In Vitro DNA Adduction Resulting from Metabolic Activation of Diosbulbin B and 8-Epidiosbulbin e Acetate

Article abstract of DOI:10.1021/acs.chemrestox.8b00071

Diosbulbin B (DBB) and 8-epidiosbulbin E acetate (EEA), belonging to furan-containing diterpenoid lactones, are the primary components of Dioscorea bulbifera L. (DB), a traditional Chinese medicine herb. Our earlier studies indicated that consumption of DBB or EEA induced acute hepatotoxicities. Both DBB and EEA were bioactivated by P450 3A4 to generate the corresponding cis-enedial reactive metabolites which are associated with the hepatotoxicities. It has been proposed that the electrophilic intermediates attack cellular nucleophiles such as protein or DNA, thought to be a mechanism of triggering toxicities. The purposes of our present study were to define the interaction of the electrophilic reactive metabolites originating from DBB and EEA with 2′-deoxyguanosine (dGuo), 2′-deoxycytidine (dCyd), and 2′-deoxyadenosine (dAdo) and to characterize DNA adducts arising from the reactive metabolites of DBB and EEA. The reactive metabolites of DBB and EEA were found to covalently bind to the exocyclic and endocyclic nitrogens of dCyd, dGuo, and dAdo to generate oxadiazabicyclo[3.3.0]octaimine adducts. The reactive metabolites of DBB and EEA also attacked dGuo, dAdo, and dCyd of calf thymus DNA. The DNA adducts possibly contribute to the toxicologies of DBB and EEA.

Full text of DOI:10.1021/acs.chemrestox.8b00071

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Article
In vitro DNA Adduction Resulting from Metabolic
Activation of Diosbulbin B and 8-Epidiosbulbin E Acetate
Dongju Lin, Weiwei Li, Xutong Tian, Ying Peng, and Jiang Zheng
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00071 • Publication Date (Web): 06 Dec 2018
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In vitro DNA Adduction Resulting from Metabolic Activation of
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Diosbulbin B and 8-Epidiosbulbin E Acetate
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Dongju Lin,† Weiwei Li,‡ Xutong Tian,† Ying Peng,§* and Jiang Zheng‡§*
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‡State Key Laboratory of Functions and Applications of Medicinal Plants, Key
Laboratory of Pharmaceutics of Guizhou Province, Guizhou Medical University,
Guiyang, Guizhou, 550025, P.R.China; Key Laboratory of Pharmaceutical Quality
Control of Hebei Province, Baoding 071002, China; College of Pharmaceutical
Sciences, Hebei University, Baoding 071002, China;§Wuya College of Innovation,
Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P.R.China.
†
Running title:
Identification of DNA adducts of DBB and EEA
Corresponding Authors:
Jiang Zheng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, 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, 550025, P.R. China
Email: zhengneu@yahoo.com
Tel: +86-24-23986361; Fax: +86-24-23986510
‡,§: The two corresponding units contributed equally to this work.
Ying Peng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang,
Liaoning, 110016, P. R. China
Email: yingpeng1999@163.com
Tel: +86-24-23986361; Fax: +86-24-23986510
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Abstract
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Diosbulbin B (DBB) and 8-epidiosbulbin E acetate (EEA), belonging to
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furan-containing diterpenoid lactones, are the primary components of Dioscorea
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bulbifera L. (DB), a traditional Chinese medicine herb.
Our earlier studies
indicated that consumption of DBB or EEA induced acute hepatotoxicities. Both
DBB and EEA were bioactivated by P450 3A4 to generate the corresponding
cis-enedial reactive metabolites which are associated with the hepatotoxicities. It
has been proposed that the electrophilic intermediates attack cellular nucleophiles
such as protein or DNA, thought to be a mechanism of triggering toxicities. The
purpose of our present study were to define the interaction of the electrophilic reactive
metabolites originating from DBB and EEA with 2’-deoxyguanosine (dGuo),
2’-deoxycytidine (dCyd), and 2’-deoxyadenosine (dAdo) and to characterize DNA
adducts arising from the reactive metabolites of DBB and EEA.
metabolites of DBB and EEA were found to covalently bind to the exocyclic and
endocyclic nitrogens of dCyd, dGuo, and dAdo to generate
oxadiazabicyclo[3.3.0]octaimine adducts. The reactive metabolites of DBB and
The reactive
EEA also attacked dGuo, dAdo, and dCyd of calf thymus DNA. The DNA adducts
possibly contribute to the toxicologies of DBB and EEA.
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Introduction
Dioscorea bulbifera L. (DB), known as Huang-Yao-Zi in Chinese, belongs to a
member of the yam family Dioscoreaceae.1
It is widely employed to treat
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carbuncles, lung abscesses, breast lumps, and goiter in China.2 Additionally, DB
possesses a series of pharmacological properties, including antitumor,3 antifeedant,4
anti-inflammation,5 and anti- salmonellal activities.6 DB and related remedies are
often used for the treatment of thyroid gland diseases and a variety of tumors.7-8 It
has been reported that DB has a wide variety of therapeutical values, but the adverse
effects of DB have also attracted attention so that this medicinal herb can be
consumed safely.9 Many cases of hepatitis have been reported to be related with the
consumption of DB and even deaths due to fulminant hepatic failure since 1976.10,11
Animal studies demonstrated that oral consumption of ethanol extracts of DB caused
increases in serum alanine transaminase (ALT), aspartate transaminase (AST), and
lipid peroxide in hepatic tissues of mice.12 DB contains varieties of diterpenoid
lactones,13 and diosbulbin B (DBB; Scheme 1) has been identified as the one in a
great abundance among the diterpenoid lactones.14 In addition, our preliminary study
found that the content of 8-epidiosbulbin E acetate (EEA; Scheme 1) varied largely in
DB and the quantity of EEA in DB relied on the commercial source.15 We found
that both DBB and EEA were capable of inducing acute liver injury.15 EEA was
much more hepatotoxic than DBB.16 Both DBB and EEA contain a 3-substituted
furan ring. Our preliminary study demonstrated that both DBB and EEA were
metabolized by cytochrome P450 3A4 to cis-enedial intermediates (1 and 2; Scheme 1)
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which are responsible for the hepatotoxicities of DBB and EEA.15,17 cis-Enedial, an
electrophilic intermediate, is chemically reactive to nucleophiles, and it easily
alkylates protein, polyamine, and DNA nucleophiles, which were considered to be a
significant mechanism of toxicities.18-21 Our previous study manifested that the
reactive intermediates of DBB and EEA could react with cysteine and lysine residue(s)
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of hepatic proteins to form protein adducts.
Furthermore, the protein covalent
binding was correlated with the hepatotoxicities of DBB and EEA.22,23
Furan is metabolized to form a chemically reactive metabolite of
cis-2-butene-1,4-dial (BDA) mediated by cytochrome P450 2E1. It is suggested that
this metabolite possibly reacts with proteins to induce cytotoxicity or attacks the
exocyclic and endocyclic nitrogens of dCyd, dGuo, and dAdo to generate
diastereomeric oxadiazabicyclo[3.3.0]octa-imine adducts.18,24
It has been
documented that furan could bind to DNA in the target organ of furan
carcinogenicity.25 DNA covalent binding has been thought to be another significant
action to trigger toxicities. We hypothesized that the reactive intermediates of DBB
and EEA can covalently bind to DNA, which may be relevant with hepatotoxicities
induced by DBB and EEA. As a consequence, the present study aimed at the
determination of the potential of reactive intermediates derived from DBB and EEA
to covalently bind to DNA.
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Materials and methods
Reagents
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Diosbulbin B (DBB) and 8-epidiosbulbin E acetate (EEA) were separated and
purified from DB rhizomes in our laboratory, based on previously reported
procedures.26,27 The structures were affirmed by mass spectrometry and NMR. The
purities of DBB and EEA were >98% assessed via high-performance liquid
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chromatography (HPLC).
2’-Deoxyadenosine (dAdo), 2’-deoxycytidine (dCyd),
2’-deoxguanosine (dGuo), thymine, DNase I, alkaline phosphatase, phosphodiesterase
I, and calf thymus DNA were purchased from Sigma (St. Louis, MO). All organic
solvents were purchased from Fisher Scientific (Springfield, NJ) with analytical or
HPLC grade.
Synthesis of dimethyldioxirane (DMDO)
Dimethyldioxirane (DMDO) was prepared, according to previously published
method [28]. In brief, 30 mL of acetone and 0.285 mol of NaHCO3 were mixed in a
1L round bottom flask containing 20 mL of distilled H2O and cooled in an water/ice
bath with stirring. After 20 min, stirring was halted, followed by addition of Oxone
(25 g, 0.041 mol). The resultant mixture was stirred for 15 min, and then a slight
vacuum was applied to the reaction assembly. The resulting pale yellow distillate
was condensed in a receiving flask, followed by cooling in an acetone/dry ice bath.
The concentration of DMDO was determined by titration, according to a reported
method.29 The concentration DMDO ranged from 0.06 to 0.065 M.
Synthesis of DBB/EEA-derived dCyd, dAdo, and dGuo adducts
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DBB (10 mg, 0.029 mmol) or EEA (10 mg, 0.026 mmol) dissolved in 2.0 mL
acetone was cooled in a dry ice bath for 1 min. The pre-prepared DMDO solution (4
mL) was dropwise added to the individual reactant solution with stirring, and the
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mixture was stirred for 10 min.
The reaction mixture was placed at room
The unreacted DMDO was removed by
temperature and left to stand for 1 h.
blowing with nitrogen gas. To the resulting mixture was dCyd, dAdo, or dGuo (20
mg for each) dissolved in PBS (2 mL, 0.1 M) added dropwise, respectively. After 1
h stirring at 60 °C, the reaction mixture was concentrated under a stream of nitrogen
gas at room temperature. The resultant solution was kept at -80 °C until purification.
The synthetic DBB-derived dCyd, dGuo, and dAdo adducts were purified by a
semipreparative HPLC system.
The synthetic products were isolated and purified by a reverse phase–HPLC
system using a YMC-Pack ODS-A C18 column (250 × 10 mm, 5 μm; YMC
Technologies, Japan, CA). The column temperature was set at 25 °C. The column
was eluted with a mobile phase system composed of water with 0.1% formic acid and
25% acetonitrile at a flow rate of 1.6 mL/min. The eluents were monitored at 210
and 254 nm.
The structures of purified products were determined by mass
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spectrometry and H NMR. The purity of DBB-derived dCyd, dAdo, and dGuo
adducts (Scheme 2) was >96%, based on HPLC analysis.
1H NMR (DMSO-d6, 600MHz) of DBB-derived dCyd adduct: δ1.10 (3H, s,
H-17), 1.52 (2H, m, H-1”), 1.74 (2H, d, J=11Hz,H-3”), 1.93 (1H, m, H-5”), 2.14 (2H,
m, H-7”), 2.21 (2H, m,H-11”), 2.24 (1H, m, H-2’), 2.39 (1H, m, H-13”), 2.45 (1H, m,
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H-10”), 2.80 (1H, d, J=4.3 Hz,H-4"), 3.53 (2H, m, H-5’), 3.80 (1H, m, H-4’), 4.21
(1H, m, H-3’) , 4.54 (1H, m, H-7), 4.61 (1H, m, H-12”), 4.72 (1H, m, H-6”), 4.76 (1H,
dd, J=5.3, 5.3 Hz, H-2”), 5.00 (1H, br s, H-5’OH), 5.20 (1H, br s, H-3’OH), 5.42 (1H,
br s, H-14”), 5.76 (1H, m, H-5), 5.98-5.84 (1H, m, H-8), 6.15 (1H, m, H-1’), 7.45 (1H,
m, H-4); 13C (DMSO-d6, 150MHz): δ15.96 (C-17”), 28.58 (C-1”), 29.26 (C-2’), 36.19
(C-10”), 37.12 (C-7”), 38.43 (C-13”), 39.24 (C-3”), 40.58 (C-11”), 42.12 (C-5”),
42.30 (C-4’’), 45.63 (C-9”), 59.35 (C-7), 61.93 (C-5’), 65.78 (C-12”), 71.20 (C-3’),
76.85 (C-2”), 78.54 (C-6”), 83.01 (C-4’),87.51 (C-1’), 96.50 (C-5), 98.96 (C-14”),
101.15 (C-8), 138.20 (C-4), 175.80 (C-16”), 176.71 (C-15”).
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1H NMR (DMSO-d6, 600MHz) of DBB-derived dGuo adduct: δ1.08 (3H, s,
H-17), 1.48 (2H, m, H-1”), 1.72 (2H, d, J=11Hz,H-3”), 1.96 (1H, m, H-5”), 2.11 (2H,
m, H-7”), 2.20 (2H, dd, J=11, 11.9 Hz,H-11”), 2.23 (1H, m, H-2’), 2.34 (1H, m,
H-13”), 2.48 (1H, m, H-10”), 2.50 (1H, m, H-2’), 2.79 (1H, d, J=4.1 Hz,H-4”),
3.61-3.42 (2H, m, H-5’), 3.80 (1H, m, H-3’), 4.31(1H, m, H-4’) , 4.56 (1H, m, H-12”),
4.73 (1H, m, H-6”), 4.78 (1H, dd, J=5.3, 5.3 Hz, H-2”), 4.88 (1H, br s, H-5’OH), 4.90
(1H, m, H-7), 5.19 (1H, br s, H-3’OH), 5.45 (1H, br s, H-14”), 6.10 (1H, m, H-1’),
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7.17 (1H, br s, H-6), 7.82 (1H, s, H-2); C (DMSO-d6, 150MHz): δ 15.98 (C-17”),
28.60(C-1”), 29.27 (C-2’), 36.20 (C-10”), 37.15 (C-7”), 38.46 (C-13”), 39.21 (C-3”),
40.53 (C-11”), 42.15 (C-5”), 42.33 (C-4”), 45.61 (C-9”), 59.30 (C-7), 61.80 (C-5’),
65.74 (C-12”), 70.53 (C-3’), 76.83 (C-2”), 78.10 (C-6”), 84.91 (C-1’), 86.27(C-4’),
88.23 (C-6), 90.12 (C-8”), 98.10 (C-14”), 138.59 (C-2),175.89 (C-16”), 176.74
(C-15”).
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1H NMR (DMSO-d6, 600MHz) of DBB-derived dAdo adduct: δ1.12 (3H, s,
H-17), 1.52(2H, d, J=5.5, 5.5 Hz, H-1”), 1.84 (2H, d, J=10.6, 11.0 Hz,H-3”), 1.98 (1H,
m, H-5”), 2.21 (2H, m, H-7”), 2.24 (2H, dd, J=11.2, 11.6 Hz,H-11”), 2.26 (1H, m,
H-2’), 2.25 (1H, m, H-13”), 2.53 (1H, m, H-10”), 2.60 (1H, m, H-2’), 2.81 (1H, d,
J=4.3 Hz,H-4”), 3.61 (2H, m, H-5’), 3.80 (1H, m, H-4’), 4.31 (1H, m, H-3’) , 4.59
(1H, m, H-12”), 4.83 (1H, m, H-6”), 4.75 (1H, m, H-7), 4.89 (1H, dd, J=5.5, 5.5 Hz,
H-2”), 4.95 (1H, br s, H-5’OH), 5.30 ((1H, m, H-14”), 5.36 (1H, br s, H-3’OH), 6.00
(1H, m, H-8), 6.17 (1H, m, H-1’), 8.16 (1H, s, H-2), 8.29 (1H, s, H-5); 13C (DMSO-d6,
150MHz): δ 16.37 (C-17”), 29.68 (C-1”), 31.54 (C-2’), 38.67 (C-13”), 37.20 (C-7”),
39.29 (C-3”), 40.63 (C-11”), 40.95 (C-10”), 42.21 (C-5”), 42.42 (C-4”), 45.82 (C-9”),
58.90 (C-7), 61.54 (C-5’), 65.84 (C-12”), 70.97 (C-3’), 77.54 (C-2”), 78.31 (C-6”),
84.89 (C-1’), 87.39 (C-4’), 90.35 (C-8”), 90.94 (C-8), 100.45 (C-14”),139.31 (C-2),
141.25 (C-5), 175.92 (C-16”), 176.86 (C-15”).
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Preparation of calf thymus DNA adducts derived from reactive metabolites of
DBB/EEA
The oxidized products of DBB and EEA prepared in accordance with the above
methods were individually mixed with ctDNA (0.3 mg dissolved in 500 μL of 50 mM
potassium phosphate, pH 7.4) at 37 °C for 8 h.
DNA hydrolysis
The resulting mixtures above were enzymatically hydrolyzed at 37 °C for 15 h
with a mixture of phosphodiesterase I (10 mg/mL), alkaline phosphatase (10 mg/mL),
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and DNase I (1.0 mg/mL) in a 1.0 mL solution of 5.0 mM CaCl2 and 5.0 mM MgCl2.
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The mixtures were centrifuged at 19,000g for 10 min.
enriched by solid phase extraction (Strata-X 30 mg cartridges, Phenomenex), eluted
with sequential water, 20% methanol, and 100% methanol (1 mL each). The
The supernatants were
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resulting eluents were then pooled and concentrated to about 100 μL under reduced
pressure. Analysis of the digestion mixture was performed using LC-MS/MS.
LC-MS/MS Analysis
An SCIEX Instruments 4000 Q hybrid triple quadrupole-linear ion trap mass
spectrometer (Applied Biosystems, Foster City, CA, USA) was equipped with an
Agilent 1260 Series Rapid Resolution LC system. In order to acquire the accurate
mass of the analytes, a microQ-TOF MS (Bruker Co., Karlsruhe, Germany) was
employed. A Symmetry reverse-phase C18 column of Waters with 3.5 μm particle
size and 4.6 × 150 mm i.d. (Waters, Milford, MA), was employed for
chromatographic separation with an elution gradient system consisting of acetonitrile
with 0.1% formic acid as mobile phase A and water with 0.1% formic acid as mobile
phase B. The gradient started with 10% A for 2 min, linearly increased to 90% A in
8 min, kept constant at 90% A for 2 min, and switched back to 10% A in 2 min. The
HPLC flow rate was set at 0.8 mL/min and the column was eluted at room
temperature. The Q TRAPTM mass spectrometer was operated in positive ion mode.
The curtain gas was set at 20 psi, GA1 and GA2 were set at 50 psi, the source
temperature was set at 650 °C.
In order to accomplish the highest sensitivity
possible, the main mass parameters were optimized in the MRM mode, such as
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declustering potential (DP), collision energy (CE), and collision cell exit potential
(CXP).. The representative ion pairs (corresponding to DP; CE; CXP) were m/z
588→472 (110, 40, 13) for DBB-derived dCyd adduct, m/z 610→494 (110, 40, 13)
for DBB-derived dGuo adduct, m/z 594→478 (110, 40, 13) for DBB-derived dAdo
adduct, m/z 632→456 (110, 40, 13) for EEA-derived dCyd adduct, m/z 654→478
(110, 40, 13) for EEA-derived dGuo adduct and m/z 638→462 (110, 40, 13) for
EEA-derived dAdo adduct. MS/MS spectra for adducts can be acquired using the
information-dependent acquisition (IDA) of the enhanced product ion (EPI). IDA
threshold was set at 5000 counts per second (cps) for the MRM scan. EPI scan rate
was 1000 amu/s and the scan for product ions ranged from 50 to 700 amu. CE was
set at 35 eV with a CE spread of 10 eV. Dynamic exclusion, defining the time for
exclusion of former target ions after acquiring an EPI scan, was set at 10 s after 3
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SCIEX AnalystTM
software (versions 1.6 and 1.6.2) was employed for data acquisition and processing.
.
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Formation of DBB-derived dCyd, dGuo, and dAdo adducts.
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DBB was oxidized by DMDO to produce cis-enedial reactive intermediate 1
which reacted with dCyd, dGuo, or dAdo. Mass spectrometric analysis (positive
mode) detected DBB-derived three adducts (A1-A3) in the microsomal reactions.
Adduct A1 determined by scanning ion pair of m/z 588→472 eluted at 6.69 min
(Figure 1A). Collision-induced dissociation (CID) of m/z 588 produced m/z 472
through neutral loss of 116 Da, suggesting the loss of 2’-deoxyribose (Figure 1C).
High resolution MS of A1 revealed exact m/z of 588.2168, responsible for the
molecular formula of C28H34N3O11 within 5 ppm (Table S1). NMR analysis showed
that the formation of DBB-derived dCyd adduct [1H δ7.45 (m, H-4), 5.98-5.84 (m,
H-8), 5.76 (m, H-5), 5.42 (m, H-14’’), 4.54 (m, H-7); 13C δ138.20 (C-4), 101.15 (C-8),
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98.96 (C-14”), 96.50 (C-5), 59.35 (C-7)] (Table S3). Additionally, the H and C
NMR data showed that the existence of 2’-deoxyribose [1H δ6.15 (m, H-1’), 5.20 (br s,
H-3’OH), 5.00 (br s, H-5’OH), 4.21 (m, H-3’), 3.75 (m, H-4’), 3.53 (m, H-5’),2.24 (m,
H-2’); 13C δ87.51 (C-1’), 71.20 (C-3’), 83.01 (C-4’), 61.93 (C-5’), 30.80 (C-2’)]
(Table S3). This led us to proposing that A1 resulted from the reactive intermediate
of DBB conjugated with one molecule of dCyd (Scheme 3).
Although a peak of adduct A2 at m/z 628 representing [M+H]+ was not detected
in the Q1 mass spectrum, the mass at m/z 610 was assigned to [M+H - H2O]+ ions.
Adduct A2 determined by scanning ion pair m/z 610→494 eluted at 6.75 min (Figure
2A). The product ion (MS2) spectrum of adduct A2 (Figure 2C), obtained via
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MRM-EPI scanning, contained the indicative characteristic fragment ion at m/z 494
which was derived from the neutral loss of 2’-deoxyribose (Figure 2C). Further
analysis by LC/Q-TOF MS showed [M+H - H2O]+ of adduct A2 at m/z 610.2120,
corresponding to the formula of C29H32N6O10 (Table S1). NMR analysis confirmed
the formation of adduct A2 [1H δ2.34 (m, H-13’’), 5.45 (br s, H-14’’), 7.82 (s, H-2),
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8.96 (s, H-5), 7.17 (br s, H-6),4.90 (m, H-7) , C δ38.46 (C-13’’), 98.10 (C-14”),
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138.59 (C-2), 88.23 (C-6), 59.30 (C-7)] (Table S4). In addition, the H and C
NMR data demonstrated that the existence of 2’-deoxyribose [1H δ6.10 (m, H-1’),
5.19 (br s, H-3’OH), 4.88 (br s, H-5’OH), 3.80 (m, H-3’), 4.31 (m, H-4’), 3.61-3.42
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(m, H-5’), 2.50 (m, H-2’), 2.23 (m, H-2’); C δ84.91 (C-1’), 70.53 (C-3’), 86.27
(C-4’), 61.80 (C-5’), 29.27 (C-2’)] (Table S4). On the basis of the observed mass
spectrometric and NMR data, we propose that A2 was composed of the reactive
intermediate of DBB and one molecule of dGuo (Scheme 3).
We failed to find adduct A3 at m/z 612 representing [M+H]+ in positive mode.
Instead, the adduct was detected by scanning of ion at m/z 594 responsible for [M+H -
H2O]+ ion. . Adduct A3 determined by scanning ion pair m/z 594→478 eluted at 7.64
min (Figure 3A). Product ion (MS2) spectrum of adduct A3 revealed a characteristic
product ion at m/z 478, originating from the loss of 2’-deoxyribose (-116Da) (Figure
2C). LC-Q-TOF MS further demonstrated [M+H - H2O]+ at m/z 594.2180 (Table S1),
corresponding to the formula of C29H32N5O9 (Table S1). NMR analysis confirmed
the formation of adduct A3 [1H δ5.30 (m, H-14’’), 2.25 (m, H-13’’), 8.16 (s, H-2),
8.29 (s, H-5), 4.75 (m, H-7), 6.00 (m H-8); 13C δ100.45 (C-14”), 38.67 (C-13”),
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139.31 (C-2), 141.25 (C-5), 58.90 (C-7), 90.94 (C-8)] (Table S5). Additionally, the
1H and 13C NMR data demonstrated that the existence of 2’-deoxyribose [1H δ6.17 (m,
H-1’), 5.36 (br s, H-3’OH), 4.95 (br s, H-5’OH), 4.31 (m, H-3’), 3.80 (m, H-4’), 3.61
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(m, H-5’), 2.60 (m, H-2’), 2.28 (m, H-2’); C δ84.89 (C-1’), 70.97 (C-3’), 87.39
(C-4’), 61.54 (C-5’), 31.54 (C-2)] (Table S5). The observed mass spectrometric and
NMR data suggest that A3 consisted of the reactive intermediate of DBB and one
molecule of dAdo (Scheme 3).
Formation of DBB-cis-enedial calf thymus DNA adducts
To determine the reactivity of the reactive intermediate of DBB to DNA, the
synthetic oxidative products of DBB was incubated with calf thymus DNA as a model
DNA. The reaction mixture was hydrolyzed using DNase I, alkaline phosphatase,
and phosphodiesterase I. The resultant mixtures were analyzed as described above.
Three adducts were observed in the digestion mixture. These adducts showed the
same chromatographic behaviors as synthetic DBB-derived dCyd, dGuo, and dAdo
adducts (Figure 1B-3B), respectively. This indicates that the reactive intermediate
of DBB could covalently bind to calf thymus DNA.
Formation of EEA-derived dCyd, dGuo, and dAdo adducts.
EEA was oxidized by DMDO to generate the corresponding cis-enedial reactive
intermediate (2), followed by reaction with dCyd, dGuo, and dAdo individually.
Mass spectrometric analysis showed three adducts (A4-A6) formed in the reaction
mixtures.. Adduct A4 determined by scanning ion pair of m/z 632→456 eluted out
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at 6.93 min (Figure 4A). Collision-induced dissociation (CID) of m/z 632 produced
m/z 516 through the neutral loss of 116 Da, suggesting the loss of 2’-deoxyribose
(Figure 4C). The major fragment ion at m/z 456 is proposed to originate from
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further loss of CH3COOH from EEA (Figure 4C).
Further analysis of A4 by
LC/Q-TOF MS demonstrated exact molecule ion of 632.2423, which is in agreement
with the formula of C30H38N3O12 (Table S2). This indicates that A4 resulted from
the reactive intermediate of EEA which is conjugated with one molecule of dCyd
(Scheme 4).
Q1 full scanning of m/z 654 in positive mode allowed us to observe adduct A5,
considered to represent [M + H - H2O]+. Adduct A5 monitored by scanning ion pair
m/z 654→478 eluted out at 7.46 min (Figure5A). The MS/MS spectrum of A5
revealed the major fragment ions arising from the neutral loss of
2’-deoxyribose,generating the product ion at m/z538 (Figure 5C).
The major
fragment ion at m/z 478 is proposed to originate from the further neutral loss of
CH3COOH from EEA (Figure 5C). Further analysis of adduct A5 by LC/Q-TOF
MS displayed its molecular ion [M + H - H2O]+ at m/z 654.2387, responsible for the
formula of C31H36N5O11 (Table S2).
On the basis of the observed mass
spectrometric data, we propose that A5 was composed of the reactive intermediate of
EEA and one molecule of dGuo (Scheme 4).
Adduct A6 was determined by Q1 full scanning of m/z 638 in positive mode,
considered to represent [M + H - H2O]+. Adduct A6 monitored by scanning ion pair
m/z 638→462 eluted out at 8.51 min (Figure 6A). The MS/MS spectrum of adduct
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A6 exhibited a product ion at m/z 522, suggesting the loss of 2’-deoxyribose (116 Da)
(Figure 6C). The major fragment ion at m/z 462 is proposed to originate from the
loss of CH3COOH from EEA and the elimination of 2’-deoxyribose from the
protonated molecular ion (Figure 6C). High resolution mass spectrometric analysis
of adduct A6, exhibited molecular ion [M + H- H2O]+ at m/z 638.2410 (Table S2),
corresponding to the formula of C31H36N5O10 (Table S2). This indicates that A6 was
generated through conjugation of the reactive intermediate of EEA with one molecule
of dAdo (Scheme 4).
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Characterization of EEA-cis-enedial calf thymus DNA adducts
Similar experiments were performed to determine adduction of calf thymus DNA
by the reactive intermediate of EEA as that for DBB. Three adducts were detected
in the digestion mixture.As expected, these adducts displayed the same
chromatographic properties as respective EEA-derived dCyd,dGuo adduct, and dAdo
adducts (Figure 4B-6B) synthesized, indicating that the reactive intermediate of EEA
could covalently modify calf thymus DNA.
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Discussion
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Hepatotoxicities of DB have been well documented in experimental animals and
humans.10-12 DBB has been reported to be the primary diterpenoid lactone in DB.14
In addition, we found that EEA was a dominant diterpenoid lactone in some varieties
of DB, and its content varied from one source of DB to another.15 Our earlier study
demonstrated both DBB and EEA could cause severe liver injuries.16,30 DBB and
EEA were biotransformed to the corresponding cis-enedial intermediates (1 and 2;
Scheme 1), and the formation of the electrophilic intermediates was associated with
the hepatotoxicities of DBB and EEA.16,30 Many furanoids have been relevant with
adverse events, including hepatotoxicity, pulmonary toxicity, and carcinogenesis,
possibly in part originating from the in situ generation of cis-enedial.31 These
reactive species are able to alkylate pivotal cellular proteins and/or DNA. Our
previous study showed that the electrophilic reactive metabolites of DBB and EEA
could modify lysine and cysteine residue(s) of hepatic proteins to form pyrrole and
pyrrolinone derivatives, possibly correlated with the hepatotoxicities of DBB and
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EEA.22,23
It has been reported that furan is bioactivated to produce
cis-2-butene-1,4-dial mediated by cytochrome P450 2E1 This metabolite could react
with the exocyclic and endocyclic nitrogens of dCyd, dAdo, and dGuo to produce
relatively stable diastereomeric oxadiazabicyclo[3.3.0]octa-imine adducts, thought to
be an important mechanism of triggering the toxicities.18,24 This encouraged us to
study the interaction of the cis-enedial intermediates derived from DBB and EEA with
DNA.
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As an initial step, we chemically synthesized the reactive intermediates of DBB
and EEA by oxidation of DBB and EEA with DMDO, followed by reaction with
dCyd, dGuo, dAdo, or thymine. DBB-derived dCyd, dGuo, and dAdo adducts
(A1-A3), EEA-derived dCyd, dGuo, and dAdo adducts (A4-A6) were observed in the
corresponding chemical reaction mixtures. However, no such adducts were detected
in the reactions of thymine with the reactive metabolites derived from DBB and EEA
were observed, presumably due to the lack of exocyclic nitrogen in thymine. Given
together, the reactive metabolites of DBB and EEA were able to react with the
exocyclic and endocyclic nitrogens of dCyd, dGuo, and dAdo to produce
oxadiazabicyclo[3.3.0]octene adducts (3, 4, 5 and 8, 9, 10; Scheme 3 and 4).
However, the dAdo and dGuo adducts rapidly rearrange by dehydration to form
substituted etheno adducts which contain a reactive aldehydic group (6, 7 and 11, 12;
Scheme 3 and 4). As a consequence, the substituted etheno adducts were found as
the primary products of the above reactions. It has been reported that reaction pH is
a major factor to determine the rearrangement of the dGuo and dAdo adducts, and
they may readily rearrange at pH 7 or 8. Thus, it is most likely that the adducts of
dGuo and dAdo mainly exist in the form of etheno adducts under physiological
conditions.19 Interestingly, for the dCyd adducts, this aldehydic form of the adduct
preferentially to produce the corresponding cyclic hemiacetal by ring closure (5 and
10; Scheme 3 and 4). These observations were consistent with the interactions of the
reactive metabolite of furan with dCyd, dGuo, and dAdo.31
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On the basis of these structures, a general reaction mechanism may be proposed
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as follows. Initially, the cis-enedial intermediates derived from DBB and EEA
reacts with the exocyclic nitrogen atom of the nucleosides (N4 of dCyd, N2ofdGuo,
and N6 of dAdo; Scheme 5) to form α, β-unsaturated aldehyde. The double bond
derived from the resulting α, β-unsaturated aldehyde reacts with the adjacent
endocyclic nitrogen atom (N3 of dCyd, and N1 of dGuo, and dAdo; Scheme 5) by
1,4-addition. The final product may be generated from the subsequent attack of the
aliphatic hydroxyl group on the second aldehyde group. Alternatively, the reaction
mechanism cannot be ruled out, which involves the reaction of the endocyclic
nitrogen with reactive intermediates of DBB and EEA, followed by reaction of the
exocyclic nitrogen with the terminal aldehyde.
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To further characterize the interaction of the cis-enedial reactive intermediates
derived from DBB and EEA with DNA, we employed calf thymus DNA as a model
DNA. Synthetic reactive intermediates derived from DBB and EEA were incubated
with calf thymus DNA, followed by treatment with hydrolytic enzymes. Three
DBB-derived adducts and three EEA-derived adducts were produced after enzymatic
hydrolysis of the resulting DNA adducts. The observed adducts shared the same
chromatographic properties as those of A1-A3 and A4-A6, respectively (Figure1C-3C
and Figure 4C-6C), suggesting the reactive metabolites of DBB and EEA could
covalently bind to calf thymus DNA.
The etheno-dGuo and etheno-dAdo adducts (6, 7 and 11, 12) with the aldehyde
group may be reactive to DNA, which possibly has important implications for the
toxicities induced by DBB and EEA.
As a result of reactions at the exposed
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aldehyde functionalities, these adducts possibly result in the formation of additional
products. It has been reported that etheno-dGuo and etheno-dAdo adducts may
induce point mutations. It was proposed that these adducts might also further react
with nucleophiles of protein and/or DNA, due to the reactive aldehyde functionality,
to form the corresponding cross-links. A number of α, β-unsaturated aldehydes and
dialdehydes can reportedly cause DNA damage by cross-linkage.32-36 In addition to
cytotoxicity, the cross-link formation may induce large scale deletions or
chromosomal rearrangements, which possibly initiates tumors.38,39 As a consequence,
the generation of etheno-dAdo and etheno-dGuo adducts is likely to contribute to the
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toxicities of DBB and EEA.
Future studies will evaluate the toxicological
importance of these adducts in animals administered DBB/EEA and the biological
activity of these adducts in mutagenesis assays.
In conclusion, the reactive metabolites of DBB and EEA covalently bind to the
exocyclic and endocyclic nitrogens of dCyd, dGuo, and dAdo.
The reactive
metabolites of DBB and EEA also modified dGuo, dAdo, and dCyd of calf thymus
DNA. Characterizing the potential DNA modifications by DBB and EEA may be an
important step in understanding DBB and EEA-induced hepatotoxicities.
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Footnotes. This work was supported in part by the National Natural Science
Foundation of China [Grant 81373471, 81430086, 81773813] and University Student
Innovation Project [2018116].
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Supporting Information Available
Description of mass spectrometric profiling data of dCyd, dGuo, and dAdo adducts
derived from DBB/EEA. Description of H NMR and 13C NMR Data of
1
DBB-derived dGuo and DBB-derived dAdo adducts in DMSO-d6. This material is
available free of charge via the Internet at http://pubs.acs.org.
Conflict of interest. The authors declare that there are no conflicts of interest.
Abbreviations
ALT,alanine transaminase; AST, aspartate transaminase; BDA, cis-2-butene-1,4-dial;
CE, collision energy; CXP, collision cell exit potential; dAdo, 2’-deoxyadenosine; DB,
Dioscoreabulbifera L.; dCyd, 2’-deoxycytidine; dGuo, 2’-deoxyguanosine; DMDO,
Dimethyldioxirane; DBB, Diosbulbin B; DP, declustering potential; EEA,
8-epidiosbulbin E acetate; NMR, nuclear magnetic resonance.
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Figure Legends
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Figure 1. LC-MS MRM analysis of A1 (m/z 588–472) in (A) DBB was oxidized by
DMDO, followed by the reaction with dCydand(B) DBB was oxidized by DMDO,
followed by the reaction withcalf thymus DNAand then DNA digestion. (C) The
MS/MS spectrum of DBB-derived dCyd adduct (A1).
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Figure 2. LC-MS MRM analysis of A2 (m/z 610–494) in (A) DBB was oxidized by
DMDO, followed by the reaction with dGuoand(B) DBB was oxidized by DMDO,
followed by the reaction withcalf thymus DNAand then DNA digestion. (C) The
MS/MS spectrum of DBB-derived dGuo adduct (A2).
Figure 3. LC-MS MRM analysis of A3 (m/z 594–478) in (A) DBB was oxidized by
DMDO, followed by the reaction with dAdoand(B) DBB was oxidized by DMDO,
followed by the reaction withcalf thymus DNA, and then DNA digestion. (C) The
MS/MS spectrum of DBB-derived dAdo adduct (A3).
Figure 4. LC-MS MRM analysis of A4 (m/z 632–456) in (A) EEA was oxidized by
DMDO, followed by the reaction with dCydand(B) EEA was oxidized by DMDO,
followed by the reaction withcalf thymus DNAand then DNA digestion. (C) The
MS/MS spectrum of EEA-derived dCyd adducts (A4).
Figure 5. LC-MS MRM analysis of A5 (m/z 654–478) in (A) EEA was oxidized by
DMDO, followed by the reaction with dGuoand(B) EEA was oxidized by DMDO,
followed by the reaction with calf thymus DNAand then DNA digestion. (C) The
MS/MS spectrum of EEA-derived dGuo adduct (A5).
Figure 6. LC-MS MRM analysis of A6 (m/z 638→462) in (A) EEA was oxidized
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by DMDO, followed by the reaction withdAdoand(B) EEA was oxidized by DMDO,
followed by the reaction withcalf thymus DNAand then DNA digestion. (C) The
MS/MS spectrum of EEA-derived dAdo adduct (A6).
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Scheme 1. Structures of DBB, EEA, DBB-cis-enedial, and DBB-cis-enedial.
Scheme 2. Structures of DBB-derived dCyd, dGuo, and dAdo adducts.
Scheme 3. Proposed reaction mechanism of DBB-derived cis-enedial with DNA
bases.
Scheme 4. Proposed reaction mechanism of EEA-derived cis-enedial with DNA
bases.
Scheme 5. Proposed mechanisms for the formation of DBB/EEA-cis-enedial-DNA
adducts
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