Mutagenicity of 5-Hydroxymethylfurfural in V79 Cells Expressing

Article pubs.acs.org/crt

Mutagenicity of 5-Hydroxymethylfurfural in V79 Cells Expressing Human SULT1A1: Identification and Mass Spectrometric Quantification of DNA Adducts Formed Bernhard H. Monien,*,† Wolfram Engst,† Gitte Barknowitz,† Albrecht Seidel,‡ and Hansruedi Glatt† †

Department of Toxicology, German Institute of Human Nutrition (DIfE) Potsdam-Rehbrücke, 14558 Nuthetal, Germany Biochemical Institute for Environmental Carcinogens, Professor Dr. Gernot Grimmer-Foundation, 22927 Grosshansdorf, Germany

‡

S Supporting Information *

ABSTRACT: 5-Hydroxymethylfurfural (HMF), a heterocyclic product of the Maillard reaction, is a ubiquitous food contaminant. It has demonstrated hepatocarcinogenic activity in female mice. This effect may originate from sulfo conjugation of the benzylic alcohol yielding 5-sulfooxymethylfurfural (SMF), which is prone to react with DNA via nucleophilic substitution. Indeed, we showed that HMF induces gene mutations in Chinese hamster V79 cells engineered for the expression of human (h) sulfotransferase (SULT)1A1 but not in parental V79 cells. In order to identify potential DNA adducts, we incubated DNA samples with SMF or HMF in aqueous solution. Modified DNA was digested and surveyed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for adducts that may be formed by nucleosides either via nucleophilic substitution at the electrophilic carbon atom of SMF or via imine formation with the aldehyde group present in HMF and SMF. The most abundant adducts formed from SMF, N6-((2-formylfuran-5-yl)methyl)-2′-deoxyadenosine (N6-FFMdAdo) and N2-((2-formylfuran-5-yl)methyl)-2′-deoxyguanosine (N2-FFM-dGuo), were synthesized, purified, and characterized by 1H NMR. Imine adducts were only detected when DNA was incubated with very high levels of HMF following reduction of the imines to corresponding secondary amines by NaBH3CN. Sensitive techniques based on LC-MS/MS multiple reaction monitoring for the quantification of the adducts in DNA samples were devised using isotope-labeled [15N5]N6-FFM-dAdo and [13C10,15N5]N2-FFM-dGuo as internal standards. Both 5-methylfurfuryl adducts were detected in DNA from V79-hSULT1A1 treated with HMF but not in DNA from V79 control cells. Considering the lack of other known mutagenic metabolites, we hypothesize that the hepatocarcinogenic potential of HMF originates from the formation of mutagenic SMF.

■

phoresis (comet assay),3 sister chromatid exchange in V79 cells,16 and micronuclei count in peripheral blood cells.10 The mechanism underlying carcinogenesis may originate from sulfotransferase (SULT)-catalyzed conversion of HMF to the highly electrophilic 5-sulfooxymethylfurfural (SMF) (Scheme 1A), a reaction that is not taken into account by standard in vitro mutagenicity tests.14 This hypothesis was supported by assays in which HMF induced his+ revertants in Salmonella typhimurium in the presence of rodent liver cytosol and 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the sulfogroup donor for SULTs.13 In addition, HMF was mutagenic in modified Salmonella typhimurium TA100 strains expressing various SULT forms, e.g., human (h) or murine SULT1A1,17 but not in the parental strain. The frequency of sister chromatid exchanges was significantly increased in V79-hCYP2E1hSULT1A1 cells compared to that observed in the parental

INTRODUCTION 5-Hydroxymethylfurfural (HMF) is a product of the Maillard reaction and occurs in foodstuffs, beverage products, parenteral solutions, and cigarette smoke.1,2 The mean daily dietary intake was estimated to be 30−150 mg of HMF per person in older studies.3,4 More recent approximations from working groups in Spain,5 Norway,2 and Germany6 were in the range from 2 to 30 mg of HMF per person. It was reported that HMF induces and promotes aberrant crypt foci in the colon of rats7 and initiates papilloma formation in mouse skin.8 Further, a single subcuteanous injection of HMF to newborn Min/+ mice increased the incidence of small intestinal adenomas and of aberrant crypt foci in the large intestine.9 Lifetime oral administration of 188 or 375 mg of HMF/kg body weight per day resulted in the induction of hepatocellular adenomas in female B6C3F1 mice.10 However, HMF lacks significant genotoxic activity as shown by negative or equivocal results from standard mutagenicity tests in bacterial10−13 and mammalian cells,3,14 the rec-assay,15 single-cell gel electro© 2012 American Chemical Society

Received: April 5, 2012 Published: May 7, 2012 1484

dx.doi.org/10.1021/tx300150n | Chem. Res. Toxicol. 2012, 25, 1484−1492

Chemical Research in Toxicology

Article

(Berlin, Germany). HPLC-grade methanol, 2-propanol, formic acid, and acetic acid were from Carl Roth GmbH (Karlsruhe, Germany). HMF and all other reagents (analytical grade) were from Sigma. Synthesis of N6-FFM-dAdo, N2-FFM-dGuo, and the Internal Standard Substances [15N5]N6-FFM-dAdo and [13C10,15N5]N2FFM-dGuo. A suspension of 120 mg (477 μmol) of dAdo and 50 mg (219 μmol) of SMF was prepared in 5 mL of 100 mM sodium phosphate buffer (pH 7) and stirred for 24 h at 37 °C. The reaction mixture was subjected to HPLC purification using a Prep LC 150 (Waters) coupled to a 996 PDA detector (Waters). The product was purified via a semipreparative column SunFire C18 OBD (5 μm; Waters), using a 20-min linear gradient starting from 5% methanol to 55% methanol in water (v/v) at a flow rate of 20 mL/min. The fractions containing N6-FFM-dAdo were pooled and freeze-dried, and the HPLC purification was repeated. The purity of N6-FFM-dAdo was >99% as determined by LC-UV-MS/MS. Isotope-labeled [15N5]N6FFM-dAdo was prepared by using 5.1 mg (20 μmol) of [15N5]dAdo starting material but otherwise in the same manner. 1 H NMR of N6-FFM-dAdo (500 MHz, dimethyl sulfoxide-d6) δ [ppm] 9.50 (s, 1H, CHO), 8.49 (bs, 1H, N-H), 8.40 (s, 1H, H2), 8.24 (s, 1H, H8), 7.45 (d, J4,5 = 1.6 Hz, 1H, H3″), 6.52 (d, 1H, H4″), 6.35− 6.37 (m, 1H, H1″), 5.29−5.30 (d, 1H, O-H3′), 5.16−5.18 (m, 1H, OH5′), 4.77 (bs, 2H, N−CH2), 4.40−4.42 (m, 1H, H3′), 3.87−3.89 (m, 1H, H4′), 3.60−3.64 (m, 1H, H5′), 3.50−3.54 (m, 1H, H5′*), 2.49− 2.75 (m, 1H, H2′), 2.24−2.29 (m, 1H, H2′*). MS (ESI+) m/z = 360.1 [M + H]+. The adduct standard N2-FFM-dGuo and the internal standard [ 13 C 10 , 15 N 5 ]N 2 -FFM-dGuo were prepared from dGuo and [13C10,15N5]dGuo analogously to N6-FFM-dAdo and [15N5]N6-FFMdAdo. 1H NMR of N2-FFM-dGuo (500 MHz, dimethyl sulfoxide-d6) δ [ppm] 10.76 (s, 1H, H1), 9.53 (s, 1H, CHO), 7.92 (s, 1H, H8), 7.49− 7.50 (d, J3,4 = 3.54 Hz, 1H, H3″), 7.09 (bs, 1H, N-H), 6.61−6.62 (d, J3,4 = 3.54 Hz, 1H, H4″), 6.12−6.15 (m, 1H, H1′), 5.25−5.27 (d, 1H, O-H3′), 4.84−4.87 (m, 1H, O-H5′), 4.60−4.62 (m, 2H, N−CH2), 4.31−4.34 (m, 1H, H3′), 3.78−3.81 (m, 1H, H4′), 3.51−3.56 (m, 1H, H5′), 3.44−3.48 (m, 1H, H5′*), 2.55−2.60 (m, 1H, H2′), 2.15−2.19 (m, 1H, H2′*). MS (ESI+) m/z = 376.1 [M + H]+. Reaction of Blank DNA with HMF or SMF. DNA (1 mg) was dissolved in 100 mM sodium phosphate buffer (pH 7) at a concentration of 1 mg/mL. After the addition of 100 μL of an aqueous solution of 4 mM SMF, the mixture was incubated at 37 °C for 24 h. The DNA was then precipitated by adding 100 μL of ice-cold 3 M sodium acetate (pH 5.2) and 700 μL of ice-cold 2-ethoxyethanol. Precipitation was completed at −80 °C for 45 min. The suspension was centrifuged at 15,000g and 4 °C for 30 min. The DNA was airdried and redissolved in 500 μL of water. The DNA concentration was determined from the absorbance at 260 nm using a nanodrop ND1000 spectrophotometer (peqlab Biotechnologie, Erlangen, Germany). The solution was stored at −80 °C. Formation of putative DNA adducts of HMF itself was tested by the addition of 100 μL of an aqueous solution of 40 mM HMF to the DNA (1 mg/mL) dissolved in 1 mL of 100 mM sodium phosphate buffer (pH 7). After 60 min at 37 °C, 10 mg NaBH3CN was added in order to reduce the imine formed by HMF and the purine exocyclic amino group to a stable secondary amine, and the DNA was precipitated as described. Enzymatic Digestion of DNA and Solid-Phase Extraction of Nucleoside Adducts. A sample containing 500 μg of DNA was dried together with 202 fmol [ 15N 5 ]N 6 -FFM-dAdo and 625 fmol [13C10,15N5]N2-FFM-dGuo under reduced pressure. The residue was taken up in 280 μL of water, 80 μL of 100 mM sodium succinate (pH 6.0), and 50 mM CaCl2. A 120 μL-aliquot of calf spleen phosphodiesterase (2.2 mU/μL) and micrococcal nuclease (110 mU/μL) was added, and the digestion mixture was incubated at 37 °C for 8 h. After adding 192 μL of 0.5 M Tris (pH 10.9) and 15 μL of shrimp alkaline phosphatase (1 U/μL), the incubation proceeded at 37 °C for 14 h. The DNA digest was diluted by the addition of 700 μL of water and centrifuged at 15,000g for 15 min. Adducts were enriched by solid-phase extraction using Oasis HLB columns (60 mg, Waters), preconditioned with 3 mL of methanol and 3 mL of water. The nucleoside mixtures were loaded onto the columns and washed with 3

Scheme 1. (A) Metabolic Conversion of HMF by SULT and Generation of DNA Adducts via Nucleophilic Substitution and (B) Condensation of an Exocyclic Amino Group in dAdo with the Aldehyde Group of HMFa

a

This condensation yields a Schiff base, which requires reduction to the amine prior to analysis.

cell line.16 Recently, we reported that SMF is formed in mice in vivo after administration of HMF.18 Here, we studied whether HMF or its metabolite SMF also induce gene mutations in mammalian cells. We conducted gene mutation tests at the hprt locus in Chinese hamster lung fibroblast (V79) cells and in a genetically modified variant expressing hSULT1A1 (V79-hSULT1A1).19,20 In addition, we studied DNA adduct formation with HMF and SMF. Potential DNA adducts may be formed by nitrogens of DNA bases either via nucleophilic substitution at the electrophilic carbon atom of SMF (Scheme 1A) or via imine formation with the aldehyde function present in HMF and SMF (Scheme 1B). Stable DNA adducts of the aldehyde moiety-bearing molecules21−26 and also from the reaction with organic sulfate esters have been identified before.27−30 We developed efficient techniques for the detection and quantification of the predominant DNA adducts by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and analyzed DNA samples of V79 cells treated with HMF and SMF.

■

EXPERIMENTAL PROCEDURES

Chemicals. SMF was synthesized as described previously.18 Shrimp alkaline phosphatase (from Pandalus borealis), micrococcal nuclease (from Staphylococcus aureus), and calf spleen phosphodiesterase were purchased from Sigma (Steinheim, Germany). Stable isotope-labeled [15N5]dAdo and [13C10,15N5]dGuo were from Campro Scientific 1485



Article

mL of water/methanol (95:5). DNA adducts were eluted with 3 mL of methanol. After evaporation of the solvents, the residue was taken up in 20 μL of water/methanol (25:75). Samples were centrifuged at 15,000g for 15 min, and the supernatant was used for mass spectrometric analysis. LC-MS/MS Analysis of N6-FFM-dAdo and N2-FFM-dGuo. Samples of DNA adducts were analyzed using an Acquity ultra performance liquid chromatography (UPLC) inlet system (Waters) with a UPLC HSS T3 column (1.8 μM, 2.1 mm × 100 mm, Waters). Water (solvent A) and acetonitrile (solvent B) were acidified with 0.25% acetic acid (v/v) and 0.25% formic acid (v/v). Samples of 8 μL were injected into a starting flow containing 98% solvent A. Nucleoside adducts were eluted with a 10-min gradient to 85% solvent A at 0.35 mL/min flow rate. The inlet system was connected to a Quattro Premier XE mass spectrometer (Waters) with an electrospray interface operated in the positive ion mode. The neutral loss of the furanose (N6-FFM-dAdo 360.1 → 244.1, N2-FFM-dGuo 376.1 → 260.1) was used for quantification (quantif ier). The fragmentation into the nucleoside and the 5-methylfurfural cation (m/z = 109.0) was used to confirm the identity of the adducts (qualif ier). The tune parameters for N6-FFM-dAdo and N2-FFMdGuo were as follows: temperature of the electrospray source, 100 °C; desolvation temperature, 450 °C; desolvation gas, nitrogen (850 L/h); cone gas, nitrogen (50 L/h); collision gas, argon (indicated cell pressure ∼5 × 10−3 mbar). For the fragmentation of N6-FFM-dAdo, collision energies were 33 and 15 eV for the transitions 360.1 → 109.0 and 360.1 → 244.1, respectively. Optimal fragmentation of N2-FFMdGuo required collision energies of 35 and 10 eV for the transitions 376.1 → 109.0 and 376.1 → 260.1, respectively. The dwell time was set to 100 ms, and the capillary voltage was set to 2 kV. The cone and RF1 lens voltages were 25 and 0.1 V, respectively. Data acquisition and handling were performed with MassLynx 4.1 software. Induction of Gene Mutations and Formation of DNA Adducts in Chinese Hamster V79 Cells. Human SULT1A1 was expressed in the V79 Chinese hamster lung fibroblast subclone V79Mz.19,20 V79 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g of D-glucose/L), supplemented with 5% fetal calf serum (v/v), 100 U of penicillin/mL, and 100 μg of streptomycin/ mL at 37 °C, under 5% CO2, and 95% atmospheric humidity. Prior to the test, cells were seeded at a density of 1.5·106 cells per Petri dish (150 cm2) in 30 mL of medium. After 18 h, the test compound dissolved in 300 μL of water was added. Incubations with HMF proceeded for 72 h. Incubations with SMF were stopped after 2 h by exchange of the medium. After 72 h, the medium was removed, and the cells were washed twice with 10 mL of PBS. Cells were harvested with 2 mL of a solution containing 0.025% trypsin (w/v) and 0.01% EDTA (w/v) in PBS. The cell number, expressed as a percentage of the corresponding value of the solvent control cultures, was used as a measure for the cytotoxicity of the treatment. The cells were subcultured in normal medium for three days and then seeded at a density of 1·106 cells per plate (6 replicate plates) in a total volume of 30 mL of DMEM containing 6-thioguanine (7 μg/mL) for the selection of mutants. The total number of colony-forming cells was determined by seeding 100 cells/22 cm2-plate in 5 mL of normal medium (3 replicate plates). After 10 days, the cultures were fixed and stained, the colonies were counted, and mutant frequencies were calculated. In order to analyze DNA adduct levels, cells were incubated with the test compounds as described in the mutagenicity assay. Cells were harvested by treatment with trypsin at the indicated time. Centrifugation at 3,000g for 10 min and two washing steps with 10 mL of PBS yielded a cell pellet, which was stored at −20 °C. The DNA was isolated from the homogenized cells and purified by a phenol/ chloroform extraction described by Gupta.31 The isolated DNA was dissolved in water and stored at −80 °C.

in V79-hSULT1A1 cells in a concentration-dependent manner (Figure 1, ■). The effect was statistically significant at

Figure 1. Mutagenicity (upper panel) and cytotoxicity (lower panel) of HMF in V79 Chinese hamster lung fibroblast cells (○) and V79 cells expressing hSULT1A1 (■). The scale of HMF concentrations is logarithmic. Values are the means and SE of five separate cultures. Student's t test: *, p < 0.05; **, p < 0.01. Lower panel: the cell number reflects HMF cytotoxicity in exposed cultures at the first subculture (expressed as a percentage from the mean cell number observed in the absence of HMF).

concentrations ≥830 μM. In various additional experiments, HMF was tested in one or two concentrations (e.g., as a positive control when testing congeners of HMF). In all these experiments, HMF induced statistically significant mutagenic effects in V79-hSULT1A1 cells (Table 1). In contrast, HMF did not demonstrate statistical significant mutagenic effects in SULT-deficient control cells (Figure 1, ○). Formation of 5-Methylfurfuryl DNA Adducts by SMF in Cell-Free Systems. DNA was incubated with SMF in aqueous solution. The modified DNA was digested, and LCMS/MS analyses techniques of parent scan and constant neutral loss were used to explore possible nucleoside adducts. The parent scan mode allowed us to identify all those molecules that cleaved off the anticipated adducted moiety, the cation of 5-methylfurfural (m/z = 109.0). The analysis is shown in Figure 2, left panel. The most pronounced signals among those molecules that split off a cation fragment with m/ z = 109.0 exhibited mass-to-charge ratios of 360.1, 376.1, and 336.1 corresponding to putative 5-methylfurfuryl adducts of dAdo, dGuo, and dCyt, respectively. These signals were absent in digests of untreated DNA (Figure S1, Supporting Information). In order to corroborate this finding, a constant neutral loss analysis was used to detect those molecules in the DNA digest that, in addition to the fragmentation of the 5methylfurfural cation (x → 109.0), underwent neutral loss of the 2′-deoxyribosyl moiety (x → x − 116.0), another characteristic collision-induced dissociation reaction of putative nucleoside adducts (Figure 2, right panel). The analyses corroborated that all those molecules found to split off a fragment of m/z = 109.0 were also losing a neutral fragment of 116.0 mass units, indicating that one adduct each was formed

■

RESULTS Mutagenicity of HMF in V79 and V79-hSULT1A1 Cells. HMF increased the number of 6-thioguanine resistant mutants 1486



Article

Table 1. Reproducibility of HMF Mutagenicity in V79-hSULT1A1 Cellsa mutants per 106 cells ± SE of n cultures (n in parentheses) exp. no. 1 2 3 4 5 6 7 8 9 10b

HMF (mM) 1.8 4.4 2.9 2.0 1.8 1.8 1.8 1.8 1.8 2.5

solvent control 1.6 3.8 2.5 2.0 3.6 2.6 3.9 3.2 8.2 2.2

± ± ± ± ± ± ± ± ± ±

0.7 1.6 0.1 0.6 0.7 1.4 0.3 0.9 0.6 0.5

(3) (3) (3) (4) (4) (4) (4) (4) (3) (5)

p, t test

HMF-treated 27.4 26.3 21.9 11.9 15.1 26.0 16.8 20.5 19.0 15.7

± ± ± ± ± ± ± ± ± ±

0.1 1.6 1.6 1.2 2.1 2.6 0.8 2.5 1.3 2.7

(2) (2) (3) (4) (4) (4) (4) (4) (2) (5)

1.1 2.5 2.6 3.3 2.3 6.8 4.2 6.0 2.9 1.2

× × × × × × × × × ×

−4

10 10−3 10−4 10−4 10−3 10−4 10−6 10−4 10−3 10−3

cell number HMF-treated cultures % of solvent control 48 71 32 62 71 71 80 109 38 60

a

HMF was tested on different occasions for mutagenicity in V79-hSULT1A1 cells, often as a positive control at a single concentration when testing congeners of HMF. Results of all these experiments are presented here. bConcentration−response curve is presented in Figure 1.

Figure 2. 5-Methylfurfuryl nucleoside adducts in plain DNA incubated with SMF in vitro. The LC-MS/MS parent scan technique (left panel) allowed detecting all molecules with daughter ions of m/z = 109.0, the characteristic mass-to-charge ratio of the positively charged 5-methylfurfuryl fragment. The extraction of parent mass chromatograms showed signals for the masses of 360.1, 376.1, and 336.1, which were indicative of 5methylfurfuryl adducts of dAdo (10.1 min), dGuo (7.7 min), and dCyt (5.1 and 5.2 min), respectively. In addition, a constant neutral loss survey (right panel) was applied to detect all those molecules in the DNA digest abstracting a molecular mass of 116.0, reflecting the loss of a 2′deoxyribosyl fragment. Gray peaks observed in the parent scan were accompanied by coeluting signals reflecting the neutral loss of 116.0 (also marked in gray), which confirms the incidence of the putative 5-methylfurfuryl adducts of dAdo, dGuo, and dCyt. The signals were absent when untreated DNA was digested and analyzed under the same conditions (Figure S1, Supporting Information).

by dAdo (10.2 min) and dGuo (7.7 min). The peaks in the trace 336.1 → 109.0 in Figure 2 suggest that two adducts were formed by dCyt. However, peak intensities were much smaller compared to signals indicative of adducts of dAdo and dGuo. The nucleoside adduct-specific transitions were also found when single 2′-deoxynucleosides were incubated with SMF alone (data not shown). The chromatogram for putative dThd adducts extracted from the parent scan (351.1 → 109.0) was devoid of appreciable signals (Figure 2). Likewise, signals

reflecting the neutral loss of the 2′-deoxyribosyl moiety were not found in incubation mixtures containing either DNA or dThd and SMF. Taken together, the screening results showed that one 5methylfurfuryl adduct of each of the nucleosides dAdo and dGuo, and two possible dCyt adducts at lower yields, were formed. Earlier reports about DNA adducts from reactions with sulfate esters showed that exocyclic nitrogens are predisposed for a nucleophilic attack at the electrophilic benzylic27,28 or 1487



Article

Figure 3. Fragmentation patterns of N2-FFM-dGuo (A) and N6-FFM-dAdo (B) observed by positive ESI-MS/MS collision-induced dissociation. Principal fragmentation ions of N2-FFM-dGuo were m/z = 260.1 (aglycone of N2-FFM-dGuo), m/z = 109.0 (cation of 5-methylfurfural), m/z = 152.0 (protonated Gua), and 117.1 (2′-deoxyribose). The predominant ions of collision-induced dissociation of N6-FFM-dAdo were as follows: m/z = 244.1 (aglycone of N6-FFM-dAdo), m/z = 109.0 (cation of 5-methylfurfural), m/z = 148.0 (N-methyladenine), and 117.1 (2′-deoxyribose).

allylic28,29 carbon atom. Thus, the main nucleoside adducts observed after incubation of blank DNA with SMF may originate from a reaction as depicted in Scheme 1A. In agreement, LC-MS/MS analyses showed that the chemically synthesized adduct standards N6-FFM-dAdo and N2-FFMdGuo coeluted with the respective adducts of dAdo and dGuo observed at 10.2 and 7.7 min, respectively, detected after incubation of DNA with SMF (Figure 2). The MS/MS scan confirmed that fragmentation patterns of the standard substances were identical to those observed in the digest of plain DNA incubated with SMF. Figure 3 depicts structures and fragmentation reactions of N2-FFM-dGuo and N6-FFM-dAdo. The 1H NMR spectra corroborated the anticipated structures of N2-FFM-dGuo and N6-FFM-dAdo (Figures S2 and S3, Supporting Information). Signal intensities of the protons at exocyclic nitrogen atoms were equivalent to single protons, indicating that 5-methylfurfuryl residues were linked to the exocyclic nitrogens, N2 of Ade or N6 of Gua, and not to other sites of the purine rings. Formation of 5-Methylfurfuryl Alcohol DNA Adducts by HMF in Cell-Free Systems. We also studied the potential formation of imine adducts (Scheme 1B). First, single nucleosides dAdo and dGuo were incubated with HMF. The expected imines were reduced by the addition of NaBH3CN as a trapping agent because imine formation is reversible in aqueous environments.32,33 The formation of putative adducts as depicted in Scheme 1B was confirmed by LC-MS/MS multiple reaction monitoring (MRM) using two fragmentation reactions: The neutral loss of the 2′-deoxyribosyl fragment (x → x − 116.0) and the cleavage of the 5-methylfurfuryl alcohol cation creating a fragment of m/z = 111.0 (Figure S5, Supporting Information). Both fragmentations gave rise to coeluting peaks (dAdo adduct, 8.6 min; dGuo adduct, 7.65 min; Figure S6, Supporting Information) indicating the formation of the two adducts N6-((2-hydroxymethylfuran-5-yl)methyl)-2′dAdo (N6-HMF-dAdo) and N2-((2-hydroxymethylfuran-5-yl)methyl)-2′-dGuo (N2-HMF-dGuo). The molecular structures are depicted in Figure S5 (Supporting Information). Neither

the peaks at 8.6 or 7.65 min nor any other signals indicative for the corresponding imines were detectable in samples of nucleosides incubated with HMF but omitting the reducing agent NaBH3CN. In order to investigate the DNA reactivity of HMF, a sample of plain DNA was incubated with HMF, and NaBH3CN was added for reduction of the imines. In a first experiment, HMF was used at a concentration of 4 mM, which is slightly higher than the maximum concentration in the mutagenicity tests. The HMF-derived adducts of dAdo and dGuo were not detected in digests of the modified DNA, neither by parent scan analysis for the fragmentation of the 5-methylfurfuryl alcohol cation (m/z = 111) nor by the neutral loss of the 2′-deoxyribosyl fragment. Likewise, when the sensitive MRM of the specific fragmentations were used, the representative peaks for dAdo and dGuo adducts did not became detectable. Only in incubations of DNA using 200 mM instead of 4 mM HMF, LC-MS/MS analysis revealed minor peaks indicating the formation of N6HMF-dAdo (Figure S7, Supporting Information). Isotope Dilution LC-MS/MS Quantification of N6-FFMdAdo and N2-FFM-dGuo. Two specific fragmentations were characteristic for 5-methylfurfuryl adducts, the neutral loss of the 2′-deoxyribosyl unit (N2-FFM-dGuo 376.1 → 260.1, N6FFM-dAdo 360.1 → 244.1) and the cleavage of the 5methylfurfural cation (N2-FFM-dGuo 376.1 → 109.0, N6-FFMdAdo 360.1 → 109.0) (Figure 3). On the basis of these particular transitions, MRM techniques for adduct detection were devised. In order to quantify N6-FFM-dAdo and N2-FFMdGuo levels in DNA samples, [15N5]N6-FFM-dAdo and [13C10,15N5]N2-FFM-dGuo were synthesized for use as internal standards. They were monitored in parallel to the analyte adducts using equivalent fragmentation reactions: the neutral loss of the 2′-deoxyribosyl moiety ([13C10,15N5]N2-FFM-dGuo 391.1 → 270.1, [15N5]N6-FFM-dAdo 365.1 → 249.1) and the cleavage of the 5-methylfurfural cation ([13C10,15N5]N2-FFMdGuo 391.1 → 109.0, [15N5]N6-FFM-dAdo 365.1 → 109.0). To determine the limit of detection (LOD) of the LC-MS/MS quantification under conditions of sample preparation, plain 1488



Article

DNA was spiked with different amounts of N6-FFM-dAdo and N2-FFM-dGuo and subjected to the entire analysis procedure. The LOD of N6-FFM-dAdo was 0.3 fmol per injection (S/N = 4.5), and the resulting calibration was linear in the range of 0.3−300 fmol (r2 = 0.998). The LOD of N2-FFM-dGuo was 1.3 fmol per injection (S/N = 8.3), and the calibration was linear between 1.3−1300 fmol (r2 = 0.998). Using 500 μg of DNA for the analyses, the LOD values corresponded to 0.08 N6-FFMdAdo and 0.3 N2-FFM-dGuo molecules per 108 2′-deoxynucleosides. Quantification of N2-FFM-dGuo and N6-FFM-dAdo in the DNA of V79 Cells. In order to study the correlation between DNA adduct formation and mutagenic activity, we analyzed N2-FFM-dGuo and N6-FFM-dAdo in DNA samples of V79 cells that were treated with HMF or SMF. For convenient quantification of DNA adducts, isotope-labeled internal standards were added to the DNA samples. After drying, digestion, and adduct enrichment by solid-phase extraction, four molecular fragmentations were analyzed by LC-MS/MS MRM, two for the analyte and two for the internal standard (Figure 4). The area ratio of the peaks from the neutral loss of the 2′-deoxyribosyl moiety were used to calculate the adduct level in the DNA sample. Initially, we analyzed adduct levels in DNA isolates of V79 cells that were treated with high concentrations of HMF. 5-Methylfurfuryl DNA adducts N2FFM-dGuo and N6-FFM-dAdo were not detected in V79 cells

incubated with HMF but were present in the DNA of V79hSULT1A1 cells (Table 2). Table 2. Levels of N6-FFM-dAdo and N2-FFM-dGuo in V79 and V79-hSULT1A1 Cells Exposed to HMF cell line

HMF (mM)

N6-FFM-dAdo per 108 nucleosidesa,b

N2-FFM-dGuo per 108 nucleosidesa,b

0 2.5 0

nd nd nd

nd nd nd

0.25 0.83 2.5

0.17 ± 0.01c 0.41 ± 0.07 0.79 ± 0.14

0.43 ± 0.13d 1.50 ± 0.35 3.32 ± 0.42

V79 V79hSULT1A1

a

nd, not detectable (LODs: 0.08 N6-FFM-dAdo molecules per 108 nucleosides and 0.3 N2-FFM-dGuo molecules per 108 nucleosides). b Means ± SD of three adduct analyses from separate experiments. c The minor N6-FFM-dAdo peaks had S/N-values from 3.1 to 5.7. d The minor N2-FFM-dGuo peaks had S/N-values from 0.7 to 4.1.

The efficiency of DNA repair was studied by incubation of V79 cells with SMF for 2 h, followed by different intervals of recovery in medium without the test compound (0, 2, 6, and 24 h). After each time interval, cells were counted. Adduct concentrations decreased during the recovery phase (Table 3). Table 3. Levels of N6-FFM-dAdo and N2-FFM-dGuo in the DNA of V79 Cells Following Exposure to 300 μM SMF for 2 h and Different Times of Recovery N6-FFM-dAdo per 108 nucleosides recovery time (h) 0 2 6 24

observed 0.46 0.43 0.47 0.24

± ± ± ±

a

0.11 0.06 0.08 0.12

expected in the absence of repairb 0.39 ± 0.10 0.40 ± 0.12 0.15 ± 0.06

N2-FFM-dGuo per 108 nucleosides

observed 4.0 3.3 3.5 1.7

± ± ± ±

a

0.1 0.3 0.3 0.9

expected in the absence of repairb 3.3 ± 0.6 3.5 ± 0.2 1.6 ± 0.3

Values are the means ± SD of three independent experiments, in which DNA adducts were analyzed from V79 cells pooled from 7 to 10 plates at each time point. The cells of each plate were counted. bEven in the absence of repair, the level of adducts would decrease due to dilution via cell replication. The expected adduct levels at certain times were estimated from the initial adduct concentration in each of the three experiments multiplied with a dilution factor, which was calculated as the mean cell number of all cultures harvested at time 0 divided by the cell count at a specific time point. Values are the means ± SD of three independent experiments. a

However, cells proliferated during this time, and no significant decrease in adduct levels was observed if the resulting dilution effect was taken into account. This finding suggests that the adducts N2-FFM-dGuo and N6-FFM-dAdo were largely resistant to DNA repair in V79 cells.

■

Figure 4. LC-MS/MS chromatograms of digested DNA from HMFtreated V79-hSULT1A1 cells. Fragmentations of N2-FFM-dGuo, 376.1 → 260.1 (first panel) and 376.1 → 109.0 (second panel), were monitored together with the transitions 391.1 → 270.1 (third panel) and 391.1 → 109.0 (fourth panel) of the isotope-labeled standard [13C10,15N5]N2-FFM-dGuo (166.7 fmol/injection). The ratio of peak areas for the transition 376.1 → 260.1 (N2-FFM-dGuo) and for the transition 391.1 → 270.1 ([13C10,15N5]N2-FFM-dGuo) was used to calculate the N2-FFM-dGuo content of the DNA. Figure S4 (Supporting Information) shows analytical chromatograms of N6FFM-dAdo in DNA samples of HMF-exposed V79-hSULT1A1 cells.

DISCUSSION DNA adducts of HMF and mutagenic SMF may be formed via two distinct reactions, either by condensation of the aldehyde group with an exocyclic nitrogen of a DNA base leading to an imine or via nucleophilic attack of a nucleophilic atom at the benzylic carbon of SMF. Various studies demonstrate that molecules with aldehyde groups, such as acetaldehyde,21 glyoxal,22 malondialdehyde,23 and glutaraldehyde,24 form 1489



Article

sulfate ester that binds to DNA and is mutagenic. At the moment, this mechanism represents the most plausible reason for hepatocarcinogenicity in HMF-treated female mice observed in the NTP study published in 2008.10 In the future, we will study the relevance of HMF sulfo conjugation for humans determining the impact of single murine and human SULTs on metabolic activation of HMF using transgenic mice expressing individual human SULTs37 or knockout animals which lack endogenous murine SULT.

DNA adducts via condensation with exocyclic nitrogens to imines. A protein adduct was detected that was formed from condensation of HMF with the N-terminal valine of hemoglobin.34 However, imines are transient in the aqueous environment, which impedes the isolation of such adducts. Some condensation reactions, e.g., that of malondialdehyde and glyoxal, eventually lead to stable ring structures.25,26 Others require the reduction of the imine via addition of a reducing agent during DNA isolation, e.g., NaBH3CN. In this case, it is essential to test for artifactual formation of the adducts during the workup because the aldehyde−DNA condensation reaction may preferentially take place under reducing conditions of the adduct isolation procedure.21 In contrast to the genotoxic aldehydes mentioned above, nucleoside adducts were not observed in incubations of plain DNA in the presence of 4 mM HMF after reduction with NaBH3CN. Only when 200 mM HMF was used, LC-MS/MS MRM analysis indicated the formation of the imine adduct of dAdo. This is in agreement with the observation that HMF-related mutagenicity in standard in vitro assays was negligible.3,10−14,16 Thus, direct genotoxicity is not a probable explanation for the rodent carcinogenicity observed for HMF. In contrast, we detected several adducts formed by the reaction of plain DNA in incubation with SMF. Two of the adducts were identified as N2-FFM-dGuo and N6-FFM-dAdo. The use of isotope-labeled dAdo and dGuo in chemical syntheses yielded the adduct standards [15N5]N6-FFM-dAdo and [13C10,15N5]N2-FFM-dGuo. The shift of fragmentational MS/MS-transitions allowed us to apply them as internal standards in sensitive and specific MRM techniques for the quantification of 5-methylfurfuryl adducts in DNA samples. Analogous to the mutagenicity of SMF, we detected N2-FFMdGuo and N6-FFM-dAdo in the DNA of V79 cells that were incubated with SMF but not in the DNA of HMF-treated conventional V79 cells. However, SMF is unstable in aqueous solution, and the passage into the cytosol may be hindered by the sulfate group. It is not known whether SMF enters the cells as it is, as 5-chloromethylfurfural14 or in the form of other reactive decomposition products. Therefore, we conducted experiments with V79 cells engineered for the expression of hSULT1A1, which allow for the study of mutagenicity and DNA modifications after intracellular formation of SMF. We used hSULT1A1 because this particular enzyme has a dominant role in the activation of other benzylic alcohols, shows a broad substrate tolerance, and is highly expressed in numerous tissues.35,36 A concentration-dependent increase in the mutagenicity of HMF was observed in V79-hSULT1A1 cells, confirming our hypothesis that HMF is bioactivated by hSULT1A1. In addition, we found that levels of N2-FFMdGuo and N6-FFM-dAdo in DNA of V79-hSULT1A1 cells increased with HMF concentration. The identity of adducts detected in incubation reactions of plain DNA with SMF in vitro and in the DNA of HMF-treated V79-hSULT1A1 as well as the correlation between DNA adduct level and mutagenicity suggest that sulfo conjugation of HMF and subsequent formation of 5-methylfurfuryl adducts induced the mutations observed in V79-hSULT1A1 cells. In summary, the mutagenicity observed in HMF-treated cell populations of V79-hSULT1A1 cells correlated with the levels of nucleoside adducts N2-FFM-dGuo and N6-FFM-dAdo, which were also detected in plain DNA incubated with chemically prepared SMF. The data verified the hypothesis that hSULT1A1 bioactivates HMF via sulfo conjugation to a

■

ASSOCIATED CONTENT

S Supporting Information *

Parent and constant neutral loss scans of a digest of untreated DNA; 1H NMR spectra of N2-MFF-dGuo and N6-MFF-dAdo; LC-MS/MS analyses of N6-FFM-dAdo in a digest of DNA isolated from V79-hSULT1A1 cells incubated in the presence of 2.5 mM HMF and LC-MS/MS MRM analyses of N2-FFMdGuo and N6-FFM-dAdo in a digested DNA sample isolated from untreated V79-hSULT1A1 cells; molecular structures of putative HMF-adducts of dGuo, N2-((2-hydroxymethylfuran-5yl)methyl)-dGuo (N2-HMF-dGuo) and of dAdo, N6-((2hydroxymethylfuran-5-yl)methyl)-dAdo (N6 -HMF-dAdo); LC-MS/MS analyses of incubation reactions containing dAdo, HMF, and NaBH3CN as well as dGuo, HMF, and NaBH3CN; LC-MS/MS MRM analyses of incubation reactions that contained DNA and 200 mM HMF following reduction by NaBH3CN. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*German Institute of Human Nutrition (DIfE) PotsdamRehbrücke, Department of Nutritional Toxicology, ArthurScheunert-Allee 114-116, 14558 Nuthetal, Germany. Phone: +49-33200-882387. Fax: +49-33200-882426. E-mail: monien@ dife.de. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Jutta Schwenk, Brigitte Knuth, and Martina Scholtyssek for their excellent technical assistance, and Dr. Heinz Frank, who died recently (Biochemical Institute for Environmental Carcinogens, Professor Dr. Gernot Grimmer Foundation, Grosshansdorf, Germany), for synthesizing SMF.

■

ABBREVIATIONS dAdo, 2′-deoxyadenosine; dGuo, 2′-deoxyguanosine; dCyt, 2′deoxycytidine; N 6 -FFM-dAdo, N 6 -((2-formylfuran-5-yl)methyl)-2′-deoxyadenosine; N2-FFM-dGuo, N6-((2-formylfuran-5-yl)methyl)-2′-deoxyguanosine; HMF, 5-hydroxymethylfurfural; LC-MS/MS, liquid chromatography tandem mass spectrometry; LOD, limit of detection; MRM, multiple reaction monitoring; SMF, 5-sulfooxymethylfurfural; SULT, sulfotransferase

■

REFERENCES

(1) Glatt, H. R., and Sommer, Y. (2006) Health Risks by 5Hydroxymethylfurfural (HMF) and Related Compounds, in Acrylamide and Other Health Hazardous Compounds in Heat-Treated Foods (Skog, K., and Alexander, J., Eds.) pp 328−357, Woodhead Publishing, Cambridge, England.

1490



Article

(2) Husoy, T., Haugen, M., Murkovic, M., Jobstl, D., Stolen, L. H., Bjellaas, T., Ronningborg, C., Glatt, H. R., and Alexander, J. (2008) Dietary exposure to 5-hydroxymethylfurfural from Norwegian food and correlations with urine metabolites of short-term exposure. Food Chem. Toxicol. 46, 3697−3702. (3) Janzowski, C., Glaab, V., Samimi, E., Schlatter, J., and Eisenbrand, G. (2000) 5-Hydroxymethylfurfural: assessment of mutagenicity, DNA-damaging potential and reactivity towards cellular glutathione. Food Chem. Toxicol. 38, 801−809. (4) Ulbricht, R. J., Northup, S. J., and Thomas, J. A. (1984) A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions. Fundam. Appl. Toxicol. 4, 843−853. (5) Rufian-Henares, J. A., and de la Cueva, S. P. (2008) Assessment of hydroxymethylfurfural intake in the Spanish diet. Food Addit. Contam., Part A 25, 1306−1312. (6) Abraham, K., Gurtler, R., Berg, K., Heinemeyer, G., Lampen, A., and Appel, K. E. (2011) Toxicology and risk assessment of 5Hydroxymethylfurfural in food. Mol. Nutr. Food Res. 55, 667−678. (7) Zhang, X. M., Chan, C. C., Stamp, D., Minkin, S., Archer, M. C., and Bruce, W. R. (1993) Initiation and promotion of colonic aberrant crypt foci in rats by 5-hydroxymethyl-2-furaldehyde in thermolyzed sucrose. Carcinogenesis 14, 773−775. (8) Surh, Y. J., Liem, A., Miller, J. A., and Tannenbaum, S. R. (1994) 5-Sulfooxymethylfurfural as a possible ultimate mutagenic and carcinogenic metabolite of the Maillard reaction product, 5hydroxymethylfurfural. Carcinogenesis 15, 2375−2377. (9) Svendsen, C., Husoy, T., Glatt, H. R., Paulsen, J. E., and Alexander, J. (2009) 5-Hydroxymethylfurfural and 5-sulfooxymethylfurfural increase adenoma and flat ACF number in the intestine of Min/+ mice. Anticancer Res. 29, 1921−1926. (10) National Toxicology Program (2008) Toxicology and carcinogenesis studies of 5-(Hydroxymethyl)-2-furfural (CAS No. 67−47−0) in F344/N rats and B6C3F1 mice (gavage studies). Natl. Toxicol. Program Tech. Rep. Ser. 554, 1−202. (11) Aeschbacher, H. U., Chappuis, C., Manganel, M., and Aeschbach, R. (1981) Investigation of Maillard products in bacterial mutagenicity test systems. Prog. Food Nutr. Sci. 5, 279−293. (12) Kasai, H., Kumeno, K., Yamaizumi, Z., Nishimura, S., Nagao, M., Fujita, Y., Sugimura, T., Nukaya, H., and Kosuge, T. (1982) Mutagenicity of methylglyoxal in coffee. Gann 73, 681−683. (13) Lee, Y. C., Shlyankevich, M., Jeong, H. K., Douglas, J. S., and Surh, Y. J. (1995) Bioactivation of 5-hydroxymethyl-2-furaldehyde to an electrophilic and mutagenic allylic sulfuric acid ester. Biochem. Biophys. Res. Commun. 209, 996−1002. (14) Surh, Y. J., and Tannenbaum, S. R. (1994) Activation of the Maillard reaction product 5-(hydroxymethyl)furfural to strong mutagens via allylic sulfonation and chlorination. Chem. Res. Toxicol. 7, 313−318. (15) Shinohara, K., Kim, E.-H., and Omura, H. (1986) Furans as the mutagens formed by amino-carbonyl reactions. Dev. Food Sci. 13, 353− 362. (16) Glatt, H. R., Schneider, H., and Liu, Y.-G. (2005) V79hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals. Mutat. Res. 580, 41−52. (17) Glatt, H. R., Schneider, H., Murkovic, M., Monien, B. H., and Meinl, W. (2011) Hydroxymethyl-substituted furans: mutagenicity in Salmonella typhimurium strains engineered for expression of various human and rodent sulphotransferases. Mutagenesis 27, 41−48. (18) Monien, B. H., Frank, H., Seidel, A., and Glatt, H. R. (2009) Conversion of the common food constituent 5-hydroxymethylfurfural into a mutagenic and carcinogenic sulfuric acid ester in the mouse in vivo. Chem. Res. Toxicol. 22, 1123−1128. (19) Kreis, P., Brandner, S., Coughtrie, M. W. H., Pabel, U., Meinl, W., Glatt, H. R., and Andrae, U. (2000) Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant. Carcinogenesis 21, 295−299. (20) Meinl, W., Pabel, U., Osterloh-Quiroz, M., Hengstler, J. G., and Glatt, H. R. (2006) Human sulfotransferases are involved in the

activation of aristolochic acids and are expressed in renal target tissue. Int. J. Cancer 118, 1090−1097. (21) Chen, L., Wang, M., Villalta, P. W., and Hecht, S. S. (2007) Liquid chromatography-electrospray ionization tandem mass spectrometry analysis of 7-ethylguanine in human liver DNA. Chem. Res. Toxicol. 20, 1498−1502. (22) Olsen, R., Ovrebo, S., Thorud, S., Lundanes, E., Thomassen, Y., Greibrokk, T., and Molander, P. (2008) Sensitive determination of a glyoxal-DNA adduct biomarker candidate by column switching capillary liquid chromatography electrospray ionization mass spectrometry. Analyst 133, 802−809. (23) Wang, M., Dhingra, K., Hittelman, W. N., Liehr, J. G., de Andrade, M., and Li, D. (1996) Lipid peroxidation-induced putative malondialdehyde-DNA adducts in human breast tissues. Cancer Epidemiol. Biomarkers Prev. 5, 705−710. (24) Olsen, R., Backman, J., Molander, P., Ovrebo, S., Thorud, S., Lundanes, E., Greibrokk, T., and Kronberg, L. (2007) Characterization of adducts formed in the reaction of glutaraldehyde with 2′deoxyadenosine. Chem. Res. Toxicol. 20, 965−974. (25) Chaudhary, A. K., Nokubo, M., Reddy, G. R., Yeola, S. N., Morrow, J. D., Blair, I. A., and Marnett, L. J. (1994) Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265, 1580−1582. (26) Chaudhary, A. K., Reddy, G. R., Blair, I. A., and Marnett, L. J. (1996) Characterization of an N6-oxopropenyl-2′-deoxyadenosine adduct in malondialdehyde-modified DNA using liquid chromatography/electrospray ionization tandem mass spectrometry. Carcinogenesis 17, 1167−1170. (27) Monien, B. H., Müller, C., Engst, W., Frank, H., Seidel, A., and Glatt, H. R. (2008) Time course of hepatic 1-methylpyrene DNA adducts in rats determined by isotope dilution LC-MS/MS and 32Ppostlabeling. Chem. Res. Toxicol. 21, 2017−2025. (28) Punt, A., Delatour, T., Scholz, G., Schilter, B., van Bladeren, P. J., and Rietjens, I. M. (2007) Tandem mass spectrometry analysis of N2(trans-isoestragol-3′-yl)-2′-deoxyguanosine as a strategy to study species differences in sulfotransferase conversion of the proximate carcinogen 1′-hydroxyestragole. Chem. Res. Toxicol. 20, 991−998. (29) Kitagawa, M., Ravindernath, A., Suzuki, N., Rieger, R., Terashima, I., Umemoto, A., and Shibutani, S. (2000) Identification of tamoxifen-DNA adducts induced by α-acetoxy-N-desmethyltamoxifen. Chem. Res. Toxicol. 13, 761−769. (30) Monien, B. H., Herrmann, K., Florian, S., and Glatt, H. R. (2011) Metabolic activation of furfuryl alcohol: formation of 2methylfuranyl DNA adducts in Salmonella typhimurium strains expressing human sulfotransferase 1A1 and in FVB/N mice. Carcinogenesis 32, 1533−1539. (31) Gupta, R. C. (1984) Nonrandom binding of the carcinogen Nhydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. U.S.A. 81, 6943−6947. (32) Godoy-Alcántar, C., Yatsimirski, A., and Lehn, J.-M. (2005) Structure-stability correlations for imine formation in aqueous solution. J. Phys. Org. Chem. 18, 979−985. (33) Wang, M., McIntee, E. J., Cheng, G., Shi, Y., Villalta, P. W., and Hecht, S. S. (2000) Identification of DNA adducts of acetaldehyde. Chem. Res. Toxicol. 13, 1149−1157. (34) Davies, R., Hedebrant, U., Athanassiadis, I., Rydberg, P., and Tornqvist, M. (2009) Improved method to measure aldehyde adducts to N-terminal valine in hemoglobin using 5-hydroxymethylfurfural and 2,5-furandialdehyde as model compounds. Food Chem. Toxicol. 47, 1950−1957. (35) Glatt, H. R., and Meinl, W. (2005) Sulfotransferases and acetyltransferases in mutagenicity testing: technical aspects. Methods Enzymol. 400, 230−249. (36) Glatt, H. R., Boeing, H., Engelke, C. E. H., Kuhlow, A., Ma, L., Pabel, U., Pomplun, D., Teubner, W., and Meinl, W. (2001) Human cytosolic sulphotransferases: genetics, characteristics, toxicological aspects. Mutat. Res. 482, 27−40. (37) Dobbernack, G., Meinl, W., Schade, N., Florian, S., Wend, K., Voigt, I., Himmelbauer, H., Gross, M., Liehr, T., and Glatt, H. R. 1491



Article

(2011) Altered tissue distribution of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine DNA adducts in mice transgenic for human sulfotransferases 1A1 and 1A2. Carcinogenesis 32, 1734−1740.

1492


Mutagenicity of 5-Hydroxymethylfurfural in V79 Cells Expressing

Recommend Documents