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Jan 2, 2012 - N7-Glycidamide-Guanine DNA Adduct Formation by Orally Ingested. Acrylamide in Rats: A Dose−Response Study Encompassing Human...
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N7-Glycidamide-Guanine DNA Adduct Formation by Orally Ingested Acrylamide in Rats: A Dose−Response Study Encompassing Human Diet-Related Exposure Levels Nico Watzek,† Nadine Böhm,† Julia Feld,† Denise Scherbl,† Franz Berger,† Karl Heinz Merz,† Alfonso Lampen,‡ Thorsten Reemtsma,‡ Steven R. Tannenbaum,§ Paul L. Skipper,§ Matthias Baum,† Elke Richling,† and Gerhard Eisenbrand*,† †

Department of Chemistry, Division of Food Chemistry and Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Strasse 52, 67663 Kaiserslautern, Germany ‡ Federal Institute for Risk Assessment, 14195 Berlin, Germany § Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ABSTRACT: Acrylamide (AA) is formed during the heating of food and is classified as a genotoxic carcinogen. The margin of exposure (MOE), representing the distance between the bench mark dose associated with 10% tumor incidence in rats and the estimated average human exposure, is considered to be of concern. After ingestion, AA is converted by P450 into the genotoxic epoxide glycidamide (GA). GA forms DNA adducts, primarily at N7 of guanine (N7-GA-Gua). We performed a dose−response study with AA in female Sprague−Dawley (SD) rats. AA was given orally in a single dosage of 0.1− 10 000 μg/kg bw. The formation of urinary mercapturic acids and of N7-GA-Gua DNA adducts in liver, kidney, and lung was measured 16 h after application. A mean of 37.0 ± 11.5% of a given AA dose was found as mercapturic acids (MAs) in urine. MA excretion in urine of untreated controls indicated some background exposure from endogenous AA. N7-GA-Gua adduct formation was not detectable in any organ tested at 0.1 μg AA/kg bw. At a dose of 1 μg/kg bw, adducts were found in kidney (around 1 adduct/108 nucleotides) and lung (below 1 adduct/108 nucleotides) but not in liver. At 10, respectively, 100 μg/kg bw, adducts were found in all three organs, at levels close to those found at 1 μg AA/kg, covering a range of about 1−2 adducts/ 108 nucleotides. As compared to DNA adduct levels from electrophilic genotoxic agents of various origin found in human tissues, N7-GA-Gua adduct levels within the dose range of 0.1−100 μg AA/kg bw were at the low end of this human background. We propose to take the background level of DNA lesions in humans more into consideration when doing risk assessment of food-borne genotoxic carcinogens.

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INTRODUCTION Acrylamide (AA) is an α,β-unsaturated aliphatic carbonyl compound generated during the heating of food. AA has been found to be formed predominantly from asparagine in the presence of reducing sugars during the Maillard reaction via Strecker type degradation.1 AA is present in many heat-treated foods, such as fried potatoes, including french fries (689−693 μg/kg), soft bread (27−37 μg/kg), and roasted coffee (225− 231 μg/kg), which are reported to be the major contributors to overall adult AA exposure.2 AA is also a component of tobacco smoke with contents in mainstream cigarette smoke reported to be around 2.3 μg per cigarette.3 In Western countries, the daily intake of AA by adults has been estimated by JECFA to range between 1 and 4 μg/kg body weight (bw).4 Margins of exposure (MOEs) of 78−310 for the induction of mammary tumors in rats have been calculated, reflecting the distance between the benchmark dose lower confidence limit of the benchmark dose associated with a modeled 10% mammary tumor incidence in rats (BMDL10, 0.31 mg AA/kg bw) and the estimated range of human exposures.4 Other calculations arrived at MOE values of 40−160.5 As a guidance, MOEs substantially © 2012 American Chemical Society

below the value of 10 000 are regarded to be of concern with respect to human health.6 Preventive measures, such as those described in the CIAA toolbox to reduce dietary exposure, only have met with limited success yet.2 AA is readily absorbed and widely distributed to tissues. It can be transferred into the breast milk and has been reported to cross the human placenta.7,8 AA itself is not reactive toward DNA under physiological conditions but avidly binds to other cellular nucleophiles, especially those present in structural and functional proteins through its Michael type reactivity. It has been suggested that this reactivity may contribute to the biological activity of AA by potentially interfering with hormonal or other regulation systems, eventually leading to malignant transformation by an epigenetic mode of action.5 In the liver, AA undergoes biotransformation into the genotoxic epoxide glycidamide (GA), primarily mediated by cytochrome P450 2E1.9 Both AA and GA have the capability to bind covalently to nucleophilic sites of biological macromolecules. Received: October 13, 2011 Published: January 2, 2012 381

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Scheme 1. Metabolic Pathways of AA in the Rat (Adapted and Modified According to Ref 25)

about 0.4.19 Moreover, whereas urinary MA excretion was practically complete 24 h after each ingestion, the simultaneously measured AA adduct at the N-terminal valine of Hb (AA-Hb) increased in direct correlation with the cumulatively ingested AA dose. Of note, in contrast to the steady increase of the AA-Hb adduct, the formation of GA-Hb adducts was not found significantly increased above base level, indicating that at this dose level, GSH coupling effectively scavenged the GA generated during the first pass in the rat liver.19 We here report results of a dose−response study in rats encompassing a low exposure range of 0.1−100 μg/kg bw. In a satellite experiment, the dose range was extended to higher exposure levels, up to 10 000 μg/kg bw. Analytical determination of AA related MAs as well as DNA adducts was achieved by HPLC-ESI-MS/MS, after application of a single dose of 14Clabeled AA for the dosage range 0.1−100 μg/kg bw and of unlabeled AA for the higher dose range.

Especially, thiol and amino groups of glutathione (GSH), hemoglobin (Hb), and other proteins appear as major targets for both compounds, whereas nucleophilic nitrogens of DNA purine bases represent targets especially for the genotoxic metabolite GA (scheme 1). AA has not been found to be mutagenic or genotoxic without metabolic activation to GA at biologically relevant concentrations.10−13 It has been classified by the International Agency for Research on Cancer (IARC) as a probable human carcinogen (class 2A). In a recent meta-analysis, it was concluded that the overall evidence from epidemiological studies does not suggest an increased risk for most types of cancer associated with dietary or occupational exposure toward AA, with the potential exception of renal cell cancer for which further confirmation is required.14 In adult mice, the formation of glycidamide-guanine DNA (N7-GA-Gua) adducts as the major type of DNA lesion and of glycidamide-adenosine DNA (N3-GA-Ade) adducts as minor DNA lesion (2 orders of magnitude lower) has been reported in liver, lung, and kidney after treatment with 50 mg AA/kg bw.15 The formation of N7-GA-Gua adducts (1 adduct/108 nucleotides) in liver of Fischer 344 rats given single oral doses of 100 μg/kg bw in drinking water (gavage) or via diet has previously been reported.16 In mammalian organisms, AA and GA are conjugated to GSH. The resulting GSH-thioethers are further biotransformed into the corresponding mercapturic acids (MA): N-acetyl-S(2-carbamoylethyl)cysteine (AAMA), N-acetyl-S-(1-carbamoyl2-hydroxyethyl)cysteine, and N-acetyl-S-(2-carbamoyl2-hydroxyethyl)cysteine (GAMA). In primary rat hepatocytes, AA-GSH adducts became detectable already after a short incubation time (30 min) with AA (2 μM), whereas GA-GSH adducts appeared much later (16 h) at a time when the metabolite GA became detectable as well, being subject to fast GSH scavenging. The data allowed us to conclude that GSH coupling in primary rat hepatocytes occurred faster than epoxidation.17 MAs excreted in the urine have been used as preferred biomarkers of AA exposure.18−22 After incubation of primary rat hepatocytes with AA, MA formation has likewise been observed, with a GAMA/AAMA ratio of roughly 0.3−0.6, depending on the AA concentration.17 In a subacute rat feeding experiment, AA was daily applied in food at a dosage of 100 μg/kg bw for up to 9 days. MA excretion reached a mean of about 51 ± 12% of the applied dose with a GAMA/AAMA ratio of

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EXPERIMENTAL PROCEDURES

Chemicals. Chemicals were of analytical grade. 1-14C-AA (25 mCi/ mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). The mercapturic acids AAMA and GAMA and the D3-labeled analogues [N-acetyl-S-(2-carbamoyl-D3-ethyl)cysteine and N-acetyl-S-(1-carbamoyl-2-hydroxy-D3-ethyl)cysteine] were available from former studies.19 D3-AA was obtained from Toronto Research Chemicals (North York, Canada). Isotope-labeled 15N5-guanosine for the synthesis of the DNA adduct was purchased from Euriso-Top (Saarbrücken, Germany). Solid-phase extraction (SPE) columns (ENV+, 100 mg, 10 mL, and ENV+, 500 mg, 6 mL) were supplied by Biotage (Uppsala, Sweden). RNase A and Proteinase K for DNA isolation and preparation were purchased from Qiagen (Hilden, Germany). Water was freshly bidistilled in an all-glass still. Preparation of N7-GA-Gua and N7-GA-15N5-Gua. N7-GA-Gua and N7-GA-15N5-Gua were synthesized according to the literature with modifications.23,24 Briefly, guanosine (200 mg, 0.7 mmol) was dissolved in 5 mL of glacial acetic acid at 75 °C under stirring. An approximately 10-fold molar excess of GA (609 mg, 7.0 mmol) was added, and the mixture was stirred and heated until the reaction was complete (∼3 h). Thereafter, the acid was evaporated (oil pump vacuum), and the oily residue was hydrolyzed in 5 mL of hydrochloric acid (1 N) by refluxing for 2 h. After neutralization with 4 N sodium hydroxide solution, the adduct precipitated. After filtration, products were characterized by mass spectrometry (MS) (turbo ionspray; direct infusion and positive ionization mode) and 1H NMR. Purity was ascertained by 1H NMR to be >95% for all reference substances. 382

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Mass spectrum [ESI+, MS2, (m/z)]: N7-GA-Gua: 239.2 (M + H)+, 152.2, 135.1, 110.1, and 58.1. N7-GA-15N5-Gua: 244.1 (M + H)+, 157.1, 139.1, 113.1, and 58.1. 1H NMR [400 MHz, 273K (ppm)]: N7GA-Gua: 4.12 (dd, 1 H, 2JHH = 13.3 Hz, 3JHH = 9.0 Hz, CH2); 4.23 (m, 1 H, CH); 4.54 (dd, 1 H, 2JHH = 13.4 Hz, 3JHH = 3.3 Hz, CH2); 5.93 (d, 1 H, 3JHH = 6.3 Hz, OH); 6.13 (s, 2 H, NH2); 7.31 (d, 2 H, 4JHH = 10.6 Hz, NH2); 7.77 (s, 1 H, H8); 10.86 (s, 1 H, NH). N7-GA-15N5-Gua: 4.01 (m, 1 H, CH); 4.03 (m, 1 H, CH2); 4.55 (m, 1 H, CH2); 5.95 (d, 1 H, 3 JHH = 4.7 Hz, OH); 6.18 (d, 2 H, 1JNH = 88.0 Hz, NH2); 7.31 (d, 2 H, 4 JHH = 14.5 Hz, NH2); 7.77 (m, 1 H, H8); 9.47 (s, 1 H, NH) Food Preparation. To keep background exposure of the animals to dietary AA as low as possible, an experimental animal diet was developed. The diet was prepared by mixing glucose (175 g/kg), corn starch (175 g/kg), wheat flour (200 g/kg), boiled potatoes (200 g/kg), lactose-reduced curd cheese (175 g/kg), tofu (50 g/kg), soy protein (10 g/kg), corn oil (15 g/kg), and a vitamin mix (6 g/kg, Multi Vital, Trixie GmbH, Tarp, Germany) and was allowed to dry at room temperature. This special diet was prepared in batches of 10−20 kg and was portioned into 125 g aliquots to be stored at −20 °C in hermetically sealed plastic bags. Each day, the rats received freshly thawed samples of this diet. Feeding of rats with the special food started two weeks prior to administration (“washout” period). A medium weight gain of 19 ± 5 g/week (10 ± 3% of bw) was observed. The AA content in this diet, determined as described below, was below the limit of detection (LOD) (“AA-free”). Study Design. Female SD rats (n = 54) were obtained from Charles River at the age of 50 days (weight about 150−170 g). At the day of the arrival, the rats were put on the experimental (AA-free) diet for two weeks, until the start of the dose−response experiments. Low Dose Experiments. Rats were randomized into five groups of eight animals each (four dosage groups and one control) and allowed to adapt to housing conditions in the radionuclide lab (day− night rhythm; 20−22 °C). Prior to the start of the experiment, rats were trained (four times for 8 h each) to accommodate to the conditions of staying in all-glass metabolism cages. To achieve doses of 0.1, 1, 10, and 100 μg 1-14C-AA/kg bw, 1-14CAA dosing solutions were administered (approximately 0.5 mL, dissolved in water) by gavage under moderate isoflurane anesthesia (about 60 s). Untreated controls were isoflurane treated in the same way. Animals were kept in metabolism cages at a constant air flow of 350 L/h, exhaled air being passed through two sequential traps containing ethanolamine and barium hydroxide solution to trap exhaled CO2. Animals had free access to drinking water and food; urine and feces were collected separately. After 16 h, animals were sacrificed by cervical dislocation, and organs were isolated. High Dose Experiments. In high dose (satellite) experiments preceding the low dose examinations, two or three animals per group received doses of 0.5, 1, 3, 6, or 10 mg non-labeled AA/kg bw by

gavage in aqueous solutions. The time dependency of the N7-GA-Gua adduct formation was examined via single dose treatment of three animals with 10 mg AA/kg bw by gavage. The animals were held and examined as described before. AA Determination. Experimental diet samples (10−15 g) were thawed, suspended in 50 mL of water, and spiked with 10 μL of D3-AA (50 μg/mL). Suspensions were stirred for 30 min and centrifuged (two times, 3000g; 30 min; 22 °C). Supernatants were applied to SPE columns (Isolute ENV+, 500 mg; 6 mL), preconditioned with methanol (4 mL) and water (2 × 2 mL). After they were washed with water (4 mL), analytes were eluted with 60% (v/v) methanol in water (2.5 mL), and the eluate was concentrated to approximately 0.5 mL under nitrogen stream. Aliquots (50 μL) of this solution were injected into the high-performance liquid chromatography electrospray ionization tandem mass spectrometry system (HPLC-ESI-MS/MS). AA contents were quantified by isotope dilution using D3-AA as stable isotope-labeled standard. The determination was performed using a system (Jasco GmbH, Groβ-Umstadt, Germany) consisting of two pumps (PU-2080), a degasser (DG-2080-53), and an autosampler (AS-2057) coupled to a SCIEX API 3200 triple quadrupole mass spectrometer (Applied Biosystems, Darmstadt, Germany). The latter was equipped with an ESI source and a valco valve, operating in the multiple reaction monitoring mode (MRM) with positive ESI (ESI+). HPLC separations were run on a reversed phase HPLC column [Phenomenex Luna C8 (2); 150 mm × 4.6 mm; 3 μm particle size] equipped with a guard column [Luna C8 (2); 4 mm × 3 mm; flow rate, 0.3 mL/min] using a gradient from 1 to 20% methanol over 20 min. The eluate from 6 to 17 min was directed into the electrospray interface. Source-dependent MS parameters were as follows: needle voltage, 5.5 kV; nitrogen as nebulizer and turbo heater gas (450 °C), 20 and 40 psi, respectively; curtain and collision gas, 10 psi; and five arbitrary units. Data were evaluated by Analyst Software (Analyst Software 1.4.2; Applied Biosystems). Compound-specific parameters were adjusted by direct flow infusion of the different compounds (10 μL/min). MS parameters, retention times, and ion transitions monitored are listed in Table 1. Calibration plots of peak area vs concentration ratios (for labeled and unlabeled compounds) were linear with an R value of at least 0.985. The LOD and limit of quantification (LOQ) were 0.14 and 0.7 pmol, respectively (absolute amounts). The interday and intraday accuracies were within 2% variations. AAMA and GAMA Determination in Urine. Urine samples were kept frozen at −80 °C until analysis. Aliquots (1−2 mL) were diluted to a total volume of 8 mL with ammonium formate buffer (50 mM; pH 2.5). D3-AAMA (doses up to 100 μg AA/kg bw, 20 μL; doses beyond 100 μg AA/kg bw, 40 μL; 10 μg/mL) and D3-GAMA (doses up to 100 μg AA/kg bw, 10 μL; doses beyond 100 μg AA/kg bw, 40 μL; 12.9 μg/mL) were added as internal standards. The samples

Table 1. Retention Times and Compound-Specific MS Parameters for the AA, Mercapturic Acid, and N7-GA-Gua Determinationsa m/z analyte

retention time (min)

Q1

Q3

DP

EP

CE

CEP

CXP

AA D3-AA AAMA 14 C-AAMA D3-AAMA GAMA 14 C-GAMA D3-GAMA N7-GA-Gua N7-14C-GA-Gua N7-GA-15N5-Gua

11.8 11.6 13.9 13.9 13.7 8.9 8.9 8.8 16.8 16.8 17.0

72.1 75.1 232.9 234.9 236.0 249.0 251.0 252.0 239.2 241.2 244.2

55.1 58.1 103.6 105.6 106.9 119.6 121.6 122.9 152.1 154.1 157.1

31.0 31.0 −30.0 −30.0 −30.0 −30.0 −30.0 −30.0 36.0 36.0 36.0

5.5 5.5 −5.0 −5.0 −5.0 −5.0 −5.0 −5.0 9.0 9.0 9.0

15.0 15.0 −15.0 −15.0 −15.0 −15.0 −15.0 −15.0 27.0 27.0 27.0

14.0 14.0 −18.1 −18.1 −18.2 −18.4 −18.4 −18.5 16.6 16.6 16.8

4.0 4.0 −55.4 −55.4 −55.4 −55.2 −55.2 −55.2 4.0 4.0 4.0

a

DP, declustering potential (V); FP, focussing potential (V); EP, entrance potential (V); CE, collision energy (V); CEP, collision cell entrance potential (V); CXP, collision cell exit potential (V); Q1, quadrupole 1; and Q3, quadrupole 3. 383

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The system (Agilent Technologies GmbH & Co.KG, Waldbronn, Germany) consisted of a binary pump, a degasser, and an autosampler (Agilent 1200 Series) coupled to a SCIEX API 5500 QTRAP triple quadrupole mass spectrometer equipped with an electrospray ionization source (ESI+), operating in the MRM mode. HPLC separations were run on a reversed phase HPLC column (Phenomenex Aqua C18; 250 mm × 4.6 mm; 5 μm particle size) equipped with a guard column (Aqua C18; 4 mm × 3 mm; flow rate, 0.3 mL/min) using a gradient from 1 to 20% acetonitrile over 17 min. Source-dependent MS parameters were as follows: needle voltage, 5.5 kV; nitrogen as nebulizer and turbo heater gas (400 °C): 30 and 30 psi, respectively; curtain and collision gas, 30 psi and six arbitrary units, respectively. Data were evaluated by Analyst Software (Analyst Software 1.5.1; Applied Biosystems). Compound-specific parameters were adjusted by direct flow infusion of the different compounds (10 μL/min). The substance-specific MS parameters, the retention times, and the ion transitions are listed in Table 1. Calibration plots of peak area vs concentration ratios (for labeled and unlabeled compounds) were linear with an R value of at least 0.999. The LOD and LOQ were 1 (0.15 adducts/108 nucleotides) and 2 fmol (0.25 adducts/108 nucleotides; absolute amounts), respectively. The interday and intraday accuracies were within 4% variations. N7-GA-Gua Determination in Liver, Kidney, and Lung (High Dose Experiment). DNA was isolated and prepared as described above. After the DNA was redissolved in 1 mL of phosphate buffer, its concentration was determined and adjusted with buffer to 0.2 mg DNA/0.3 mL. The samples were spiked with 20 μL of N7-GA-15N5Gua (25 ng/mL). N7-GA-Gua adducts were cleaved from DNA by NTH (15 min, 95 °C). The samples were cooled down to room temperature and eluted through an Amicon Ultra 3 kDa molecular mass centrifugal size exclusion filter device (Millipore, Schwalbach, Germany) at 12 000g (60 min; 22 °C).15 For adduct analysis, 100 μL aliquots were injected into the HPLC-ESI-MS/MS system (cf. MA determination). The N7-GA-Gua adducts were quantified by isotope dilution analysis (IDA) using the above-mentioned stable isotope-labeled standard in the MRM mode with positive electrospray ionization (ESI+). HPLC separations were run on a reversed phase HPLC column (Phenomenex Aqua C18; 250 mm × 4.6 mm; 5 μm particle size) equipped with a guard column (Aqua C18; 4 mm × 3 mm; flow rate: 0.3 mL/min) using a gradient from 1 to 20% acetonitrile over 17 min. The eluate from 10 to 24 min was directed into the electrospray interface. Source-dependent MS parameters were as follows: needle voltage, 5.5 kV; nitrogen as nebulizer and turbo heater gas (400 °C), 30 and 30 psi, respectively; curtain and collision gas, 30 psi and seven arbitrary units. Data were evaluated by Analyst Software (Analyst Software 1.4.2; Applied Biosystems). Compound-specific parameters were adjusted by direct flow infusion of the different compounds (10 μL/min). The substance-specific MS parameters, the retention times, and the ion transitions are listed in Table 1. Calibration plots of peak area vs concentration ratios (for labeled and unlabeled compounds) were linear with an R value of at least 0.999. The LOD and LOQ were 8 (1 adduct/108 nucleotides) and 17 pmol (3 adducts/108 nucleotides; absolute amounts), respectively. The interday and intraday accuracies were within 3% variations. Statistics. Data were analyzed by unpaired two-sample Student's t test. Linear and non-linear curve fitting was performed using Microcal Origin Software (Northampton, MA).

were shaken, and the pH was adjusted to 2.5 by acidification with 4 N hydrochloric acid. After centrifugation (3000g; 15 min), supernatants (7.5 mL) were applied to an Isolute ENV+ SPE column (Biotage), preconditioned with methanol (4 mL), water (2 × 2 mL), and hydrochloric acid, and adjusted to pH 2.5 (2 mL). The column was washed with hydrochloric acid (2 mL; pH 2.5) and hydrochloric acid (pH 2.5) with 10% (v/v) methanol (1 mL) and subsequently dried under vacuum. Analytes were eluted from the column with methanol containing 1% (v/v) formic acid (1.85 mL). The eluate was evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in water with 0.1% (v/v) formic acid (0.3 mL), and 50 μL aliquots were injected into the HPLC-ESI-MS/MS system (see AA Determination). AAMA and GAMA contents in urine were determined according to Boettcher et al.,25 with modifications. Briefly, HPLC separations were run isocratically on a reversed phase HPLC column [Phenomenex Luna C8 (2); 150 mm × 4.6 mm; 3 μm particle size] equipped with a guard column [Luna C8 (2); 4 mm × 3 mm; flow rate: 0.3 mL/min]. The eluate cut between 5 and 30 min was directed into the electrospray interface (ESI−) of the mass spectrometer (SCIEX API 3200). Source-dependent MS parameters were as follows: needle voltage, −3.5 kV; nitrogen as nebulizer and turbo heater gas (475 °C), 30 and 45 psi, respectively; and curtain gas, 30 psi. Data were evaluated by Analyst Software (Analyst Software 1.4.2; Applied Biosystems). Compound-specific parameters were adjusted by direct flow infusion of the different compounds (10 μL/min). The substance-specific MS parameters, the retention times, and the ion transitions are listed in Table 1. Calibration plots of peak area ratios vs concentration ratios (for labeled and unlabeled compounds) were linear with R values of at least 0.998. LODs and LOQs were 4 and 8 pmol, respectively, for AAMA and 8 and 12 pmol, respectively, for GAMA (absolute amounts). The interday and intraday accuracies were within 5% variations. N7-GA-Gua Determination in Liver, Kidney, and Lung (Low Dose Experiment). DNA was prepared from slices (about 500 mg) of all liver lobes and the entire lung and kidney tissues. DNA was isolated according to a procedure adapted and modified from protocols reported previously.26,27 Briefly, frozen tissue was thawed and homogenized in 4 mL of lysis buffer [25 mM EDTA (pH 8.0), 0.3 M NaCl, 0.2 M Tris HCl, 2% Proteinase K, and 0.2% RNase A] with a tissue grinder (Buddeberg, Frankfurt a. M., Germany), and the homogenate was incubated at 37 °C for 3 h under shaking. After the addition of 4 mL of a mixture of phenol, chloroform, and isoamyl alcohol (25:24:1), the homogenate was extracted twice by thorough mixing (15 mL polypropylene tubes) and centrifugation (4500g at 4 °C). To remove organic residues from the nucleic acid solution, they were extracted with chloroform (4 mL). DNA was precipitated by the addition of 3 vol of absolute ethanol and 0.1 vol of 5 M NaCl, freezing (30 min at −80 °C), and centrifugation (4500g at 4 °C). The DNA pellet was washed with 2 mL of 70% ethanol (−20 °C) and redissolved in 1 mL of phosphate buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.1). DNA concentration was determined spectrophotometrically (NanoDrop, Erlangen, Germany) and adjusted with phosphate buffer to 1 mg/mL. The purity of the isolated DNA was checked by monitoring A260/A280 ratios (all samples exceeded the required ratio of 1.8). After DNA samples were spiked with 10 μL of N7-GA-15N5Gua (25 ng/mL), N7-GA-Gua (cf. N7-14C-GA-Gua) adducts were cleaved from DNA by neutral thermal hydrolysis (NTH). This was performed by heating and shaking the samples at 100 °C for 15 min in a thermomixer (Eppendorf, Hamburg, Germany).28 After centrifugation (12 000g; 1 min), samples (1 mL, about 1 mg of DNA) were applied to an Isolute ENV+ SPE column, preconditioned with methanol (5 mL) and water (2 × 2 mL). The column was washed with 2 mL of water, followed by water with 10% (v/v) methanol (1 mL). Thereafter, it was dried under vacuum. Analytes were eluted from the column with water containing 60% (v/v) methanol (1 mL). The eluate was evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved in phosphate buffer (0.3 mL; 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.1), and 100 μL aliquots were injected into the HPLC-ESI-MS/MS system.

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RESULTS AA Content in the Animal Diet. In four commercially available pelleted diets, AA contents in the range of 6.0−11.0 μg/kg were found. These contamination levels would have resulted in a calculated daily background AA uptake of 0.9−1.7 μg/kg bw (200 g bw; 30 g food uptake/day). The AA level in the experimental diet prepared in-house was below the LOD (0.5 μg/kg), resulting in a maximum daily AA uptake from this diet equivalent to 0.1 μg/kg bw (200 g bw; 30 g food uptake/ day).

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dx.doi.org/10.1021/tx200446z | Chem. Res. Toxicol. 2012, 25, 381−390

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AAMA and GAMA in Urine. Total MA (AAMA plus GAMA) excretion (within 16 h) in the untreated control was 0.84 ± 0.12 nmol. Treatment groups showed dose-dependent increases at 1 μg AA/kg bw (p < 0.01) and higher dosages (p < 0.001). At all dose levels, GAMA/AAMA ratios were found to be about constant over the whole dose range with no indication of a dose-related shift (0.4 ± 0.1). At the lowest dose level (0.1 μg AA/kg bw), MA concentrations were not significantly different from untreated control values, showing the same GAMA/AAMA ratio (0.4) and an apparent 80% excretion of the given dose. For the other dosages, 37.0 ± 11.5% of the dose given were recovered as MAs in urine after 16 h, with 20−30% at doses up to 100 μg AA/kg bw and about 40% at doses exceeding 500 μg AA/kg bw (Table 2). At 500 μg AA/kg bw, total MA excretion was somewhat higher (57%); yet, as can be seen from Table 3, N7-GA-Gua adduct levels at this dose did not appear to deviate from the semilog dose−response regression curve (within the 90% confidence interval) (Figure 1). N7-GA-Gua in Liver, Kidney, and Lung Tissues. The values of N7-GA-Gua adducts found after 16 h in liver, kidney,

and lung are presented in Table 3. A 16 h postapplication period was used because in a pilot experiment peak adduct contents were observed in all tissues tested at 16 h after dosage (Figure 2). At the 1 μg/kg bw dose, the formation of N7-GAGua adducts became measurable in kidney (mean ± SD; 1.1 ± 0.4 adducts/108 nucleotides) and lung (0.6 ± 0.1 adducts/108 nucleotides) with a significant increase of the adduct formation as compared to the nontreated control group (p < 0.001) but not in liver (LOD 0.2 adducts/108 nucleotides). Between 1 and 10 μg AA/kg bw, treatment groups showed no significant dosedependent increase in kidney and lung. At dosages exceeding 10 μg/kg bw, the increase in adduct formation was found to be significant over the whole dose range (p < 0.001) in liver, kidney, and lung (Table 3). At all dose levels, amounts of N7GA-Gua adducts in kidney and lung were about comparable, whereas in liver invariably lower values were found.

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DISCUSSION In this dose−response study, rats received AA orally via gavage in a wide concentration range (0.1−10 000 μg AA/kg bw), the low end dose (0.1 μg/kg bw) being below current average consumer exposure (range 1−4 μg/kg bw).4 Gavage under slight isoflurane anesthesia had previously been shown to have no influence on biomarker response.19 To achieve optimal sensitivity, background exposure to AA had to be kept as low as possible. Four commercially available pelleted rodent diets were tested. They were found AA contaminated to varying degrees, accounting for a calculated daily AA uptake of 0.9−1.7 μg/kg bw. We therefore prepared our own experimental diet, using ingredients considered practically free of AA and avoiding any heat treatment. As a worst case assumption, a maximum daily AA uptake from this diet was assessed, based on the LOD value (0.5 ppb), equivalent to 0.1 μg/kg bw (200 g bw; 30 g food uptake/day). Because DNA adduct determination was considered the limiting factor in terms of analytical sensitivity, we set the tissue sampling time at 16 h. At this time point, peak levels of N7-GAGua adduct were found in all tissues tested after an oral dose of 10 mg AA/kg bw (figure 2). At 24 h sampling time, DNA repair, imidazole ring-opening of N7-GA-Gua into the formamidopyrimidine isomer as well as spontaneous depurination may already have contributed to somewhat reduce the yield of N7-GA-Gua liberated from DNA by NTH.29 At 16 h, MA formation and excretion are not yet complete, reaching a mean of 37.0 ± 11.5% of a given AA dose (Table 2). The GAMA/AAMA ratio was 0.4 ± 0.1 over the whole dose range with no indication of a dose-related shift. This compares to data from a previous rat study, where 51 ± 12% of the oral applied AA dose (100 μg AA/kg bw) were found excreted as MA within 24 h, with a GAMA/AAMA ratio of 0.8.19 The same GAMA/AAMA ratio of 0.8 has been reported also in another study.30 Differences to the data of the present study can be explained by the different urine collecting periods. The excretion of AAMA and GAMA in rats follows separate kinetics, reflected by peak concentrations in urine (tmax) for AAMA at 10.9 h and for GAMA at 17.2 h.30 In humans, tmax (AAMA) was 11.5 h and tmax (GAMA) was 22.1 h.22 The observed 5 h difference of GAMA tmax between rat and human may be indicative for somewhat slower P450-mediated epoxidation kinetics in humans. This assumption is further supported by the difference in the excreted molar amount of GAMA after uptake of 100 μg AA/kg bw in rats (24 h; 20.1%), as compared to that reported for humans31 after ingestion of

Table 2. MAs in Urine Collected for 16 h after Dosing Expressed as an Absolute Excreted Amount, GAMA/AAMA Ratio, and % of the Given Dosea dosage (μg AA/ kg bw) control 0.1 1 10 100 500 1 000 3 000 6 000 10 000

AAMA (nmol) 0.6 0.8 1.3 4.8 49.5 499 851 2630 5299 9871

± ± ± ± ± ± ± ± ± ±

0.1 0.3 0.3* 0.6* 11.1* 24* 51* 376* 150* 1507*

GAMA (nmol) 0.2 0.3 0.5 2.1 17.9 309 368 1215 2673 4138

± ± ± ± ± ± ± ± ± ±

0.03 0.1 0.1* 0.3* 4.1* 68* 14* 203* 173* 1101*

GAMA/ AAMA

ΣMA (% of the given dose)

± ± ± ± ± ± ± ± ± ±

80.2 33.3 19.7 20.7 56.6 40.1 44.2 41.9 39.4

0.41 0.41 0.37 0.44 0.37 0.62 0.43 0.46 0.48 0.41

0.07 0.07 0.07 0.08 0.10 0.15 0.01 0.04 0.01 0.06

a Values represent mean values (0.1−100 μg AA/kg bw, n = 8; 500− 10 000 μg AA/kg bw, n = 2−3) of each treatment group ± SD; *significantly different from control (p < 0.001).

Table 3. N7-GA-Gua Adducts in Tissues 16 h after Dosing Expressed as Adducts Per 108 Nucleotides Determined with HPLC-ESI-MS/MSa dosage (μg AA/kg bw)

liverb

kidney

lung

control 0.1 1 10 100 500 1 000 3 000 6 000 10 000