Glutathione Conjugation and Protein Adduction by Environmental Pollutant 2,4-Dichlorophenol in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.chemrestox.0c00118

2,4-Dichlorophenol (2,4-DCP), an environmental pollutant, was reported to cause hepatotoxicity. The biochemical mechanisms of 2,4-DCP induced liver injury remain unknown. The present study showed that 2,4-DCP is chemically reactive and spontaneously react

Full text of DOI:10.1021/acs.chemrestox.0c00118

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Glutathione Conjugation and Protein Adduction by Environmental
Pollutant 2,4-Dichlorophenol In Vitro and In Vivo
Qingmei Li, Wei Li, Jiaxing Zhao, Xiucai Guo, Qian Zou, Zixin Yang, Ruixue Tian, Ying Peng,*
and Jiang Zheng*
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ABSTRACT: 2,4-Dichlorophenol (2,4-DCP), an environmental
pollutant, was reported to cause hepatotoxicity. The biochemical
mechanisms of 2,4-DCP induced liver injury remain unknown. The
present study showed that 2,4-DCP is chemically reactive and
spontaneously reacts with GSH and bovine serum albumin to form
GSH conjugates and BSA adducts. The observed conjugation/
adduction apparently involved the addition of GSH and departure
of chloride via the ipso substitution pathway. Two biliary GSH
conjugates and one urinary N-acetyl cysteine conjugate were observed
in rats given 2,4-DCP. The N-acetyl cysteine conjugate was chemically
synthesized and characterized by mass spectrometry and NMR. As
expected, 2,4-DCP was found to modify hepatic protein at cysteine
residues in vivo by the same chemistry. The observed protein
adduction reached its peak at 15 min and revealed dose dependency.
The new findings allowed us to better understand the mechanisms of the toxic action of 2,4-DCP.
other research, the geometric mean of 2,4-DCP was 0.14 μg/L
in Korean urine samples.10
1. INTRODUCTION
2,4-Dichlorophenol (DCP) is a hazardous pollutant with
widespread exposure via industrial and indoor air pollution,
diet, and the use of pesticides and herbicides.1 Besides, as a
byproduct of chlorination in industrial wastewater, 2,4-DCP is
widely distributed in the environment because of its stability. It
was reported that 2,4-DCP showed uneven distribution in the
surface water of China with 50 ng/L as median concentration.
In addition the Yellow River, one of the two longest river in
China, suffered heavy pollution from 2,4-DCP with a
maximum concentration of 20 μg/L.2 In some other countries,
the differences of 2,4-DCP distribution in surface water
Liver is an important organ for metabolism and
detoxification, and the liver damage caused by 2,4-DCP is of
particular concern. Previous studies in vitro have shown that
2,4-DCP resulted in primary hepatocyte apoptosis and
embryonic fibroblasts in mice6 and decreased cell viability
and inhibited formation of a colony in human liver 7702
cells.11 2,4-DCP was also found to increase liver weights of
rats7 and to cause liver histopathological changes and elevated
ALT and AST levels in mice.11 Severe endoplasmic reticulum
stress was responsible for the hepatotoxicity induced by 2,4-
DCP.11 Mitochondria-dependent pathway was reported to play
an undeniable role in apoptosis caused by 2,4-DCP in primary
hepatocytes.6
presented similar levels with concentrations from 1 to 5 μg/
3
̂
L in the Rhone River Delta and 0.1 to 6 μg/L in the Gulf of
Gdansk.4
Dehalogenation reactions are often involved in the
metabolism of halogen-containing hydrocarbons. For example,
N-acetyl-S-(pentachlorophenyl) cysteine was detected in urine
of rats, mice and rabbits given fungicide hexachlorobenzene
(HCB)12 that reportedly induced liver cancer and hepatic
2,4-DCP was reported to induce DNA hypermethylation5
and liver injury6 along with immunotoxicity7 in animals and to
cause olfactory dysfunction1 and allergy8 in humans. In
consideration of the various toxic effects caused by 2,4-DCP,
biological monitoring was performed for better assessment of
potential risks. In a recent study, the levels of urinary 2,4-DCP
were determined in nine countries and territories around the
world, and 2,4-DCP displayed the highest detection rate
(92.7%) compared with that of other chlorophenols. The
median concentration in all regions was 0.34 ng/mL, with the
highest median concentration of 1.61 ng/mL in China.9 In
Received: March 27, 2020
Published: August 11, 2020
https://dx.doi.org/10.1021/acs.chemrestox.0c00118
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porphyria.13,14 This implies the loss of a chlorine atom from
HCB during its metabolic process in vivo. Dechlorination of
2,4-DCP has also been reported, and Mehmood and co-
workers found that 2,4-DCP was metabolized to a p-quinone
intermediate in microsomal fractions and whole-cells of
Saccharomyces cerevisiae containing human cytochrome P450
3A4.15 This indicates that oxidation was involved in the
observed 2,4-DCP dechlorination. Here we report a novel
dechlorination pathway of 2,4-DCP and related GSH
conjugation and protein adduction.
(time-dependent experiment, n = 3 for each time point). The
harvested liver tissues (0.2 g) were homogenized in 2.0 mL of
phosphate buffers (pH 7.4), followed by centrifugation at 19 000g for
10 min. The resulting homogenates were heated at 60 °C for 1 h and
centrifuged at 19 000g for 10 min. The resultant pellets were
reconstituted in 100 mM ammonium bicarbonate and subjected to
proteolytic digestion.
In a separate study, mice were intraperitoneally treated with 2,4-
DCP dissolved in corn oil at dose of 50 mg/kg body weight. Blood
was harvested at 10 min (n = 3) after the administration. Vehicle
group was treated intraperitoneally with corn oil. Blood samples were
allowed to stand at room temperature for 4 h and centrifuged at
19 000g for 10 min. The supernatants were analyzed by an LC-MS/
MS system for determination of the GSH conjugates.
2. MATERIALS AND METHODS
2.4. Chemical Synthesis of 2,4-DCP-Derived NAC and
Cysteine Conjugates (7 and 10). Synthesis of 2,4-DCP-derived
NAC conjugates was started with by diazotization of 2-amino-4-
chioroanisole, followed by reaction with NAC and demethylation
(Scheme 2). Briefly, concentrated hydrochloric acid (6.25 mL, 75
2.1. Chemicals and Materials. 2,4-DCP (purity >98%) was
obtained from Aladdin Industrial Technology Co., Ltd. (Shanghai,
China). N-Acetylcysteine (NAC), cysteine, chymotrypsin, DL-
dithiothreitol (DTT) and bovine serum albumin (BSA) were
purchased from Sigma-Aldrich Co. (St. Louis, MO). Pronase E with
a purity of 98% was acquired from Shanghai Yuanye Biological
Technology Co., Ltd. (Shanghai, China). All reagents and solvents
were of either analytical or HPLC grade.
Scheme 1. Proposed Pathways of GSH Conjugate
Formation Derived from Dechlorination of 2,4-DCP
2.2. Incubations of 2,4-DCP with GSH/Cysteine In Vitro. Rat
(Sprague−Dawley) liver microsomes (RLMs) and mouse (Kun-
Ming) liver microsomes (MLMs) were prepared as described by our
laboratory.16 2,4-DCP (0.1 mM) was mixed with RLMs (1.0 mg
protein/mL), MgCl2 (3.2 mM), and GSH (10 mM) in 250 μL of
phosphate buffer (PBS, pH 7.4). The incubations were initiated by
addition of NADPH (1.0 mM) and terminated by addition of equal
volume of ice-cold acetonitrile after 1 h incubation at 37 °C. Control
samples were NADPH excluded. RLMs-free incubations contained
2,4-DCP (0.5 mM) and GSH or cysteine (20 mM) in 200 μL of PBS
(pH 7.4). As the blank group, GSH (or cysteine) was replaced by
PBS. In a separate study, RLMs were replaced by MLMs for NADPH-
free incubations. The corresponding control samples excluded MLMs.
The mixtures were then incubated for 1 h at 37 °C. The resulting
incubation mixtures were votexed for 3 min and centrifuged at
19 000g for 10 min, followed by LC-MS/MS analysis. Each incubation
was performed in triplicate.
mmol) was added dropwise to 100 mL of water at 0 °C, followed by
addition of 2-amino-4-chioroanisole (4.7 g, 30 mmol) and then
dropwise addition of NaNO2 (2.2 g, 31.5 mmol) dissolved in 10 mL
of water with stirring. During the process, the reaction solution
gradually turned yellow. After 15 min stirring, the cold mixture was
then added to a stirred solution of NAC (9.8 g, 60 mmol) dissolved in
50 mL of water at 90 °C. The resulting brown-yellow viscous solid
was dissolved in 20 mL of ethyl acetate. After 2 h stirring, the mixture
was cooled to room temperature and extracted with ethyl acetate. The
ethyl acetate layer was washed twice with water and saturated aqueous
solution of NaCl respectively, dried with anhydrous sodium sulfate,
condensed to dryness under reduced pressure, and submitted to a
silica gel column for purification. The purified product (3′, Scheme 2)
was characterized by mass spectrometry and NMR.
2.3. Animal Experiments. Male Sprague−Dawley rats (200 20
g, 6−7 week old) and male Kun-Ming mice (18−22 g, 5−6 week old)
were purchased from the Animal Center of Shenyang Pharmaceutical
University (Shenyang, China). All animals had free access to food and
water and were housed in a temperature-controlled (22
4 °C)
facility with a 12-h dark/light cycle for at least 5 days after receipt and
before treatment. All animal studies were performed, according to
procedures approved by the Ethics Review Committee for Animal
Experimentation of Shenyang Pharmaceutical University.
One group of rats (n = 3) were anesthetized with chloral hydrate,
and their bile ducts were cannulated with PE-10 tubing. 2,4-DCP
dissolved in corn oil was administered intraperitoneally at dosage of
100 mg/kg body weight, and bile samples were collected for 2 h
following dosing. Blank bile samples were collected before the
treatment. The other group of rats (n = 3) administered with the
same dose of 2,4-DCP and were individually placed in metabolism
cages. Urine samples were collected during the time periods of 0−12
and 12−24 h after administration. The control rats treated with
vehicle were included. During the experiment, the rats were allowed
free access to food and water. The resulting bile and urine samples
were mixed with triple volumes of acetonitrile and centrifuged at
19 000g for 10 min. The resultant supernatants were concentrated to
dryness under a stream of nitrogen gas, reconstituted with 200 μL of
10% acetonitrile in water, and centrifuged at 19 000g for 10 min. The
supernatants (5.0 μL) were analyzed by an LC-MS/MS system.
In a separate study, mice were treated intraperitoneally with 2,4-
DCP dissolved in corn oil at doses of 0, 15, 50, or 100 mg/kg body
weight, and livers were harvested at 15 min after the treatment (dose-
dependent experiment, n = 3 for each dose). In parallel, animals were
treated with 2,4-DCP at dose of 15 mg/kg body weight, and livers
were harvested at 5, 10, 15, 20, 30, 45, and 60 min after the treatment
The resulting product (3′, 0.1 g, 0.33 mmol) was slowly mixed with
3.5 mL of a BBr3 solution (1.0 M) in dichloromethane at −30 °C with
stirring, and the resulting mixture was further stirred at room
temperature overnight.17 The reaction was terminated by addition of
5 mL of cold water, and the resultant mixture was extracted with
dichloromethane. The organic phase was washed three times with
water and twice with saturated aqueous solution of NaCl, and
condensed under reduced pressure. Product 7 (Scheme 2) was
purified by a semipreparative HPLC system. Colorless crystals (0.011
g, 11% in yield) were obtained and characterized by mass
1
spectrometry and H NMR.
The related cysteine conjugate (10, Scheme 3) was synthesized by
enzymatic hydrolysis of synthetic NAC conjugate 7 (Scheme 4).
Colorless crystals (1.0 mg) dissolved in 50 μL of acetonitrile mixed
with a solution (0.5 mL) of Pronase E (2.0 mg/mL) and CaCl2 (5.0
mM). The mixture was incubated at 37 °C for 4 h, and then the
digestion mixtures were centrifuged at 19 000g for 10 min, and the
supernatants (5.0 μL) were subjected to an LC-MS/MS system for
analysis.
B
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Scheme 2. Synthetic Routes of NAC Conjugate 7
Scheme 3. Proposed Pathways of GSH/NAC and Protein Adduct Formation by 2,4-DCP In Vivo
2.5. Proteolytic Digestion. Protein samples were mixed with a
DTT solution (final concentration: 5.0 mM, final volume: 0.5 mL).
After 1 h incubation at 60 °C, the resulting protein samples were
treated with a mixture of chymotrypsin (1.0 mg/mL) and Pronase E
(2.0 mg/mL) in the presence of 5.0 mM CaCl2 with continuous
incubation at 37 °C for 15 h, followed by addition of an acetonitrile
solution (0.5 mL) containing propranolol (internal standard, final
concentration: 10 ng/mL). The resulting mixtures were centrifuged at
16 000g for 10 min, and the supernatants were subjected to LC-MS/
MS for analysis.18
C
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(50, 28, and 5) for cysteine conjugates, and m/z 260/116 (70, 25, and
5) for propranolol (internal standard).
Scheme 4. Synthesis of Cysteine Conjugate 10 by Enzymatic
Hydrolysis of 7
Furthermore, MS/MS spectra were obtained from an AB SCIEX
Instruments 4000 Q-Trap (Applied Biosystems, Foster City, CA)
interfaced inline with an Agilent 1260 Series Rapid Resolution LC
system (Agilent Technologies, Santa Clara, CA). The information-
dependent acquisition (IDA) method was utilized to trigger the
enhanced product ion (EPI) scans by analyzing MRM signals. The
EPI scan was run in positive mode at a scan range for productions
from m/z 50 to 460. The CE was set at 35 eV with a spread of 5 eV.
All data were processed using the AB SCIEX Analyst 1.6.2 software
(Applied Biosystems).
2.6. Reaction of 2,4-DCP with Bovine Serum Albumin (BSA).
2,4-DCP (1.0 mg) was dissolved in 10 μL of acetonitrile and mixed
with 3.2 mg of BSA dissolved in 200 μL of PBS buffer. The mixture
was incubated for 1 h at 37 °C.19 The resulting sample was submitted
to proteolytic digestion as described above.
2.7. LC-MS/MS Methods. All samples were analyzed on an AB
SCIEX Instruments 5500 triple quadrupole mass spectrometer
(Applied Biosystems, Foster City, CA) interfaced inline with a 1260
infinity system (Agilent Technologies, Santa Clara, CA). Chromato-
graphic separation was achieved on an Eclipse Plus-C18 column (4.6
× 150 mm, 5 μm; Agilent Technologies, Santa Clara, CA). Gradient
elution was performed with acetonitrile containing 0.1% formic acid
(solvent A) and water containing 0.1% formic acid (solvent B) at a
constant flow rate of 0.8 mL/min. The gradient started at 10% A for 2
min, ramped to 90% A over 6 min, returned to 10% A in 2 min, and
then held at 10% A for 2 min. The column temperature was kept at 25
°C.
Samples with a volume of 5 μL were analyzed by LC-MS/MS with
multiple-reaction monitoring (MRM) scanning in positive ion mode.
The optimized MS instrument parameters obtained after tuning were
as follows: ion spray voltage (IS) was set at 5500 V, and ion source
temperature (TEM) was at 650 °C. Curtain gas, gas 1, and gas 2 were
20, 50, and 50 psi, respectively. The characteristics of product ion
pairs (corresponding to declustering potential DP, collision energy
CE, and collision cell exit potential CXP) were m/z 450/321 (100,
30, and 10) and m/z 434/202 (100, 33, and 10) for GSH conjugates,
m/z 290/159 (50, 30, and 10) for NAC conjugates, m/z 248/159
3. RESULTS
3.1. GSH Conjugates Derived from 2,4-DCP In Vitro.
As an initial step, 2,4-DCP was incubated in rat liver
microsomes fortified with NADPH and GSH. Two GSH
conjugates (3 and 4, Scheme 1A) monitored by acquiring of
ion pair m/z 450/321 were detected with retention time at
6.52 and 6.81 min, respectively (Figure 1B). The obtained
MS/MS spectra (Figure 1C), along with the detected
molecular ion, allowed us to propose that 3 and 4 were
GSH conjugates derived from a p-quinone metabolite (2,
Scheme 1A) of 2,4-DCP. No such conjugates were detected
(Figure 1A) in an NADPH-free microsomal incubation system.
Besides, two GSH conjugates, namely 5 and 6, with
retention times at 6.77 and 6.95 min were detected (Figure
2B) by acquiring of ion pair m/z 434/202. The MS/MS
spectra displayed the indicative characteristic fragment ions of
the GSH moiety (Figure 2D), such as m/z 359 (loss of 75 Da,
glycinyl portion) and 305 (loss of 129 Da, glutamyl portion).
The ions at m/z 202 (loss of 232 Da) and 185 (loss of 249 Da)
were considered to result from the cleavage of the GSH
moiety. The observed molecular ion and MS/MS spectra
suggest the formation of 5/6 (Scheme 1B), monochlorine
Figure 1. Characterization of GSH conjugates 3 and 4 derived from oxidative dechlorination of 2,4-DCP. Extracted ion (m/z 450/321)
chromatograms obtained from LC-MS/MS analysis of RLMs incubations containing 2,4-DCP and GSH in the absence (A) or presence (B) of
NADPH. MS/MS spectra of 3/4 obtained from studies B (C) described above.
D
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Figure 2. Characterization of GSH conjugates 5 and 6 derived from 2,4-DCP. Extracted ion (m/z 434/202) chromatograms obtained from LC-
MS/MS analysis of RLMs incubations containing 2,4-DCP and GSH in the absence (A) or the presence (B) of NADPH, and incubations
containing GSH and 2,4-DCP (C). MS/MS spectra of 5/6 obtained from study B (D) described above.
Figure 3. Characterization of GSH conjugates 5 and 6 derived from 2,4-DCP. Extracted ion (m/z 434/202) chromatograms obtained from LC-
MS/MS analysis of bile samples obtained from rats before (A) or after (B) treatment with 2,4-DCP at 100 mg/kg. MS/MS spectra of 5/6 obtained
from study B (C) described above.
E
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Figure 4. Characterization of NAC conjugate 7 derived from 2,4-DCP. Extracted ion (m/z 290/159) chromatograms obtained from LC-MS/MS
analysis of urine samples obtained from rats before (A) or after (B) treatment with 2,4-DCP at 100 mg/kg, and synthetic adduct (C). MS/MS
spectra of NAC conjugate 7 obtained from studies B (D) and C (E) described above.
substituted GSH conjugates. Unlike 3 and 4, 5 and 6 were also
detected in NADPH-free microsomal incubations (Figure 2A,
RLMs; Figure S2B, MLMs). Conjugates 5 and 6 were also
detected in an incubation mixture containing 2,4-DCP and
GSH dissolved in PBS solution (Figure 2C).
3.2. Biliary GSH Conjugates Derived from 2,4-DCP in
Rats. GSH conjugates are considered to be biomarkers of
exposure to electrophilic species and are often secreted in bile.
Bile samples were collected from rats treated with 2,4-DCP
and analyzed by LC-MS/MS. As expected, 5 and 6 were
detected in the bile, but not in blank bile samples (Figure 3A).
The two biliary GSH conjugates showed the same chromato-
graphic and mass spectrometric properties (Figure 3B,C) as 5/
6 detected in the above in vitro studies (Figure 2B,D).
3.3. Urinary NAC Conjugates Derived from 2,4-DCP
in Rats. Similar to GSH conjugates, NAC conjugates
(mercapturic acids) are regarded as another biomarkers of
the exposure of electrophiles and often excreted in urine. Urine
samples of rats given 2,4-DCP were collected and analyzed by
LC-MS/MS. As shown in Figure 4B, a 2,4-DCP-derived
metabolite (7, Scheme 3) with retention time of 7.81 min was
detected by acquiring of ion pair m/z 290/159. No such
metabolite was observed in blank urine (Figure 4A). The MS/
MS spectrum displayed product ions m/z 159, 185 and 244,
along with molecular ion at m/z 290 (Figure 4D). Ion m/z 159
possibly resulted from the cleavage of the C−S bond within
NAC portion. The fragment ions at m/z 185 (C8H6ClOS) and
244 (C10H11ClNO2S) were related to the cleavage of NAC
moiety, implying that NAC was involved in the generation of
7. Conjugate 7 was likely a NAC conjugate derived from
dechlorinated 2,4-DCP.
3.4. Identification of Synthetic NAC Conjugate. The
proposed conjugate 7 was synthesized and characterized byLC-
MS/MS and NMR. The synthetic product shared the same
chromatographic (Figure 4C) and mass spectral identities
(Figure 4E) as those of 7 detected in urine samples (Figure
1
4B,D). H NMR (400 MHz, DMSO-d6): δ 1.83 (3H, s,
−CH3), 3.04 (1H, dd, J = 8.44, 13.5 Hz, −CH2−), 3.25 (1H,
dd, J = 5.13, 13.6 Hz, −CH2−), 4.31 (1H, dt, J = 4.97, 8.12 Hz,
−CH), 6.82 (1H, d, J = 8.57 Hz, Ar−H), 7.07 (1H, dd, J =
2.57, 8.63 Hz, Ar−H), 7.22 (1H, d, J = 2.56 Hz, Ar−H), 8.27
(1H, d, J = 7.8 Hz, Ar−H; Figure S1). The 1H NMR spectrum,
along with the designed synthesis route, indicates that a
molecule of NAC was attached to position C2 of 2,4-DCP,
implying the loss of a chlorine atom at C2. The results
provided solid evidence for metabolic dechlorination pathway
of 2,4-DCP.
3.5. Serum GSH Conjugates Derived from 2,4-DCP.
Blood glutathione conjugates could be a biomarker for
exposure to electrophilic species. Serum GSH conjugates
were determined in mice given 2,4-DCP. As expected, two
metabolites with similar chromatographic and mass spectral
properties of 5 and 6 detected in rat liver microsomal
incubations (Figure 2B) were found in sera of mice treated
with 2,4-DCP (Figure S3B). No such GSH conjugates were
observed in blank serum samples (Figure S3A).
3.6. Adduction of Bovine Serum Albumin by 2,4-DCP.
Bovine serum albumin was employed as a model to define the
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Figure 5. LC-MS/MS (m/z 248/159) chromatograms of proteolytic digestion of native BSA (A) or 2,4-DCP-treated BSA (B), incubations
containing cysteine and 2,4-DCP (C), and incubations fortified with Pronase E in the presence of adduct 7 (D). MS/MS spectra of 9/10 obtained
from studies B (E), C (F), and D (G) described above.
interaction between 2,4-DCP and protein. BSA was mixed with
2,4-DCP and incubated in a PBS solution, followed by
proteolytic digestion and LC-MS/MS analysis. As shown in
Figure 5B, two cysteine conjugates (9 and 10) with retention
time at 6.56 and 6.91 min respectively were detected by
acquiring of ion pair m/z 248/159. No such adducts were
detected in the digestion reaction of native BSA (Figure 5A).
The MS/MS spectra of both 9 and 10 showed fragment ions
m/z 159 and 185 as well as molecular ion at m/z 248 (Figure
5E). Fragment ion m/z 159 was associated with the cleavage of
the C−S bond within cysteine portion. And fragment ion m/z
185 (loss of CH5NO2) was related to the cleavage of the
cysteine moiety. Thus, 9 and 10 were most likely cysteine
conjugates derived from dechlorinated 2,4-DCP.
chromatographic and mass spectrometric behaviors (Figure
5C,F) as 9/10 detected in hydrolysates of 2,4-DCP-treated
BSA (Figure 5B,E). In addition, synthetic 7 (NAC conjugate,
Scheme 2) was hydrolyzed with Pronase E (Scheme 4),
according to our early work,20 and analyzed by LC-MS/MS.
The resulting product showed the same chromatographic and
mass spectrometric behaviors (Figure 5D,G) as those of 10.
This provided strong evidence for the modification of BSA at
cysteine residues by 2,4-DCP.
3.8. Modification of Hepatic Proteins by 2,4-DCP in
Mice. Mice were administered with 2,4-DCP, and liver tissues
were harvested, followed by proteolytic digestion and LC-MS/
MS analysis. As expected, the two cysteine conjugates were
detected in the resulting digestion mixtures, with the same
chromatographic and mass spectrometric properties (Figure
6B,C) as 9/10. No such products were observed in the
digestion mixture obtained from mice treated with vehicle
(Figure 6A).
3.7. Identification of Cysteine Conjugates. To verify
the structure of the cysteine adducts (9 and 10), proposed 9
and 10 were synthesized by reaction of 2,4-DCP with cysteine.
As expected, two cysteine conjugates were detected by LC-
MS/MS. The synthetic cysteine conjugates displayed the same
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Figure 6. LC-MS/MS (m/z 248/159) chromatograms of proteolytic digestion of liver tissues obtained from mice before (A) or after (B) treatment
(i.p.) with 2,4-DCP at 15 mg/kg. MS/MS spectra of cysteine residues-derived conjugates obtained from study B (C) described above.
We examined the time course of the hepatic protein
modification in mice. The most abundant cysteine-based
adduction detected in mouse liver was normalized to 100%. As
shown in Figure 7, hepatic protein adduction reached its peak
Figure 8. Changes in cysteine-based protein adduction in mice
administered (i.p.) with 2,4-DCP at 0, 15, 50, and 100 mg/kg (n = 3).
The present study was initiated with rat liver microsomal
incubations to determine GSH conjugation with the quinone
metabolite. As expected, two GSH conjugates (3 and 4, Figure
1B) derived from quinone metabolite 2 were detected in the
incubation reaction. Unexpectedly, two new GSH conjugates
(5 and 6) were observed in the incubations (Figure 2B).
Conjugates 5 and 6 were also detected in NADPH-absent
microsomal incubations (Figure 2A), suggesting P450 enzymes
were not involved in the conjugation reaction. Additional
incubation containing GSH and 2,4-DCP in PBS was executed,
and 5 and 6 were detected in the reaction (Figure 2C). This
indicates that the formation of 5 and 6 resulted from a
spontaneous chemical reaction. The MS/MS spectra of the
two conjugates suggest that a molecule of GSH was
incorporated into 2,4-DCP, along with the loss of a chlorine.
Similar incubations were performed with mouse liver micro-
somes where GSH conjugates 5 and 6 were also detected
(Figure S2B). Apparently, no species difference was observed
in metabolism of 2,4-DCP in rats and mice.
Figure 7. Time-course changes in cysteine-based protein adduction
examined at 5, 10, 15, 20, 30, 45, and 60 min in mice treated (i.p.)
with 2,4-DCP at 15 mg/kg (n = 3).
at 15 min after the administration at dose of 15 mg/kg.
Furthermore, protein adduction showed dose dependency.
Elevated protein adduction was observed with the increase in
doses of 2,4-DCP administered in mice (Figure 8).
4. DISCUSSION
2,4-DCP is widely used in industry and agriculture. However, it
was also known as an environmental pollutant and showed
toxic effects to goldfish,5 mammals11 and humans. More and
more attention has been paid to the hepatotoxicity caused by
2,4-DCP and the biochemical mechanisms involved. Dechlori-
nation is a common metabolic pathway for halogenated
hydrocarbons. Mehmood reported an oxidative dechlorination
pathway of 2,4-DCP by P450 enzymes,15 and a p-
benzoquinone metabolite (2, Scheme 1A) was generated
resulting from the dechlorination.
The GSH conjugates were also observed in bile (Figure 3B)
and blood (Figure S3B) in 2,4-DCP-treated animals. GSH
conjugates often undergo sequential metabolism to cysteine
conjugates and then acetylation to NAC conjugates
(mercapturic acids) which are virtually excreted in urine
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(Scheme 3). One NAC conjugate (7, Figure 4B) was detected
in urine samples of rats administered with 2,4-DCP. The
structure of 7 was confirmed by chemical synthesis (Figure
4C). NMR analysis showed NAC was attached to position C2
of 2,4-DCP in replacement of the chlorine atom. The
identification of the urinary NAC conjugate confirmed the
novel pathway of the GSH conjugation of 2,4-DCP.
after exposure to 2,4-DCP, which was more pronounced with
the dosage.22 Apparently, the observed dose-dependent protein
adduction is consistent with the dose-dependence of the
documented hepatotoxicity of 2,4-DCP.
There are multiple nucleophilic sites in protein, such as
cysteine thiols and lysine amines, which are likely to be targets
for electrophilic species. Extensive research showed that
exogenous substances often modify protein by forming stable
covalent binding, which has been considered to be an
important mechanism responsible for hepatotoxicity.23 The
observed protein modification by 2,4-DCP might trigger the
development of hepatotoxicity of 2,4-DCP, although further
mechanistic work on the toxic action is in need.
The in vitro and in vivo studies, particularly the observed
direct conjugation of GSH with 2,4-DCP, indicate that 2,4-
DCP is an electrophilic species. Clearly, 2,4-DCP was reactive
to GSH, and a spontaneous GSH conjugation took place with
a loss of chlorine. We propose that an ipso mechanism is
involved in the formation of GSH conjugates. As shown in
Scheme 3, the sulfhydryl attacks C2 to form a negative charged
intermediate, and the electronegative chlorine stabilizes the
developing ipso intermediate by electron withdrawing effect.
The sequential departure of the chloride anion as a leaving
group results in dechlorination and the formation of conjugate
6. Similar mechanism is proposed for the formation of isomer
5 resulting from GSH conjugation and dechlorination of 2,4-
DCP (Scheme 3). Hepatic cytosol was found to accelerate the
GSH conjugation with 2,4-DCP (Figure S2), possibly resulting
from the catalysis by cytosolic glutathione S-transferases.
Two GSH conjugates (5 and 6) were detected in in vitro
and in vivo studies, while only one urinary NAC conjugate (7)
was apparently observed in rats given 2,4-DCP. The formation
GSH conjugate 6 was found to dominate that of GSH
conjugate 5 (Figure 3B). It is most likely that NAC conjugate
7 is derived from GSH conjugate 6, according to their relative
abundance. We failed to detect the NAC conjugate 8
responsible for 5, possibly due to the limited sensitivity of
the mass spectrometry.
The in vitro observation of GSH conjugation with 2,4-DCP
clearly indicates that 2,4-DCP is an electrophilic species and
reactive to the sulfhydryl group of GSH. The observed direct
reaction of GSH with 2,4-DCP made us anticipate that
cysteine residues of protein would react with 2,4-DCP by the
same chemistry to form protein covalent binding. BSA was
employed as a model protein to determine the interaction of
cysteine residues with 2,4-DCP. Two cysteine conjugates (9
and 10, Figure 5B,E) were detected in proteolytic digestion of
2,4-DCP-exposed BSA, and the same conjugates were found in
incubations of cysteine with 2,4-DCP as well (Figure 5C,F).
Clearly, 2,4-DCP is reactive not only to GSH (small peptide)
but also to BSA (macromolecule).
Modification of hepatic protein at cysteine residues was
investigated in mice (Scheme 3). As expected, the two cysteine
conjugates were also detected in liver protein hydrolysates
obtained from mice given 2,4-DCP (Figure 6B,C). Such
protein adduction occurred in time- and dose-dependent
manners (Figures 7 and 8). Fu and co-workers reported that
2,4-DCP at a dose of 120 mg/kg caused endoplasmic
reticulum stress in mice, leading to hepatotoxicity.11 In the
present study, significant protein modification was observed in
mice treated with 2,4-DCP at a single dose of 15 mg/kg. The
protein adduction was detected in 5 min and reached its peak
at 15 min after administration. The observed peak time is
consistent with that of hepatic contents of 2,4-DCP reported in
rats.21 Furthermore, protein adduction increased with the dose
(0, 15, 50, and 100 mg/kg) administered in mice. Previous
studies showed that 2,4-DCP markedly increased serum ALT
and AST levels of mice at a dose of 120 mg/kg.11 Erdogan and
co-workers found changes in liver tissue morphology of rats
5. CONCLUSION
In conclusion, 2,4-DCP is an electrophilic species which can
react with GSH and protein to form GSH conjugates and
protein covalent binding. The conjugation/adduction possibly
starts with the reaction of a sulfhydryl group with the carbon
with chlorine to form an ipso intermediate, followed by the
departure of the chloride. The pathway newly found allows us
to better understand the mechanisms of toxic action of 2,4-
DCP.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.chemrestox.0c00118.
■
sı
Additional figures as discussed in the text (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Jiang Zheng − State Key Laboratory of Functions and
Applications of Medicinal Plants, Key Laboratory of
Pharmaceutics of Guizhou Province, Guiyang, Guizhou
550004, P. R. China; Key Laboratory of Environmental
Pollution, Monitoring and Disease Control, Ministry of
Education, Guizhou Medical University, Guiyang 550025, P. R.
China; Wuya College of Innovation, Shenyang Pharmaceutical
University, Shenyang 110016, China; orcid.org/0000-
0002-0340-0275; Phone: (206) 986-7651;
Email: zhengneu@yahoo.com; Fax: (206) 987-7660
Ying Peng − Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China;
Phone: +86-24-23986361; Email: yingpeng1999@163.com;
Fax: +86-24-23986510
Authors
Qingmei Li − Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China
Wei Li − Wuya College of Innovation, Shenyang Pharmaceutical
University, Shenyang 110016, China
Jiaxing Zhao − Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China
Xiucai Guo − Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China
Qian Zou − Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China
Zixin Yang − Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China
Ruixue Tian − Wuya College of Innovation, Shenyang
Pharmaceutical University, Shenyang 110016, China
I
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(13) Cabral, J., Mollner, T., Raitano, F., and Shubik, P. (1979)
Carcinogenesis of Hexachlorobenzene in Mice. Int. J. Cancer 23, 47−
51.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.0c00118
(14) D’Amour, M., and Charbonneau, M. (1992) Sex-related
difference in hepatic glutathione conjugation of hexachlorobenzene in
the rat. Toxicol. Appl. Pharmacol. 112, 229−234.
Funding
This work was supported in part by the National Natural
Science Foundation of China (Nos. 81830104, U1812403, and
81773813).
(15) Mehmood, Z., Kelly, D. E., and Kelly, S. L. (1997) Cytochrome
P450 3A4 mediated metabolism of 2,4-dichlorophenol. Chemosphere
34, 2281−2291.
Notes
(16) Lin, G., Tang, J., Liu, X. Q., Jiang, Y., and Zheng, J. (2007)
Deacetylclivorine: A Gender-Selective Metabolite of Clivorine
Formed in Female Sprague-Dawley Rat Liver Microsomes. Drug
Metab. Dispos. 35, 607−613.
(17) Watanabe, T., Hashimoto, H., and Tobita, H. (2004) Selective
and Stepwise Bromodemethylation of the Silyl Ligand in Iron(II) Silyl
Complexes with Boron Tribromide. Organometallics 23, 4150−4153.
(18) Lin, D., Wang, K., Guo, X., Gao, H., Peng, Y., and Zheng, J.
(2016) Lysine- and cysteine-based protein adductions derived from
toxic metabolites of 8-epidiosbulbin E acetate. Toxicol. Lett. 264, 20−
28.
The authors declare no competing financial interest.
ABBREVIATIONS
■
2,4-DCP, 2,4-dichlorophenol; BSA, bovine serum albumin;
GSH, glutathione; NAC, N-acetyl cysteine; NADPH, reduced
nicotinamide adenine dinucleotide phosphate; HCB, hexa-
chlorobenzene; DTT, DL-dithiothreitol; AST, aspartate trans-
aminase; ALT, alanine transaminase; LC-MS/MS, liquid
chromatography−tandem mass spectrometry; MRM, multi-
ple-reaction monitoring; MS/MS, tandem mass spectrometry;
CE, collision energy; CXP, cell exit potential; DP, declustering
potential; IDA, information-dependent acquisition; EPI,
enhanced product ion
(19) Li, W., Wang, K., Lin, G., Peng, Y., and Zheng, J. (2016) Lysine
Adduction by Reactive Metabolite(s) of Monocrotaline. Chem. Res.
Toxicol. 29, 333−341.
(20) Wang, H., Feng, Y., Wang, Q., Guo, X., Huang, W., Peng, Y.,
and Zheng, J. (2016) Cysteine-Based Protein Adduction by Epoxide-
Derived Metabolite(s) of Benzbromarone. Chem. Res. Toxicol. 29,
2145−2152.
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