TOXICOLOGICAL SCIENCES, 149(1), 2016, 192–201
doi: 10.1093/toxsci/kfv228
Advance Access Publication Date: October 9, 2015
Research Article
Conversion of Suspected Food Carcinogen
5-Hydroxymethylfurfural by Sulfotransferases and
Aldehyde Dehydrogenases in Postmitochondrial
Tissue Preparations of Humans, Mice, and Rats
Benjamin Sachse,*,† Walter Meinl,† Hansruedi Glatt,‡ and
Bernhard H. Monien*,‡,1
*Research Group Genotoxic Food Contaminants, German Institute of Human Nutrition (DIfE) PotsdamRehbrücke, 14558 Nuthetal, Germany; †Department of Molecular Toxicology, German Institute of Human
Nutrition (DIfE) Potsdam-Rehbrücke, 14558 Nuthetal, Germany; and ‡Department of Food Safety, Federal
Institute for Risk Assessment (BfR), 10589 Berlin, Germany
1
To whom correspondence should be addressed at Federal Institute for Risk Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany.
Fax: þ49-30-18412-3003. E-mail: [email protected]
ABSTRACT
The food contaminant 5-hydroxymethylfurfural (HMF) is formed by heat- and acid-catalyzed reactions from carbohydrates.
More than 80% of HMF is metabolized by oxidation of the aldehyde group in mice and rats. Sulfo conjugation yields
mutagenic 5-sulfoxymethylfurfural, the probable cause for the neoplastic effects observed in HMF-treated rodents.
Considerable metabolic differences between species hinder assessing the tumorigenic risk associated with human dietary
HMF uptake. Here, we assayed HMF turnover catalyzed by sulfotransferases or by aldehyde dehydrogenases (ALDHs) in
postmitochondrial preparations from liver, kidney, colon, and lung of humans, mice, and rats. The tissues-specific
clearance capacities of HMF sulfo conjugation (CLSC) and ALDH-catalyzed oxidation (CLOX) were concentrated to the liver.
The hepatic clearance CLSC in mice (males: 487 ml/min/kg bw, females: 2520 ml/min/kg bw) and rats (males: 430 ml/min/kg bw,
females: 198 ml/min/kg bw) were considerably higher than those in humans (males: 21.2 ml/min/kg bw, females: 32.2 ml/min/
kg bw). The ALDH-related clearance rates CLOX in mice (males: 3400 ml/min/kg bw, females: 1410 ml/min/kg bw) were
higher than those of humans (males: 436 ml/min/kg bw, females: 646 ml/min/kg bw) and rats (males: 627 ml/min/kg bw,
females: 679 ml/min/kg bw). The ratio of CLOX to CLSC was lowest in female mice. This finding indicated that HMF sulfo
conjugation was most substantial in the liver of female mice, a target tissue for HMF-induced neoplastic effects, and that
humans may be less sensitive regarding HMF sulfo conjugation compared with the rodent models.
Key words: 5-hydroxymethylfurfural; metabolism; sulfotransferase; aldehyde dehydrogenase
5-Hydroxymethylfurfural (HMF) is formed by heat- and acidinduced dehydration of hexoses. It is present in a variety of
foodstuffs resulting in an average daily uptake of 4–30 mg HMF
per person (Abraham et al., 2011; Husoy et al., 2008; Murkovic
and Pichler, 2006). Recently, HMF attracted considerable interest
as a sustainable building block for the synthesis of polymers,
such as furanic polyesters, polyamides, and polyurethanes and as
alternative transportation fuel (Binder and Raines, 2009; Bredihhin
et al., 2013; Buntara et al., 2011). Many efforts were directed towards
the preparation of HMF from the catalytic turnover of C6-carbohydrates in biomass as a renewable source (Choudhary et al., 2013;
Pagan-Torres et al., 2012; Roman-Leshkov et al., 2006). Also, the
C The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.
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SACHSE ET AL.
pharmacological properties of HMF were studied. It was found to
increase the oxygen affinity of human hemoglobin and to reduce
sickling of erythrocytes in patients with sickle cell disease
(Abdulmalik et al., 2005). A phase I clinical trial initiated by AesRx
(Newton, Massachusetts) was completed evaluating the effect of
single HMF doses against sickle cell anemia (ClinicalTrials.gov
Identifier: NCT01597401).
Health concerns have to be taken into consideration when
HMF is to be used as pharmacon or as a large-scale chemical
building block. HMF induced neoplasia in a series of animal experiments (Surh et al., 1994; Svendsen et al., 2009; Zhang et al.,
1993). For example, it caused liver adenoma in female mice in a
2-years bioassay conducted by the National Toxicology Program
(2008). The relevance of this finding for humans has been questioned because liver adenomas were also detectable in control
animals and a dose-response relationship was not observed
(Abraham et al., 2011). Studies conducted in various standard
test systems, eg, a bacterial reverse mutation test using
Salmonella typhimurium strains (Lee et al., 1995a), an HPRT test
and an in vitro comet assay in V79 cells (Janzowski et al., 2000), a
sister chromatid exchange assay in V79 cells (Glatt et al., 2005),
and the micronucleus test in peripheral blood cells of mice
(National Toxicology Program, 2008), showed that HMF is barely
genotoxic. However, 5-sulfoxymethylfurfural (SMF) formed
from sulfo conjugation of HMF was mutagenic (Lee et al., 1995b;
Surh et al., 1994). Also, HMF was mutagenic in V79 cells
expressing human (h) sulfotransferase (SULT) 1A1, which was
accompanied by formation of specific DNA adducts N6-((2formylfuran-5-yl)methyl)-20 -deoxyadenosine (N6-FFM-dA) and
N2-((2-formylfuran-5-yl)methyl)-20 -deoxyguanosine
(N2-FFMdG) (Monien et al., 2012). Thus, it is possible that neoplastic
effects of HMF originate from SULT-catalyzed bioactivation.
The extent of DNA damage caused by HMF exposure in a
particular tissue depends partially on the expression of certain
SULT forms and their specific catalytic efficiencies for HMF
turnover. Recent studies of HMF sulfo conjugation by individual
SULT forms showed that primarily SULT1A1 orthologues from
humans, mice, and rats contribute to HMF bioactivation (Sachse
et al., 2014). However, due to the lack of consistent data about
SULT expression in all 3 species the reported enzyme activities
do not allow a comparative quantitative assessment of HMF
sulfo conjugation. Also, the internal dose of SMF may be influenced by various other species- and tissue-dependent factors,
such as blood perfusion and detoxification of HMF and SMF. For
an improved understanding of the hazard potential related to
human HMF exposure, we studied HMF sulfo conjugation and
oxidative turnover to 5-hydroxymethyl-2-furoic acid (HMFA),
the most important pathway of HMF metabolism (Germond
et al., 1987), in liver, kidney, lung, and colon of humans, mice,
and rats. Michaelis-Menten parameters KM and VMAX of HMF
turnover allowed estimating tissue-specific clearance rates of
HMF sulfo conjugation and HMF oxidation in all 3 species
(Obach et al., 1997).
MATERIALS AND METHODS
Chemicals. HPLC-grade methanol, 2-propanol, formic acid, and
acetic acid were from Carl Roth GmbH (Karlsruhe, Germany), and
HPLC-grade water was prepared using a Milli-Q Integral Water
Purification System from Millipore Merck (Darmstadt, Germany).
[13C6]D-glucose was purchased from Silantes GmbH (Munich,
Germany). HMFA was from Toronto Research Chemicals
(Toronto, Canada) and HMF and all other reagents (analytical
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193
grade) were from Sigma-Aldrich (Steinheim, Germany). SMF was
synthesized as described recently (Monien et al., 2009).
Synthesis of [13C6]HMF. The [13C6]D-glucose was converted to
[13C6]HMF via a biphasic reaction consisting of an aqueous
phase for the Lewis acid-catalyzed dehydration and an organic
layer for continuous extraction of the product (Pagan-Torres
et al., 2012). A portion of 2.7 mg (20 mmol) AlCl3 was dissolved in
4 ml saturated brine and 203 mg (1091 mmol) [13C6]D-glucose was
added. The solution was mixed with 8 ml 2-sec-butylphenol and
the mixture was refluxed at 160 C for 24 h. The 2-sec-butylphenol was removed and diluted with 60 ml hexane. The organic
phase was extracted 3 times with 20 ml water. The water fractions were pooled and extracted 3 times with 100 ml tetrahydrofuran. The organic phase was carefully removed at 50 mbar and
50 C. Caution: [13C6]HMF may evaporate as well at lower pressure or at higher temperatures. The analysis of the [13C6]HMF by
ultra performance liquid chromatography (UPLC)-UV-MS/MS
showed that the yield was 43.5%. The [13C6]HMF was employed
for sulfation without further purification. 13C NMR (600 MHz,
dimethyl sulfoxide-d6) d ¼ 178.0 (CO, d), 161.7–162.6 (C5, t), 151.2–
152.2 (C2, t), 124.2–124.9 (C3, t), 109.3–110.1 (C4, t), 55.7–56.1 (COH, d). The 13C NMR spectrum is shown in Supplementary
Figure S1 of the supplementary data.
Synthesis of [13C6]SMF. The sulfation of [13C6]HMF was basically
performed as described (Monien et al., 2009). Briefly, 14 mg
(107 mmol) of [13C6]HMF was dissolved in 2 ml dimethylformamide (DMF) under argon. The solution was cooled down to 0 C
and 34 mg (214 mmol) pyridine:SO3 was added. The solution was
stirred for 5 h at 0 C. Subsequently, 642 ml of 500 mM NaOMe
(321 mmol) in methanol was added. After stirring for 30 min, the
mixture was added dropwise to 30 ml of cold ether. The suspension was centrifuged and the precipitate was washed 2 times
with 10 ml cold ether and dried for 60 min under high vacuum.
[13C6]SMF was subjected to preparative HPLC using a Prep LC 150
(Waters, Eschborn, Germany) with a SunFire Prep C18 OBD column (5 mm, 19 150 mm; Waters) coupled to a photo diode array
detector 996 (Waters). The eluents were 5 % methanol in water
(solvent A) and methanol (solvent B). A 20-min linear gradient
was applied starting from 100% solvent A to 50% solvent A at a
flow rate of 20 ml/min. In order to minimize the loss of the reactive [13C6]SMF (t1/2 ¼ 120 min at 37 C in the presence of water)
the eluents were refrigerated to 4 C and glass tubes in the fraction collector were kept on ice. The fractions containing
[13C6]SMF were pooled. The purity of [13C6]SMF was >99% as
indicated by UPLC-UV-MS/MS. A standard solution of 428 nM
[13C6]SMF in 2-propanol was prepared and stored at 80 C.
Synthesis of [13C6]HMFA. Three milligrams (22.7 mmol) [13C6]HMF
was dissolved in 7 ml of an aqueous solution of 100 mM glycine/
sodium hydroxide (pH 10.0), 20 mM b-nicotinamide adenine
dinucleotide (NADþ) and 7.7 mg cytosolic protein prepared from
transgenic S. typhimurium TA100 expressing human (h) aldehyde
dehydrogenase (ALDH) 3A1 (Kollock et al., 2009). The reaction
mixture was incubated at 37 C for 24 h, diluted with 7 ml methanol and centrifuged at 15 000 g for 10 min. The clear supernatant was concentrated under reduced pressure and the
remaining aqueous solution was subjected to purification using
the preparative HPLC described above with a Delta Pak C4 column (15 mm, 19 300 mm; Waters). The product was eluted with
water (solvent A) and methanol (solvent B) using a 20-min linear
gradient starting from 100% solvent A to 60% solvent A at a flow
rate of 15 ml/min. Both solvents were acidified with 0.25% acetic
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acid and 0.25% formic acid. After removal of the solvents under
reduced pressure, [13C6]HMFA was purified again using a 20-min
linear gradient starting from 100% solvent A (2% methanol) to
100% solvent B (75% methanol) at a flow rate of 15 ml/min. The
product fractions were pooled and the purity (> 99%) was determined via UPLC and UV detection. Following dilution with
methanol the concentration (1.69 mM [13C6]HMFA) was determined using a standard calibration line of commercially available HMFA, which was linear in the range between 20 nM and
50 mM.
Postmitochondrial supernatants of tissue samples. Samples of
human liver, lung, kidney, and colon, 5 of each organ and each
gender, were generously provided by Biopredic International
(Rennes, France). The samples contained healthy tissue from the
margins around sites of tumor resections, whereas colon tissue
samples were mainly from patients with diverticular disease.
After surgical intervention the samples were stored at 80 C.
The studies conducted were acknowledged by the ethic committee of the University of Potsdam (Germany). Animal tissues (liver,
lung, kidney, and colon) were obtained from FVB/N mice and
Wistar rats (Charles River Laboratories, Sulzfeld, Germany).
Tissue samples of 0.2–1.0 g were homogenized in 3 ml KCP
medium per gram of tissue (150 mM potassium chloride, 10 mM
sodium phosphate buffer, pH 7.4) using a Potter-Elvehjem
homogenizer. Homogenates were centrifuged at 9000 g for
20 min and the protein content of the supernatant (postmitochondrial supernatants [PMS]) was determined. Aliquoted volumes between 100 and 500 ml were stored at 80 C.
HMF sulfo conjugation in the presence of PMS. Sulfo conjugation of
HMF was determined in reaction mixtures of 100 ml containing
25–200 mg protein, 50 mM potassium phosphate buffer (pH 7.4),
5 mM MgCl2, 50 mM 30 -phosphoadenosine-50 -phosphosulfate
(PAPS) and different concentrations of HMF. Previous experiments ensured that measurements were recorded under
“linear” conditions, ie, doubling of incubation time or protein
concentration was doubling the amount of SMF formed. The
reactions were stopped after 15 min by adding 300 ml of a solution of 428 nM [13C6]SMF in cold 2-propanol. The mixture was
vortexed briefly and centrifuged at 15 000 g for 10 min. The
clear supernatants were analyzed by UPLC-MS/MS. Incubations
were performed in duplicate.
Quantification of SMF by UPLC-MS/MS. SMF and [13C6]SMF were
analyzed using an Acquity UPLC system (Waters) coupled to a
tandem-quadrupole mass spectrometer Quattro Premier XE
(Waters). Samples of 4 ml were injected onto a SunFire C18 column (5 mm, 3 150 mm; Waters). The mobile phase consisted of
2 solvents: 10 mM ammonium acetate/methanol (95:5, solvent
A) and acetonitrile/methanol (95:5, solvent B). The gradient for
elution of SMF and subsequent reequilibration of the column
was as follows: 0–1 min, 95% A; 1–3 min, 95–20% A; 3–4 min, 5%
A; 4–6 min, 95% A. The flow rate was 0.5 ml/min and the column
was kept at room temperature (20 C). The mass spectrometer
was operated in the negative ion mode. A multiple reaction
monitoring (MRM) method was devised for specific detection
and quantification of SMF using the fragmentation transitions
m/z ¼ 204.9 ! 95.9 and m/z ¼ 204.9 ! 80.9. The corresponding
transitions were used to monitor the internal standard
[13C6]SMF, m/z ¼ 210.9 ! 95.9 and m/z ¼ 210.9 ! 80.9. The tune
parameters were: temperature of the electrospray source 120 C;
desolvation temperature 475 C; desolvation gas: nitrogen (800 l/
h); cone gas: nitrogen (100 l/h); collision gas: argon (indicated
cell pressure 5103 mbar). For the fragmentation of SMF
([13C6]SMF), collision energies were 18 and 23 eV for the transitions m/z ¼ 204.9 ! 80.9 (m/z ¼ 210.9 ! 80.9) and for m/z ¼ 204.9
! 95.9 (m/z ¼ 210.9 ! 95.9), respectively. The dwell time was set
to 100 ms, and capillary voltage was set to 0.35 kV. The cone and
RF1 lens voltages were 32 V and 0.1 V, respectively.
HMF oxidation in the presence of PMS. The reactions were performed in 100 ml mixtures containing 2.5–20 mg protein, 50 mM
potassium phosphate buffer (pH 7.4), 5 mM NADþ, and different
concentrations of HMF. Initial experiments ensured that the
incubation conditions were in a linear range. After incubation
for 15 min at 37 C, the reactions were stopped by adding 200 ml
methanol containing 1.69 mM [13C6]HMFA. The samples were
centrifuged for 10 min at 15 000 g and the HMFA content of the
supernatant was determined by UPLC-MS/MS. Incubations were
performed in duplicate.
Quantification of HMFA by UPLC-MS/MS. HMFA and [13C6]HMFA
were analyzed with the UPLC-MS/MS system described above.
Here, an HSS T3 column (1.8 mm, 2.1 100 mm; Waters) was
used and the analytes were eluted with a 3-min linear gradient
of water (solvent A) and acetonitrile (solvent B) starting from
98% solvent A to 50% solvent A and a flow rate of 0.35 ml/min.
Both solvents were acidified with 0.25% acetic acid and 0.25%
formic acid. Three specific fragmentations for HMFA
(m/z ¼ 143.1 ! 125.1, m/z ¼ 143.1 ! 97.1 and m/z ¼ 143.1 ! 79.1)
were monitored together with the corresponding transitions of
[13C6]HMFA (m/z ¼ 149.1 ! 131.1, m/z ¼ 149.1 ! 102.1 and
m/z ¼ 149.1 ! 84.1) using collision energies of 10, 15, and 20 eV,
respectively. The transitions of m/z ¼ 143.1 ! 79.1 and
m/z ¼ 149.1 ! 84.1 were used as quantifiers whereas the presence of the other fragmentations ensured the specificity of
detection. The tune parameters were as follows: temperature of
the electrospray source 100 C; desolvation temperature 490 C;
desolvation gas nitrogen (850 l/h); cone gas nitrogen (50 l/h); collision gas argon (indicated cell pressure 5 10-3 mbar). The
dwell time was set to 50 ms and capillary voltage was set to
0.75 kV. The cone voltage and the RF1 lens voltage were set to
18 V and 0.1 V, respectively.
HMF clearance by sulfo conjugation and by oxidation. Kinetic parameters were usually derived by fitting of the HMF concentration
dependent turnover rates with the conventional MichaelisMenten model using the nonlinear regression algorithm in
Sigma Plot 11 from Systat Software (Erkrath, Germany). The
HMF sulfo conjugation in PMS of some tissue samples was
described better under consideration of substrate inhibition
(Houston and Kenworthy, 2000):
V ¼ VMAX =ð1 þ KM =½HMF þ ½HMF=KI Þ
(1)
with the rate of the reaction V, the substrate concentration
[HMF], the Michaelis-Menten constant KM, the maximum rate
VMAX and the substrate inhibition constant KI.
The tissue-specific clearance rates (CL) were estimated from
the Michaelis-Menten parameters by scaling up (Obach et al.,
1997; Soars et al., 2002):
CL ¼
VMAX
protein in PMS
tissue
ðmg=gÞ ðg=kgÞ
tissue
body weight
KM
(2)
For a consistent evaluation, we used the initial slope of the
HMF turnover rates determined at the 3 lowest concentrations
SACHSE ET AL.
as intrinsic clearance VMAX/KM. The relative tissue weight of
human liver was 24.7 and 25.4 g/kg body weight for males and
females, respectively, and of human lung 18.3 and 17.5 g/kg
body weight for males and females, respectively. The kidney
weight was 4.7 g/kg body weight for both sexes (de la
Grandmaison et al., 2001). Yamanaka et al. (2007) reported 30 g/
kg body weight for colon from both sexes. The tissue weights of
FVB/N mice and of Wistar rats were determined when the
organs were removed. The data for the calculation of the clearance rates are summarized in Supplementary Tables S3 and S4
(mice) and Supplementary Tables S5 and S6 (rats) of the supplementary data.
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195
the analyte concentration corresponding to the arithmetic mean
area of the background signal from 10 control samples plus 3 SD.
The LOD, independent from the tissue, was 12 nM SMF, which
corresponds to 50 fmol SMF per injection.
HMF Sulfo Conjugation in PMS From Tissues Homogenates
The rates of sulfo conjugation in the presence of PMS prepared
from liver, kidney, colon, and lung of human, mouse and rat
were measured at different HMF concentrations. Figure 3 shows
HMF turnover kinetics by hepatic PMS preparations. The turnover rate decreased at concentrations higher than about 10–
RESULTS
Quantification of SMF by Isotope-Dilution UPLC-MS/MS
In previous studies, we used UPLC-MS/MS and external calibration lines of SMF to assess HMF sulfo conjugation by different
SULT forms in vitro (Sachse et al., 2014) and in FVB/N mice in vivo
(Monien et al., 2009). To reduce the technical inaccuracy we synthesized [13C6]SMF as an internal standard for the quantification
of the reaction product. Analogous to the unlabeled substance,
the daughter scan of [13C6]SMF showed 2 collision-induced fragmentations: the dissociation of the sulfate ion radical (m/
z ¼ 210.9 ! 95.9) and of the protonated sulfonate ion (m/
z ¼ 210.9 ! 80.9) (Figure 1). Based on these transitions an MRM
method for the specific detection of [13C6]SMF and the simultaneous recording of SMF (m/z ¼ 204.9 ! 95.9 and m/z ¼ 204.9 !
80.9) was developed.
The solution of [13C6]SMF in 2-propanol was added to terminate the incubation reactions and the clear supernatants were
directly injected for MRM analysis. Figure 2 shows the chromatography of SMF and [13C6]SMF in a sample containing 100 mg
mouse kidney protein and 10 mM HMF. The background of the
chromatograms was essentially free of interfering signals. The
limit of detection (LOD) for the quantification of SMF was estimated from the background integral of the “quantifier” trace (the
total ion current of both transitions) at the precise retention time
in incubations without HMF (‘controls’). The LOD was defined as
FIG. 2. LC-MS/MS chromatograms of an incubation with mouse kidney protein
(1 mg/ml of PMS) and HMF (10 mM) fortified with the sulfo group donor PAPS
(50 mM). After stopping the incubation, fragmentations of SMF, m/z ¼ 204.9 !
95.9 and m/z ¼ 204.9 ! 80.9 (upper panels), were monitored together with the
transitions m/z ¼ 210.9 ! 95.9 and m/z ¼ 210.9 ! 80.9 (lower panels) of the isoFIG. 1. Fragmentation pattern of [13C6]SMF observed by negative ESI-MS/MS collision-induced dissociation. The asterisks denote the presence of
13
tope-labeled standard [13C6]SMF (1.28 pmol/injection). The ratio of the sums of
C. Principal
both peak areas was used to calculate the SMF content in the incubation.
fragment ions of [13C6]SMF (parent ion with m/z ¼ 210.9) were the sulfonate ion,
HSO3- (m/z ¼ 80.9) and the sulfate ion radical, SO4, (m/z ¼ 95.9).
Control samples without HMF did not show a signal at the retention time of
SMF.
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(1.6 mM) was about 2-fold lower compared with that in male
liver (3.0 mM), while the VMAX was about 2-fold higher. In rats,
the KM values of hepatic HMF sulfo conjugation in male and
female rats were similar, but the VMAX was more than 2-fold
higher in male liver (766 pmol/mg/min) compared with that in
female liver (308 pmol/mg/min).
Quantification of HMFA by Isotope-Dilution UPLC-MS/MS
Previous studies showed that 80%–90% of a dose of [14C]HMF
was metabolized via oxidation of the aldehyde group in rats and
mice (Godfrey et al., 1999). In order to compare the ALDH-catalyzed turnover of HMF to HMFA in different tissues we devised
an in vitro assay in which PMS samples were incubated with
HMF and NADþ. The formation of HMFA was monitored by
three characteristic mass spectrometric fragmentations (m/
z ¼ 143.1 ! 125.1, m/z ¼ 143.1 ! 97.1 and m/z ¼ 143.1 ! 79.1).
The simultaneous detection of the internal reference compound
[13C6]HMFA using the analogous transitions (m/z ¼ 149.1 !
131.1, m/z ¼ 149.1 ! 102.1 and m/z ¼ 149.1 ! 84.1) allowed quantifying the HMFA content in the incubation mixtures. The mass
spectrometric daughter scan of [13C6]HMFA is shown in
Supplementary Figure S2 of the supplementary data. Exemplary
chromatograms for the detection of HMFA and of [13C6]HMFA
are depicted in Supplementary Figure S3 of the supplementary
data. The LOD was calculated from the arithmetic mean area of
the background noise at the precise retention time of HMFA
from 10 incubations without HMF plus 3 SD. The resulting LOD
was 50 nM HMFA regardless of the tissue, which corresponds to
200 fmol HMFA per injection.
FIG. 3. HMF sulfo conjugation in PMS preparations of liver tissues from human
(A), mouse (B), and rat (C). The rates at single HMF concentrations are
means 6 SE of 5 different samples of males (solid squares) and females (open
circles). Fitting of the data with the Michaelis-Menten model including a substrate inhibitory effect at elevated HMF concentrations yielded values for the
catalytic parameters KM and VMAX, which are summarized in Table 1.
25 mM HMF, which was observed for most of the tissue samples
included in this study. Assuming a substrate inhibitory effect,
apparent KM and VMAX values (Table 1) were derived by approximations of the kinetic data with the extended MichaelisMenten equation (equation 1). Values of R2 > 0.98 (> 0.90)
showed the goodness of 77% (98 %) of the 120 fittings (10 data
sets of 4 tissues from three species) with the Michaelis-Menten
model.
The KM and VMAX values for HMF sulfo conjugation in hepatic PMS preparations from human males (20.6 mM and
262 pmol/mg/min) were similar to those from females (21.8 mM
and 317 pmol/mg/min) (Figure 3A and Table 1). In contrast, we
observed pronounced sexual dimorphisms in mice and rats
(Figs. 3B and C and Table 1). The KM in female liver of mice
HMF Oxidation in PMS From Tissue Homogenates
The oxidation rates were determined at concentrations between
1 and 1250 mM HMF in the presence of PMS samples from
humans, mice and rats (Figure 4). An inhibitory effect was not
detected up to 1250 mM HMF. Most of the data sets were
described well with the standard Michaelis-Menten equation.
The coefficients of determination (R2) reflected the goodness of
the fittings. The data resulting from incubations of particular
tissue PMS were not approximated adequately with the
Michaelis-Menten equation, ie, the HMF turnover with cytoplasmic protein from human kidney (R2 ¼ 0.701–0.851), human liver
(R2 ¼ 0.826–0.929; compare Figure 4A) and human lung
(R2 ¼ 0.791 – 0.970). Also, with hepatic PMS of female mice the
fitting curve deviated to some extent from the data points
(R2 ¼ 0.924–0.979, compare Figure 4B). In these cases, fittings
with a Michaelis-Menten model including 2 components,
V ¼ (VMAX,1 þ [HMF]/(KM,1[HMF])) þ (VMAX,2 þ [HMF]/(KM,2 [HMF]),
yielded good approximations (data not shown). The KM,1 values
of the high binding affinity components were in the order of
magnitude of the apparent KM values derived from the approximations with the standard Michaelis-Menten equation. The low
binding affinity component (KM,2, VMAX,2) described the slow
increase in the turnover rate becoming apparent in the range of
> 100 mM HMF (Figure 4A). Due to the limitations of our data sets
with 1250 mM as the highest HMF concentration it was not possible to derive KM,2 values with sufficient confidence. To ensure a
consistent evaluation of all data sets, we used the standard
Michaelis-Menten model. The resulting values for KM and VMAX
are summarized in Table 2.
It was evident that the KM values of ALDH-catalyzed HMF oxidation in human tissues were usually lower than those of the
other species. A sexual dimorphism was observed in mice. The
apparent KM of hepatic HMF oxidation in male animals (9.4 mM)
was about 7-fold lower compared with that in females (72.5 mM).
SACHSE ET AL.
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TABLE 1. Kinetic Parameters (KM and VMAX)a and Clearance Rates (CLSC)b of HMF Sulfo Conjugation in Different Tissues of Human, Mouse, and
Rata
Males
KM
mM
Human
Liver
Kidney
Lung
Colon
Mouse
Liver
Kidney
Lung
Colon
Rat
Liver
Kidney
Lung
Colon
Females
VMAX
pmol/mg/min
CLSC
ml/min/kg bw
20.6 6 1.7
17.7 6 3.4
25.5 6 3.9
37.5 6 9.6
262 6 69
15.1 6 3.0
37.7 6 10.2
134 6 53
21.2 6 6.3
0.14 6 0.02
0.60 6 0.15
1.9 6 0.6
3.0 6 0.1
10.8 6 1.0c
7.6 6 1.3
9.7 6 0.6c
360 6 23
540 6 21c
9.9 6 1.7
290 6 24c
5.3 6 0.5
3.8 6 0.2
23.4 6 5.6
27.6 6 2.3
766 6 61
28.8 6 7.0
2.1 6 0.5
45.7 6 8.4
KM
mM
VMAX
pmol/mg/min
CLSC
ml/min/kg bw
21.8 6 2.6
15.0 6 2.5
17.0 6 2.4
21.4 6 2.2
317 6 72
17.5 6 6.3
30.5 6 9.5
41.7 6 19.7
32.2 6 8.9
0.17 6 0.06
0.81 6 0.17
0.69 6 0.18
487 6 21
48.8 6 3.0
0.80 6 0.13
7.7 6 0.9
1.6 6 0.1
11.4 6 0.2c
16.9 6 6.6
10.1 6 0.3c
717 6 71
644 6 50c
11.6 6 4.6
293 6 26c
2520 6 220
41.4 6 1.6
0.57 6 0.14
8.6 6 0.7
430 6 34
3.2 6 0.7
0.02 6 0.00
0.32 6 0.06
5.2 6 0.1
14.8 6 2.4c
6.4 6 1.4
14.9 6 2.0
308 6 17
16.3 6 1.4c
2.1 6 0.6
22.5 6 7.9
198 6 14
0.53 6 0.06
0.09 6 0.03
0.46 6 0.15
a Values of apparent KM and VMAX are means 6 SE of measurements from 5 tissue samples. The HMF sulfo conjugation rates were fitted with the Michaelis-Menten
model including a substrate inhibitory effect observed at high HMF concentrations (compare Figure 3) as described in the method section (equation 1).
b CLSC values (means 6 SE of 5 different samples) were calculated separately for all individual PMS samples from human, mouse and rat. The parameters for the calculation according to equation. 2 in the method section are summarized in Tables S1 to S6 of the supplementary data.
c Due to the absence of an inhibitory effect at high HMF concentrations the sulfo conjugation was best fitted with the standard Michaelis-Menten model.
In contrast, the HMF oxidation in male human liver (KM ¼ 10.3 mM
and VMAX ¼ 2370 pmol/mg/min) was similar to that in females
(KM ¼ 8.4 mM and VMAX ¼ 2420 pmol/mg/min), and, also there was
no detectable difference of HMF oxidation in the presence of hepatic protein from male and female rats (Figure 4C).
Clearance of HMF by Sulfo Conjugation and Aldehyde Oxidation
The intrinsic clearance VMAX/KM was used to estimate the
capacities of SULT- and ALDH-catalyzed HMF turnover (CLSC
and CLOX) in each tissue according to equation 2. The clearance
rates for HMF sulfo conjugation (CLSC) are summarized in Table
1. In all three species the liver showed the highest HMF sulfo
conjugation capacity. For example, in human males the hepatic
clearance was about 151-fold, 35-fold, and 11-fold greater compared to that in kidney, lung, and colon, respectively. The overall relative contributions of kidney, lung, and colon to HMF
clearance by sulfo conjugation in the 4 tissues studied were
11.0% and 4.9% for male and female humans, respectively, and
10.5% and 2.0% for male and female mouse, respectively. In the
rat, the nonhepatic tissues investigated in this work contributed
only 0.8% and 0.5% to the overall HMF sulfo conjugation in PMS
from males and females, respectively. Comparing sulfo conjugation between species indicated that HMF is bioactivated more
efficiently in mice and rats than in humans. For example, the
hepatic clearance rates in male and female mice were 23-fold
and 78-fold greater than in male and female humans, respectively. Also, the capacity of hepatic sulfo conjugation in male
and female rats was about 20-fold and 6-fold greater compared
to that in male and female humans, respectively (Table 1).
The tissue-specific clearance rates of ALDH-mediated HMF
oxidation (CLOX) are summarized in Table 2. The contribution of
the liver to the overall HMF turnover determined in the 4 tissues
was about 80% and 86% in male and female humans, respectively, and about 80% and 85% in male and female rats, respectively. In mice, the hepatic turnover of HMF was 99% and 97% of
the overall ALDH-related oxidative capacity in male and female
animals, respectively. A species comparison suggested that
humans and rats may be similar regarding ALDH-catalyzed
HMF clearance, especially in the liver (Table 2). The capacity of
hepatic HMF oxidation in mice exceeded that in the other species. In male mice, the oxidative clearance was 7.8-fold and 5.4fold higher compared with humans and rats, respectively, and
in female mice it was 2.2-fold and 2.1-fold higher compared
with humans and rats, respectively.
DISCUSSION
The metabolic detoxification of HMF is primarily determined by
the oxidation of the aldehyde moiety, which accounts for > 80%
of the overall HMF turnover. The main metabolites observed in
urine of rats or mice within 24 h after administration of
[14C]HMF were HMFA (77.5%–84.9%), N-(5-hydroxymethyl-2furoyl)glycine (1.3%–7.9%), and 2,5-furan dicarboxylic acid
(2.0%–5.9%) (Godfrey et al., 1999). Also in humans exposed to
high amounts of HMF by consumption of dried plum juice or
dried plums all of the urinary metabolites detected were either
HMFA or conjugates thereof (Prior et al., 2006). Sulfo conjugation
contributes less to HMF metabolism. When HMF was intravenously injected in male mice, only about 500 ppm of the dose
was converted into SMF reaching the plasma (Monien et al.,
2009). Comparable data on HMF sulfo conjugation are not available for rats, humans, and female mice. For the assessment of
the human hazard potential associated with the internal SMF
exposure it is desirable to compare the capacities of HMF sulfo
conjugation and HMF aldehyde oxidation in humans with that
in mice and rats, the model organisms used in previous HMF
carcinogenicity studies. To this end, we determined clearance
rates for HMF sulfo conjugation (CLSC) and oxidation (CLOX) in 4
different tissues from humans, mice, and rats, ie, liver (target
tissue of HMF-induced neoplasia in the mouse [National
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TOXICOLOGICAL SCIENCES, 2016, Vol. 149, No. 1
FIG. 4. HMF oxidative turnover to HMFA in PMS preparations of liver tissues
from human (A), mouse (B), and rat (C). The rates at single HMF concentrations
are means 6 SE of 5 different samples of males (solid squares) and females
(open circles). Applying the Michaelis-Menten equation yielded satisfactory fittings for HMF oxidation by hepatic protein samples from male mouse
(R2 ¼ 0.987) and male (R2 ¼ 0.986) as well as female rats (R2 ¼ 0.987) but not for the
data sets of male humans (R2 ¼ 0.888), female humans (R2 ¼ 0.886), and female
mouse (R2 ¼ 0.962), which deviated from the classic Michaelis-Menten curve in
the range of HMF > 100 mM. In order to ensure a consistent evaluation, we used
the initial slope of all data sets as intrinsic clearance VMAX/KM for the calculation
of CLOX.
Toxicology Program, 2008]), kidney (SMF-induced renal toxicity
in the mouse (Bauer-Marinovic et al., 2012)), lung, and colon
(induction of aberrant crypt foci was reported in rats [Zhang
et al., 1993] and in mice [Svendsen et al., 2009]).
In all three species most of the capacities of HMF sulfo conjugation were concentrated in the liver (Table 1). This is primarily due to high expression levels of different SULT forms (Glatt,
2002) but also due to the relative weight of the liver, which
exceeds that of the other tissues (de la Grandmaison et al.,
2001). The comparison of apparent KM values of hepatic HMF
sulfo conjugation with the KM values of individual SULT forms
(Sachse et al., 2014) indicated that HMF turnover in PMS samples
may be catalyzed by several different SULT forms. In some
cases, the apparent KM values in PMS were similar to the KM values reported for the HMF turnover catalyzed by individual SULT
forms highly expressed in liver. For instance, the KM of hepatic
HMF sulfo conjugation in female mice (1.6 6 0.1 mM) was in
agreement with the observation that Sult1a1 (KM ¼ 2.0 6 0.3 mM)
is the principal SULT form in mouse liver (Alnouti and Klaassen,
2006; Honma et al., 2001). However, the HMF sulfo conjugation
in a particular tissue may be catalyzed usually by various different SULT forms. In humans, for example, the KM values of hepatic HMF sulfo conjugation, 20.7 mM in males and 18.5 mM in
females, may not be explicable by hSULT1A1-mediated catalysis
alone. Other important SULT forms expressed at considerable
levels in human liver besides hSULT1A1 (53 %, KM ¼ 3.2 mM) are
hSULT2A1 (27 %, KM > 50 mM) and SULT1B1 (14 %, KM ¼ 13.3 mM)
(Riches et al., 2009; Sachse et al., 2014). Yet, the HMF turnover in
most of the tissue PMS was usually well described by a standard, 1-component Michaelis-Menten equation (Figure 3). The
resulting apparent KM values may be regarded as average values
resulting from the contribution of different SULT forms.
The most important reaction of HMF detoxification is probably
the ALDH-catalyzed turnover to HMFA (Godfrey et al., 1999). In the
ALDH superfamily three classes contribute primarily to xenobiotic
metabolism in mammals: ALDH1 (cytosolic), ALDH2 (mitochondrial) and ALDH3 (cytosolic). Isoenzymes of hALDH1 and hALDH2,
which are expressed constitutively in the liver at high levels
(Sladek, 2003; Stewart et al., 1996), have relatively low KM values in
the micromolar range for many aliphatic and aromatic aldehydes
(Klyosov, 1996; Wang et al., 2009). In contrast, hALDH3 was
described as a class of isoenzymes with relatively high KM values
(Yin et al., 1989). To our knowledge kinetic data about HMF oxidation by single ALDH forms were not published. Other representative substrates for small aromatic aldehydes are benzaldehyde and
2-furaldehyde, a structural congener of HMF. Freshly isolated
ALDH1 from human liver oxidized benzaldehyde (KM ¼ 0.25 mM)
and 2-furaldehyde (KM ¼ 4.8 mM) (Wang et al., 2009). Purified
recombinant hALDH1A1 catalyzed the turnover of benzaldehyde
(KM ¼ 0.4 mM) and 2-furaldehyde (KM ¼ 5.8 mM) with similar efficiencies (Solobodowska et al., 2012). In contrast, hALDH3A1 was active
at much higher concentrations of benzaldehyde (KM ¼ 148 mM) or 2furaldehyde (KM > 1000 mM (Solobodowska et al., 2012); KM ¼ 20 mM
(Yin et al., 1989)). The current data suggested that the liver may be
the most important tissue also for HMF oxidative turnover catalyzed by ALDH and that at least 2 ALDH forms with distinct kinetic
properties may contribute to hepatic HMF oxidation to HMFA in
humans. The KM values resulting from the fitting of ALDH-catalyzed HMF turnover in human liver with a standard MichaelisMenten equation (Table 2) were determined primarily by the
increase in turnover rates in the range of low substrate concentrations (Figure 4). The KM values are in the same range than those of
the hALDH1A1-catalyzed turnover of benzaldehyde and 2-furaldehyde (Solobodowska et al., 2012). This observation suggests that
hALDH1A1 may be the high affinity component of HMF oxidation
and that the low binding affinity component, which was not
adequately described by our experiments, may be hALDH3A1.
Initial experiments showed that mitochondria did not contribute
significantly to HMF turnover (Supplementary Table S13 in the
supplementary data). Especially in mice and in rats the ALDH-catalyzed HMF turnover observed in the presence of mitochondrial
preparations was minute in comparison to that in the PMS
samples.
Various unknown parameters hinder drawing comparative
conclusions on the overall HMF sulfo conjugation in the three
species. For example, the influence of SMF detoxification by glutathione conjugation, the quantitative impact of the tissue-specific
SACHSE ET AL.
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199
TABLE 2. Kinetic Parameters (KM and VMAX)a and Clearance Rates (CLOX)b of HMF Oxidation in Different Tissues of Human, Mouse, and Rat
Males
KM
mM
Human
Liver
Kidney
Lung
Colon
Mouse
Liver
Kidney
Lung
Colon
Rat
Liver
Kidney
Lung
Colon
Females
VMAX
pmol/mg/min
CLOX
ml/min/kg bw
KM
mM
VMAX
pmol/mg/min
CLOX
ml/min/kg bw
10.3 6 1.1
12.0 6 5.4
13.0 6 7.9
8.8 6 1.4
2370 6 130
1420 6 80
717 6 129
614 6 110
436 6 57
30.5 6 6.2
36.7 6 7.9
40.8 6 10.3
8.4 6 0.6
9.3 6 1.3
6.9 6 1.0
15.5 6 8.9
2420 6 230
1490 6 110
464 6 118
704 6 172
646 6 116
34.5 6 3.3
33.3 6 7.4
37.3 6 11.1
9.4 6 0.7
1960 6 240
31.2 6 3.4
96.4 6 8.6
11900 6 200
7100 6 960
662 6 150
3420 6 270
3400 6 210
4.2 6 0.7
11.6 6 1.9
8.8 6 0.6
72.5 6 9.8
1490 6 130
14.7 6 2.0
107 6 15
9240 6 440
8660 6 1390
366 6 64
3970 6 180
1410 6 100
19.9 6 4.5
16.0 6 2.8
13.3 6 1.7
87.7 6 24.4
64.6 6 4.2
51.4 6 6.3
79.5 6 11.0
9900 6 390
10600 6 300
2680 6 210
1330 6 80
627 6 112
144 6 6
10.6 6 2.0
3.9 6 0.7
100 6 37
105 6 8
39.7 6 3.5
77.5 6 8.9
9360 6 510
8990 6 160
2900 6 190
1330 6 110
679 6 127
102 6 1
15.6 6 1.9
5.7 6 0.4
a Values are means 6 SE of measurements from 5 tissue samples. The HMF oxidation rates were fitted with the standard Michaelis-Menten model (compare Figure 4)
as described in the method section.
b CLOX values (means 6 SE of 5 different samples) were calculated separately for all individual PMS samples. The parameters for the calculation according to equation 2
in the method section are summarized in Tables S7 to S12 of the supplementary data.
directed import and export of SMF (Bakhiya et al., 2009), and
important physiologic parameters, eg, blood-perfusion of single
tissues, were not considered. It is also not clear whether the
experimental conditions of the in vitro study were suitable for the
simulation of the enzymatic turnover of HMF in vivo. However,
the marked differences between HMF sulfo conjugation and aldehyde oxidation detected in this study allowed for some plausible
conclusions. The bioactivation and detoxification of HMF was
compared by predicting values of CLSC and CLOX from the intrinsic
clearance (VMAX/KM) of sulfo conjugation and aldehyde oxidation
in PMS samples prepared from human, mouse, and rat tissues
(Obach et al., 1997). Generally, the ALDH-related clearance rates
CLOX were about 3 orders of magnitude greater compared with
those of HMF sulfo conjugation CLSC. This is in accordance to previous reports about HMF metabolism in rodents in vivo (Godfrey
et al., 1999; Monien et al., 2009). The CLOX in liver of male and
female mice was about 7000- and 560-times greater, respectively,
than the CLSC. The data from rats indicated an opposite sexdependency. In male and female rats, the hepatic CLOX values
exceeded those of CLSC by factors of about 1460-fold and 3400fold, respectively. In humans, there was no sexual dimorphism
regarding the HMF turnover. The ratio of CLOX to CLSC values
determined in hepatic PMS samples of male and female humans
were 20 600 and 20 100, respectively. The data suggested that
there are prominent species differences in HMF sulfo conjugation
and detoxification. The ratio of CLOX to CLSC was lowest in liver of
female mice indicating a relatively high quantitative importance
of HMF sulfo conjugation. This correlates with the observation
that oral exposure to 188 mg HMF/kg body weight and 375 mg
HMF/kg body weight for 104 weeks led to increased incidences of
hepatocellular adenomas in female but not in male mice and also
not in rats (National Toxicology Program, 2008). In humans the
ratio of hepatic CLOX to CLSC was about 36-times greater compared with that in female mice suggesting that ALDH-mediated
HMF detoxification may be quantitatively more important in
humans than in female mice. If the neoplastic effects of HMF
were due to the formation of SMF, the current data would indicate
that female mice may be the most sensitive organism for HMFinduced tumorigenicity when compared with rats and humans.
This finding supports the notion that the carcinogenic risk resulting from human dietary exposure to HMF may be of minor
importance.
FUNDING
This work was supported by the German Research
Foundation [grant number MO 2520/1-1].
ACKNOWLEDGMENTS
The authors thank Martina Scholtyssek and Elke Thom for
their excellent technical assistance.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.
oxfordjournals.org/.
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