Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats

Food and Chemical Toxicology 86 (2015) 1e8

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Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats M.I. Churchwell, R.C. Scheri, L.S. Von Tungeln, G. Gamboa da Costa, F.A. Beland, D.R. Doerge* Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 July 2015 Received in revised form 27 August 2015 Accepted 29 August 2015 Available online 11 September 2015

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (~6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-20 deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 108 nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo. Published by Elsevier Ltd.

Keywords: Furan Hepatotoxicity Hepatocarcinogen Mass spectrometry Pharmacokinetics

1. Introduction Furan is an important thermal processing contaminant present in a number of common foods, including canned meats, vegetables, soups, and brewed coffee, at levels that can exceed 100 ppb (U.S. Food and Drug Administration, 2007). The lack of correlation between furan levels and food composition is consistent with the complexity associated with many formation pathways, which is corroborated by results from model pyrolytic systems showing that ascorbate, reducing sugars with and without amino acids, and polyunsaturated fatty acids can serve as precursors to furan (PerezLocas and Yaylayan, 2004). Dietary intake assessment, derived from measured levels of furan in foods and consumption estimate distributions, shows that mean exposure to furan for the U.S. population above the age of 2 is 0.25 mg/kg body weight (bw)/day and 90th percentile exposure is 0.61 mg/kg bw/day, of which approximately one half is provided by coffee (U.S. Food and Drug Administration, 2007; Morehouse et al., 2008). Morehouse et al.

* Corresponding author. E-mail address: [email protected] (D.R. Doerge). http://dx.doi.org/10.1016/j.fct.2015.08.029 0278-6915/Published by Elsevier Ltd.

(2008) also reported remarkably similar dietary intake assessments for potentially susceptible population groups, including boys and girls (age 2e5 years, 0.23 mg/kg bw/day) and women of childbearing age (15e45 years, 0.24 mg/kg bw/day). Similar estimates of furan intake worldwide were reported by the World Health Organization/Food and Agriculture Organization Joint Expert Committee on Food Additives (JECFA), in which mean intake in European countries was estimated at 0.27e1.17 mg/kg bw/day and high consumers at 0.60e2.22 mg/kg bw/day (JECFA, 2011). The presence of furan in foods is a toxicological concern because sufficient doses of furan are hepatotoxic in rodents (Moser et al., 2009; Gill et al., 2010) and chronic lifetime exposure increases the incidences of tumors in male and female rats and mice, most notably in the liver (National Toxicology Program, 1993; Moser et al., 2009). Furan is rapidly and extensively metabolized in the liver (Burka et al., 1991; Kedderis et al., 1993), primarily via cytochrome P450 (CYP) 2E1, following either oral or inhalational administration to Fischer 344 (F344) rats. Following a single gavage dose of 14Clabeled furan to F344 rats, 14% of administered 14C was exhaled as furan, another 26% was exhaled as CO2, 25% was excreted in the urine, and 20% in the feces during the first 24 h (Burka et al., 1991). Most of the radiolabel remaining in the animal after 24 h was found

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in the liver (13% of dose) and the preponderance of that radiolabel could not be extracted by organic solvents, which is consistent with extensive macromolecular binding. The absence of detectable radiolabel associated with the resulting nucleic acid fraction of liver homogenates was interpreted as resulting from the binding of reactive furan metabolites predominantly to proteins. Even though binding to DNA was undetectable, the low specific activity of the 14 C-furan (10 mCi/kg bw for 8 mg/kg bw or 0.085 mCi/mmol) make lower bound estimates of DNA adduct formation quite high by current standards of DNA adduct analysis using LC/MS/MS (i.e., assuming 1 fmol binding per mg DNA or 32 adducts per 108 nucleotides, only 0.2 dpm per mg DNA would have resulted). Scheme 1 shows that CPY 2E1 converts furan to the highly reactive biselectrophilic species, cis-2-butene-1,4-dial (BDA, Byrns et al., 2002), which covalently modifies nucleophilic groups in amino acids (Chen et al., 1997) and nucleic acid bases (Byrns et al., 2006) under proper conditions in vitro. Furan administration does not appear to be directly mutagenic in rat liver in vivo (McDaniel et al., 2012; Ding et al., 2012), and it has long been proposed that furan exerts its hepatocarcinogenicity via a primary mechanism involving protein damage and cytotoxicity, with secondary effects including inflammation, oxidative stress, and cell proliferation (Kedderis et al., 1993; Moser et al., 2009; Dong et al., 2015); however, some evidence for DNA-associated binding from accelerator mass spectrometric measurements of rat tissue DNA (Neuwirth et al., 2012) and for formation of DNA-protein crosslinks in ovo (Jeffrey et al., 2012) suggests that directly genotoxic mechanisms cannot be ignored. The current study sought to identify toxicokinetic factors that predispose the liver as the primary site of toxicity following furan administration, with a focus on the oral route, by evaluating the time courses for furan in male F344 rat serum and liver following a single gavage dose. Information about this route of exposure extends and complements that available for inhalation (Kedderis et al., 1993), which is directly relevant to furan exposures through either cigarette smoke (Grill et al., 2015) or possible industrial

CYP 2E1 O

O

O O

O

Furan

cis-2-butene-1,4-dial (BDA) DNA

Proteins GSH

O

HO

N N

N

BDA-Adducts (biomarkers)

N

O

dR

BDA-dC O

HO

N N

N

2. Experimental Water and methanol were Optima LC/MS grade purchased from Fisher Scientific (Pittsburgh, PA). Furan (redistilled and stored at 20  C, d4 efuran, adenosine deaminase, alkaline phosphodiesterase, ammonium acetate, Bis-Tris, ethylenediaminetetraacetic acid (EDTA), 20 -deoxycytidine, 2,5-dimethoxy-2,5-dihydrofuran (cis and trans mixture), DNase I, methoxylamine hydrochloride, sodium phosphate, snake venom phosphodiesterase, and sodium dodecyl sulfate (SDS) were purchased from SigmaeAldrich Chemical Company (St. Louis, MO). Ammonium hydroxide was purchased from JT Baker, Phillipsburg (NJ). 2.1. Preparation of standards Stock standards were prepared in methanol at 50 nmol/mL and 500 pmol/mL and stored at 20  C for up to four weeks. Working standards were prepared daily in water at 5 pmol/mL and kept on ice or at 4  C. Due to the small but measurable content of unlabeled furan (d0 content 0.01%), internal standard blanks were analyzed for each sample set and the calculated concentrations were subtracted from all samples in the set (range 0.4e1.5 pmol/mL). 2.2. Animal handling procedures Procedures involving care and handling of rats were reviewed and approved by the National Center for Toxicological Research (NCTR) Institutional Animal Care and Use Committee. Male F344 rats were obtained from the NCTR colony at approximately 50 days of age. All furan dosing was performed by gavage in corn oil and the furan concentrations in all dosing solutions were quantified using headspace gas chromatography/mass spectrometry (GC/MS) prior to use. A dose of 0.92 mg/kg bw was used for the single dose toxicokinetic study. The requirement to collect a large volume of whole blood (1 mL) for furan time course analysis and the need for timed removals of livers made it necessary to conduct the toxicokinetic study by analyzing 6 rats at each time point, rather than taking serial blood samples from individual rats. A furan dose-adduct formation study was conducted using single doses of 0.92e9.2 mg/kg bw with a common 24 h post-dose exposure time for liver removal. A repeat furan dosing study was conducted using a common dose of 4.4 mg/kg bw/day (n ¼ 6 rats per dose) for 45e360 days of daily gavage treatment (5 days per week) with liver removal 24 h after the last dose. A corn oil vehicle-treated group of rats was included in each sub-study. 2.3. Furan quantitation method development

N

N

exposures (National Toxicology Program, 1993). Secondly, this study evaluated the possibility of liver DNA adduct formation in vivo following single or repeated oral dosing of F344 rats with furan by validating sensitive analytical methodology to quantify the major, and most stable, nucleoside adduct formed in vitro by reaction of 20 -deoxycytidine (dC) and BDA (Gingipalli and Dedon, 2001).

dR

BDA-dA O

HO

N

N

O N H

N

N dR

BDA-dG

Scheme 1. Metabolic activation of furan.

The method of Nyman et al. (2006) was adapted for quantifying furan in whole blood and liver. Various GC columns were evaluated, with a PLOT-Q column giving the best chromatographic results. Also, both split and splitless injection modes were evaluated, and a 10:1 split ratio gave the best response. Autosampler agitation time and incubation temperatures were evaluated from 40 to 80  C and 5e30 min, with 10 min at 40  C giving the best response. The effect of adding salt to the headspace vial was evaluated by the addition of 500 mg ammonium sulfate per mL to spiked blood or liver samples;

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however, since no improvement in recovery of furan was noted and because the addition of salt tended to plug the sampling needle, no salt was used for subsequent analyses. For preparation of liver samples, homogenization using either sucrose or SDS was evaluated. Since there was an apparent furan contamination in the commercially available sucrose, SDS was chosen for homogenization. Since homogenization in SDS was prone to form an emulsion, it was necessary to add 2.6 mL of liver homogenate to the vial in order to obtain a minimum homogenate volume of 2.5 mL from approximately 500 mg of liver. The volume was verified after analysis by measuring the final volume after uncapping the vials. 2.4. Whole blood preparation Whole blood was collected by cardiac puncture in 3 mL purple top EDTA Vacutainer tubes (BectoneDickinson, Franklin Lakes, NJ). The tubes were filled completely, mixed well, and immediately cooled. Blood samples were analyzed on the same day as collection. A 1 mL aliquot of whole blood was added to a 10 mL headspace vial and spiked with 100 pmol of d4 efuran internal standard. The internal standard was kept on ice during sample preparation. The vial was then sealed with a Teflon lined crimp top cap and the sample analyzed by headspace GC/MS. 2.5. Liver preparation Livers were collected after cardiac puncture and stored briefly in aluminum foil under dry ice until transfer to a 80  C freezer for storage. Approximately half of each liver was used during sample preparation. Working on ice, a 4 g portion was cut up into to 10 to 15 pieces. The pieces were added to a 50 mL centrifuge tube containing 20 mL of cold 1% SDS. The liver pieces were quickly homogenized with a Polytron. This work was also conducted on ice. A 2.6 mL aliquot of the emulsion (equal to 500 mg of liver) was added to a 10 mL headspace vial and spiked with 100 pmol of d4-furan internal standard. The internal standard was kept on ice during sample preparation. The vial was then sealed with a Teflon lined crimp top cap and the sample analyzed by headspace GC/MS. 2.6. Headspace gas chromatography Sampling was done with a TriPlus autosampler (Thermo Scientific, Waltham, MA). Samples were agitated at 40  C for 10 min. The agitator cycled on and off in 5 s increments. The sample syringe was held at 45  C and the injection volume was 2 mL. A Trace Ultra GC (Thermo Scientific) equipped with a HP-PLOT-Q column (15 m  32 mm  20 mm film, Agilent Technologies, Palo Alto, CA) was used for chromatography. The GC temperature program consisted of the following: 40  C for 1 min, a temperature gradient from 40  C to 225  C at 10  C/min, and then held for 5 min. Split mode injection was utilized with a 10:1 split ratio and an injector temperature of 200  C. The helium flow was constant at 1.7 mL/ min. 2.7. GC/MS A DSQII single quadrupole mass spectrometer (Thermo Scientific) was used in the 70 eV electron impact positive ionization single ion monitoring mode. The mass spectrometer was autotuned with perfluorotributylamine. The transfer line and source temperature were set at 250  C. The start delay was 3.5 min. Two ions were monitored for both furan and d4-furan, quantitation ions of m/z 68.1 and 72.1 and confirmation ions of m/z 39.1 and 42.1, respectively. The dwell times were all 100 msec.

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2.8. Furan quantification in blood and liver 2.8.1. Method validation in whole blood A linear calibration curve (r2 > 0.999) was generated over the range of 0e500 pmol furan/mL of blood. The method was validated over two days using control rat blood and spiked control rat blood. Control rat blood was spiked at 50 pmol/mL. Intra- and inter-day precision ranged from 1.5 to 6.4% relative standard deviation (RSD). Intra- and inter-day accuracy (%) ranged from 81 to 90%. Replicates of internal standard blanks were analyzed with each sample set because small background peaks associated with the small amount of unlabeled furan present in the internal standard were consistently observed (range 0.4e1.5 pmol/mL). In addition, daily limits of detection (LOD) were determined. If the sample value after subtraction of the internal standard blank was not higher than the daily calculated LOD, it was reported as 0.999) was generated over the range of 0e4000 pmol furan/g of liver. The method was validated over two days using control rat liver and spiked control rat liver. The control rat liver was spiked at 200 pmol/g. Intra- and inter-day precision ranged from 4 to 7% RSD. Intra- and inter-day accuracy ranged from 91 to 94%. Replicates of internal standard blanks were analyzed with each sample set of livers, which ranged from 1.4 to 2.2 pmol/g, and were subtracted daily. Daily LODs above the internal standard blank value were estimated as described above for blood samples and averaged 1.0 pmol/g. 2.9. Furan-DNA adduct synthesis and characterization 2.9.1. Synthesis of 20 -deoxycytidine-oxadiazabicyclo(3.3.0) octaimine (BDA-dC) 2,5-Dimethoxy-2,5-dihydrofuran (438 mmol) was reacted with dC (0.18 mmol) in 50 mM sodium phosphate, pH 7.0, for 18 h at 55  C in a total volume of 1 mL (Chen et al., 1995). The resulting BDA-dC was collected using HPLC (Acquity, Waters Inc., Milford, MA) and concentrated under reduced pressure. The resulting precipitate was dissolved in Optima water and stored at 20  C. The HPLC was equipped with a Capcell Pak AG C18 column (4.6  250 mm, 5 mm, Shiseido, Phenomenex, Torrance, CA) and a Security C18 guard column (4.6  2.0 mm, Phenomenex) maintained at 55  C. Purified BDA-dC standard concentration was quantified using 1H NMR spectroscopy with t-butyl alcohol as the internal reference. An extinction coefficient (10,200 M1 cm1) was determined from the UV absorbance at 280 nm (lmax) on a Thermo Scientific Nanodrop 8000 spectrophotometer. Salmon sperm DNA was similarly reacted with varying amounts of BDA (prepared as described above from 2,5-dimethoxy-2,5dihydrofuran), starting with 1 mg BDA per mg DNA and subsequent serial 1/10 dilutions to 1010 mg BDA per mg DNA in 5 mM BisTris, 0.1 mM EDTA, pH 7.1. After overnight incubation at 37  C, the DNA was precipitated with 0.1 volume of 5 M NaCl and 2 volumes of ethanol. The resulting precipitate was washed with 5 mL of 70% ethanol and redissolved in the Bis-Tris buffer. Concentrations of BDA-dC as the nucleoside adduct were determined using HPLC with QTOF-MS/MS detection after enzymatic hydrolysis as described below. Adduct elution was accomplished using an isocratic gradient (95% 50 mM ammonium acetate, pH 7.0e5% acetonitrile) at a flow rate of 1 mL/min with 0.1 mL/min split to a quadrapole time-offlight (QTOF) mass spectrometer (Synapt, Waters Inc.) equipped with an electrospray ionization source (ESI) operated in the positive

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ion mode. The capillary, cone, and extraction voltages were 3.0 kV, 10 V, and 3.5 V, respectively. The desolvation gas was nitrogen with a temperature of 300  C and a flow rate of 750 l/hr. The source block temperature was 110  C. The trap gas was argon with a flow rate of 1.5 mL/min. Trap and transfer collision energies were 3.0 and 0.0 eV, respectively, with a scan time of 1.0 s. 2.9.2. Methoxylamine-derivatization of 20 -deoxycytidine oxadiazabicyclo(3.3.0)octaimine adducts (MA-BDA-dC) Derivatization was accomplished by reacting BDA-dC (2 nmol) with methoxylamine hydrochloride (5 mmol) in 5.0 mM Bis-Tris, 0.1 mM EDTA, pH 7.1 for 18 h at 30  C in a total volume of 1 mL in the presence of similar amounts of 20 -deoxyadenosine (dA), 20 deoxyguanosine (dG), 20 deoxythymidine (dT), and dC. The samples were cleaned up/desalted using solid phase extraction (SPE, Oasis HLB, 96-well plate format, 10 mg/30 mm, Waters Inc.). After SPE, multiple samples were combined, solvent was evaporated, and the residue redissolved in 1.0 mL water. The final derivatized adduct concentration (24 mM) was estimated by spectrophotometry, the efficiency of the derivatization reaction (>95%, based on the substrate peak area remaining) and the SPE recovery efficiency (~70%, based on the derivatized adduct chromatographic peak areas obtained before and after SPE). An Acquity UPLC BEH C18 column (2.1  100 mm, 1.7 mm, Waters Inc.) was used at a flow rate of 0.4 mL/min and temperature of 55  C. Elution was performed with: 4% acetonitrile in 0.1% ammonium hydroxide for the first 2 min, followed by a linear gradient of 4e30% acetonitrile for 4 min; a step to 60% acetonitrile for 2 min; and then stepped back down to 4% acetonitrile for column equilibration. The column eluent was fed directly into the Synapt ESI source for analysis in the positive ion mode. The capillary, cone, and extraction voltages were 3.0 kV, 20 V, and 3.5 V, respectively. The desolvation gas was nitrogen with a temperature of 350  C and a flow rate of 850 L/hr. The source block temperature was 110  C. The trap gas was argon with a flow rate of 1.5 mL/min. Trap and transfer collision energies were 4.0 and 0.0 eV, respectively. A scan time of 0.5 s was used. 2.10. DNA isolation and purification Whole rat livers (whenever available) or right lateral lobes were homogenized and the nuclei isolated according to the method of Basler et al. (1981). DNA was prepared using the method of Beland et al. (1984) and dissolved in 5.0 mM Bis-Tris, 0.1 mM EDTA, pH 7.1 for analysis. DNA quantification was performed using UV spectroscopy (260 nm). 2.11. DNA hydrolysis and BDA-dC derivatization Aliquots containing 100 mg of DNA (in 100e200 mL 5.0 mM BisTris, 0.1 mM EDTA, pH 7.1) were initially hydrolyzed with DNase I, followed by snake venom phosphodiesterase and alkaline

Table 1 Quantification of BDA-dC Adducts in Chemically Modified Salmon Sperm DNA. DNA (1 mg/mL) was treated with BDA at concentration ratios ranging from 1 mg per mg DNA to 0.1 fg per mg DNA and purified as described in the Experimental section. Levels of BDA-dC were quantified for the lower range of adducts following enzymatic hydrolysis to nucleosides, methoxylamine derivatization, and LC/MS/ MS using external standard calibration. BDA/DNA ratio

BDA-dC (adducts/108 nucleotides)

10,000 pg/mg 100 pg/mg 10 pg/mg 1 pg/mg Control DNA

19336 166 18 1.2 1.1

[Furan] (pmol/mL in blood, pmol/g in liver)

4

Blood Liver

1000

100

10

1 0

2

4

6

8

time (h) Fig. 1. Time Courses for Furan in Blood and Liver after a Single Oral Dose of Furan (0.92 mg/kg bw). Blood and liver concentrations of furan were measured using GC/MS at various times after dosing (log scaled values shown represent means ± SD, n ¼ 6 rats per time point).

phosphatase. After hydrolysis, adenosine deaminase was used to convert the 20 -deoxyadenosine in the hydrolysate to 20 -deoxyinosine in order to avoid chromatographic interference with the derivatized BDA-dC adduct. Finally, 50 mL of 50 mM methoxylamine hydrochloride was added and the total volume brought up to 500 mL with water. Samples were then incubated for 18 h at 30  C. 2.12. Derivatized BDA-dC cleanup The methoxylamine-derivatized BDA-dC was desalted/cleaned up using SPE with Oasis HLB sorbent in a 96 well plate format. Optimal results were obtained with SPE at a pH of 10. The pH of the samples after hydrolysis and derivatization was around 7; therefore, 125 mL of 50 mM ammonium acetate was added to each sample to adjust the pH. The HLB sorbent was conditioned with methanol, washed with water, and equilibrated with 10 mM ammonium acetate, pH 10, prior to sample addition. The samples were washed with 10 mM ammonium acetate, pH 10, eluted with methanol, and then evaporated in a heated 96 well nitrogen evaporation system (SPEdry, Biotage Inc., Charlotte, NC). Samples were dissolved in 0.1% ammonium hydroxide before HPLC and mass spectrometry. 2.13. HPLC-tandem mass spectrometry of derivatized BDA-dC in rat liver DNA hydrolysates An Alliance Model 2795 HPLC (Waters Inc.) was used equipped with a Luna C18 column (2.0  150 mm, 5 mm, 100 Å Phenomenex) and a SecurityGuard C18 cartridge (4  2 mm, Phenomenex) run at a flow rate of 0.2 mL/min at room temperature. An injection volume of 50 mL was used. Elutions were performed with 5% acetonitrile in 0.1% ammonium hydroxide for the first 5 min, followed by a linear gradient of 5e50% acetonitrile for 15 min, and then a return to 5% acetonitrile for column equilibration. The eluent was fed directly into the ESI source of a Quattro Ultima triple quadrupole (Waters Inc.) mass spectrometer for quantification. External standards consisted of varying concentrations of methoxylamine-derivatized BDA-dC dissolved in 0.1% ammonium hydroxide. Mass spectrometry acquisition parameters were as follows: the capillary voltage was set to 1.0 kV and the cone voltage to 40 V; source block and desolvation gas temperatures were 100  C and 400  C, respectively; the desolvation and cone gases were nitrogen,

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Fig. 2. MRM Chromatograms for BDA-dC Standard in Rat Liver DNA. Panel A. Chromatograms for major and minor MRM transitions for vehicle-treated rat liver DNA (100 mg) with the blank signal corresponding to 2.4 BDA-dC per 108 nucleotides. Panel B. The same vehicle-treated rat liver DNA sample spiked with BDA-dC standard (29 fmol in 100 mg DNA, equivalent to 9 adducts per 108 nucleotides.

run at flow rates of 750 L/hr and 100 L/hr, respectively; argon was the collision gas, at a collision cell pressure of 2.5  103 mbar; data were acquired in selected reaction monitoring (SRM) mode (the resolution was set to give 0.9 Th at full width half maximum for the precursor and product ions with a dwell time of 0.2 s) for the analysis of positive ions. The optimal transition (Table 1) for the methoxylamine-derivatized BDA-dC that maximized the MS response [m/z 341 / 225] was monitored, along with a secondary confirmatory transition [m/z 341 / 176]. The collision energies used for the main and confirmatory transitions were 12 and 26 eV, respectively. Control DNA (salmon sperm or vehicle-treated rat liver) blanks were analyzed concurrently.

3% vs. 65% across different locations on the liver. The mean level of liver furan was maximal at the second sampling time (Cmax ¼ 547 ± 993 pmol/g at Tmax ¼ 0.5 h) and the kinetics showed two log-linear phases for distribution (half-time ¼ 0.55 h) and elimination (half-time ¼ 0.62 h, Fig. 1). Undetectable levels of furan were present in both blood and liver by 8 h after dosing. Liver concentrations of furan were consistently greater than the corresponding blood concentration, with an overall mean ratio of 6.2. The area under the timeeconcentration curve, extrapolated to infinity, was 772 pmol*hr *g1 for liver and 139 nmol *hr* L1 for blood (AUC ratio ¼ 5.6). 3.2. Furan-DNA adduct quantification

3. Results 3.1. Toxicokinetics of furan in blood and liver Blood and liver concentrations of furan were measured in rats (n ¼ 6 rats per time point) at various times (0.25e8 h) after a single gavage dose of furan (0.92 mg/kg bw). Furan levels were
Chemically modified salmon sperm DNA samples were prepared by reacting with varying amounts of BDA (0.1 fg to 1 mg of BDA per mg of DNA, in 10-fold increments). An initial survey of samples analyzed by high resolution MS showed that BDA-dC adducts were detectable in hydrolyzed DNA samples and the increases in BDA-dC adduct levels were proportionate to the orders of magnitude reactant increments (not shown). Quantitative analysis of BDA-dC in these DNA samples was performed using a more sensitive derivatization LC/MS/MS methodology in a lower range of adduct levels (1e100,000 adducts per 108 nucleotides, Table 1). The value obtained for the 1 pg of BDA per mg DNA was quantitatively similar to the background level observed in untreated DNA (~1 adduct per 108 nucleotides) and 10-, 100-, and 10,000-fold higher reaction ratios produced similarly augmented BDA-dC adduct levels (Table 1). The levels of BDA-dC were quantified in control rat liver DNA using the derivatization LC/MS/MS method, with quantification by

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Fig. 3. The Effect of Dose on BDA-dC Levels in Furan-Treated Rat Liver DNA. Rats were treated with different single doses of furan and after 24 h, liver DNA was extracted and analyzed for BDA-dC levels using LC/MS/MS as described in the Experimental section. Panel A, vehicle treatment; B, 0.92 mg/kg bw; C, 2 mg/kg bw; D, 4.4 mg/kg bw; E, 9.2 mg/kg bw. The major MRM transition was used to quantify the BDA-dC present and responses were similar for all doses and the vehicle control.

the method of standard addition (i.e., spiking a known amount of BDA-dC into a DNA hydrolysate at 1.5e29 fmol/mg, or 0.45e9 adducts in 108 nucleotides, prior to derivatization with methoxylamine; Fig. 2). The method was validated by spiking a pooled control rat liver DNA sample with 0.45, 0.9, or 9 BDA-dC adducts per 108 nucleotides on two different days. The response for the major MRM transition (m/z 341 / 225) was linear (r2 ¼ 0.995) for the three different spike levels with precision of 3.2e4.2% RSD. The LOD, based on a signal/noise ratio of 3 for the major transition for BDA-dC, was estimated to be 0.4 adducts per 108 nucleotides above the background signal. The background level of BDA-dC in vehicletreated rat liver DNA samples, using standard addition on three separate days, was determined to be 1.2e2.4 adducts per 108 nucleotides. Since this background signal was consistently present in rat liver DNA samples, regardless of the magnitude of furan dose or duration of dosing, and was also observed in commercial salmon sperm DNA blank samples (Table 1), it was deemed likely that trace level contamination by the synthetic adduct occurred during processing of all DNA samples. When liver DNA was analyzed for BDAdC levels from rats 24 h after being administered single dose gavage treatments with furan (0.92e9.2 mg/kg bw), no differences in BDAdC were observed above the level present in vehicle-treated rats (Fig. 3). Similarly, when rats were repeatedly gavaged daily with 4.4 mg/kg bw furan for different lengths of time (45e360 days), no differences in BDA-dC levels were observed above the level present in vehicle-treated rats (Fig. 4). 4. Discussion The presence of carcinogenic compounds, like furan, in cooked foods is an ongoing concern for food regulatory authorities

worldwide (Benford et al., 2010). Heterocyclic aromatic amines (e.g., PhIP), polycyclic aromatic hydrocarbons (PAH, e.g., benzo[a] pyrene), acrylamide, and fatty acid esters of chloropropanols (e.g., MCPD) and glycidol (Bakhiya et al., 2011) are all formed during typical thermal food preparation processes and sufficient doses of these chemicals produce tumors in chronic rodent bioassays. While the contribution of such “cooking carcinogens” to the etiology and incidence of human cancer is highly uncertain, these observations implicate typical human diets as a significant hazard, which could contribute to the long-standing estimate that at least one-third of cancer deaths are potentially avoidable by changes in the diet (Doll and Peto, 1981). While the variety of cooked foods typically consumed in a human diet is enormously complex, risk assessment of the carcinogenic components typically proceeds using a reductionist strategy of one component at a time. While this strategy is clearly of limited value for assessing risks/benefits at the whole diet level, it can be nonetheless useful to compare relative risks of individual carcinogens (Benford et al., 2010). The current study examines the metabolism and disposition of furan in a context applicable to its consumption in food to provide additional information regarding the mode of action in rat liver. This toxicokinetic study shows that after oral exposure of male F344 rats to furan, their livers quickly achieve furan concentrations consistently above those in blood (~6-fold) prior to rapid elimination from the body within 8 h. These observations differ from blood and liver concentrations resulting from inhalation exposure to male F344 rats, in which liver concentrations of furan were consistently lower than those in blood (~1/10th, Kedderis et al., 1993). These results from oral administration emphasize the importance of portal vein delivery from the gut to the liver in determining peak

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Fig. 4. The Effect of Repeated Dosing on BDA-dC Levels in Furan-Treated Rat Liver DNA. Rats were treated with a common dose of 4.4 mg/kg bw daily for different times (A, vehicle; B, 45 days; C, 90 days; D, 180 days; E, 360 days) and 24 h after the last dose, liver DNA was extracted and analyzed for BDA-dC levels using LC/MS/MS as described in the Experimental section. The major MRM transition was used to quantify the BDA-dC present and responses were similar for all dosing times and the vehicle control.

levels of furan in the target tissue. First pass extraction of furan, by virtue of its lipophilic nature (log Kow ¼ 1.3) and high-affinity blood flow-limited metabolism by CYP 2E1 (Kedderis et al., 1993), can account for the primacy of hepatotoxicity associated with oral exposures to furan and the hepatocarcinogenicity observed after chronic exposure (National Toxicology Program, 1993). A key factor in the risk assessment of furan carcinogenicity is the mechanism whereby the chemically reactive furan metabolite, BDA, interacts with biological macromolecules, especially DNA. The reactivity of BDA with sulfur- and nitrogen-containing amino acids nucleophiles gives rise to several well-characterized protein- and glutathione-derived adducts that have been validated as biomarkers in rats and humans (Kellert et al., 2008; Grill et al., 2015). While BDA-GSH-derived adducts are clearly products of detoxification, the BDA-protein adducts can be considered potential biomarkers of cytotoxicity (Kellert et al., 2008; Grill et al., 2015). Similarly, BDA reacts with nucleosides in vitro to produce a series of diastereomeric hemiacetal adducts, of which the BDA-dC adducts are much more stable than the corresponding dA and dG purine adducts (Byrns et al., 2006). While specific BDA adducts with dC and dA have been quantified in bacterial DNA after treatment of S. typhimurium strain TA104 with 1.42e2.86 mM BDA for 30 min (Byrns et al., 2006), treatment of rats with up to 2 mg/kg bw furan for 28 days showed undetectable levels of these adducts (LOD for the dC adduct, <3.3 in 108 nucleotides and the dA adduct, <6.6 in 108 nucleotides; Neuwirth et al., 2012). The current study, with its analysis and quantification of BDA-dC adducts by LC/MS/MS in liver DNA from rats treated with doses up to 9.2 mg/kg bw or treated daily with a dose of 4.4 mg/kg bw for up to 360 days, showed no evidence for BDA-dC adduct formation (LOD ~0.4 in 108 nucleotides) above a background level present in commercial control DNA

and vehicle-treated rat liver DNA (~1e2 BDA-dC in 108 nucleotides). These results suggest that while nucleoside adducts can form in bacterial cells treated with high concentrations of BDA, nucleoside adduct formation in rat liver is negligible, even after extremes of furan dosing. The observation that specific BDA-protein adducts, but not nucleoside adducts, are produced in rats by doses of furan sufficient for hepatotoxicity and hepatocarcinogenicity after chronic exposure implies a dichotomy in accessibility and/or reactivity of metabolically produced BDA between cellular components such that nuclear DNA is apparently spared. This conclusion must be tempered by the observation from accelerator mass spectrometry that two unidentified peaks containing 14C-radiolabel from furan (a single dose of 2 mg/kg bw, 2 h post-dosing) were released by enzymatic hydrolysis from rat liver DNA (Neuwirth et al., 2012); however, a caveat to interpretation of that finding is the observation of similar levels of 14C binding in liver (33 ± 21 adducts in 108 nucleotides) vs. the kidney (13 ± 5 adducts in 108 nucleotides), a non-target tissue for carcinogenesis (National Toxicology Program, 1993) or histological changes in this dose range (Gill et al., 2010). Similarly, injection of furan into turkey eggs containing a functional liver, at concentrations in the range of approximately 25e250 mM (2e20 mmol/85 g egg), produced changes in comet assay results consistent with formation of DNAprotein crosslinks (Jeffrey et al., 2012); however, Neuwirth et al. (2012) found no evidence for such DNA-protein crosslinks by using LC/MS/MS-based screening of furan-dosed rat liver (see above). A related observation to be considered is the apparent ability of furan-treatment to induce, in some cases irreversibly, epigenetic modifications (e.g., global and specific DNA methylation, specific histone methylation and acetylation) that can lead to specific gene expression changes in the liver of male F344 rats over the same

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dose range that produces hepatotoxicity and hepatocarcinogenicity (de Conti et al., 2014; de Conti et al., 2015). So, while direct covalent modification of DNA to form mutagenic BDA-nucleoside adducts is unlikely to occur in a dose range relevant to rodent bioassays, it is possible that other modifications related to BDA-protein reactivity could influence DNA transcription. The apparent absence of specific BDA-nucleoside adducts in liver DNA from rats treated with hepatotoxic/hepatocarcinogenic doses of furan (>2 mg/kg bw/day) is consistent with the general absence of evidence for in vivo genotoxicity of furan in Big Blue rats at similar doses (McDaniel et al., 2012). This body of evidence argues against furan and its reactive metabolite acting as a typical “genotoxic carcinogen”. Furthermore, it is not clear that any mechanism derived from such doses relates to the effects of typical dietary exposures, which are <1 mg/kg bw/day, since a noobserved-adverse-effect level for histological endpoints of 30 mg/ kg bw/day was reported in a recent 90-day study (Gill et al., 2010). As a further cautionary note, the predominant source of furan exposure in the adult diet is coffee, for which a broad body of epidemiological findings supports a protective association between consumption of up to several cups of coffee per day and indicators of liver pathology (Morisco et al., 2014), and even incidences of hepatocellular carcinoma (Petrick et al., 2015). The significant uncertainties surrounding the mode of action, extrapolation of toxico/ pharmacodynamics from high controlled dosing in animal models to low dietary doses in humans, and inter-species differences, all point to caution in the assessment of potential risks and benefits from consumption of cooked foods containing furan. Acknowledgments This study was funded by an Interagency Agreement between FDA and NIEHS/NIH (FDA IAG # 224-12-0003/NIEHS IAG # AES12013). RCS acknowledges support of a fellowship from the Oak Ridge Institute for Science and Education, administered through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The authors are grateful for helpful discussions with Drs. Ronald Lorentzen and Michael DiNovi, U.S. FDA Center for Food Safety and Applied Nutrition. The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration or National Toxicology Program. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.08.029. References Bakhiya, N., Abraham, K., Gürtler, R., Appel, K.E., Lampen, A., 2011. Toxicological assessment of 3-chloro-1,2-propanediol and glycidol fatty acid esters in food. Molec. Nutr. Food Res. 55, 509e521. Basler, J., Hastie, N.D., Pietras, D., Matsui, S.-I., Sandberg, A.A., Berezney, R., 1981. Hybridization of nuclear matrix attached deoxyribonucleic acid fragments. Biochemistry 20, 6921e6929. Beland, F.A., Fullerton, N.F., Heflich, R.H., 1984. Rapid isolation, hydrolysis and chromatography of formaldehyde-modified DNA. J. Chromatogr. 308, 121e131. Benford, D., Bolger, P.M., Carthew, P., Coulet, M., DiNovi, M., Leblanc, J.-C., Renwick, A.G., Setzer, W., Schlatter, J., Smith, B., Slob, W., Williams, G., Wildemann, T., 2010. Application of the Margin of Exposure (MOE) approach to substances in food that are genotoxic and carcinogenic. Food Chem. Toxicol. 48, S2eS24. Burka, L.T., Washburn, K.D., Irwin, R.D., 1991. Disposition of [14C]furan in the male F344 rat. J. Toxicol. Environ. Health 34, 245e257. Byrns, M.C., Predecki, D.P., Peterson, L.A., 2002. Characterization of nucleoside adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan. Chem. Res. Toxicol. 15, 373e379. Byrns, M.C., Vu, C.C., Neidigh, J.W., Abad, J.-L., Jones, R.A., Peterson, L.A., 2006. Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-

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