Electron Deficiency of Nitro Group Determines Hepatic Cytotoxicity of Nitrofurantoin

Article abstract of DOI:10.1021/acs.chemrestox.8b00362

Nitrofurantoin (NFT) is a widely used antimicrobial agent in the treatment of specific urinary tract infections (UTIs). Many adverse effects associated with NFT use have been reported, including hepatotoxicity. A structure-toxicity relationship study was

Full text of DOI:10.1021/acs.chemrestox.8b00362

Subscriber access provided by University of Glasgow Library
Article
Electron deficiency of nitro group determines
hepatic cytotoxicity of nitrofurantoin
Hui Li, Zhengyu Zhang, Xiaojing Yang, Xu Mao, Ying Wang, Jian Wang, Ying Peng, and Jiang Zheng
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00362 • Publication Date (Web): 19 Feb 2019
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Electron deficiency of nitro group determines hepatic cytotoxicity of
nitrofurantoin
†
†
†
†
║
§
†*
Hui Li , Zhengyu Zhang , Xiaojing Yang , Xu Mao , Ying Wang , Jian Wang , Ying Peng ,
Jiang Zheng†,‡*
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†Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning,
‡
1
1016, P. R. China; State Key Laboratory of Functions and Applications of Medicinal
Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical
§
University, Guiyang, Guizhou, 550004, P. R. China;
School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenyang, Liaoning, 11016, P. R.
║
China;
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, Shenyang, Liaoning, 11016, P. R. China
*Corresponding Author:
Jiang Zheng, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang,
Liaoning, 110016, P. R. China. State Key Laboratory of Functions and Applications of
Medicinal Plants, Key Laboratory of Pharmaceutics of Guizhou Province, Guizhou
Medical University, Guiyang, Guizhou, 550004, P. R. China.
Email: zhengneu@yahoo.com
Tel: +86-24-23986361; Fax: +86-24-23986510.
Ying Peng, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang,
Liaoning, China.
Email: yingpeng1999@163.com
Tel: +86-24-23986361; Fax: +86-24-23986510.
†
,‡: the two corresponding units contributed equally to this work.
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Abstract
Nitrofurantoin (NFT) is a widely used antimicrobial agent in the treatment of
specific urinary tract infections (UTIs). Many adverse effects associated with NFT
use have been reported, including hepatotoxicity. A structure-toxicity relationship
study was performed to gain the insight into the mechanisms of toxic action of NFT.
The toxic effects of NFT and its nine analogues or constituent moieties (1-9) designed
and synthesized by structural manipulation of NFT were evaluated in rat liver
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microsomes and primary rat hepatocytes.
A decrease in ability to deplete
glutathione (GSH) was found in the following order: nitrofuran-containing
compounds (NFT and 1-3) > nitrobenzene-containing compounds (4-5) > nitro-free
compounds (6-9).
Similar pattern was observed in the cytotoxicity of these
The potential for reduction (electron
compounds as that of GSH depletion.
deficiency) of nitro groups of the nitro-containing test compounds (NFT, 1-5)
decreased with the decrease in the ability to deplete GSH and the intensity of their
cytotoxicity. The corresponding nitroso and hydroxylamine intermediates resulting
from metabolic reduction of NFT were found to be reactive to GSH for the first time.
Additionally, nitro-containing compound 4 (a model compound) was much more
cytotoxic than the corresponding analine (4a). The findings allowed us not only to
define the mechanism of toxic action of NFT but also to provide medicinal chemists
with instructive guidance for rational design of nitro-containing pharmaceutical
agents.
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Introduction
Nitrofurans are commonly used as antibiotics and food additives in many
countries. The potentially mutagenic and carcinogenic effects and the ambiguous
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mechanisms have drawn broad attention.1
nitrofurantoin
As a 5-nitrofuran derivative,
(NFT),
(1-{[(5-nitro-2-furanyl)methylene]amino}-2,4-imidazolidinedione),
is
an
antimicrobial agent and has been widely used for almost seven decades in the
first-line treatment of uncomplicated urinary tract infections (UTIs).2,3
However,
various adverse effects have been observed arising from the long-term use of NFT,
including hepatotoxicity which was characterized by acute hepatitis, granulomatous
reaction, cholestasis, autoimmune-mediated hepatitis and chronic active hepatitis.4,5
And NFT-associated liver injury is also known as one of the most common causes of
drug induced liver injury (DILI).6 Numerous liver injury cases due to NFT use have
been reported, even including a number of deaths.7-12
Thus, clarifying the
mechanisms is very urgent for assessment of its adverse effects for its safe use.
As a member of 5-nitrofuran family, NFT contains a nitrofuran group and a
hydantoin side chain (Figure 1, NFT).
-nitrofuran group is considered as a basic requirement for mutagenicity and
carcinogenicity.13 Reduction of the nitro group is a major metabolic pathway of
NFT. It is presumed that the corresponding nitroso and hydroxylamine
intermediates are likely to form covalent binding with bio-macromolecules, which
According to the previous studies, the
5
may trigger toxicities.14,15
The reactive intermediates of NFT via metabolic
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reduction, however, have not been identified. Besides, the furan ring can also be
bioactivated via epoxidation and the resulting electrophilic epoxide metabolite can
react with nucleophilic sites of bio-macromolecules.16-19 It is worth noting that the
toxicities and mutagenicities of various nitrofurans are different by several orders,
which appears that the hydantoin side chain of NFT may be responsible for the
toxicities as well, not merely due to the common nitrofuran group.20 Nevertheless,
which structural element within NFT plays a key role in liver injury is still largely
unknown. The objectives of the present study were to define the critical element
responsible for NFT-induced hepatotoxicity by dissecting the structure of NFT, to
determine the importance of electrophilicity of nitro groups in cytotoxicity of
nitroaromatic compounds, and to characterize reactive metabolites responsible for
NFT-induced toxicities.
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Materials and methods
.1 Chemicals and materials
2
NFT (>98%), furfural, benzaldehyde, 5-nitro-2-furaldehyde, 3-nitrobenzaldehyde,
p-nitrobenzaldehyde, hydrazine monohydrate, methylhydrazine sulfate and tin
chloride dihydrate (SnCl ·2H O) were supplied by Aladdin Industrial Corporation
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(Shanghai, China). Hydantoin (99%) was gained from J&K Scientific Ltd. (Beijing,
China). 1,1-Dimethylhydrazine hydrochloride was offered by Sam Chemical
Technology Co., Ltd. (Shanghai, China). Glutathione (GSH), S-hexylglutathione,
NADPH, oxidized glutathione (GSSG), monobromobimane (mBrB), collegenase Ⅱ
and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Sigma
Aldrich (St. Louis, MO). 1-Aminohydantoin hydrochloride (>98%), percoll and rat
tail tendon collagen type Ⅰ were obtained from Dalian Meilun Biotechnology Co.,
LTD. (Dalian, China). Thiazolyl blue tetrazolium bromide (MTT) was provided by
Acros organics (Morris Plains, NJ).
Sprague-Dawley, male) were prepared in our laboratory, according to a previously
published method.21 All organic solvents were purchased from Fisher Scientific
Springfield, NJ). All of the reagents and solvents were of analytical or HPLC
Rat liver microsomes (RLMs)
(
(
grade.
2.2 Chemical synthesis
2
.2.1 General chemistry
All commercial reagents and solvents were used directly without further
purification. 1H NMR spectra were obtained on a Bruker 600 MHz spectrometer.
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Spectral data were recorded as follows: chemical shift (δ, as ppm referenced to TMS),
multiplicity (s = singlet, d = doublet, dd = double doublets, t = triplet, m = multiplet),
coupling constant (J, as Hz), and integration value. High resolution mass spectra
(HRMS) were obtained with a hybrid quadrupole time-of-flight (Q-TOF) mass
spectrometer (Bruker microQ-TOF, Germany) with an ESI source in positive mode.
Purity of the test compounds was estimated by HPLC-analysis with an Eclipse Plus
C18 column (5 μm, 150 mm×4.6 mm; Agilent, USA) and UV detection at 254 nm on
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a 1260 infinity system (Agilent Technologies, Santa Clara, CA).
Analytical
conditions were as follows: solvent A: acetonitrile with 0.1% (v/v) formic acid;
solvent B: water with 0.1% (v/v) formic acid; flow rate: 0.8 ml/min; linear gradient
program, 10% A 1-2 min, 10-90%A 2-10 min, 90%A 10-12 min, 90-10%A 12-14
min, 10%A 14-15 min; column temperature, 25 °C.
The purity of all tested
compounds was at least 95%. Reported yields were not optimized, since the focus is
on purity of products rather than quantity.
2
.2.2. General procedure for the synthesis of compound 1-7
Aldehydes (1.0 equiv) dissolved in methanol and the corresponding hydrazines
(1.1-2.0 equiv) in water were mixed, and the resultant mixtures were stirred at room
temperature in the dark, respectively. The resulting precipitates were filtered,
washed twice with ice-cold methanol and water, and finally, dried at room
temperature to give 1-7.
5-Nitro-2-furancarboxaldehyde hydrazone (1)
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The reaction was carried out as previously reported, with some modifications，as
the general procedure described above.22,23 Compound 1 (yield 66%) was obtained
1
as a yellow solid. H NMR (600 MHz, DMSO-d ): δ8.01 (s, 2H), 7.71 (d, J=4.0 Hz,
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+
1H), 7.57 (s, 1H), 6.75 (d, J= 4.0 Hz, 1H). HRMS: [M + Na] m/z calcd 178.0223,
found 178.0225, err -1.0 ppm.
5-Nitro-2-furancarboxaldehyde methylhydrazone (2)
The title compound (yield 42%) as an orange solid was prepared, according to
the general procedure with an additional neutralization step of methylhydrazine
1
sulfate with NaOH before the reaction.
H NMR (600 MHz, DMSO-d ): δ8.65 (q,
6
J=4.0 Hz, 1H), 7.72 (d, J=4.0 Hz, 1H), 7.24 (s, 1H), 6.71 (d, J= 4.0 Hz, 1H), 2.87 (d,
+
J=3.8 Hz, 3H). HRMS: [M + Na] m/z calcd 192.0380, found 192.0383, err -2.0
ppm.
5
-Nitro-2-furancarboxaldehyde dimethylhydrazone (3)
The methods reported by Kevin T. Potts et al. and Etienne Pair et al. were
followed with some modifications, as the general procedure described above.24,25
Compound 3 (yield 75%) was obtained as a red solid.
1H NMR (600 MHz,
DMSO-d ): δ7.72 (d, J=4.0 Hz, H), 7.11 (s, 1H), 6.69 (d, J=4.0 Hz, 1H), 3.09 (s, 6H).
6
+
HRMS: [M + Na] m/z calcd 206.0536, found 206.0544, err -4.1 ppm.
1-[[(4-Nitrophenyl)methylene]amino]-2,4-imidazolidinedione (4)
Compound 4 (yield 80%) as a white solid was prepared following the general
1
procedure. H NMR (600 MHz, DMSO-d ): δ11.39 (s, 1H), 8.32 (d, J=8.9 Hz, 2H),
6
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+
7.94 (d, J= 8.9 Hz, 2H), 7.92 (s, 1H), 4.38 (s, 2H). HRMS: [M + Na] m/z calcd
2
71.0438, found 271.0444, err -2.2 ppm.
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1
-[[(3-Nitrophenyl)methylene]amino]-2,4-imidazolidinedione (5)
Compound 5 (yield 76%) as a white solid was prepared following the general
1
procedure. H NMR (600 MHz, DMSO-d ): δ11.36 (s, 1H), 8.50 (s, 1H), 8.25 (dd,
6
J=8.1, 2.20Hz, 1H), 8.12 (d, J=7.8 Hz, 1H), 7.97 (s, 1H), 7.76 (t, J=8.0, 8.0 Hz 1H),
+
4
.37 (s, 2H). HRMS: [M + Na] m/z calcd 271.0438, found 271.0433, err 1.7 ppm.
1
-[(2-Furanylmethylene)amino]-2,4-imidazolidinedione (6)
Compound 6 (yield 51%) as a white solid was prepared following the general
1
procedure. H NMR (600 MHz, DMSO-d ): δ11.24 (s, 1H), 7.82 (d, J=1.2 Hz, 1H),
6
7.69 (s, 1H), 6.83 (d, J= 3.4 Hz, 1H), 6.62 (dd, J=3.4, 1.8 Hz, 1H), 4.31 (s, 2H).
+
HRMS: [M + Na] m/z calcd 216.0380, found 216.0388, err -3.9 ppm.
1
-[(2-Phenylmethylene)amino]-2,4-imidazolidinedione (7)
Compound 7 (yield 73%) as a white solid was prepared following the general
1
procedure. H NMR (600 MHz, DMSO-d ): δ11.25 (s, 1H), 7.80 (s, 1H), 7.70 (m,
6
+
2H), 7.43 (m, 3H), 4.36 (s, 2H). HRMS: [M + Na] m/z calcd 226.0587, found
2
26.0597, err -4.5 ppm.
2
.2.3 Synthesis of
1-[[(4-Aminophenyl)methylene]amino]-2,4-imidazolidinedione(4a)
Compound 4a was obtained by reduction of 4 (30 mg, 0.12 mmol) with
SnCl ·2H O (110 mg, 0.49 mmol) in concentrated HCl (2 mL) as previously
2
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reported.26 Compound 4a (3 mg, 11%) was obtained as a yellow solid. 1H NMR
(
600 MHz, DMSO-d ): δ11.09 (s, 1H), 7.60 (s, 1H), 7.36 (d, J= 8.5 Hz, 2H), 6.57 (d,
6
+
J= 8.5 Hz, 2H), 5.59 (s, 2H), 4.30 (s, 2H). HRMS: [M + Na] m/z calcd 241.0696,
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2
.3 Microsomal incubations of NFT
The incubation mixtures containing 100 μM NFT, 1.0 mg/mL rat liver
microsomal protein, 10.0 mM GSH and 3.2 mM MgCl were prepared in potassium
2
phosphate buffer (PBS, pH 7.4) in the presence or absence (control) of NADPH.
The reactions were initiated by addition of NADPH (final concentration: 1.0 mM) to
the mixture. After incubation for 30 min at 37 °C, the reactions were quenched by
mixing with an equal volume of ice-cold acetonitrile, followed by vortexing for 1 min
and centrifuging at 19 000 g for 10 min at 4 °C to remove the precipitated proteins.
A 5 μL aliquot of the resulting supernatants was loaded into liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS) system for analysis. Stock
solution of NFT was prepared in DMSO.
2.4 GSH and GSSG assessment in microsomal incubations
The GSH and GSSG assays in microsomal incubations were performed as
previously reported.27,28 The incubation mixtures (final volume: 250 μL) contained
1.0 mM individual test compounds (NFT, 1-7 or 4a), 2.0 mg/mL rat liver microsomal
protein, 200 μM GSH and 3.2 mM MgCl . NADPH (1.0 mM) was added to start the
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reaction, followed by incubating for 30 min at 37 °C.
The amount of GSH
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remaining in the protein-free supernatants was measured by the method established by
our lab, with some modifications.29 Briefly, aliquots of the resulting incubation
mixtures (30 μL) were moved to another Eppendorf vials containing 50 μL ice-cold
acetonitrilic mBrB (2.2 mM) to stop the reactions together with chemical
derivatization. Particularly, the derivatization reactions were conducted in the dark
at room temperature for 1 h to produce the mBrB-derived GSH conjugate.30 The
resulting derivatization samples were spiked with 110 μL PBS and 10 μL of
S-hexylglutathione (in acetonitrile, internal standard, final concentration: 0.3 μM).
After centrifuging at 19,000g for 10 min, the supernatants (5 μL) were subjected to
LC-MS/MS for analysis.29 The results were corrected for loss of GSH (formation of
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GSSG) resulting from auto-oxidation.
Comparisons were made relative to the
control and contents of GSH, GSSG or loss of GSH, and expressed as the percentage
of the control, respectively.
2.5. Isolation and culture of primary rat hepatocytes
The study was approved by the Institutional Animal Ethical Committee of
Shenyang Pharmaceutical University. Male Sprague-Dawley rats (280-300 g) were
fed with a standard chow diet and water ad libitum. Primary rat hepatocytes were
prepared, according to a two-step collagenase perfusion method.31 The viability was
always higher than 90% as determined by trypan blue exclusion.
Isolated
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hepatocytes were seeded at a density of 1.5×10 cells/mL on collagen-precoated
96-well plates, followed by culture for 4 h at 37 °C under a humidified atmosphere
(
95% O and 5% CO ) in DMEM which contains 20% fatal bovine serum (FBS), 200
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U/ml penicillin-streptomycin, 0.09 M insulin and 0.1 M dexamethasone.
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.6 Cytotoxicity measured by the MTT assay
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Cells were incubated for 4 h in serum-free DMEM containing NFT, 1-9 or 4a
(500 μM). Microplates were then rinsed with EDTA-free PBS (pH 7.4) and the cell
viability was determined using the MTT assay, according to the method described by
Mosmann.32 Stock solutions of individual test compounds (0.5 M) were prepared in
DMSO (final concentration in the media, 0.1%), and vehicle control was performed
using DMSO (0.1%) only.
2.7 Computational details
All theoretical calculations were carried out using the SYBYL Versions 6.91
®
(SYBYL 6.9.1 Tripos Inc., St. Louis, USA) molecular modeling software. All
molecular electrostatic potential (MEP) calculations and visualization were performed
using the MOLCAD program. Partial atomic charges of the nitro-containing test
compounds were calculated by the Gasteiger-Hückel method.
2
.8 LC-MS/MS method
NFT-derived GSH conjugates were analyzed on an AB SCIEX Instruments 5500
triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA)
interfaced online with a 1260 infinity system (Agilent Technologies, Santa Clara,
CA). Chromatographic separation was performed from an Eclipse Plus C18 column
(5 μm, 150 mm×4.6 mm; Agilent, USA) by gradient elution with a flow rate of 0.8
mL/min. The mobile phases were composed of 0.1% (v/v) formic acid in
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acetonitrile (A) and 0.1% (v/v) formic acid in water (B). The linear gradient was as
follows: 10% A at the first 2 min; linear gradient from 10% to 90% A in 8 min; 90%
A step for 2 min; back to 10% A in 2 min; 10% A step for 1 min. The total analysis
time was 15 min. Conjugates were analyzed in positive ion mode by MRM mode.
The mass spectrometric instrument parameters were set as follows: ion source
temperature (TEM) was at 650°C; ion spray voltage (IS) and entrance potential (EP)
were 5500 and 10 V, respectively; curtain gas (CUR), gas 1 (GS1) and gas 2 (GS2)
were 35, 50 and 50 psi, respectively. The characteristics of ion pairs (corresponding
to declustering potential DP, collision energy CE, collision cell exit potential CXP)
were m/z 530/308 (90, 30, 13) and 514/162 (100, 35, 14) for the GSH conjugates.
Additionally, AB SCIEX Instruments 4000 Q-TrapTM (Applied Biosystems,
Foster City, CA) interfaced online with a 1260 infinity system (Agilent Technologies,
Santa Clara, CA) were used and the NFT-derived GSH conjugates were analyzed by
multiple-reaction monitoring-information-dependent acquisition-enhanced product
ion (MRM-IDA-EPI) scan mode. IDA was used to trigger acquisition of EPI spectra
for ions exceeding 500 cps with exclusion of former target ions after three
occurrences for 10 s. The EPI scan was also run in positive mode at a scan range for
product ions from m/z 50 to 550. The CE was set at 40 eV with a spread of 15 eV.
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Other instrument parameters were equivalent to those described above.
The
operation conditions for determining the contents of GSH and GSSG were in
accordance with the method described previously with an addition of the ion pair m/z
of 613.1/355.0 (115, 30, 3) for GSSG.29 All data were analyzed using the AB
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SCIEX Analyst 1.6.3 software (Applied Biosystems).
A fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer
(Bruker Solarix 7.0T, Germany) with an ESI source equipped with an Agilent 1260
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LC system was also employed to characterize the NFT-derived GSH conjugates.
The optimized parameters of electrospray ionization-mass spectrometry (ESI-MS)
were as follows: nebulizer gas pressure (4.0 bar), dry gas flow rate (8.0 L/min) and
temperature (200°C), ion accumulation time (0.15 s), time of flight (0.6 ms), capillary
voltage (4.5 kV), and end plate offset (500 V). The full scan MS data were acquired
over a range of m/z 100-1000 in positive mode. LC conditions were similar to those
described above. Data Analysis Software (Bruker, Germany) was used for data
analysis.
2
.9 Statistical Analysis.
Data are expressed as the mean±S.D. of triplicate samples.
Statistical
significance of differences between treatment groups in these studies was evaluated
with the Student’s t test and differences were considered significant at p<0.05.
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Results
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.1 GSH depletion by NFT and 1-9 in rat liver microsomal incubations.
The levels of GSH and GSSG in microsomes after 30 min incubation of
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individual test compounds (NFT, 1-9) in the presence of NADPH are shown in Figure
As for the full range of incubation systems, the decrease of GSH accompanied
2
.
with the formation of GSSG and the lower the contents of GSH remained, the more
the GSSG detected, suggesting that the observed GSH depletion resulted from the
formation of GSSG. Besides, the levels of GSH showed an increasing tendency in
NFT, 1-9 treated samples, ranging from 14.0% to 98.5% when compared with control.
In other words, a decrease in ability to deplete GSH was found in the following order:
nitrofuran-containing compounds (NFT and 1-3) > nitrobenzene-containing
compounds (4-5) > nitro-free compounds (6-9) (Figure 1 and Figure 3A).
Nitro-containing compounds NFT and 1-5 were as much as 1.75-58 fold more potent
to deplete GSH than nitro-free compounds 6-9. The potencies of NFT and 4 or 5 for
GSH depletion were about 4 and 30 or 23 times higher than that of the corresponding
nitro-free 6 and 7, respectively.
Furthermore, 83%, 45% and 35% GSH were
depleted when treated with NFT, 4 and 5, respectively, indicating that the replacement
of furan ring with a phenyl ring resulted in approximately 50% loss of the ability to
deplete GSH.
3
.2 Cytotoxicity of NFT and 1-9 in primary rat hepatocytes
Cytotoxicities of NFT and 1-9 were evaluated in primary rat hepatocytes. Cells
were exposed to the individual test compounds at 500 μM, a cytotoxic concentration
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of NFT.33 Nitrofuran derivatives NFT and 1-3 were found to be more toxic than
nitrobenzene derivatives 4-5 (Figure 1 and 4A), which coincides with that of the GSH
depletion assay (Figure 1, 3A and 4A).
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.3 Computer simulation
To gain a better understanding of the relationship between the chemical
reactivity of nitro-containing compounds (NFT, 1-5) and their toxicities, MEP and
partial atomic charges on nitrogen of the nitro groups of the test compounds were
calculated. MEP contour maps show that the region near the nitrogen atom of nitro
group of NFT is more positive than those of the corresponding 4 and 5, a range of
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2.7-29.9 kcal/mol versus 1.2-8.4 kcal/mol (Figure 5), indicating higher nucleophilic
susceptibility of NFT. As for the other three nitrofuran-containing compounds, 1-3,
the positive regions are merely over nitrogen atom of the nitro moieties of them,
respectively, ranging from 29.9 to 51.4 kcal/mol, which show high electrophilic
reactivity. Besides, the regions over furan rings of nitrofuran-containing compounds
NFT and 1-3 are more positive than those of the phenyl rings of
nitrobenzene-containing compounds 4 and 5. Partial atomic charges on nitrogen of
the nitro groups of NFT and 1-5 are shown in Figure 6. As expected, partial positive
charges on nitrogen of the nitro groups of the listed compounds decreased in the
following order: nitrofuran-containing compounds (1-3 and NFT)
nitrobenzene-containing compounds (4 and 5), coinciding with MEP calculation.
>
3
.4 Toxic role of nitro group of NFT
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3.4.1 Formation of NFT-derived GSH conjugates in microsomal incubations.
Identification of reactive metabolites is a key step to define the mechanisms of
toxic action of NFT. Reactive intermediates of NFT were trapped by GSH in
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microsomal incubations.
The formation of the resulting GSH conjugates was
monitored by LC-MS/MS. Two metabolites (M1 and M2) were detected and the
formation of the two both appeared to be NADPH- and cytochrome P450 (P450)-
dependent (Figure 7A, B, D and E).
M1, with the retention time at 2.27 min, was detected by scanning of the ion pair
of m/z 530→308. The tandem mass (MS/MS) spectrum of M1 obtained through
MRM-IDA-EPI scanning in positive mode is shown in Figure 7C. Major fragment
ions at m/z 308.0, 223.0, 179.0, and 162.1 all resulted from the cleavage of GSH
moiety, indicating the participation of GSH in the formation of M1. In detail,
product ions at m/z 308.0 and 223.0 were generated from the protonation of GSH and
a total loss of GSH (-307 Da) from m/z 530, respectively, arising from the cleavage of
the N-S bond of M1. Another major ion at m/z 179.0 was derived from a loss of the
γ-glutamyl moiety (-129 Da) from GSH and a sequential loss of NH generated
3
fragment ion at m/z 162.1.
M2, eluted at a retention time of 2.30 min, was detected by acquiring ion pair at
m/z 514→162 in positive mode. The MS/MS spectrum of M2 exhibited the major
fragment ions associated with fragmentation of the GSH moiety, signaling the
participation of GSH (Figure 7F).
Product ions at m/z 439.1 and 385.1 were
obtained from the loss of the glycinyl moiety (-75 Da) and γ-glutamyl moiety (-129
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Da) from m/z 514, respectively, and 367.1 was gained from an elimination of H O
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from m/z 385.1. The observed fragment ion at m/z 162.2 was generated as that of
M1 described above.
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Additionally, the incubation mixtures were also analyzed by high resolution
mass spectrometry to characterize the elemental compositions of M1 and M2. The
protonated molecular ions of these two conjugates were found to be 530.13174 and
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14.13619, matching the formulas of C H N O S and C H N O S, respectively
18 24 7 10 18 24 7 9
(Figure 8 and Table 1). And the accurately measured mass in the high resolution
mass spectrometry system was in accordance with the corresponding theoretical mass
within 5 ppm based on the predicted formula and the similarity scores of the detected
and predicted isotope patterns of M1 and M2 were both found to be 100, making the
data more reliable (Table 1).
3
.4.2 GSH depletion by 4 and 4a in microsomal incubations
To define the mechanisms of NFT-induced toxicities, the ability to deplete GSH
of 4a, an aniline analogue of 4, was evaluated. Figure 3B shows that 45% GSH was
depleted by 4, whereas 4a showed little GSH depletion property (only 0.6%). In
other words, reduction of 4 to the corresponding aniline resulted in 99% decreased
capability of GSH depletion.
3
.4.3 Cytotoxicity of 4 and 4a in primary rat hepatocytes
Cytotoxicity study of 4 and 4a was conducted in primary hepatocytes by the
MTT assay. As expected, less cytotoxicity was found when cells were treated with
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4a rather than 4, as exhibited in Figure 4B, consistent with that of the GSH depletion
assay.
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Discussion
NFT, a nitrofuran derivative, has been applied for the first-line treatment of
specific UTIs since 1950s.2,3 However, since its introduction into clinical use, NFT
has been associated with various adverse effects, including hepatotoxicity.4,5
Though many efforts have been made to explore the insight into NFT-induced liver
injury, little is known about the mechanisms, and even the group which plays a key
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role in this adverse drug reaction.
In the present study, we conducted a
structure-toxicity relationship study by structural dissection to determine the essential
functional group(s) responsible for NFT-induced hepatotoxicity. A total of nine
compounds were rationally designed and synthesized to probe the mechanism of toxic
action of NFT. Basically, the structure-toxicity relationship study was designed to
answer three questions, including 1) whether the hydantoin moiety plays a role in
NFT toxicity; 2) whether the nitro group is essential for NFT toxicity; and 3) whether
the furan ring is required for NFT toxicity.
GSH plays a crucial role in cytoprotection, acting as a nucleophile capture for
reactive metabolites generated from xenobiotics. GSH depletion is known as an
early biomarker of cytotoxicities.
As an initial step of toxicity evaluation, we
determined the ability of NFT and 1-9 to deplete GSH in microsomal incubation
systems. The GSH depletion-based microsomal incubation system is often used to
investigate metabolism-mediated mechanisms of toxic action.27,28
The GSH
reactivity associated with these test compounds ranked in the following order: 1-3 and
NFT > 4-5 > 6-9. It is interesting to find that these compounds can be divided into
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three groups: nitrofuran-containing compounds (1-3, NFT), nitrobenzene-containing
compounds (4-5), and nitro-free compounds (6-9), based on their ability to deplete
GSH. In other words, the results showed that the nitrofuran-containing compounds
demonstrated the greatest GSH depletion property, indicating that 5-nitrofuran moiety
of NFT most likely elicited toxicities rather than the hydantoin side chain. The
nitro-containing compounds (NFT, 4/5) produced much higher responses than those
of the corresponding nitro-free compounds (6, 7) (approximately 4, 30/23 times
higher), respectively, suggesting that nitro group played the essential role in NFT
toxicity. Besides, when the furan moiety of NFT was replaced by a phenyl ring, that
is, the nitrobenzene-containing compounds, the small structural alternation resulted in
dramatic decrease in ability to deplete GSH. This indicates that the furan ring may
be another factor regarding the toxicities.
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As the second step, toxicity of the test compounds was evaluated in primary rat
hepatocytes.
observed in the GSH depletion study, which solidified the evidence obtained from the
GSH depletion study. However, compound 6, a nitro-free furan-containing
The cytotoxicity of these compounds revealed similar pattern as
compound, was found to be more cytotoxic than nitro-containing compound 5. Our
early studies demonstrated nitro-free furan-containing compounds underwent
oxidation to form cis-enedials. The formation of oxidative metabolites is most likely
responsible for their toxicities.21,34,35 These furan compounds did not show much
GSH depletion effects.21,35 Therefore, we were not surprised by the exceptional
finding. Additionally, no significant difference was observed in the degrees of GSH
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depletion induced by NFT and compounds 1-3 (Figure 3A), whereas the cellular
viability of NFT and compound 3 were both significantly higher than that of 1-2
(Figure 4A). Interestingly, compounds 1 and 2 appeared to be more toxic than NFT
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and compound 3. This may suggest that the numbers of methyl substituents at the
terminal nitrogen atom may play some role in the observed cytotoxicity besides the
nitro group, and the addition of hydantion in the side chain made NFT less cytotoxic.
We speculated that the electron deficiency on the nitrogen of the nitro groups
which can be dictated by the properties of neighboring functional groups
(furan/phenyl ring) would determine the potential of GSH depletion and the potency
of cytotoxicity. An attempt was made to estimate the electron deficiency by MEP
and partial atomic charges calculation. The calculated MEP contour maps and the
partial charges on nitrogen of the nitro groups of the test compounds showed that the
nitrogen of the nitro groups of the nitrofuran-containing compounds possessed more
positive potential than that of the nitrobenzene-containing compounds, indicating
higher electrophilic reactivity of the nitrofuran-containing compounds than that of the
nitrobenzene-containing compounds. This explains the observed greater cytotoxicity
of the nitrofuran-containing compounds than that of the nitrobenzene-containing
compounds.
The rates of reductive reactions are known to depend on the electron density of
the reactants, and electron deficiency favors reductive reactions.
Additionally,
similar trends were found for the formation of GSSG, the contents of which were
monitored at the same time in the GSH depletion study, revealing that the observed
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GSH depletion resulted in the formation of GSSG.
We proposed that these nitro-containing compounds mainly undergo
nitro-reductive metabolic pathways to deplete GSH, as shown in Scheme 1. It is
likely that the nitro group is firstly reduced to a nitroso mediated by P450, and the
GSH applied attacks at the nitrogen atom of the nitroso to form the resulting GSH
conjugate, followed by nucleophilic attack through a second molecule of GSH to the
hydroxylamine with a release of GSSG. The hydroxylamine may also react with a
third molecule of GSH to form another GSH conjugate, and then undergoes sequential
attack by the fourth molecule of GSH to the amine, releasing a second GSSG
molecule. The more GSH is depleted, the more GSSG formed.
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We characterized reactive metabolites of NFT trapped with GSH in microsomal
incubations and monitored the formation of the corresponding GSH conjugates.
Two products (M1 and M2) were detected by LC-MS/MS, which appeared to be
NADPH and P450 dependent, suggesting that P450 enzymes played an important role
in the metabolic reduction of the nitro group of NFT. There were two possible
chemical structures of M1 we proposed at first, since the hydroxyl group of the
semi-mercaptal may migrate to the sulfur atom to form a sulphinamide. However,
the observed product ions of M1 at m/z 308.0 and 223.0, which were generated from
the cleavage of the N-S bond, indicate that the hydroxyl group attaches to the nitrogen
atom, and the formation of sulphinamide isomer of M1 is unlikely. Additionally,
MS spectrum and elemental analysis demonstrated M2 contained one oxygen atom
less than M1, suggesting M2 is a sulfenamide.
This surprised us, since
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semi-mercaptals may rearrange to the sulphinamides which are apparently more
stable.
Besides, aryl sulfenamides are notoriously unstable, while the
semi-mercaptal form (M1) and sulfenamide form (M2) of NFT appeared to be
unexpectedly stable. The characteristic fragment ions (mainly derived from the
cleavage of GSH) and elemental compositions (characterized by high resolution mass
spectrometry) confirmed the proposed structures of M1 and M2. NFT-induced
decrease in cellular GSH contents has been documented, along with proposed
metabolic reduction.36-39 However, the mechanisms of the observed GSH depletion
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remain unknown.
We succeeded in identifying the nitroso and hydroxylamine
(Scheme 1) as the reactive intermediates responsible for the GSH depletion. The
findings encouraged us to hypothesize that metabolic reduction of the nitro group to
nitroso and hydroxylamine initiated GSH depletion and the development of toxicities.
Besides, GSH depletion can also be attributed to redox recycling of the NFT nitro
radical anion, which may trigger oxidative stress and lead to cytotoxicity as a
consequence (Scheme 1). 36-39
We made a great effort to chemically reduce NFT to the corresponding nitroso
and amine, since we wanted to manipulate the electron density of the nitro group of
NFT and to compare the relative toxicity of these compounds to provide additional
evidence for our hypothesis. Unfortunately, we failed due to the unstability of the
anticipating products. Then we successfully reduced 4 to its aniline analogue (4a)
and employed 4 and 4a for our mechanistic studies. As expected, 4a was less toxic
than 4, which was found in both GSH depletion and cytotoxicity studies, suggesting
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that the reductive process of nitro to the amine induces cytotoxicity and the nitro
group plays a key role in cytotoxicity. It is likely that NFT underwent similar
reductive metabolic pathway with the nitroaromatic moiety as that of 4 to trigger
cytotoxicity. This also supports our hypothesis, since the more electron deficient
nitro group is, the easier be reduced to the nitroso and hydroxylamine, and the faster
to deplete GSH, followed by an increase in GSSG formation and cytotoxicity.
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Conclusions
In conclusion, the key moiety in contribution to NFT cytotoxicity was the nitro
group. The development of NFT cytotoxicity required metabolic reduction of its
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nitro group.
The resulting nitroso and hydroxylamine intermediates were
responsible for the observed GSH depletion and cytotoxicity induced by NFT. The
electron deficiency of the nitrogen of the nitro group favored the cytotoxicity of
nitroaromatic compounds. The findings of this study facilitate the understanding of
the mechanisms of toxic action of nitroaromatic compounds and the development of
nitroaromatic new drugs.
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Funding Information
This work was supported in part by the National Natural Science Foundation of China
[Grants 81773813, 81430086, 81830104].
Compliance with ethical standards
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Conflict of interest
The authors declare that they have no conflict of interest.
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Abbreviations list
CE, collision energy; CUR, curtain gas; CXP, collision cell exit potential; DILI,
drug induced liver injury; DMEM, Dulbecco's Modified Eagle Medium; DP,
declustering potential; EP, entrance potential; ESI-MS, electrospray ionization-mass
spectrometry; FBS, fatal bovine serum; FT-ICR, fourier transform-ion cyclotron
resonance; GSH, glutathione; GS1, gas 1; GS2, gas 2; GSSG, oxidized glutathione; IS,
ion spray voltage; LC-MS/MS, liquid chromatography coupled with tandem mass
spectrometry; mBrB, monobromobimane; MEP, molecular electrostatic potential;
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MRM-IDA-EPI,
multiple-reaction
monitoring
information-dependent
acquisition-enhanced product ion; MS/MS, tandem mass; MTT, thiazolyl blue
tetrazolium bromide; NFT, nitrofurantoin; P450, cytochrome P450; PBS, potassium
phosphate buffer; Q-TOF, hybrid quadrupole time-of-flight; RLMs, rat microsomes;
SnCl •2H O, tin chloride dehydrate; TEM, ion source temperature; UTIs, urinary tract
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