Chem. Res. Toxicol. 2006, 19, 683-691
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Bioactivation of Carboxylic Acid Compounds by UDP-Glucuronosyltransferases to DNA-Damaging Intermediates: Role of Glycoxidation and Oxidative Stress in Genotoxicity Benedetta C. Sallustio,*,†,‡ Yvette C. DeGraaf,†,‡ Josephine S. Weekley,† and Philip C. Burcham§ Department of Cardiology and Clinical Pharmacology, The Queen Elizabeth Hospital, WoodVille 5011, Australia, Discipline of Pharmacology, School of Medical Sciences, The UniVersity of Adelaide, Adelaide 5005, Australia, and Pharmacology Unit, School of Medicine and Pharmacology, The UniVersity of Western Australia, Nedlands 6009, Australia ReceiVed February 1, 2006
Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin . gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 KruskalWallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51× and 2.57× control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10× more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidationdependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes. Introduction (UGTs1)
UDP-glucuronosyltransferases metabolize a wide range of endogenous and xenobiotic substrates, typically forming pharmacologically inactive, polar glucuronide conjugates. Because these are readily excreted into urine or bile, glucuronidation is conventionally viewed as facilitating the toxicological deactivation and excretion of xenobiotics. However, this paradigm is not universally applicable because the glucuronidation of carboxylic acid compounds in particular can generate reactive acyl glucuronides that may contribute to drug-induced toxicity (1, 2). The reactivity of acyl glucuronides largely reflects their electrophilic ester function. In one major class of reactions, * To whom correspondence should be addressed. Tel.: 61 8 8222 6510. Fax: 61 8 8222 6033. E-mail:
[email protected]. † The Queen Elizabeth Hospital. ‡ The University of Adelaide. § The University of Western Australia. 1 Abbreviations: BHT, butylated hydroxytoluene; CFAG, clofibric acid glucuronide; CuDIPS, copper bis-3,5-diisopropylsalicylate; DFOM, desferrioxamine mesylate; LC50, concentration causing 50% cell death; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bomide; UDPGA, uridine 5′-diphosphoglucuronic acid; UGT, UDP-glucuronosyltransferase.
commonly termed nonenzymatic transacylation reactions, the ester is attacked by nucleophilic centers within proteins, generating adducts in which the aglycone is covalently bound to the protein (3, 4). In a second class of reactions, the ester participates in intramolecular reactions with hydroxyl groups on the glucuronic acid moiety, facilitating migration of the aglycone from the 1-O-β position to the C-2, -3, or -4 positions on the sugar (5, 6). This rearrangement re-exposes the hemiacetal function of glucuronic acid, allowing a series of classic reactions typical of monosaccharides, including anomerization (5, 6) and protein glycation (3, 4). These various reactions of acyl glucuronides lead to modification of a range of proteins in plasma and tissues such as liver and kidney (7-9). Such damage is implicated in hepatotoxicity and hypersensitivity reactions in patients receiving a number of COOH-containing drugs (1, 2). Although the reactivity of acyl glucuronides with proteins is quite well understood, the possibility that these substances might also damage DNA has received less attention. The first suggestion of this prospect emerged from early work in our laboratory, in which direct exposure of single-stranded bacteriophage DNA to the glucuronides of clofibric acid and gemfibrozil
10.1021/tx060022k CCC: $33.50 © 2006 American Chemical Society Published on Web 04/26/2006
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Sallustio et al.
Figure 1. Potential mechanisms for the induction of DNA damage and strand-nicking by glucuronide metabolites formed from COOH-containing drugs. See the Introduction for the description of each pathway.
produced large declines in transfection efficiency in E. coli (10). In contrast, exposing the DNA to parent aglycones had no effect on transfection efficiencies (10). In the same study, incubation of double-stranded pSP189 plasmid DNA with either clofibric acid glucuronide or gemfibrozil glucuronide produced extensive strand-nicking as detected by agarose gel electrophoresis (10). In more recent work, we demonstrated that clofibric acid undergoes glucuronidation-dependent bioactivation to DNAdamaging species in cultured mouse hepatocytes (11). The genotoxicity occurred at clinically relevant drug concentrations and, as in our earlier in vitro experiments, involved extensive DNA strand-nicking (11). These findings have established that glucuronidation can convert chemically innocuous acyl compounds into DNA-damaging intermediates. In a broader sense, they also suggest that because routine genotoxicity screening assays commonly employ cell lines with limited glucuronidation capacity, their ability to detect DNA damage by acyl glucuronides may be inadequate (10). At present, the chemical basis for the induction of DNA damage by acyl glucuronides is unknown. However, a number of potential mechanisms can be envisaged on the basis of the known reactivity of acyl glucuronides with proteins and of monosaccharides with proteins (12) and DNA (13, 14). As with the reaction with proteins, one route to DNA damage might involve transacylation of nucleophiles within the pyrimidines and purines present in DNA, an outcome designated Mechanism 1 in Figure 1. Alternatively, as highlighted by Mechanism 2, the reactive aldehyde possessed by open chain rearrangement isomers might undergo glycation reactions with primary amines in DNA, forming imines and Amadori rearrangement products (Figure 1). If the latter exhibit reactivity similar to monosaccharide-derived DNA adducts, in the presence of O2 and transition metals they could be expected to form advanced glycation endproducts and DNA-damaging dicarbonyls as highlighted in
Mechanism 3 (12, 14). Finally, assuming that acyl glucuronides possess the same capacity for autoxidative reactions as exhibited by glucose, they may also induce oxidative DNA damage (Mechanism 4) (12, 14). Such autoxidation reactions are accelerated by transition metals and appear to involve redox-cycling by the open chain R-hydroxy-aldehyde forms of sugars (15). Because acyl glucuronide rearrangement isomers could also conceivably exist as R-hydroxy-aldehydes, they may also promote oxygen radical formation by this route (Mechanism 4). The aim of this study was to clarify the chemical and biochemical mechanisms underlying acyl glucuronide-mediated DNA damage. Furthermore, because our previous observations have been made with acyl glucuronides formed from two fibrate hypolipidemics, we sought to determine whether genotoxicity is induced by glucuronides generated by a broader range of drugs. Having selected a series of COOH-containing drugs for which glucuronidation is known to be a major in vivo fate, we then assessed their ability to induce DNA damage in mouse hepatocytes. Because it is well suited to detect the DNA-nicking that is characteristic of acyl glucuronide genotoxicity, the singlecell gel electrophoresis assay (Comet assay) was used to detect DNA damage in exposed cells (11). The role of UGT-dependent bioactivation in the genotoxicity of a subset of active aglycones was confirmed using the glucuronidation inhibitor borneol. Finally, the mechanism(s) contributing to DNA damage were evaluated using reagents that interfere with potential genotoxic mechanisms, including aminoguanidine to interfere with glycation/glycoxidation chemistry, and several compounds that modulate cellular responses to oxidative stress, including the lipid peroxidation inhibitors Trolox and butylated hydroxytoluene (BHT), the superoxide dismutase mimetic copper bis-3,5diisopropylsalicylate (CuDIPS), the metal chelating agent desferrioxamine mesylate (DFOM), and the hydroxyl radical scavenger mannitol (16).
UGT-Dependent DNA Damage by Carboxylic Acid Drugs
Experimental Procedures Chemicals. Clofibric acid, bezafibrate, gemfibrozil, probenecid, diclofenac, ketoprofen, uridine 5′-diphosphoglucuronic acid (UDPGA), glucuronic acid, collagenase (Type IV), aminoguanidine, l-phenylalanine, D-mannitol, and o-, m-, and p-tyrosine isomers were purchased from Sigma Chemical Co. (Castle Hill, NSW, Australia). Nafenopin and DFOM were obtained from Ciba-Geigy (Pendle Hill, NSW, Australia), benoxaprofen from Lilly (Indianapolis, IN), (-)borneol and Trolox from Aldrich (Castle Hill, NSW, Australia), BHT from ICN Biochemicals (Cleveland, OH), and CuDIPS from Calbiochem (EMD Biosciences, San Diego, CA). Clofibric acid glucuronide (CFAG) was biosynthesized using the method described for the biosynthesis of gemfibrozil glucuronide (17), with minor modifications, and stored at -20 °C until use. The identity and purity of CFAG was determined by mass spectral analysis as well as by generation of parent clofibric acid following β-glucuronidase or NaOH hydrolyses. RPMI-1640 media was purchased from Gibco BRL Life Technologies (Cergy Pontoise, France). SYBR Green I was purchased from Molecular Probes Inc. (Eugene, OR). Doublestranded M13mp19 DNA was purchased from Pharmacia (Sydney, NSW, Australia), while double-stranded pSP189 DNA was a generous gift from Prof. L. J. Marnett (Vanderbilt University, TN). All other reagents were of analytical grade or better and were obtained from standard commercial suppliers. DNA Damage in Mouse Hepatocytes. 1. Hepatocyte Cultures. Adult male albino Swiss mice (30-35 g) were obtained from the Adelaide University Animal Breeding Facility (Waite Institute, Adelaide, SA). Hepatocytes were prepared by collagenase digestion of mouse liver (18). Cells were washed three times in CaCl2supplemented Krebs Henseleit solution, resuspended in RPMI-1640 media supplemented with 0.2% BSA, 0.3% L-glutamine, gentamicin (50 µg/mL), and layered onto 35 mm collagen-coated culture dishes (1 × 106 cells/dish). Following incubation for 2-3 h to allow attachment (37 °C, 5% CO2), nonviable cells were removed by washing monolayers three times with phosphate-buffered saline with subsequent addition of fresh media (without BSA). Carboxylic acid compounds were added to the culture medium at noncytotoxic concentrations ranging from 10 to 1000 µM, and cells were incubated for a further 18 h (37 °C, 5% CO2). Solutions of clofibric acid, nafenopin, bezafibrate, probenecid, and benoxaprofen were prepared in 0.1 M NaOH. Gemfibrozil solutions were prepared in 50% (v/v) methanol, while ketoprofen was prepared in 50% (v/v) ethanol. The role of UGT-mediated bioactivation was investigated by pretreating hepatocytes with borneol (1 mM) for 30 min prior to the addition of carboxylic acids. Stock solutions of borneol were prepared in 40% (v/v) ethanol. Diclofenac was prepared in MilliQ water. Final concentrations of NaOH, ethanol, or methanol in the culture medium did not exceed 2.5 mM, 1.0%, and 1.0%, respectively. Negative controls were prepared by culturing cells with NaOH (2.5 mM) or ethanol (1.0%) or methanol (1.0%) alone. 2. Cytotoxicity Assay. The cytotoxicity of all test compounds added to hepatocyte cultures was assessed using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bomide (MTT) viability assay using 96-well microtiter plates layered with 3 × 104 cells/well in 100 µL of RPMI-1640 culture medium (19). Briefly, following a 2-3 h attachment period, the culture medium was discarded and hepatocytes were overlaid with fresh medium containing increasing concentrations of clofibric acid (25-5000 µM), nafenopin (1-1000 µM), gemfibrozil (500-10 000 µM), bezafibrate (10-10 000 µM), probenecid (10-10 000 µM), benoxaprofen (1-1000 µM), diclofenac (1-1000 µM), ketoprofen (1-1000 µM), or borneol (10-2500 µM). Plates were incubated for 18 h (37 °C, 5% CO2), and subsequently 100 µL of MTT solution (0.5 mg/mL in RPMI-1640 medium (BSA free)) was added to each well. Plates were incubated for a further 2-3 h, the medium was removed, and cells were lysed by the addition of 100 µL of DMSO to each well. The optical density of the cell contents was then measured at an absorbance of 570 nm using a microplate reader (Polarstar Galaxy, BMG Labtechnologies GmbH, Germany). Concentrations causing 50% cell death (LC50) were estimated from the
Chem. Res. Toxicol., Vol. 19, No. 5, 2006 685 concentration-response curves using nonlinear regression (GraphPad Software Inc., San Diego, CA). 3. Comet Assay. Prior to determining DNA damage in cultured hepatocytes, cell viability was assessed at the end of each incubation via trypan blue exclusion, and only preparations with >75% of cells excluding the dye were used for Comet assays. The Comet assay was performed using the method of Singh et al. (20) adapted for mouse hepatocytes as we have previously described (11, 21). Slides were photographed at 200× magnification under a fluorescence microscope (Olympus BX 50, Tokyo, Japan) equipped with a 450-490 nm reflector and a 530-550 nm barrier filter set. Between 50 and 100 images of randomly selected nuclei from nonoverlapping fields on duplicate slides were acquired using a Peltier cooled CCD SPOT RT video camera (Diagnostic Instruments, MI). Comet tail moments were measured using public domain “NIH Scion Image” software (Scion Corporation, MD) and a Comet analysis macro kindly provided by Prof. Geller (Department of Pharmacology, Robert Wood Johnson Medical school, NJ). Comet moment was determined for typically 100-250 cells per treatment, from 2-4 individual mouse hepatocyte preparations. Mechanism of DNA Damage. 1. In Vitro DNA StrandNicking. Double-stranded plasmid DNA (5 µg) was incubated for 4-6 days at 37 °C in 50 µL volumes of filter-sterilized sodium phosphate buffer (0.1 M, pH 7.0) in the presence of 0-20 mM concentrations of CFAG or 0-50 mM glucuronic acid. At the end of the reaction period, 0.8-1.0 µg of DNA was loaded onto a 0.8% agarose gel containing 0.5 µg/mL ethidium bromide. The DNA was then resolved for 2 h at 70 V after which it was visualized under UV light. Supercoiled (Form I), relaxed (Form II), and linearized (Form III) DNA were quantitated via densitometry using a Kodak DC40 digital camera and Digital Science electrophoresis analysis software (22). In a separate series of experiments, double-stranded DNA was incubated with the hydroxyl radical scavenger mannitol (100-500 mM), aminoguanidine (10-100 mM), and either CFAG (5 mM) or glucuronic acid (200 mM). 2. In Vitro Hydroxyl Radical Formation. The hydroxyl radicalgenerating activity of CFAG was investigated using the phenylalanine hydroxylation assay, which measures several positional isomers formed via hydroxyl radical attack on phenylalanine (i.e., ortho-, meta-, and para-tyrosine) (23). Briefly, a 4 mM solution of phenylalanine was prepared in 50 mM filter-sterilized sodium phosphate buffer (pH 7.0). CFAG was added to obtain final concentrations of 1, 5, or 10 mM, and then the tubes were sealed and placed in a 37 °C incubator for 5 days. For comparative purposes, tubes were also prepared that contained glucuronic acid at final concentrations of 10, 25, 50, or 75 mM. The effects of mannitol (500 mM) were also examined in incubations with 5 mM CFAG or 50 mM glucuronic acid. At the end of the reaction, the levels of tyrosine isomers were measured in 45 µL aliquots of reaction mixture via reverse phase HPLC using a published procedure (23). Data from the UV detector were acquired and analyzed using Delta Junior chromatography software (Digital Solutions Pty. Ltd., Margate, Qld., Australia). Levels of ortho-, meta-, and para-tyrosines were determined from a standard curve constructed over a 0.1-1.0 nmol range using authentic compounds dissolved in phosphate buffer. 3. Effect of Aminoguanidine and Antioxidants on DNA Damage in Mouse Hepatocytes. Mouse hepatocytes were incubated, as described above, with clofibric acid (100 µM) following pretreatment for 30 min with aminoguanidine (10 mM), Trolox (1 mM), BHT (10 µM), CuDIPS (5 µM), or DFOM (5 mM). DNA damage was assessed using the Comet assay, as described above. Neither aminoguanidine nor the antioxidant compounds produced any significant cytotoxicity or DNA damage when incubated alone with mouse hepatocytes (data not shown). 4. Statistical Analyses. All Comet data were analyzed using the nonparametric Kruskall-Wallis test with Dunn’s post-hoc analysis (GraphPad Software). DNA strand-nicking data were analyzed by one-way or two-way analysis of variance (GraphPad Software) with Dunnett’s or Bonferroni’s post-hoc analysis, respectively. P values diclofenac, benoxaprofen, nafenopin . gemfibrozil, probenecid > bezafibrate > clofibric acid. To avoid induction of cytotoxicity by the NSAIDS, initial screening for DNA-damaging potency using the Comet assay (Table 1) was carried out with a single concentration (100 µM) of each carboxylic acid, which was below the LC50 for all compounds. At this drug concentration, neither diclofenac, ketoprofen, gemfibrozil, nor bezafibrate induced statistically significant levels of DNA damage (Table 1). However, Comet tail moments were strongly induced by clofibric acid and probenecid (Table 1). For both compounds, the 100 µM concentration at which they exhibited strong genotoxicity was considerably below their respective LC50 (Table 1). Nafenopin and benoxaprofen also significantly increased tail moments, although the effect was not as strong as that for clofibric acid and probenecid (Table 1). Collectively, the findings that probenecid (a uricosuric benzoic acid derivative), benoxaprofen (a 2-arylpropionoic acid NSAID), as well as the fibric acid hypolipidemics clofibric acid and nafenopin all exhibit DNA-damaging activity in glucuronidation-proficient mouse hepatocytes suggest glucuronides formed from a diverse range of drugs can induce DNA damage. The concentration-response for the induction of DNA damage was then assessed for the two compounds that exhibited greatest activity in the screening experiment, clofibric acid and probenecid (Figure 2). Both drugs produced concentrationdependent increases in Comet tail moment, with 10 µM concentrations of clofibric acid and probenecid increasing median tail moment by 1.40× (P < 0.001) and 1.63× (P < 0.05) relative to control, respectively (Figure 2). A maximum increase in median Comet tail moment of 4.10× (P < 0.001) control was observed in nuclei from hepatocytes incubated with 1000 µM clofibric acid, whereas the response to probenecid
Figure 2. Concentration-dependent induction of DNA damage by (A) 0-1.00 mM clofibric acid and (B) 0-1.00 mM probenecid in primary mouse hepatocyte cultures. Results are shown normalized by the median Comet tail moment in control (NaOH) incubations. The box and whiskers plots indicate 0, 25, 50, 75, and 100 percentiles (*p < 0.05, ***p < 0.001, cf., NaOH control).
appeared to plateau at a drug concentration of 50 µM with a maximum increase of 2.40× (P < 0.001) control. Consistent with a role for glucuronides in the induction of DNA damage, pretreatment of the cells with the glucuronidation inhibitor borneol completely abolished the genotoxocity of 500 µM concentrations of both drugs (Figure 3). Mechanisms of DNA Damage - In Vitro Strand-Nicking and Hydroxyl Radical Formation. We then explored the possible mechanisms underlying induction of DNA damage by the most genotoxic glucuronide-forming acyl compound we have identified to date, clofibric acid. First, to establish whether acyl glucuronides might induce DNA damage by generating free radicals (Mechanism 4 in Figure 1), we examined the effect of the hydroxyl radical scavenger mannitol on DNA-nicking by CFAG in a plasmid relaxation assay. In keeping with our prior observations (10), both CFAG and glucuronic acid caused concentration-dependent DNA-nicking after an extended incubation at neutral pH and 37 °C (Figure 4), evidenced as a loss of supercoiled Form I DNA and a corresponding increase in Form II DNA (Figure 4A and B). As in our preceding
UGT-Dependent DNA Damage by Carboxylic Acid Drugs
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Figure 3. Inhibitory effect of borneol on the DNA damage induced by clofibric acid (CFA) and probenecid (PB) in mouse hepatocytes. Results are shown normalized by the median Comet tail moment in control incubations. The box and whiskers plots indicate 0, 25, 50, 75, and 100 percentiles (*p < 0.05, **p < 0.01, cf., NaOH control; ##p < 0.01, cf., probenecid; and ###p
