Plant Uptake and Metabolism of Nitrofuran Antibiotics in Spring Onion Grown in Nitrofuran-Contaminated Soil

Article abstract of DOI:10.1021/acs.jafc.7b01050

Environmental pollution caused by the discharge of mutagenic and carcinogenic nitrofurans to the aquatic and soil environment is an emerging public health concern because of the potential in producing drug-resistant microbes and being uptaken by food crops. Using liquid chromatography-tandem mass spectrometry analysis and with spring onion (Allium wakegi Araki) as the plant model, we investigated in this study the plant uptake and accumulation of nitrofuran from a contaminated environment. Our study revealed for the first time high uptake and accumulation rates of nitrofuran in the edible parts of the food crop. Furthermore, results indicated highly efficient plant metabolism of the absorbed nitrofuran within the plant, leading to the formation of genotoxic hydrazine-containing metabolites. The results from this study may disclose a previously unidentified human exposure pathway through contaminated food crops.

Full text of DOI:10.1021/acs.jafc.7b01050

Article
pubs.acs.org/JAFC
Plant Uptake and Metabolism of Nitrofuran Antibiotics in Spring
Onion Grown in Nitrofuran-Contaminated Soil
Yinan Wang, K. K. Jason Chan, and Wan Chan*
Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
S
* Supporting Information
ABSTRACT: Environmental pollution caused by the discharge of mutagenic and carcinogenic nitrofurans to the aquatic and soil
environment is an emerging public health concern because of the potential in producing drug-resistant microbes and being
uptaken by food crops. Using liquid chromatography−tandem mass spectrometry analysis and with spring onion (Allium wakegi
Araki) as the plant model, we investigated in this study the plant uptake and accumulation of nitrofuran from a contaminated
environment. Our study revealed for the first time high uptake and accumulation rates of nitrofuran in the edible parts of the
food crop. Furthermore, results indicated highly efficient plant metabolism of the absorbed nitrofuran within the plant, leading to
the formation of genotoxic hydrazine-containing metabolites. The results from this study may disclose a previously unidentified
human exposure pathway through contaminated food crops.
KEYWORDS: nitrofuran, root uptake, plant metabolism, green liver, spring onion, LC−MS/MS
Despite being prohibited from use in food animal production
in many countries, the use of nitrofuran in some developing
countries is still allowed and illegal use of nitrofuran exists.5,13
Furthermore, only few studies reported the potential plant
uptake of nitrofuran from a contaminated environment, and no
information on their plant metabolism is available in the
literature.14,15 Therefore, it is still of high importance to
monitor food for nitrofuran contamination as a result of their
adverse effect on human health.16,17 Currently, meat products
are routinely sampled by regulatory authorities for nitrofuran
residues by detecting their protein-bound metabolites (semi-
carbazides or carbazates) in the muscles (Figure 1).18,19
It is known that most antibiotics, nitrofuran included, are
poorly absorbed through the intestines and are largely excreted
unchanged.20 For example, it was reported that over 60% of
furazolidone was excreted through urine and feces.21 These
residual nitrofurans in the excrements, together with those used
in the aquaculture fisheries, may end up being discharged into
the aquatic environment and soil through improper wastewater
handling or when applying the animal excreta as fertilizer for
food crop production,14,22,23 leading to concerns that residual
antibiotics may be taken up by food crops and enter the human
food chain.24 In this study, we tested the hypothesis that
humans may be exposed to nitrofuran through consumption of
contaminated food crops grown in polluted soil that have
uptaken nitrofuran through the roots and accumulated
nitrofuran in their edible parts. This could be an existing
“secondary exposure” pathway to nitrofuran, of which we have
been unaware.
INTRODUCTION
Nitrofuran (Figure 1) is a class of broad-spectrum veterinary
drugs for the treatment of microbial infections, with furaltadone
■
Figure 1. Chemical structures of nitrofurans and their corresponding
protein-bound metabolites.
(1), furazolidone (2), nitrofurantoin (3), and nitrofurazone (4)
being the most widely used nitrofurans.1−5 They are highly
effective in suppressing and killing most kinds of bacteria, fungi,
and protozoa, which led them to be used extensively in
aquaculture and livestock farming as growth promoters until
they were observed to be carcinogenic and mutagenic to
humans.6−10 Currently, the use of nitrofuran in food-producing
animals has been banned in many countries, including China,
U.S.A., and many European countries.11,12
Using a liquid chromatography−tandem mass spectrometry
(LC−MS/MS) method and with spring onion (Allium wakegi
Received: March 7, 2017
Revised: April 24, 2017
Accepted: May 11, 2017
Published: May 11, 2017
© XXXX American Chemical Society
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Araki) as the plant model, we investigated in this study the
uptake and accumulation of nitrofuran from nitrofuran-
contaminated soil. The in vitro and in vivo metabolisms of
nitrofuran in spring onion homogenate and spring onion,
respectively, were also studied for the first time. Furthermore,
the study also evaluated the plant uptake efficiency and
biogeochemical stability of nitrofuran from the soil environ-
ment.
Figure 2. Acid-catalyzed release of the protein-bound metabolites of
nitrofuran and simultaneous derivatization of the released metabolites
with 2-nitrobenzaldehyde to produce hydrophobic derivatives of
enhanced chromatographic performance for LC−MS/MS analysis.
MATERIALS AND METHODS
■
Reagents. Compounds 1, 2, 3, and 4, 3-amino-5-morpholino-
methyl-2-oxazolidinone (1′), 3-amino-2-oxazolidinone (2′), 1-amino-
hydantoin hydrochloride (3′), semicarbazide hydrochloride (4′),
[13C,15N2]-semicarbazide ([13C,15N2]-4′), 2-nitrobenzaldehyde, and
5-nitro-2-furaldehyde were purchased from Sigma-Aldrich (St. Louis,
MO). High-performance liquid chromatography (HPLC)-grade
acetonitrile, ethyl acetate, hexane, and methanol were obtained from
Tedia (Fairfield, OH). Isotope-labeled compound 4 ([13C,15N2]-4)
was synthesized by reacting [13C,15N2]-4′ with 5-nitro-2-furaldehyde,
as described previously.25 Using a similar strategy, the 2-nitro-
benzaldehyde derivative of [13C,15N2]-4′ was also synthesized by
reacting [13C,15N2]-4′ with 2-nitrobenzaldehyde.
LC−MS/MS Analyses. LC separation of both nitrofurans and their
metabolites was performed on a 150 × 2.1 mm inner diameter, 5 μm,
GraceSmart RP 18 (Grace, Deerfield, IL) powered by an 1100 HPLC
system (Agilent Technologies, Palo Alto, CA). The sample extracts (5
μL) were injected into the column eluted using 20 mM ammonium
acetate in water (A) and methanol (B) as the mobile phase at a flow
rate of 0.3 mL/min. The gradient elution was programmed with a
linear gradient increasing from 10% (20% for nitrofuran metabolites)
to 100% B (v/v) in 10 min and holding for 5 min, before being re-
equilibrated to initial conditions for 7 min.
The LC eluate was directed to an API 4000 QTRAP LC−MS/MS
system (Toronto, Ontario, Canada) operated in positive electrospray
ionization (ESI) mode. Quantitative analysis was performed by the
multiple reaction monitoring (MRM) mode of the MS/MS system
using the optimized instrumental parameters. The m/z values for the
qualifying and quantifying MRM transition of the target analytes and
internal standard are listed in Table 1, with the dwell time for each
transition set to 100 ms.
In Vitro Study. The in vitro experiment was performed by
incubating a mixture of compounds 1, 2, 3, and 4 (1 μg/mL) with the
homogenate of spring onion leaf/root bulb at 25 °C for an extended
period of 12 h (n = 3). Aliquots of the solution were sampled at 0 and
30 min and 1, 2, 4, 8, and 12 h and processed for nitrofuran and
associated metabolite (1′, 2′, 3′, and 4′) analyses using the methods
described below. Using a similar strategy, the control experiment was
conducted by incubating a mixture of compounds 1, 2, 3, and 4 (1 μg/
mL) with homogenate that was pretreated by heating at 100 °C.
In Vivo Study. The in vivo experiment was conducted by
cultivating spring onion in nitrofuran-contaminated soil (4 mg/kg).
In brief, to the nitrofuran-spiked soil samples that were left in the
ambient environment for 2 days, spring onions were cultivated. The
plants were left in an ambient environment with a natural day/night
cycle and were watered once per week. After growing in the soil for 7,
14, 28, and 56 days, the leaves and root bulbs were harvested
separately for analysis (n = 3). The control experiment was performed
by analyzing spring onion grown on soil with no nitrofuran
contamination and analyzing nitrofuran-contaminated soil with no
spring onion cultivated.
Sample Preparation. For Nitrofuran Analysis. The leaf and root
bulb samples were processed for nitrofuran analysis using a method
described previously.26 In brief, to the homogenized samples (200 mg)
was added ethyl acetate (5 mL); the mixture was mixed by vigorous
shaking for 5 min and then centrifuged; and the supernatant was
collected and dried under a stream of nitrogen. To the dried sample
extract, acetonitrile (5 mL) and hexane (3 mL) were added and shaken
for 5 min. After centrifugation [4200 relative centrifugal force (rcf)]
for 5 min, the acetonitrile fraction was isolated and dried under
nitrogen before the internal standard [13C,15N2]-4 (0.3 μg/mL in
methanol, 50 μL) and aqueous 50% methanol (450 μL) were added to
the residues for LC−MS/MS analyses.
Table 1. MS/MS Transitions Used To Monitor Nitrofurans
and Their Protein-Bound Metabolites, Together with the
Log(Kow) Values and Chromatographic Migration Time of
the Nitrofuran
log(Kow
)
migration time (min) MRM transitions (m/z)
Nitrofuran
1
0.20
−0.04
−0.47
0.23
8.6
325/252 and 325/281
226/122 and 226/139
239/95 and 239/139
199/156 and 199/182
202/158
2
8.0
3
7.7
4
7.9
[13C,15N2]-4
7.9
a
Metabolites
1′
2′
3′
4′
8.3
7.2
7.0
7.3
7.3
335/262 and 335/291
236/104 and 236/134
249/104 and 249/134
209/166 and 209/192
212/168
[13C,15N2]-4′
a
After chemical derivatization with 2-nitrobenzaldehyde.
For Metabolite Analysis. Using a previously reported method,27 the
leaf and root bulb samples were also processed for protein-bound
metabolite analysis. In brief, hydrochloric acid (0.125 M, 5 mL) and 2-
nitrobenzaldehyde (50 μg/mL in methanol, 200 μL) were added to
ground leaf or root bulb samples (200 mg). After vortex mixing, the
solutions were incubated at 37 °C overnight to liberate and derivatize
the protein-bound metabolites (Figure 2). Sodium hydroxide solution
(1 M, 400 μL) was then added to adjust the sample solutions to pH
7.0 ( 0.3) before the solutions were washed with n-hexane (3 mL)
and extracted with ethyl acetate (2 mL). The ethyl acetate extracts
were combined and dried under a stream of nitrogen before the
derivatized [13C,15N2]-4′ internal standard (0.6 μg/mL in methanol,
50 μL) and aqueous 50% methanol (450 μL) were added to the
residue for LC−MS/MS analyses.
Calibration. Working standard solutions of nitrofuran were
prepared by serial dilution of the stock solution mixture of compounds
1, 2, 3, and 4 (2 μg/mL) with blank sample extract. To 450 μL of the
working standard solution mixtures with concentrations ranging from
0.01 to 0.5 μg/mL, 50 μL of the internal standard ([13C,15N2]-4, 0.3
μg/mL) was added. After vortex mixing, the standard solutions were
analyzed by the LC−MS/MS method described above. Calibration
curves were established by plotting the peak area ratio of individual
nitrofuran to that of the internal standard against the concentration of
the nitrofuran in the working standards.
A spring onion sample with no detectable amount of compounds 1′,
2′, 3′, and 4′ was used to prepare the matrix-matched calibration
curves of compounds 1′, 2′, 3′, and 4′. In brief, to the homogenized
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samples (200 mg), 100 μL of the standard mixture of 1′, 2′, 3′, and 4′
(from 0.01 to 1.0 μg/mL; n = 3) was added. After vortex mixing, 2-
nitrobenzaldehyde (50 μg/mL in methanol, 200 μL) and hydrochloric
acid (0.125 M, 5 mL) were added and allowed to react at 37 °C
overnight. The derivatized working standard solutions were then
extracted with ethyl acetate, added with an internal standard (2-
nitrobenzaldehyde derivative of [13C,15N2]-4′), and then analyzed by
LC−MS/MS, as described above.
Method Validation. The methods were validated for recovery and
limit of detection (LOD). From analysis of nitrofurans and their
metabolties at the lowest calibration concentration (0.01 μg/mL) and
using a previously reported method,28−30 the LOD was estimated as
the amount of analyte that generated a signal 3 times the noise level in
analyzing nitrofuran and metabolites.
The recovery of nitrofuran was evaluated by analyzing homogenized
samples (200 mg) that were spiked with 450 μL of a nitrofuran
mixture of compounds 1, 2, 3, and 4 (0.01, 0.25, and 0.5 μg/mL; n =
3). The nitrofuran-fortified samples were vortex-mixed and allowed to
stand at room temperature for 5 min before being extracted using ethyl
acetate, added with an internal standard, and analyzed using the
method described above. The recovery was established by dividing the
recovered nitrofuran to that of the spiked amount. Using a similar
method, the recovery of the nitrofuran metabolites was also evaluated.
In brief, to 200 mg of homogenized samples, 100 μL of a standard
mixture of compounds 1′, 2′, 3′, and 4′ (0.1, 0.5, and 1.0 μg/mL; n =
3) was added. After vortex mixing, 2-nitrobenzaldehyde (50 μg/mL in
methanol, 200 μL) and hydrochloric acid (0.125 M, 5 mL) were added
and allowed to react at 37 °C overnight. The sample solutions were
then adjusted to pH 7.0 using sodium hydroxide, washed with n-
hexane (3 mL), and extracted with ethyl acetate before the
concentration of the metabolites in the sample extracted was analyzed
using the method described above.
Table 2. Calibration and Validation Parameters of the LC−
MS/MS Method for Nitrofuran and Associated Metabolite
Analyses
linear range
(μg/mL)
LOD
(ng/g)
recovery
(%)
slope
intercept
R2
Nitrofuran
0.1506
1
2
3
4
0.01−0.5
0.01−0.5
0.01−0.5
0.01−0.5
0.0321
0.0066
0.0023
0.0033
0.9967
0.9981
0.9993
0.9929
0.2
0.2
0.8
0.7
86.2−87.9
86.8−94.4
87.2−90.5
87.3−92.9
0.0653
0.0392
0.0588
a
Metabolites
0.3809
1′
2′
3′
4′
0.01−1.0
0.01−1.0
0.01−1.0
0.01−1.0
0.0258
0.0079
0.0023
0.0036
0.9978
0.9979
0.9983
0.9963
0.2
0.4
0.7
0.7
90.9−92.0
88.4−91.5
86.5−92.9
89.0−91.8
0.1693
0.0663
0.1119
a
After chemical derivatization with 2-nitrobenzaldehyde.
contaminated soil into their root bulbs in a time-dependent
manner from 0 to 28 days (Figure S1 of the Supporting
Information). The presence of nitrofuran was also detected in
the leaves in a similar concentration pattern but at lower
concentrations, indicating that transport of nitrofuran from the
root bulb up to the leaves occurs efficiently by transpiration.
Shown in Figure 3 are typical chromatograms obtained from
LC−MS/MS of the nitrofuran in leaf samples.
Data Analysis. The half-life (t1/2) of individual nitrofuran in in
vitro incubations with plant tissue homogenates were estimated by
plotting ln([NFt]/[NFi]) versus time,31 where [NFt] and [NFi]
represent the concentrations of individual nitrofuran at the time t and
the beginning of the experiment, respectively. The concentration
factors for leaves and root bulbs were calculated by dividing the
concentrations of nitrofuran in plant tissues to that in the soil samples
at the harvesting time (day 56).32
RESULTS AND DISCUSSION
■
Root Uptake and Accumulation of Nitrofuran in
Spring Onion. Emerging evidence suggests that antibiotics
are a new class of environment pollutants.26,28 The discharged
antibiotics in the environment originate from both human
medicinal sources and food-producing animal facilities.24 For
example, the contamination with nitrofuran as well as many
other types of antibiotics is now widespread in the environ-
ment.23 It is likely that antibiotics may contaminate the human
food supply through root uptake into food crops from a
contaminated environment,33−36 in addition to tainted poultry
or seafood products.37
Among the commonly used antibiotics, the mutagenic and
carcinogenic nitrofurans are of particular concern to human
health.5 Previous studies focused on detecting the protein-
bound nitrofuran metabolites in meat products,13,17 with only
one study investigating the uptake of compound 1 by algae
from contaminated seawater.26 We hypothesize that food crops
grown in nitrofuran-contaminated soil may similarly uptake
nitrofuran through the roots and become contaminated.
Using a calibrated and validated LC−MS/MS method (Table
2), we investigated in this study the plant uptake of nitrofuran
from a contaminated environment. Choosing spring onion as
our plant model and four commonly used nitrofurans 1, 2, 3,
and 4, as model compounds, we revealed that food crops do
indeed uptake and accumulate these compounds from
Figure 3. Reconstructred chromatograms from LC−MS/MS analysis
of (A) nitrofuran and (B) nitrofuran metabolites (after chemical
derivatization with 2-nitrobenzaldehyde) in the leaf of spring onion
grown in nitrofuran-contaminated soil.
Data showed a slight decrease in nitrofuran concentrations,
in both the root bulbs and leaves from days 28 to 56 (Figure 4);
this decrease is parallel to the gradual depletion of nitrofuran in
the soil (Figure 4). Nevertheless, the concentrations of the four
nitrofurans in both the root bulbs and leaves decreased in the
order of 2 > 3 > 1 > 4, which is in good agreement with their
decreasing solubility in water [increasing log(Kow); Table 1].
The close correlation of the log(Kow) values with the
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higher accumulation levels in the root bulbs compared to the
leaves.
Using green algae (Ulva lactuca) as their plant model, Leston
et al. reported that, when the concentration of compound 1 was
monitored in seawater over a 5 day period of aquaculture,
roughly 70% of compound 1 in the seawater was degraded by
microbes and/or sunlight and 30% was removed by plant
uptake.26 Our study using spring onion demonstrated a
comparatively lower uptake and degradation rate of nitrofuran.
Specifically, over an extended period of 56 days, we observed
that less than 20% of nitrofurans were degraded by microbial/
photodegradation (Figure S2 of the Supporting Information),
while approximately 50% (except for compound 4 with 34.6%
being absorbed) has been absorbed by the plant (Figure 4).
This observed difference in uptake and degradation efficiency
could be attributed to using different plant species and media
for the experiments. Nevertheless, the results from our study
could demonstrate that nitrofuran released into the soil
environment would likely be more persistent than in the
aquatic environment.
Determination of Protein-Bound Metabolites of
Nitrofuran in Spring Onion. After demonstration of the
ability of spring onion to uptake nitrofuran from a
contaminated environment, the study was extended to detect
several previously identified protein-bound metabolites from
animal and plant tissues.13,16 These protein-bound, genotoxic
metabolites of nitrofuran, carbazates 1′ and 2′ and semi-
carbazides 3′ and 4′ in particular (Figure 1), are hydrolytic
products of the imine bond in nitrofuran. They were used
extensively to detect nitrofuran contamination in muscle
samples.17,18 However, the identification of these metabolites
in vegetable food crops has not been reported in the literature
to this date, to the best of our knowledge. It was proposed that
plants are able to perform some xenobiotic metabolism similar
to that observed in hepatic metabolism through enzymes
present in plant cells.38,39 Using a validated LC−MS/MS
method (Table 2), we tested in this study the hypothesis that
the absorbed nitrofuran may be metabolized within the plant
cells of spring onion.
In Vitro Study. The goal of this study was to test the
feasibility of using the previously identified protein-bound
metabolites of nitrofuran as marker molecules for the detection
of nitrofuran contamination in food crops.13,16−19 We carried
out in vitro incubations of nitrofurans 1, 2, 3, and 4 with the
homogenates of leaf and root bulb tissue from spring onion
separately (n = 3). The incubated mixtures were sampled at
different times, then processed, and analyzed for the presence
of nitrofurans and their protein-binding metabolites using the
methods described above (Figure 2).
Figure 4. Time-dependent changes in the concentrations of nitrofuran
in (A) soil, together with that of (B) nitrofuran and (C) associated
metabolites in the leaf of spring onion grown in nitrofuran-
contaminated soil.
absorption efficiency strongly suggests that nitrofurans were
uptaken into the plants as the roots uptake water from the soil
containing dissolved nitrofuran. The absorbed nitrofurans were
then transported up the leaves through transpiration. Figure 5
summarized the bioconcentration factors of nitrofuran in the
leaves and root bulbs of spring onion, also illustrating a
decreasing uptake efficiency in the order of 2 > 3 > 1 > 4 and
Surprisingly, the metabolite analysis revealed, in both the
incubated leaf and root bulb tissue homogenates, a similar and
time-dependent increase in the concentrations of nitrofuran
metabolites, i.e., compounds 1′, 2′, 3′, and 4′ (Figure 6 and
Figures S3 and S4 of the Supporting Information). The parallel
nitrofuran analysis also detected a corresponding time-depend-
ent decrease in nitrofuran concentrations during the
incubations (Figure 6), with over 60% of nitrofurans converted
into their metabolites during a 4 h period of incubation. Table 3
summarizes the half-lives of nitrofuran when incubated with leaf
and root bulb homogenates; it showed that the metabolic rates
in root bulb homogenate are generally faster compared to the
leaf homogenate, indicating that, in the whole plant, the
metabolic rate would likely also be faster in the root bulb than
Figure 5. Concentration factors of nitrofurans 1, 2, 3, and 4 in the leaf
and root bulb of spring onion grow in nitrofuran-contaminated soil.
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persistence of the metabolites was also observed in the animal
tissues from nitrofuran-treated animals in previous studies.18,42
Because both nitrofurans and their metabolites were identified
as secondary species capable of being transferred through
contaminated food sources,5 it is feasible that through
bioaccumulation and biomagnification up the food chain, the
risk of human exposure to these toxic substances through plants
grown in tainted soil would be enhanced.
Using LC−MS/MS methods, we investigated in this study
the environmental stability, root uptake, and plant metabolism
of nitrofuran, a group of carcinogenic and mutagenic
antibiotics, in spring onion. Our results demonstrated for the
first time the high persistence of nitrofurans in the soil
environment and their highly efficient uptake into food crops.
With nitrofuran antibiotics emerging as a new class of
environmental pollutants, this study disclosed important but
previously unrealized human exposure pathways to nitrofuran.
Furthermore, the study revealed an interesting, novel
phenomenon of plant metabolism in which nitrofurans were
efficiently degraded to their genotoxic metabolites by the living
plant cells.31,43 It is expected that the study will alert food
scientists and regulatory authorities to a potential new source of
human exposure to nitrofuran.
Figure 6. Time-dependent changes in the concentrations of
nitrofurazone and its protein-bound metabolites, i.e., semicarbazide,
in homogenized fresh leaf samples as well as that of the concentration
of nitrofurazone in heat-treated tissue homogenate.
Table 3. Half-Life (t1/2) of Nitrofuran in In Vitro Incubations
with Leaf and Root Bulb Homogenates of Spring Onion
in leaf homogenate
in root bulb homogenate
a
a
(h)
(h)
furaltadone (1)
furazolidone (2)
nitrofurantoin (3)
nitrofurazone (4)
1.4 0.2
0.8 0.1
1.3 0.2
1.1 0.1
0.9 0.1
0.7 0.1
1.1 0.1
0.9 0.1
a
ASSOCIATED CONTENT
A total of 200 mg of homogenized plant tissue samples was used in
■
both experiments. Data represent the mean 3 times the standard
derivation.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.7b01050.
the leaves. A half-life of around 1 h was observed for all four
nitrofurans, indicating that the plant enzymes can process
structurally different nitrofurans efficiently. No noticeable
change in the nitrofuran concentration was observed in the
control experiments using heat-denatured plant tissue homo-
genates (Figure 6), which strongly suggests that the
degradation of nitrofuran is indeed mediated through enzymes,
e.g., nitroreductases, which were previously observed to play a
key role in the metabolism of nitrofuran in bacterial and
mammalian cells.40,41
Time-dependent changes in the concentration of nitro-
furan and associated metabolites in root bulb of spring
onion grown in nitrofuran-contaminated soil (Figure S1),
time-dependent changes in the concentrations of nitro-
furan in soil without spring onion cultivation (Figure S2),
time-dependent changes in the concentration of nitro-
furans and their protein-bound metabolites in in vitro
incubation with leaf and root bulb homogenates (Figures
S3 and S4) (PDF)
In Vivo Study. Having successfully detected the protein-
bound metabolites of nitrofuran in in vitro incubations, we
extended the study to the living whole plant and tested for the
presence of metabolites 1′, 2′, 3′, and 4′ in the leaf and root
bulb samples of spring onion grown in nitrofuran-dosed soil.
To our surprise, our study detected all of the targeted
nitrofuran metabolites in both the leaf (Figure 4) and root
bulb (Figure S1 of the Supporting Information) samples and
with the leaf/bulb concentration ratio (∼1:2) similar to that
observed for the parent drugs. No nitrofuran metabolites were
detected in any of the soil samples, indicating that the only
source of these metabolites was from plant metabolism and
ruling out their formation by soil microorganisms or chemical
decomposition. Depicted in Figure 3 are typical chromatograms
obtained from LC−MS/MS of the metabolites in leaf samples.
To the best of our knowledge, this is the first report of
nitrofuran metabolism taking place in a plant.
Interestingly, a similar concentration pattern (2′ > 3′ > 1′ >
4′) as that displayed by the parent drugs (2 > 3 > 1 > 4) was
also observed (Figure 4), indicating a similar rate of metabolism
of the four nitrofurans by the enzymes in the plant.
Nevertheless, the metabolite concentration showed an
increasing trend throughout the entire experiment (from day
0 to day 56), indicating these metabolic products are highly
persistent and will accumulate in the plant. A similar high
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: +852-2358-7370. Fax: +852-2358-1594. E-mail:
chanwan@ust.hk.
ORCID
Wan Chan: 0000-0001-8479-3172
Author Contributions
Yinan Wang and Wan Chan designed the research; Yinan Wang
performed the research; Yinan Wang, K. K. Jason Chan, and
Wan Chan analyzed the data; and Wan Chan wrote the paper.
Funding
Wan Chan expresses his sincere gratitude to Hong Kong
University of Science and Technology for a Startup Fund
(Grant R9310).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank AB Sciex for providing the LC−MS/MS
system for this study.
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