxy1-4-naphthoquinone (DMNQ) and MNQ, two electron reduction to the dihydronaphthoquinone (H2DMNQ and H2MNQ respectively) appears to be the major route of quinone activation leading to toxic effects (16). Similar data was obtained with β-lapachone in A549 cells and in vivo in rats for nephrotoxicity mediated by 2-amino-1,4-naphthoquinone confirming that two electron nqo1 mediated quinone reduction is an activation route for at least some quinone species (17, 18). However, for the therapeutically important quinones doxorubicin and daunorubcin, two electron reduction by nqo1 does not appear to be an activating event as doxorubicin is a poor substrate for nqo1 (19, 20). Due to their reactive nature, many ROS species, particularly the hydroxyl radical, can also interact with lipids and DNA (21). Nucleophilic centers within DNA are susceptible to attack, in

10.1021/tx800472z CCC: $40.75  2009 American Chemical Society Published on Web 03/27/2009

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particular deoxyguanosine, resulting in the formation of an 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adduct, one of the major markers for oxidative stress on DNA (22, 23). 8-oxodG can lead to mutation with consequent downstream events such as cell transformations (24, 25). In 1988, Gant et al. first reported the use of DMNQ as an effective redox cycling naphthoquinone in vitro (26). DMNQ possesses a redox activity similar to that of MNQ but does not react with soft nucleophiles because the second and third positions on the naphthoquinone moiety are blocked by methoxy groups (27). Since this initial report, DMNQ has been extensively used for the investigation of redox mechanisms mostly in in vitro systems (28). In vitro redox activity induced by DMNQ leads to cell signaling events (29), cell necrosis and apoptosis (30), and DNA single strand breaks (31). One of the most extensive studies on the in vitro effects of DMNQ showed that the cellular effects were defined by the pro-oxidant level, with low levels causing proliferation, intermediate levels intracellular polyamine depletion, and high levels DNA strand breaks, calcium overload, and necrosis (32). Here, we show for the first time that the major route of DMNQ metabolism in vivo is via two electron reduction and conjugation. In the liver, redox damage measured using the biomarkers 8-oxodG and oxidized glutathione (GSSG) appears limited, and there were no redox associated changes in gene transcription. Liver metabonomic data indicated sustained decrease in oxidative metabolism.

Experimental Procedures Synthesis of DMNQ and Animal Treatment. DMNQ was synthesized according to the method published by Gant et al. (26). The purity was estimated at greater than 99% by HPLC (single peak) and structure verified by 1H NMR (250.13 MHz, CDCl3) δ 4.12 (s, 6H, C-2-COCH3, C-3-COCH3), 7.71 (s, 2H C-6-H, C-7H), 8.10 (s, 2H, C-5-H, C-8-H), and mass spectrometry (highest mass peak 218). MNQ was obtained from Sigma Chemicals (Poole, Dorset, UK). The N-acetyl-S-(2-methyl-1,4-naphthoquinonyl-3) cysteine standard (MNQ-MA) was synthesized by incubation of MNQ with N-acetyl-L-cysteine using methods similar to those of Li et al. (33) and the structure confirmed by 1H NMR (400.13 MHz, methanol-d4) δ 1.83 (s, 3H, C(O)CH3), 2.34 (s, 3H, C-2-CH3), 3.46 (dd, 1H, CH2S, 2JH,H ) 14.16 Hz, 3JH,H ) 7.63 Hz), 3.76 (dd, 1H, CH2S, 2JH,H ) 14.16 Hz, 3JH,H ) 4.43 Hz), 4.59 (dd, 1H, CHNH, 3 JH,H ) 7.63 Hz, 3JH,H ) 4.43 Hz), 7.75-7.80 (m, 2H, C-6-H, C-7H), 8.05-8.10 (m, 2H, C-5-H, C-8-H). All other materials were from established suppliers and of the highest grade available. C57B6/AJ (male, 8 weeks, 21-25 g) mice were obtained from an inbred colony maintained in the facility. All animal experiments were carried out under UK home office project number 80/1690. DMNQ and MNQ were administered using the intraperitoneal (i.p.) route in arachis oil to a final dose of 25 mg/kg in a volume of 10 mL/kg. This dose was just below the threshold for overt toxicity. Between three and five animals were used in each treatment group. Means and standard deviations are shown in the figures and group numbers ranging from 3-5. To prepare the DMNQ and MNQ solutions in arachis oil, the arachis oil with the naphthoquinone was gently heated until dissolution occurred and then cooled to room temperature before use. The naphthoquinone remained in solution after cooling. Freshly prepared solutions were used for each experiment. Blood was withdrawn by cardiac puncture without thoractomy under isofluorane induced anesthesia. After withdrawal of blood, a laparotomy was performed, the descending vena cava cut, and organs perfused in situ with PBS from the heart to ensure that there was no blood contamination of the organs used for determination of tissue naphthoquinone concentrations. To collect urine following injection of naphthoquinone, mice were placed in

Parry et al. metabolism cages and allowed access to water ad libitum but not food. The urine samples were collected in polypropylene tubes on dry ice. Determination of Tissue Quinone Concentration. Tissues were homogenized in 2:1 (w/v) water at room temperature. Plasma samples and tissue homogenates were mixed with acetonitrile (2:1 v/v) for 30 min at room temperature and centrifuged to remove any precipitate. The supernates were dried and extracts dissolved in 50 µL of 50% v/v methanol per 100 mg or 100 µL of original starting tissue/plasma. The methanol solutions were centrifuged briefly, and the supernatant loaded into HPLC vials for analysis. HPLC was performed on a 150 × 2.1 mm C18 3.5 µm column (WAT106005, Waters) using a 2695 HPLC Analyzer with a 2487 Dual Wavelength Detector (Waters). Buffer A was HPLC grade water, and Buffer B was HPLC grade methanol. The flow rate was set at 150 µL/min, the column temperature was maintained at 30 °C, and the sample temperature was 10 °C. The initial conditions were 60% Buffer A and 40% Buffer B. A linear gradient was run over 15 min to a final mix of 10% Buffer A and 90% Buffer B. This was held for 5 min before re-equilibrating the column at 60% Buffer A and 40% Buffer B. The total run time was 35 min, and the injection volume was 10 µL. Naphthoquinone was monitored at 276 nm and the amount of naphthoquinone in each sample determined by calibration of the system with standards of known concentration. The mean of naphthoquinone exposure profiles were imported into WinNonlin version 1.4 (Pharsight Corporation) and the data analyzed using noncompartmentalized analysis. Liquid Chromatography/Mass Spectrometry and Metabolite Identification. For liquid chromatography (LC), an Agilent 1100 G1312A pump with an Agilent 1100 Autosampler and a HyperClone 3 µm C8-BDS column, 150 × 2 mm, with a corresponding guard cartridge (Phenomenex, Aschaffenburg, Germany) were used. The mobile phase was 10 mM ammonium acetate at pH 5.2 (A) and acetonitrile (B) with a flow rate of 190 µL/min. A linear gradient was used from 2 to 32% B in 30 min, 32 to 52% B in 10 min, 52 to 92% B in 10 min, 92% B for 5 min, 92 to 2% B in 1 min, and re-equilibration at 2% B for 10 min. For the analysis of mouse urine, 10 µL of mouse urine diluted with water (1:1) was injected. The column eluate was introduced into the MS/MS system consisting of a TURBO-ionspray source operated in the negative ion mode and a hybrid quadrupole linear ion trap (QTRAP, Applied Biosystems/MDS Sciex, Concord, Ontario). The instrument parameters were source voltage, -4.2 kV; vaporizer temperature, 400 °C; curtain gas, 30 psi; nebulizer gas, 45 psi; turbogas, 50 psi; CAD gas, high; declustering potential, -35 V; and entrance potential, -7 V. For the analysis of bile, each gall bladder was put into 100 µL of H2O/acetonitrile 1:1 on ice, and the contents were squeezed with tweezers. The sample was centrifuged, the supernatant transferred to a fresh tube, and the treatment repeated with the pellet. The combined supernatants were lyophilized. The residue was dissolved in 20 µL of H2O/acetonitrile 1:1, and 10 µL was injected. For MS/MS analysis in positive ion mode and using NL 129, the instrument parameters were as follows: source voltage, 4.5 kV; declustering potential, 36 V; entrance potential, 6.5 V; collision energy, 19 V; collision cell exit potential, 6 V. The solvents for LC were H2O and acetonitrile, both containing 0.1% formic acid. For the survey scan, the enhanced mass scan mode (EMS) was used. This is the full-scan mode where all ions from 160 to 600 m/z for urine and 160 to 1200 m/z for bile were trapped in Q3 during 20 ms prior to detection at a scan rate of 1000 amu/s. MS/ MS data of the most abundant ion were collected by the enhanced product ion scan mode (EPI). When the EMS signal for a specific ion exceeded the threshold (2500 counts), Q1 filtered this ion for fragmentation in Q2 with a collision energy of -30 V. Fragment ions were trapped in Q3 before they were scanned from m/z 50 to 580 for urine and to 1180 for bile at a scan rate of 4000 amu/s. This precursor ion was then excluded from fragmentation for 60 s. Data acquisition was achieved using the Analyst 1.4.1 software (Applied Biosystems, Darmstadt, Germany). The data were analyzed with Metabolite ID software version 1.3 (Applied Biosystems/MDS

Naphthoquinone ADME and Biological Consequence Sciex, Concord, Ontario). This software compares the data of the treated sample with the data of the control sample and lists the peaks that were only found in the treated sample. Differences found by the software were confirmed by visual inspection of the manually extracted ion chromatograms. Determination of 8-oxodG in Urine. A 100 µL aliquot of each urine sample was diluted with 890 µL of HPLC grade water and spiked with 10 pmol of the stable isotope internal standard, [15N5]8oxodG (1 pmol/ µL), which was synthesized as described previously (34). The 8-oxo-dG in the urine was then determined using a mass spectrometry method as previously described (35). Determination of Urine Specific Gravity and Creatinine Concentration. Urine specific gravity was determined using a Reichert TS 400 refractometer (Reichert Analytical Instruments, Depew, USA). Creatinine was determined using an LC-MS/MS assay with [2H3]-creatinine as an internal standard (35). Determination of Reduced and Oxidized Glutathione. Liver, kidney, brain, or heart was homogenized in 10-20 µL of chilled 5% w/v metaphosphoric acid (MPA) per mg of wet tissue and centrifuged to remove any precipitate. Supernatants were placed on ice and then stored at -80 °C until required. Reduced glutathione (GSH) and GSSG concentrations were determined using the Bioxytech GSH/GSSG-412 Assay (OxisResearch, Foster City, CA). A total glutathione (GSHt) sample was prepared by diluting 10 µL of MPA lysate 1/10 to 1/40 with GSH Assay Buffer. A GSSG sample was prepared by incubating 50 µL of MPA lysate with 10 µL of Assay Scavenger solution (2-methyl-2-vinyl-pyridium trifluorosmethane sulfonate [M2VP]) at 4 °C for 30 min prior to diluting 1/3 to 1/10 in GSSG Assay Buffer. Samples were loaded onto a SPAce Autoanalyzer and mixed with equal volumes of Chromogen solution (5,5′-dithiobis-(2-nitrobenzoic acid [DNTB]), enzyme solution (glutathione reductase), and NADPH solution as supplied. Absorbance was monitored at 408 nm for 5 min and the rate of reaction (which is proportional to the GSH/GSSG present in the MPA solution) measured. GSHt and GSSG concentrations were determined from standard curves (as supplied) that were assayed on the same day. To derive the concentration of GSH within a tissue, 2 × GSSG value was subtracted from the respective GSHt value. Both GSH and GSSG results were expressed in terms of nmol per mg of wet tissue. 1 H NMR. Mouse liver tissue was stored at -80 °C until needed for analysis. On removal from the storage freezer, samples were kept on dry ice to prevent deterioration. A 20-30 mg portion of each liver tissue sample was dissected from the main tissue mass, weighed, and immediately placed back on dry ice in eppendorf tubes. The area of the organ taken was kept constant for all samples. Dissection instruments were thoroughly cleaned between sample preparations to prevent carry-over contamination. For sample homogenization, 400 µL of a MeOH/H2O mixture (3:1) was added to each eppendorf, and the sample tubes immediately transferred to a TissueLyser (Qiagen, Crawley, UK), whereupon the samples were mechanically disrupted (5 min, 25 Hz) using stainless steel beads (5 mm). Following the removal of the steel beads from each sample tube, 200 µL of CHCl3 was added and the samples mixed using a benchtop vortexer (1 min) to extract aqueous and organic components of the samples, which were then centrifuged (16000g, 10 min) and the two extraction components separated. For each extraction sample, the upper aqueous layer was transferred into a clean eppendorf tube, and the lower organic layer was transferred into a clean glass vial. A second, identical MeOH/ H2O/CHCl3 extraction of the remaining material in the sample tube was conducted (without the TissueLyser step) to improve metabolite extraction efficiency. Both the aqueous and organic extracts were left in a fume hood overnight (RT) to allow volatile solvents to evaporate. The aqueous extracts were lyophilized (12 h) and the dry material reconstituted in deuterated sodium phosphate buffer (0.2 M, 1 mM TSP, and 3 mM NaN3) using a vortexer (1 min) before transfer to a clean glass 5 mm NMR tube for analysis. The dried organic extract was retained for future use. 1 H NMR: Metabolic Profile Acquisition. High-resolution, onedimensional, 1H NMR spectra were acquired at a field strength of

Chem. Res. Toxicol., Vol. 22, No. 4, 2009 719 14.1 T (600.13 MHz 1H frequency) using a Bruker DRX600 spectrometer fitted with a 5 mm broadband-inverse tube probehead (Bruker Biospin, Rheinstetten, Germany) at 300 K. Samples were introduced to the instrument using a BACS 60 automated sample changer, and acquisitions controlled using Xwin-NMR and IconNMR (Bruker Biospin). Gradient shimming was used to improve the magnetic field homogeneity prior to all acquisitions. Carr-Purcell-Meiboom-Gill (CPMG) 1H spectra were also acquired for all aqueous samples (media, aqueous cell extracts) using the pulse sequence (RD-90°-(t-180°-t)n-AQ). The fixed echo time, t was set to 400 µs, giving a total spin-echo time of 64 ms. During the acquisition period (AQ, 2.73 s), the free induction decay (FID) was recorded into 64 k data-points in the time domain, with a spectral width of 20 ppm. Typically, spectra were recorded as the sum of 128 transients following 16 dummy scans. In both cases, suppression of the water resonance centered at δH ) 4.7 ppm was achieved by the application of a presaturation pulse during the relaxation delay (RD, 3 s). An exponential function equivalent to a 0.3 Hz linebroadening was applied to each FID prior to Fourier transformation to give frequency-domain spectra. 1 H NMR: Data Processing and Analysis. 1H NMR spectra obtained from each sample were phased, baseline corrected, calibrated to the TSP internal standard resonance (δH ) 0.00 ppm), and imported into Matlab (release 2007b, The MathWorks, USA) using a proprietory script written by Rachel Cavill, Hector Keun, and Tim Ebbels (Imperial College London) running in the Matlab computing environment. Interpolation was used to give the resulting data set a resolution of 32 k datapoints in the frequency domain between -1 and 10 ppm. The data was normalized by the medianfold change to a reference spectrum generated by calculation of the median of all spectra. Discriminant analysis was used to define resonances in the spectra that were correlated with treatment, which were then assigned using literature data. The statistical significance of the changes in the peak area of these resonances was tested using a two-tailed Student’s t-test. Transcriptomics. Transcriptomics data was obtained using Affymetrix mouse 430_2 microarrays according to the manufacturer’s protocol. Data was normalized by RMA using JustRMA from the Bioconductor Affy Library. Pathway analysis was carried out by EASE (36, 37).

Results Absorption, Distribution, and Excretion. Initial experiments established a dose level of 25 mg/kg for both DMNQ and MNQ in C57B6/AJ mice as close to the maximum tolerated dose for this formulation and dosing route. Gross toxicity at dose levels above 25 mg/kg appeared quickly after dosing i.p. in arachis oil. The toxicity resolved after approximately 45 min following dosing. This correlated with the pharmacokinetics of the parent compound, which was almost entirely eliminated by 60 min (Figure 1A). For DMNQ, the Tmax (plasma) was reached in 15 min, and Cmax was 41 nM (Table 1 and Figure 1A). Distribution occurred to all the tested organs (Figure 1B to E) including the brain where a Cmax of 6 nM was achieved. MNQ showed a reduced distribution to tissues compared with DMNQ and faster time to plasma Cmax but slower time to tissue Cmax. Concentrations of MNQ in the liver were difficult to consistently determine, which may indicate that there was some binding to protein occurring. Contamination of the tissue samples with blood was avoided by first perfusing the mice with 10 mL of PBS under anesthesia. The concentrations of naphthoquinone measured in the organs therefore represent true tissue concentrations. A mass balance analysis would be required to determine if some of the dose was being retained, and given the variability in the liver concentrations with MNQ, but not DMNQ, this is a possibility. Addition-elimination reactions between MNQ and proteins have been previously demonstrated (33).

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Figure 1. Tissues and plasma concentrations of DMNQ and MNQ with time. Mice (C57B6/AJ male) were treated with DMNQ or MNQ at 25 mg/kg in 10 mL/kg arachis oil i.p. at time 0 and groups taken at time points thereafter. Blood was collected into heparinized tubes by cardiac puncture under terminal isofluorane anesthesia and tissues after perfusion with PBS via the left ventricle to clear residual blood. The plasma and tissues were analyzed for naphthoquinone concentrations by HPLC. (A to E), Linear plots of the concentration of DMNQ with time after dosing (A), plasma; (B), liver; (C), kidney; (D), brain; (E), heart. Closed symbols, DMNQ; open symbols, MNQ. Mean and SD are shown from groups of three animals except for MNQ in the liver, which was variable, and one is shown.

Metabolite Profiling by LC-ESI-MS/MS. A quadrupolelinear ion trap mass spectrometer was used to detect and characterize the metabolites in urine treated with DMNQ or arachis oil. This allows in a single run a sensitive survey scan in full scan mode combined with an enhanced product ion (EPI) scan for each signal detected. Figure 2 shows full scan chromatograms of urine from animals treated with DMNQ (Figure 2A) or arachis oil (Figure 2B). Data from both sets of samples was compared using metabolite ID software. This resulted in a list of peaks (m/z and RT) that were only present in the DMNQ treated sample, i.e., potential metabolites. Extracted ion chromatograms were generated for the most prominent m/z values, 299, 395, 475, and 571, and an overlay is presented in Figure 2C and D for DMNQ or arachis oil treated samples, respectively. The two main metabolites had m/z 299 (RT 24.1) and m/z 395 (RT 18.1 min). Information on the structure of the compounds was obtained by inspection of the fragmentation pattern in the EPI spectrum. The metabolite at m/z 299 (Figure 2E) showed the characteristic fragments for a sulfate conjugate of the hydronaphthoquinone (H2DMNQ; loss of 80 Da from the parent ion, namely, 299 f 219) and a fragment of m/z 80 (38). The metabolite at m/z 395 was the glucuronide of H2DMNQ (Figure 2F) with a loss of 176 Da from the parent ion (395 f 219) and fragments of m/z 175, 113, and 85 (39). Among the minor metabolites were double conjugates. The EPI spectrum of the sulfate-glucuronide double conjugate of H2DMNQ (m/z 475, RT 11.8) is shown in Figure 2G. It indicates the characteristic losses and fragments of

Parry et al.

glucuronic acid and sulfate. The EPI spectrum of the double glucuronide (m/z 571, RT 3.3) is given in Figure 2H. No double sulfate conjugate was observed. Minor metabolites included demethylation products at the methoxy group of DMNQ followed by reduction to the H2DMNQ and conjugations in a manner similar to the parent compound (data not shown). The full scan chromatogram for MNQ and control treated mice is shown in Figure 3A and B and an overlay of the extracted ion chromatograms of the main metabolites in Figure 3C and D. Two peaks were apparent at m/z 349 eluting at RT 13.8 and 15.5 min, showing in the EPI spectrum (Figure 3F) the characteristic fragmentation of monoglucuronides at the first or fourth hydroxyl group. The monosulfates eluted at 22.1 min with m/z 253, and the EPI spectrum is shown in Figure 3E. Among the minor metabolites were double conjugates as seen with DMNQ. On the basis of published data for MNQ metabolism in vitro, one would have expected the formation of a glutathione conjugate and excretion into urine as a mercapturic acid (MA). The extracted ion chromatograms for m/z 332 or 334, corresponding to the naphthoquinone- or hydronaphthoquinone-derived MA of MNQ, did not show treatment related peaks. As this was unexpected, a MNQ-MA standard was synthesized following a published method (33). The NMR data (given in the Experimental Procedures section) were in accordance with this report. The EPI spectrum of the negative molecular ion m/z 332 showed the fragments 203 (loss of N-acetylcysteine), 185, 171, 159, and 145. A sensitive multiple reaction method (MRM) using the specific transition 332/203 was established. Using the method, it was possible to detect a small peak in the MNQ-treated mouse urine showing the same RT (25.3 min) and EPI spectrum as those of the standard. These data indicated the MNQ-MA to be a present but a very minor metabolite of MNQ. Following metabolism in the liver, secretion into bile is an important route of excretion of acidic conjugates. If GSH conjugation of MNQ represented a major metabolic pathway, GSH conjugates would be expected to be measurable in gall bladder extracts. No peaks were found that could have been MNQ-GSH conjugates using negative ionization, while the ions m/z 349 and m/z 253 of the glucuronide and the sulfate hydronaphthoquinone conjugates of MNQ were clearly detected. Previously, a low limit of detection of GSH conjugates in bile has been achieved by (i) using positive ionization and (ii) a neutral loss of 129 Da (glutamic acid) survey scan (40). We therefore determined the optimal MS/MS parameters in positive ion mode by infusion of the S-hexyl-GSH conjugate standard. Analysis of the extracts of gall bladders of mice treated with arachis oil (control) or MNQ showed GSH (m/z 308) and GSSG (m/z 613) with the corresponding EPI spectra (data not shown). However, no GSH conjugate of MNQ could be detected. These findings confirm the conclusions drawn from the urine data that GSH conjugation of MNQ only represents a very minor pathway in vivo. Evidence of Redox Activity. 8-oxodG was measured in the urine of animals treated with a single dose of 25 mg/kg DMNQ, and urine was collected over 4 h and frozen during collection. In order to generate equal variance, the data was transformed by taking the log2 prior to using a two sample unpaired two tail t-test to assess significance. A significant increase in excretion of 8-oxodG (p ) 0.008) was detected in the urine of the DMNQ treated animals but not those treated with MNQ (Figure 4A). However, the DMNQ treated animals also had a significant increase in urine specific gravity and in urinary creatinine (Figure 4B and C), which did not occur with MNQ (Figure 4B

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Table 1. Summary of the Kinetic Data for DMNQ and MNQ after a Single i.p. Dose of the Naphthoquinone at 25mg/kg in Arachis Oila analyte

DMNQ

MNQ

tissue

Cmax (ng/mL)

Cmax (nM)

Tmax (min)

AUClast (ng.min/mL)

AUCinf (ng.min/mL)

AUC Extrap (%)

half-life (min)

brain heart kidney liver plasma kidney liver plasma

1.35 2.74 18.7 9.75 8.97 5.91 3.31 11.1

6.2 12.6 85.7 44.7 41.1 34.3 19.2 64.5

10 10 10 10 15 30 20 10

51.6 159 757 287 246 171 119 498

57 197 789 300 250 172 135 507

9.4 19.4 4.0 4.2 1.9 1.0 11.7 1.8

70 126 122 51 18 15 24 20

a Cmax is the highest concentration of the naphthoquinone achieved at Tmax. The final column shows that the half-life of the naphthoquinone and the AUC is calculated in two ways, either the AUC to the last measured time point or after extrapolation to zero (AUCinf). The percentage difference between these figures is given in the penultimate column.

Figure 2. Urinary metabolites of DMNQ excreted within 4 h in the male C57B6/AJ mouse following an i.p. dose of DMNQ 25 mg/kg in arachis oil. Metabolites were detected by full scan LC-ESI-MS and characterized by enhanced product ion scan (EPI). Total ion chromatograms of DMNQ (A) and control arachis oil treatment (B); overlay of the extracted ion chromatograms for m/z 299, 395, 475, and 571 for DMNQ treatment (C) and control (D); (E to H) product ion spectra of the peaks shown in C and proposed structure. Intensity of the signal (cps) is shown on the vertical axis with the multiplication factor shown for each graph.

and C). These data indicated potential kidney damage occurring with DMNQ, although kidney damage was not confirmed by urine metabonomic analysis (data not shown). Therefore, it was not clear that increased 8-oxodG in response to DMNQ was a real response because the DMNQ animals had more concentrated urine. Measurement of GSSG in the liver showed a significant increase at 5 and 10 min time points for both DMNQ and MNQ (Figure 4D, insert), but not at the later time points (Figure 4D). There was no change in the concentration of liver GSH with either naphthoquinone with time (Figure 4E). For the other organs, no change in the concentrations of GSH or GSSG was detected. 1H NMR showed an increase in plasma

Figure 3. Urinary metabolites of MNQ excreted within 4 h in the male C57B6/AJ mouse following an i.p. dose of MNQ 25 mg/kg in arachis oil. Metabolites were detected by full scan LC-ESI-MS and characterized by enhanced product ion scan (EPI). Total ion chromatograms of MNQ (A) and control arachis oil treatment (B); overlay of the extracted ion chromatograms for m/z 349 and m/z 253 for MNQ treatment (C) and control (D) on the same scale. Product ion spectra (E,F) and proposed structures of the peaks shown in C. Intensity of signal (cps) is shown on the vertical axis with the multiplication factor shown for each graph.

tyrosine following DMNQ at both the level of the spectra (Figure 4F) and after normalization and quantization (Figure 4G). The hypothesis for redox action in the liver following DMNQ was further tested by real time PCR analysis of Sod1, Sod2, Cat, GPX1, Gpx2, HO1, and Gst3 by real time PCR at 0, 10, 30, 60, 120, and 300 min. No change in expression was detected (data not shown). Effect on Respiration of DMNQ Exposure. As there was a lack of global redox effect in the liver, we addressed the question of whether there was any effect on oxidative metabolism or other biochemistry in the liver using metabonomics. 1H NMR on liver 96 h after a single 25 mg/kg dose of DMNQ showed an increase in lactate (Figure 5A), decreased glucose (Figure 5B), and increased creatine (Figure 5C).

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Figure 4. Urinary 8-oxodG, specific gravity, and creatinine. (A) 8-oxodG was determined in the urine using mass spectrometric techniques as described in the Experimental Procedures. The urine for analysis was collected over 4 h from a single treatment with 25 mg/kg DMNQ i.p. The urine was collected onto dry ice. The data was transformed by taking log2 to ensure that the distribution was normal and that the Shapiro-Wilcoxan test was satisfied in this respect. Variance of the two samples was tested and found not to be statistically different, and therefore, the difference of the means was tested using a standard unpaired t-test with two tails, which returned a p-value of 0.008. (B) Specific gravity of the urine. (C) Creatinine content of the urine was measured using a LC-MS/MS technique. (D) GSSG in the liver with time (insert an expansion of the 5-20 min time points); open circles, Arachis oil; closed circles, DMNQ; closed triangles, MNQ. (E) GSH in the liver with time. Significance (*) from the arachis oil control was determined by Dunnett’s test. (F and G) Tyrosine in the plasma measured by 1H NMR 96 h after a single dose of 25 mg/kg DMNQ. (F) H1-NMR spectra for control (solid line) and DMNQ (broken line). (G) Data from F after normalization.

Transcriptomics. Transcriptomic profiling for DMNQ showed that the majority of gene expression changes occurred at 10 min, which corresponded to the Cmax for the compound. The majority of these differential gene expression had resolved to baseline levels by 30 min (Figure 5D). Pathway analysis showed monooxygenases to be upregulated (Table 2). For the downregulation, there was no one predominant pathway, and only those downregulated at 10 min of elog2 - 0.7 are shown in Table 3.

Discussion Quinones are of fundamental importance in biological processes that involve the carriage of electrons, such as respiration and photosynthesis. Many quinones are toxic primarily via two routes, chemical reactivity and redox cycling

Parry et al.

Figure 5. 1H NMR data for liver lactate (A), glucose (B), and creatine (C) 96 h after a single dose of DMNQ (25 mg/kg ip). In each case, the upper panel indicates the resonance for the control (solid line) and the DMNQ treated (broken line) sample. The lower panel in each case shows the relative normalized intensity. (D) Average transcriptomic data for up (black line) and down (gray line) differentially regulated genes at 10 min postdosing.

(26). Quinone groups are necessary for the pharmacology of some drugs such as doxorubicin. Naphthoquinone species have been used extensively in vitro to investigate the effects of redox cycling. However, in vitro systems for redox cycling investigations do not necessarily represent the in vivo situation because the oxygen tension in vitro is high compared with that in vivo, and xenobiotic metabolic capacity can be compromised. For these reasons, here we have investigated the in vivo pharmacokinetics, metabolism, and redox chemistry of the model naphthoquinones DMNQ and MNQ. After administration of either DMNQ or MNQ (25 mg/ kg) i.p. in an arachis oil solution, concentrations of the parent compound were measured in plasma, brain, heart, liver, and kidneys over a time period of 5 h. For both naphthoquinones, 25 mg/kg represented close to the maximum tolerated dose. DMNQ was widely distributed, reached the heart, and crossed the blood-brain barrier (Figure 1). This was in contrast to MNQ that was not so extensively distributed but found at a higher concentration in the plasma (Figure 1). Concentrations of MNQ in the liver were difficult to determine and variable, which may indicate that there was some retention on cellular macromolecules occurring. Measurement of liver DMNQ concentrations had much better reproducibility. For both DMNQ and MNQ metabolism occurred by two electron reduction to the H2DMNQ and H2MNQ, respectively, and subsequent conjugation of the hydroxyl groups with excretion into the urine (Figure 2 and 3). These data were in accordance with findings previously published for MNQ in vivo (41, 42). These data supported the hypothesis of two electron reduction as a detoxifying route and explain why nqo1 knockout mice

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Table 2. Gene Expression Changes in Liver Where There Was an Upregulation at 10 mina probeset ID 1417017_at 1449525_at 1419590_at 1422257_s_at 1443147_at 1438007_at 1450883_a_at 1443906_at 1418059_at 1417023_a_at 1418654_at 1448416_at 1453345_at 1428547_at 1419663_at 1428896_at 1419700_a_at a

gene bank accession Hugo gene symbol NM_007809 NM_008030 NM_010000 NM_009998 BB505010 BB432758 BB534670 BE686894 BC017134 NM_024406 NM_019545 NM_008597 AK014427 AV273591 BB542051 AK004179 NM_008935

Cyp17a1 Fmo3 Cyp2b9 Cyp2b10 AI851790 Cd36 Cd55 Eltd1 Fabp4 Hao3 Mgp Npal1 Nt5e Ogn Pdgfrl Prom1

description

10 min 30 min 60 min 300 min

cytochrome P450, family 17, subfamily a, polypeptide 1 flavin containing monooxygenase 3 cytochrome P450, family 2, subfamily b, polypeptide 9 cytochrome P450, family 2, subfamily b, polypeptide 10 acyl-CoA thioesterase 3 expressed sequence AI851790 CD36 antigen CD55 antigen EGF, latrophilin seven transmembrane domain containing 1 fatty acid binding protein 4, adipocyte hydroxyacid oxidase (glycolate oxidase) 3 matrix Gla protein NIPA-like domain containing 1 5′ nucleotidase, ecto Osteoglycin platelet-derived growth factor receptor-like prominin 1

0.6 0.6 0.7 0.7 0.6 0.5 0.5 0.5 0.6 0.6 0.8 0.5 0.7 0.6 0.5 0.6 0.5

-0.2 -0.1 0.3 0.0 0.4 -0.4 0.0 -0.4 -0.2 -0.2 -0.1 -0.1 -0.2 -0.3 0.0 -0.2 0.2

0.0 -0.2 -0.2 0.0 0.4 -0.4 -0.3 -0.3 -0.3 -0.4 -0.1 -0.4 0.1 -0.3 -0.3 -0.3 -0.3

-0.3 -0.5 0.1 0.3 -0.4 -0.5 -0.2 0.0 -0.1 -0.1 -0.9 -0.1 -0.3 -0.3 -0.2 -0.2 -0.3

Values shown are the log2 ratio of the DMNQ treated over the control. Genes in the oxidoreductase family are shown directly above the empty row.

Table 3. Gene Expression Changes Where Downregulation Occurred at 10 mina probeset ID

gene bank accession

Hugo gene symbol

description

10 min

30 min

60 min

300 min

1451204_at 1438558_x_at 1442656_at 1456388_at 1458701_at 1437218_at 1459601_at 1423493_a_at 1450252_at 1444037_at

BC016096 AV009267 BG066936 AV378604 BB550273 BM234360 AI648260 BB315728 NM_008262 BE981934

Scara5 Foxq1 D3Ertd343e Atp11a Prei4 Fn1 Snf1lk Nfix Onecut1 Lman1

scavenger receptor class A, member 5 (putative) forkhead box Q1 DNA segment, Chr 3, ERATO Doi 343, expressed ATPase, class VI, type 11A preimplantation protein 4 fibronectin 1 SNF1-like kinase nuclear factor I/X one cut domain, family member 1 lectin, mannose-binding, 1

-1.1 -1.0 -0.9 -0.9 -0.8 -0.8 -0.8 -0.8 -0.7 -0.7

-0.4 0.4 0.4 -0.1 0.1 0.4 0.0 0.2 0.2 0.1

0.3 0.0 0.1 0.1 0.0 0.0 0.0 0.3 0.0 0.3

0.2 -0.1 -0.1 0.6 0.2 0.0 0.0 0.1 -0.1 0.1

a Values shown are the log2 ratio of the DMNQ treated over the control, and only data where the differential expression at 10 minutes was -0.7 or greater is shown.

are more sensitive to naphthoquinone toxicity (15). In cells, however, nqo1 appears to be activating (16), indicating that under certain conditions there is the possibility for two electron cycling and damage via nqo1. To determine if there was generalized redox damage in the DMNQ or MNQ treated mice, we measured 8-oxodG in the urine and showed an apparent increase in 8-oxodG (Figure 4A) with DMNQ but not MNQ, which would suggest redox activity with DMNQ. However, for DMNQ, there was also an increase in the specific gravity of urine (Figure 4B), which when normalized to the 8-oxodG, canceled out the increase seen. Furthermore, increased creatinine in the urine following DMNQ (Figure 4C) suggested kidney damage, but this diagnosis was unsupported by the 1H NMR data that showed no increase in metabolites associated with nephrotoxicity (data not shown). Additionally, the increased specific gravity of the urine following DMNQ (Figure 4B) is not consistent with kidney damage. Measurement of GSSG in the liver indicated some redox activity at the early time points for both DMNQ and MNQ, but the effect was small (Figure 4D), and there was no change in GSH concentrations from 5 to 300 min (Figure 4E). In the plasma, increased tyrosine 96 h after the single dose of DMNQ suggested generic tissue damage (Figure 4F and G). As the dose of DMNQ used here was close to the maximum tolerated dose, it was not possible to administer more DMNQ to test the hypothesis whether redox damage occurred when the reduction routes to H2DMNQ and/ or conjugation were saturated. We used a metabonomic approach to investigate if there was any longer term biochemical change in the liver resulting from the DMNQ exposure and in particular related to mitochondrial respiration working on the hypothesis that

localized one or two electrons (from NADH oxidase or mitochondrial nqo1) could specifically damage mitochondria. These data showed increased lactate (Figure 5A), decreased glucose (Figure 5B), and increased creatine (Figure 5C) consistent with a change in the tissue status to a more anaerobic state, which could result from mitochondrial damage. Using a hypothesis that mitochondrial damage would lead to a specific alteration in gene expression associated with the glycolytic pathways, we undertook transcriptomic analysis. The data did not support the hypothesis but did show a maximal effect on a proportion of the global transcriptome at the Cmax of DMNQ in the liver consistent with a compound related effect. Some oxidoreductases were specifically induced (Table 2), while repressions of gene expression at 10 min did not appear to indicate a specific pathway response but rather one due to general cell stress (Table 3). These data indicate that the longer term damage from acute DMNQ exposure is likely to be subtle and at a physical level affecting the respiratory state of the hepatocyte and probably arises through mechanisms other than redox stress.

Conclusions We have established in this study the in vivo kinetics and metabolism of DMNQ and MNQ. DMNQ is extensively distributed and crosses the blood-brain barrier in contrast to MNQ, which is less extensively distributed to the tissues and not found outside of the liver and kidney. For both naphthoquinones, metabolism takes place primarily through two electron reduction with subsequent conjugation. Glutathione conjugation of MNQ is only a very minor route of metabolism. For both naphthoquinones, there is little liver general redox stress

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occurring over the longer time frame, though a small transient increase in GSSG occurs in the liver a few minutes after dosing. However, 96 h after a single dose of DMNQ glucose is decreased in the liver accompanied by increased lactate and creatine, suggesting a change to a less aerobic metabolic status potentially indicative of a sustained mitochondrial damage or decreased tissue oxygen tension arising through mechanisms other than redox stress. Acknowledgment. We thank Petra R. Baus for synthesis of the DMNQ, Rachel Cavill for assistance with NMR data processing, Shu-Dong Zhang for transcriptomics data processing scripts, Tim Marczylo for mass spectrometry advice, and Professor G. M. Cohen for helpful comments on the manuscript. T.J.A. and P.H.C. are funded by the European Union carcinoGENOMICS FP6 project (Contract No: PL037712).

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