Novel o-Toluidine Metabolite in Rat Urine Associated with Urinary Bladder Carcinogenesis

Article abstract of DOI:10.1021/acs.chemrestox.0c00098

o-Toluidine (o-Tol), a monocyclic aromatic amine, causes bladder cancer in humans and experimental animals and is therefore classified as a Group 1 carcinogen (IARC) in which the carcinogenicity of o-Tol is involved in metabolic activation, DNA damage, and DNA adduct formation. In the DNA adduct formation mechanism, o-Tol is metabolized by N-hydroxylation, N-acetoxylation, and then deacetoxylation to produce an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8. Therefore, dG-C8-o-Tol is thought to be a plausible DNA adduct of o-Tol exposure. However, direct detection of dG-C8-o-Tol in biological samples has not been reported yet. Here, we show that a novel o-Tol metabolite, 2-methyl-N1-(2-methylphenyl)benzene-1,4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in o-Tol-exposed rat urine. MMBD was also detected in a reaction of o-Tol and S9 mix, indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD showed a potent stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains,and cytotoxicity in human bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells compared with o-Tol. Furthermore, a DNA adduct (m/z 478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA with rat urine containing MMBD, and also in hepatic DNA of rats treated with o-Tol. These results therefore suggested that o-Tol-induced bladder carcinogenesis could be at least partly attributed to MMBD formation. The possible dimerization of monocyclic aromatic amines should be considered in the evaluation of the risk of bladder carcinogenesis after exposure.

Full text of DOI:10.1021/acs.chemrestox.0c00098

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Article
A novel o-toluidine metabolite in rat urine
associated with urinary bladder carcinogenesis
Yuya Tajima, Takeshi Toyoda, Yuichiro Hirayama, Kohei Matsushita, Takanori Yamada, Kumiko Ogawa,
Kenji Watanabe, Takeji Takamura-Enya, Yukari Totsuka, Keiji Wakabayashi, and Noriyuki Miyoshi
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.0c00098 • Publication Date (Web): 28 Apr 2020
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A novel o-toluidine metabolite in rat urine associated
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with urinary bladder carcinogenesis
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Yuya Tajima†, Takeshi Toyoda‡, Yuichiro Hirayama†, Kohei Matsushita‡, Takanori
Yamada‡, Kumiko Ogawa‡, Kenji Watanabe†, Takeji Takamura-Enya§, Yukari
Totsuka║, Keiji Wakabayashi†, Noriyuki Miyoshi† *
† Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University
of Shizuoka, Shizuoka, Japan
‡ Division of Pathology, National Institute of Health Sciences, Japan.
§ Department of Chemistry, Kanagawa Institute of Technology, Japan
║ Division of Carcinogenesis and Cancer Prevention, National Cancer Center
Research Institute, Japan
Key Words: ortho-toluidine, bladder cancer, metabolic activation, DNA adduct
*Corresponding author: Noriyuki Miyoshi
Graduate School of Integrated Pharmaceutical and Nutritional Sciences
University of Shizuoka
52-1 Yada, Suruga, Shizuoka 422-8526, Japan
Email: miyoshin@u-shizuoka-ken.ac.jp
Tel: +81-54-264-5531
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Abstract
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ortho-toluidine (o-Tol), a monocyclic aromatic amine, causes bladder cancers in
human and experimental animals, and is therefore classified as a Group 1 carcinogen
(IARC), in which the carcinogenicity of o-Tol is involved in metabolic activation, DNA
damage and DNA adduct formation. In the DNA adduct formation mechanism, o-Tol is
metabolized by N-hydroxylation, N-acetoxylation, and then deacetoxylation to produce
an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8.
Therefore dG-C8-o-Tol is thought to be a plausible DNA adduct of o-Tol exposure.
However direct detection of dG-C8-o-Tol in biological samples has not been reported yet.
Here we show that a novel o-Tol metabolite, 2-methyl-N1-(2-methylphenyl) benzene-1,
4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in
o-Tol-exposed rat urine. MMBD was also detected in a reaction of o-Tol and S9 mix,
indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD
showed a potent stronger mutagenicity in N-acetyltransferase overexpressed Salmonella
typhimurium strains, and cytotoxicity in human bladder carcinoma T24 cells and human
spleen lymphoblastoid TK6 cells compared with o-Tol. Furthermore, a DNA adduct (m/z
478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA
with rat urine containing MMBD, and also in hepatic DNA of rats treated with o-Tol.
These results therefore suggested that o-Tol-induced bladder carcinogenesis could be at
least partly attributed to MMBD formation. The possible dimerization of monocyclic
aromatic amines should be considered in the evaluation of the risk of bladder
carcinogenesis following exposure.
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Introduction
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A causal relationship between occupational exposure to o-Tol and urinary
bladder cancer has been well studied and reported in several animal experiments and
epidemiological studies 1,2, and therefore o-Tol is listed as a Group 1 carcinogen
(“carcinogenic to humans”) by the International Agency for Research on Cancer (IARC
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2012) . Recently, a high frequency of bladder cancers was reported in Japanese male
workers at plants that produce organic dyes and pigment intermediates owing to their
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occupational exposure to aromatic amines . Patients had been exposed primarily to
ortho-toluidine (o-Tol) and partially to aniline, p-Tol, 2,4-xylidine, and o-anisidine 3.
In animal experiments, high dose (1,000~32,000 ppm) exposure to o-Tol for
mice or rats for long periods (~2 years) induced tumorigenesis in several tissues including
the urinary bladder 1,2. Most studies reported that o-Tol was not mutagenic in Salmonella
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typhimurium, other studies showed positive responses in the same strains . IARC
summarizes the carcinogenic mechanism of o-Tol as involving metabolic activation,
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formation of DNA adducts, and induction of DNA damaging effects . In general, the
major metabolic pathways of o-Tol are the oxidation by cytochrome p450, which
produces hydroxy o-Tol (amino cresols) and positional isomers, some of which are
further converted by acetylation and/or sulfate/glucuronide conjugations and then
excreted in urine. Both N-hydroxylation and the subsequent N-acetoxylation are thought
to be important pathways that produce active metabolites, which induce DNA damage
and DNA adduct formation. Therefore, a plausible mechanism through which o-Tol
metabolism forms DNA adducts is that in which o-Tol first oxidizes in the liver to form
N-hydroxy-o-Tol, and is then acetylated in the bladder to form N-acetoxy-o-Tol. The N-
acetoxy-o-Tol is an unstable metabolite, which covalently modifies a DNA base, such as
the guanine C8 position, to form dG-C8-o-Tol. Many reports speculate that the major
DNA adduct of o-Tol is dG-C8-o-Tol. However, direct detection of dG-C8-o-Tol by LC-
MS or GC-MS has not yet been reported. Instead of dG-C8-o-Tol, o-Tol cleaved from
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hydrolyzed DNA was analyzed using GC-MS in a previous study of the o-Tol-DNA
adduct 4.
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Recently we reported that phosphorylated histone H2AX (γ-H2AX), a well-
established biomarker for DNA damage and especially DNA double-strand breaks 5, was
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strongly induced in urinary bladders of F344 rats fed a 0.8% o-Tol diet for 2 days .
Therefore, in this study F344 rats were exposed to o-Tol for 2 days during which urine
samples were collected for the analysis of o-Tol metabolites associated with
carcinogenesis. We identified both the novel o-Tol metabolites and the bioactivities
associated with the carcinogenesis.
Experimental procedures
Animal experiment
The experimental design was approved by the Animal Care and Utilization
Committee of the National Institute of Health Sciences, Japan, and the animals were cared
for in accordance with institutional guidelines as well as the Guidelines for Proper
Conduct of Animal Experiments (Science Council of Japan, June 1, 2006). o-Tol
hydrochloride was purchased from Sigma-Aldrich (St Louis, MO, USA; lot no. S85883V;
99.1%). Five-week-old male 344 rats were obtained from Charles River Japan
(Yokohama, Japan), and housed in plastic cages with free access to tap water and standard
chow (CRF-1) under controlled humidity (50±5%), light (12/12h light/dark cycle),
temperature (23±1ºC), and ventilation (air change rate 20 times/h) conditions. After a 1
week adaptation, rats were randomly allocated to control (n=5) and o-Tol (n=5) groups
based on body weight and were then placed into metabolic cages and administered CRF-1
or CRF-1 containing 0.8% o-Tol for 2 days. Urinary samples were collected every 24 h.
After rats were euthanized on day 2 by exsanguination under deep anesthesia caused by
inhalation of isoflurane, the liver and urinary bladder, as well as blood samples, were
collected from each rat.
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LC-MS analysis for rat urine samples
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An aliquot (50 μL) of urine was added to diethyl ether (150 μL), vortexed, and
centrifuged at 21,500 g for 10 min at 4ºC, and the organic phase was collected in a new
tube. A residual aqueous phase was re-extracted using diethyl ether. The combined
organic phase was evaporated to dryness in vacuo. The residues dissolved in 50 μL of
50% methanol were subjected to LC-MS consisting of AQUITY UPLC (Waters, Milford,
MA) coupled with micrOTOFQII (Bruker Daltonics, Bremen, Germany). UPLC
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separation was performed with a BEH C18 column (1.7 μm, 50 mm × 2.1 mm i.d.,
Waters) at 40°C, using solvent A (0.1% formic acid in water) and solvent B (MeCN
containing 0.1% formic acid). Samples were eluted from the column using a linear
gradient of 1% solvent B from 0 min to 99% solvent B at 10-12 min. The flow rate of the
mobile phase was 0.4 ml/min. The TOF-MS was operated in the ESI positive mode. The
mass ranged between m/z 100 and 1000. All analyses were performed using a sodium
formate solution to accurately calibrate the mass. MS peak data from UPLC-TOF-MS
analyses were subjected to Compass Data Analysis (Bruker) and Signpost (Reifycs,
Tokyo Japan) for peak detection and integration, and Mass Profiler Professional software
(Agilent Technologies) for statistical analysis.
Preparation of o-Tol dimers
2-Methyl-N1-(2-methylphenyl) benzene-1,4-diamine (MMBD), a o-Tol dimer
by head-to-tail binding, was prepared according to a previous report 9. Briefly, 3-methyl-
4-nitoroaniline (9.0 mmol, 4.56 g), 2-iodotoluene (7.5 mmol, 633.5 µL), CuO
nanoparticle 20 mg, KOH (7.5 mmol, 420 mg), 3.75 mL t-butanol, and 1.25 mL dimethyl
sulfoxide (DMSO) were stirred at 110ºC overnight. The reaction mixture was cooled, and
combined with 75 mL ethyl acetate and 25 mL ultrapure water. After the extraction and
evaporation, the residue was purified by silica-gel chromatography (hexane:ethyl acetate
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= 9:1) to obtain 3-methyl-4-nitro-N-(o-tolyl)aniline (1.5 g). Aliquots of 3-methyl-4-nitro-
N-(o-tolyl) aniline (4.7 mol, 1 g), 10% Pd/C 100 mg, and ethanol 10 mL were stirred at
room temperature overnight. After filtration, the solution was evaporated to obtain
MMBD (800 mg, purity 99.7%).
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2,2’-Dimethylhydrazobenzene (DHB), a o-Tol dimer by head-to-head binding,
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was synthesized according to a previous report . 2,2’-Azoxytoluene (5 mmol, 1.13 g;
Sigma-Aldrich), magnesium turnings (1.2 mmol, 29 mg), hydrazine sulfate (6 mmol, 780
mg), and 2.5 mL ultrapure water were stirred at room temperature overnight. After
filtration, the solution was evaporated and purified by silica-gel chromatography
(hexane:ethyl acetate = 49:1) to obtain 2,2’-dimethylazobenzene (570 mg). Then, aliquots
of 2,2’-dimethylazobenzene (0.5 mmol, 105mg), magnesium turnings (1.2 mmol, 29 mg),
hydrazine sulfate (1.2 mmol, 156 mg), and 500 mL ultrapure water were stirred at room
temperature overnight. After filtration, the solution was evaporated and purified by silica-
gel chromatography (methanol) to obtain DHB (22.6 mg).
3,3’-Dimethylbenzidine (DBZ), tail-to-tail binding of o-Tol, was obtained from
Tokyo Chemical Industry (Tokyo, Japan).
In vitro reaction of aromatic amines with S9 mix
S9 mix used in this study was obtained from Kikkoman Biochemifa Co., Japan
(Cat# S-9MIXTS、Lot# CAM201806A), which was prepared from S9 fraction (25.59
mg protein/mL) of SD rat (male, 7 week-old) liver induced by the combination of
phenobarbital and 5,6-benzoflavone. The product of S9 mix included 4 µmol/mL NADP.
49 μL of S9 mix and 1 μL of 500 mM monocyclic aromatic amines (o-Tol, aniline, p-Tol,
2,4-xylidine, or o-anisidine) were incubated at 37ºC for 20 min. Samples were extracted
with diethyl ether, evaporated to dryness in vacuo, dissolved in 50 μL of 50% methanol
and then subjected to LC-MS.
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Assay for cytotoxicity and γ-H2AX
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Human bladder cancer T24 cells (RIKEN Cell Bank, Ibaraki, Japan) were
maintained in 10% FBS/DMEM supplemented with 50 U/ml penicillin and 50 µg/ml
streptomycin and grown in an atmosphere of 95% air and 5% CO2 at 37°C. Human spleen
lymphoblastoid TK6 cells were kindly gifted by Dr. Takeda Shunichi of Kyoto University
and cultured in 5% HS/RPMI supplemented with 50 U/ml penicillin and 50 µg/ml
streptomycin. Cytotoxicity of o-Tol and MMBD was determined using an AramarBlue
assay as described previously 11,12. Levels of phosphorylated histone H2A.X (γ-H2AX)
were evaluated by a flowcytometry using AlexaFluor 488 rabbit phospho-histone H2A.X
(Ser139) antibody (Cell Signaling Technology, Beverly, MA).
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Ames test
Mutagenicity of MMBD was evaluated by pre-incubation method of Ames test
employing Salmonella strains TA98, TA100, YG1027, and YG1029 13. Briefly, 100 μL
of MMBD in DMSO and 500 μL of S9 mix or 0.1M phosphate buffer (pH 7.4) was added
into the pre-grown Salmonella culture, vortexed and then incubated at 37°C for 20 min.
Soft agar, 2 mL, pre-warmed at 45°C were added to each sample, which were then plated
on minimal glucose agar. After incubation at 37°C for 2 days, revertant colonies were
counted. Data were obtained with triplicate plates at each dose. When the samples induced
two-fold increases over the average yield of spontaneous revertants and showed well-
behaved concentration-response patterns, the samples were judged to be positive.
DNA adductome
Fifty microliters of urine from the control and o-Tol groups collected in the last
24 h of the metabolic caging were incubated with 50 μL of S9 mix and 50 μL of 1 μg/μL
calf thymus DNA at 37ºC for 24 h, then deproteinated with PCIA
(phenol/chloroform/isoamyl alcohol 25:24:1) and washed with ethanol precipitation.
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DNA samples dissolved in ultrapure water, 50 μL, were digested according to a
previously described method with slight modification 14. Briefly, DNA (50 μL) were
added to 5 µl of 50 mM Tris-HCl (pH 7.4) and 2 µl of deoxyribonuclease I (229 U/µl)
and incubated at 37ºC for 12 h. Then, 1 µl of 0.15 N HCl, 3 µl of nuclease P1 (1.6 U/µl),
2 µl of 0.3M sodium acetate (pH 5.3), and 2 µl of 1 M ZnCl2 were added and incubated
at 37ºC for 6 h. Finally, 2 µl of 0.5 M Tris base, 2 µl of alkaline phosphatase (150 U/ml),
and 2 µl of phosphodiesterase I (20 U/ml) were added and incubated at 37ºC for 12 h.
Samples concentrated by a centrifugal evaporator to ~20 µl were added to 500 µl
methanol, vortexed, and centrifuged (21,500 g for 10 min at 4ºC). The supernatants were
collected into new tubes, and the precipitations were washed again with methanol. The
supernatants gathered in the tubes were evaporated to dryness in vacuo. The residues
dissolved in 50 μL of 50% methanol were subjected to LC-MS. LC-MS/MS was
performed on an Agilent 1290 series HPLC system using an Atlantis T3 column (3 µm,
150 × 2.0 mm, Waters, Milford, MA, USA) at 40°C and an Agilent G6410B triple
quadrupole tandem mass spectrometer with an electrospray ionization device running in
the positive ionization mode. The detection of compounds was performed using the
multiple reaction monitoring (MRM) mode set between m/z 281.1>112.1 and
627.1>511.1, in which the neutral loss of deoxyribose (∆116.0) in the nucleoside was
monitored. In terms of the DNA adductome of rat liver, DNA extraction and purification
were performed as described previously 15. LC-MS/MS of digested DNA was performed
using a SHIMADZU prominence HPLC system with a Synergi Fusion-RP column (2.5
µm, 100 × 2.0 mm, SHIMADZU GLC, Japan) at 40ºC and a Triple TOF 6600 (AB
SCIEX) with an electrospray ionization device running in the positive ionization mode.
Data processing was performed using PeakView for peak detection and integration, and
MarkerView for PCA as described previously 15. dG-C8-o-Tol was synthesized according
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to a previous report 10,16
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Results
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Exploration of urine metabolites in rats exposed to o-Tol
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Our previous report showed that γ-H2AX, a hallmark of DNA damage, in rat
urinary bladders was significantly induced by exposure to o-Tol for 2 days 6. Therefore,
to explore the o-Tol metabolites associated with DNA damage in the urinary bladder,
urine samples from control or o-Tol treated F344 rats were collected on day 2 and then
subjected to LC-MS (Fig. 1A). Ninety-four peaks were observed after peak detection
(intensity > 3000) and integration using software, which were then filtered by the
frequency (100% of samples in at least one experimental group) to extract 53 MS ions.
The profile data of the 53 MS ions were subjected to statistical analysis (an unpaired t-
test with multiple testing correction using the Bonferroni family-wise error rate), which
further extracted 24 MS ions. Among them, one (m/z 146.1 RT = 2.3 min) was only
detected in the control group, another one (m/z 302.3 RT = 5.5 min) was detected in both
groups (Supplemental Table 1), and four were fragmented ions derived from their
precursor. Therefore, 18 MS ions were finally filtered out, which were considered to be
plausible o-Tol metabolites or o-Tol itself (Table 1). As shown in Fig. 1A and B, among
these 18 MS ions, peak 1 (m/z 108.08, C7H10N) was o-Tol, and peaks 2 and 3 were well-
known metabolites; 2 was oxidized o-Tol (m/z 124.08, C7H10ON, amino cresol) and 3
was acetyl amino cresol (m/z 166.09, C9H12O2N). These 3 peaks were predominantly
detected as o-Tol group-specific peaks (supplemental Fig. 1A), and therefore would be
the main compounds excreted, although peaks 2 and 3 included some structural isomers.
To further explore the o-Tol metabolites, the molecular formula and structures of
plausible o-Tol metabolites were predicted (Table 1). Among them, peak 4 was the most
noticeable, in which m/z was 213.14, which corresponds to o-Tol dimer (C14H16N2). The
molecular formula of 5 ~ 10 were estimated using accurate mass, and their chemical
structures were also predicted (Table 1), all of which were o-Tol dimers with chemical
modifications such as oxidation and/or acetylation (Supplemental Fig. 1B).
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Identification of o-Tol dimer detected in rat urine
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To identify 4, possible o-Tol dimers including 3,3’-dimethylbenzidine (DBZ),
2-methyl-N1-(2-methylphenyl)
benzene-1,4-diamine
(MMBD),
and
2,2’-
dimethylhydrazobenzene (DHB) formed by tail-to-tail, head-to-tail and head-to-head
binding, respectively, were prepared, and these authentic standards were injected into LC-
MS. As shown in Fig. 2, DBZ, MMBD, and DHB were eluted at 1.5, 3.0, and 6.2 min,
respectively. The elution time and MSMS spectrum of 4 was matched with MMBD (Fig.
2), and was therefore identified as MMBD. This is the first report to detect MMBD in
biological samples. Quantitative analysis revealed that urinary levels of o-Tol and
MMBD were 3.02±0.6 mM and 110.1±39.6 μM, respectively (Supplemental Table 2).
MMBD formation was confirmed by the in vitro reaction of o-Tol with S9 mix, but not
heat-inactivated S9 mix (Fig. 3). Additionally, the dimerization of other monocyclic
aromatic amines, including aniline, p-Tol, 2,4-xylidine, and o-anisidine was evaluated.
As a result, the reaction of other aromatic amines with S9 mix showed that aniline and o-
anisidine formed dimers, but that p-Tol, and 2,4-xylidine did not (Supplemental Fig. 2).
Taken together, these results suggest that MMBD is one of the o-Tol metabolites, which
are enzymatically formed in animals.
Assays of o-Tol and MMBD bioactivities
Because MMBD showed a stronger cytotoxicity than that of o-Tol in human
urinary bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells (Fig.
4A), flowcytometry for γ-H2AX and Ames assay was performed. When the T24 cells
were treated with o-Tol (2.5 mM) or MMBD (25 µM) for 4 h, MMBD markedly increased
γ-H2AX-positive cells, although o-Tol showed almost no activity (Fig. 4B). The
mutagenicity was determined by using S. typhimurium TA98 and TA100 and their
acetyltransferase overproducing derivatives YG1024 and YG1029 13 at a MMBD range
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of 0 to 100 μg/plate (Fig. 4C). Although MMBD with and without S9 mix did not show
clear activities in the TA 98 and TA 100 strains, MMBD with S9 mix showed strong
mutagenicity in YG1024 and YG1029 but only with S9 mix activation. The mutagenic
potencies of MMBD were high, in that it induced 8,773 revertants/mg in YG1024 with
S9 mix and 5,067 revertants/mg in YG1029 with S9 mix. These findings demonstrated
for the first time that MMBD was an active metabolite, which required metabolic
activation and acetylation.
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DNA adductome of o-Tol metabolites
Rat urine collected from the control and o-Tol groups was incubated with calf
thymus DNA (ctDNA) in the presence of S9 mix, and then DNA adductome analysis was
performed. As shown in Fig. 5A, the adductome map indicated that several chemically
modified nucleosides were detected in the control and o-Tol groups. dG-C8-o-Tol (m/z
373.2 > 257.2), a plausible DNA adduct of o-Tol, was under the detection limit. In
contrast, the strongest peak signal detected in the o-Tol group was m/z 478.1 (Fig. 5B),
which corresponded to the dG adduct with o-Tol dimer (dG-MMBD). The level of this
adduct was estimated at 1.31/ 103 nucleoside. The DNA adductome of the urinary
bladders of the control and o-Tol group rats was not completed because not enough DNA
was obtained. When hepatic DNA of the control and o-Tol group rats were subjected to
DNA adductomics, the level of m/z 478.1 corresponding to dG-MMBD was significantly
higher in the o-Tol group (Fig. 6A). The level of this adduct was estimated at 2.6/105
nucleoside. In the MSMS analysis the m/z 478.1 induced several fragmented ions
including m/z 152.1, which corresponds to the guanine base (Fig. 6B). Although synthesis
of authentic dG-MMBD was not completed in this study, m/z 478.1 detected in in vitro
and in vivo could be the dG adduct at the C8, N2 or N7 position with MMBD.
Discussion
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o-Tol is a carcinogen that induces urinary bladder cancer, and carcinogenicity of
o-Tol requires metabolic activation to induce DNA damage. A plausible mechanism of
o-Tol induced DNA damage is assumed as follows; an electrophilic nitrenium ion is
formed by the deacetoxylation of N-acetoxy-o-Tol, which then binds to a DNA base to
form dG-C8-o-Tol. However, dG-C8-o-Tol has not been directly detected in biological
samples. As the mechanisms of carcinogenicity of o-Tol are not fully understood, we
investigated the o-Tol metabolites associated with the carcinogenesis. In the current study,
we identified a novel o-Tol metabolite MMBD for the first time in urine samples of rats
after o-Tol exposure. MMBD was identified as a dimer of o-Tol formed by head-to-tail
binding. Additionally, a possible DNA adduct of MMBD was detected in an in vitro
reaction of rat urine from the o-Tol group with calf thymus DNA, and also in the liver of
rats treated with o-Tol. Moreover, we observed that MMBD induced strong cytotoxicity
and DNA damage in mammal cells and mutagenicity in an Ames test using salmonella
strains. These results suggest that dimerization of o-Tol could be an important metabolic
process that contributes to o-Tol-induced bladder cancer.
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Although the head-to-tail dimer MMBD was detected in rat urine from the o-Tol
group, the head-to-head and tail-to-tail dimers DHB and DBZ, respectively, were under
the detection limit (Fig. 2). MMBD was also formed by the in vitro reaction of o-Tol with
S9 mix, but not with the heat-inactivated S9 mix (Fig. 3), indicating o-Tol is dimerized
by the enzymatic reaction. In contrast, tail-to-tail dimer DBZ (also called o-tolidine) is
possibly carcinogenic to humans and is therefore classified as Group 2B carcinogen by
the IARC. Although DBZ was under the detection limit in rat urine, a trace amount of
DBZ was observed in the in vitro reaction of o-Tol with S9 mix. As benzidine and dye
metabolized to benzidine are carcinogenic (Group 1), we cannot exclude the possible
associations of these compounds with o-Tol-induced bladder carcinogenesis.
Interestingly it was reported that o-anisidine (Group 2B; IARC) was also metabolized to
a head-to-tail dimer that was catalyzed by mammal peroxidases 17. The 32P-postlabeling
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assay revealed that the o-anisidine metabolites formed DNA adducts that were targeted
to dG, although the structures of the DNA adducts were not identified 17. o-Anisidine is
one of the chemicals associated with occupational exposure-induced bladder
carcinogenesis 3. Additionally, our previous animal experiments also showed that o-Tol
and o-anisidine, but not other aromatic amines including aniline, p-Tol, and 2,4-xylidine,
significantly induced γ-H2AX in the rat urinary bladder 6. In the in vitro reaction, o-Tol
and o-anisidine were metabolized to form a dimer (Supplemental Figure 2). These results
suggest that dimerization might be a critical step in the carcinogenicity of monocyclic
aromatic amines. Further, the existence of a functional group at the para position of
monocyclic aromatic amines is one of the essential factors for the dimerization followed
by carcinogenicity.
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MMBD formation was confirmed by the in vitro reaction of o-Tol with S9 mix
(Fig. 3). In this in vitro experimental condition, 4.83 µM MMBD was produced from 10
mM o-Tol, therefore almost 0.05% of o-Tol was converted. In contrast, the in vivo
experiment showed that the amount of o-Tol administered for 24 h was estimated as
672±16 μmol, and that of MMBD excreted in urine was determined to be 604±222 nmol,
therefore about 0.1~0.2% of o-Tol was finally detected as MMBD in the urine samples
(Supplemental Table. 2). These results suggest that less than 1% of o-Tol seems to be
converted to MMBD. Additionally, samples were prepared by diethyl ether extraction for
the LC-MS analysis of MMBD, since many metabolites including MMBD and o-Tol
were extractable by ether. In contrast, acid-conjugate metabolites were extractable by the
methanol. Therefore, using methanol extraction, sulfate/glucuronide conjugates of
MMBD and MMBD derivatives could be detectable in urine. Additionally, in the current
experimental condition, o-Tol trimer in the urinary samples was under the detection limit
in the LC-MS analysis.
In the DNA adduct analysis, m/z 478.1 corresponding to dG-MMBD was the
predominant adduct formed in the reaction of rat urine from the o-Tol group and ctDNA
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(Fig. 5). However, the predominant peak was not identified as the synthesis of the
authentic standard of dG-MMBD was not completed. In general, the molecular
mechanism of the DNA adduct formation of aromatic amines is as follows; 1) N-
hydroxyation forms hydroxylamine, 2) further acetoxylation of the hydroxylamine occurs,
and 3) deacetoxylation produces an electrophilic nitrenium ion, which is able to bind to a
DNA base, such as dG-C8, dG-N2 or dA-C8. Additionally, MMBD is structurally
analogous to 4-aminobiphenyl (Group1; IARC), a well-studied bladder carcinogen.
Therefore, the DNA adduct at the position of C8 with MMBD may also be formed by
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equal mechanism to other carcinogenic aromatic amies including 4-aminobiphenyl .
These results suggest that the DNA adduct formed with the dimer rather than the
monomer of monocyclic aromatic amines might contribute to bladder carcinogenesis.
Further analysis must be performed to prepare an authentic standard to identify the DNA
adduct corresponding to dG-MMBD.
Taken together, in this study we detected and identified MMBD as a o-Tol
metabolite, which possibly forms DNA adducts for the first time. Furthermore, MMBD
showed strong cytotoxicity, DNA damage and mutagenicity. Therefore o-Tol-induced
bladder carcinogenesis could be partly attributed to the MMBD metabolite. Further
analysis is required to evaluate the carcinogenicity of MMBD exposure.
Acknowledgements
This study was supported in part by a Health and Labor Sciences Research Grant
for the Research on Risk of Chemical Substances from the Ministry of Health, Labour
and Welfare, Japan. The authors thank Dr. Takeda Shunichi of Kyoto University for his
kindly given human spleen lymphoblastoid TK6 cells. The authors also thank Dr.
Sugiyama Keiichi of National Institute of Health Sciences, Japan and Dr. Yamada
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Masami of National Defense Academy of Japan for providing Salmonella strains for the
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Ames test.
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Table 1. Compounds specifically detected in o-Tol group
RT
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Compound#
m/z
Molecular formula
Structure (*)
[o-Tol]
(min)
1
2
1.02 108.0808
0.29 124.0751
1.32 166.0848
3.04 213.1366
2.26 255.1472
3.07 227.1155
4.15 255.1476
4.70 241.1315
4.92 240.1365
6.33 227.1155
1.32 124.0752
1.99 151.0852
2.05 120.0440
2.81 176.0693
3.42 209.1151
3.94 251.1160
6.22 330.3337
6.44 340.2237
C7H9N
C7H9NO
[o-Tol] + [O]
[o-Tol] + [O] + [Ac]
[o-Tol]2
3
C9H11NO2
C14H16N2
C16H18N2O
C14H14N2O
C16H18N2O
C15H16N2O
C16H17NO
C14H14N2O
C7H9NO
4
5
[o-Tol]2 + [Ac]
[o-Tol]2 - [2H] + [O]
[o-Tol]2 + [Ac]
[o-Tol]2 + [Ac] - [Me]
[o-Tol]2 + [Ac] - [NH2]
[o-Tol]2 - [2H] + [O]
[o-Tol] + [O]
N.D.
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C8H10N2O
C7H5NO
N.D.
C10H9NO2
C12H16O3
C16H14N2O
C20H43NO2
C22H29NO2
N.D.
N.D.
N.D.
N.D.
N.D.
* Compounds 1, 4, and 10 were identified using authentic standard. Other structures of
o-Tol metabolites were predicted by accurate mass and following experiment findings.
N.D., not determined.
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Figure 1
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Fig. 1. LC-MS analysis of rat urinary metabolites of o-Tol. Male F344 rats were fed
standard chow (CRF-1) or CRF-1 containing 0.8% o-Tol for 2 days. Urinary samples
collected during the last 24 h of metabolic caging from control (n=5) and o-Tol groups
(n=5) were extracted with diethyl ether, and then injected into LC-MS. (A) Base peak ion
chromatograms of control (n=5, gray) and o-Tol group rats (n=5, black) were overlaid.
(B) Extracted ion chromatograms for the indicated m/z of control (n=5, gray) and o-Tol
group rats (n=5, black) were overlaid. Peak 1 was o-Tol ([o-Tol]). Peaks 2 and 3
corresponded to modified o-Tol with oxidation [O] and acetylation [Ac]. Peak 4 was o-
Tol dimer [o-Tol]2. Chemical structures of 1 ~ 10 were predicted and shown in
Supplemental Fig. 1.
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Figure 2
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Fig. 2. LC-MS and MS/MS analysis of peak 4 (o-Tol metabolite in rat urine) and o-Tol
dimers. (A) Rat urine from the o-Tol group and the authentic standard of o-Tol dimers
including DBZ, MMBD, and DHB prepared as described in the Materials and Methods
section were injected into LC-MS. Extracted chromatograms (EIC) for m/z 213.1 and
MS/MS spectra obtained by CID 50 eV are shown.
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Figure 3
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Fig. 3. MMBD formation by the reaction of o-Tol with S9 mix. o-Tol (final 10 mM) was
incubated with or without S9 mix, boiled S9 mix, or BSA at 37ºC for 20 min. Samples
extracted with diethyl ether were injected into LC-MS for MMBD quantification. Values
are mean±SD (n=3).
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Fig. 4. Bioactivities of MMBD. (A) Cytotoxicities of o-Tol (□) and MMBD (●). These
compounds were exposed to T24 cells (left) and TK6 cells (right) for 24 h, then cell
viabilities were determined by AlamarBlue assay. Values are mean±SD (n=3). (B) γ-
H2AX was determined using a flowcytometer. T24 cells were exposed to 2.5 mM o-Tol
or 25 µM MMBD in the presence of S9 mix for 4 h, then stained with AlexaFluor 488
rabbit phosphor-histone H2A.X (Ser139) antibody. Histograms of o-Tol and MMBD are
shown by a solid line, but controls are denoted by gray shadow. (C) Mutagenicities of
MMBD in the presence (●) or absence (○) of S9 mix were determined by Ames test
using TA98, TA100, YG1024, and YG1029. Values are mean±SD (n=3).
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Fig. 5. DNA adduct in calf thymus DNA treated with S9 mix activated rat urine
metabolites. (A) DNA adductome maps. Calf thymus DNA was incubated with rat urine
in the presence of S9 mix, digested to nucleosides, then injected into LC-MS. In the DNA
adductome maps, bubbles represent chemically modified nucleosides detected by LC-MS
analysis, in which their positions were determined by retention time (min) and m/z, and
bubble size represents the peak area. Data are mean±SD (n=3). (B) Peak area and m/z of
37 DNA adducts specifically detected only in the o-Tol group are summarized.
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Fig. 6. DNA adductome of rat liver. Hepatic DNA extracted from male F344 rats fed
CRF-1 (control, n=5) or CRF-1 containing 0.8% o-Tol (o-Tol, n=5) for 2 days were
digested and injected into LC-MS. (A) Data were subjected to PCA analysis. (B) MS/MS
spectrum of m/z 478 and proposed structures of C8-dG, N2-dG, and N7-dG adduct are
shown.
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Supporting information
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Supplemental Table S1 List of 24 MS ions detected by GC-MS
Supplemental Table S2 Urinary concentration of o-Tol and MMBD
Supplemental Figure S1 Metabolome map of urine samples from o-Tol group rats
Supplemental Figure S2 Dimerization of monocyclic aromatic amines by the reaction
with S9 mix
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