Dose-Dependent Response to 3-Nitrobenzanthrone Exposure in Human Urothelial Cancer Cells

Article abstract of DOI:10.1021/acs.chemrestox.7b00174

A product of incomplete combustion of diesel fuel, 3-nitrobenzanthrone (3-NBA), has been classified as a cancer-causing substance. It first gained attention as a potential urinary bladder carcinogen due to the presence of its metabolite in urine and formation of DNA adducts. The aim of the present study was to characterize the dose-response relationship of 3-NBA in human urothelial cancer cell line (RT4) exposed to concentrations ranging from 0.0003 μM (environmentally relevant) to 80 μM by utilizing toxicological and metabolomic approaches. We observed that the RT4 cells were capable of bioactivation of 3-NBA within 30 min of exposure. Activity measurements of various enzymes involved in the conversion of 3-NBA in RT4 cells demonstrated NAD(P)H:quinone oxidoreductase (NQO1) as the main contributor for its bioactivation. Moreover, cytotoxicity assessment exhibited an initiation of adaptive mechanisms at low dosages, which diminished at higher doses, indicating that the capacity of these mechanisms no longer suffices, resulting in increased levels of intracellular reactive oxygen species, reduced proliferation, and hyperpolarisation of the mitochondrial membrane. To characterize the underlying mechanisms of this cellular response, the metabolism of 3-NBA and metabolomic changes in the cells were analyzed. The metabolomic analysis of the cells (0.0003, 0.01, 0.08, 10, and 80 μM 3-NBA) showed elevated levels of various antioxidants at low concentrations of 3-NBA. However, at higher exposure concentrations, it appeared that the cells reprogrammed their metabolism to maintain the cell homeostasis via activation of pentose phosphate pathway (PPP).

Full text of DOI:10.1021/acs.chemrestox.7b00174

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Article
Dose-dependent response to 3-nitrobenzanthrone
exposure in human urothelial cancer cells
Mario Pink, Nisha Verma, Anna Zerries, and Simone Schmitz-Spanke
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00174 • Publication Date (Web): 18 Sep 2017
Downloaded from http://pubs.acs.org on September 19, 2017
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Dose-dependent response to 3-nitrobenzanthrone exposure
in human urothelial cancer cells
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Mario Pinka,b,*, Nisha Vermaa, Anna Zerriesa, and Simone SchmitzꢀSpankea
a
Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine,
University of ErlangenꢀNuremberg, Schillerstr. 25/29, 91054 Erlangen, Germany
b
Postgraduate Course for Toxicology and Environmental Toxicology, Institute for Legal
Medicine, University of Leipzig, Johannisallee 28, 04103 Leipzig, Germany
*Corresponding author
Mario Pink, Institute and Outpatient Clinic of Occupational, Social and Environmental
Medicine, University of ErlangenꢀNuremberg, Schillerstraße 25/29, Dꢀ91054 Erlangen,
Germany
Eꢀmail: mario.pink@fau.de
Phone: +49ꢀ9131ꢀ8526102
Running Title: 3ꢀnitrobenzanthrone and bladder cancer
Keywords: 3ꢀnitrobenzanthrone (3ꢀNBA); bladder toxicity; bioactivation; metabolomic
profiling; adaptive response
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ABSTRACT
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A product of incomplete combustion of diesel fuel, 3ꢀnitrobenzanthrone (3ꢀNBA), has been
classified as a cancerꢀcausing substance. It first gained attention as a potential urinary bladder
carcinogen due to the presence of its metabolite in urine and formation of DNA adducts. The
aim of the present study was to characterise the doseꢀresponse relationship of 3ꢀNBA in
human urothelial cancer cell line (RT4) exposed to concentrations ranging from 0.0003 µM
(environmentally relevant) to 80 µM by utilising toxicological and metabolomic approaches.
We observed that the RT4 cells were capable of bioactivation of 3ꢀNBA within 30 minutes of
exposure. Activity measurements of various enzymes involved in the conversion of 3ꢀNBA in
RT4 cells demonstrated NAD(P)H:quinone oxidoreductase (NQO1) as the main contributor
for its bioactivation. Moreover, cytotoxicity assessment exhibited an initiation of adaptive
mechanisms at low dosages, which diminished at higher doses, indicating that the capacity of
these mechanisms no longer suffices ꢀ resulting in increased levels of intracellular reactive
oxygen species, reduced proliferation and hyperpolarisation of the mitochondrial membrane.
To characterise the underlying mechanisms of this cellular response, the metabolism of 3ꢀ
NBA and metabolomic changes in the cells were analysed. The metabolomic analysis of the
cells (0.0003, 0.01, 0.08, 10, and 80 µM 3ꢀNBA) showed elevated levels of various
antioxidants at low concentrations of 3ꢀNBA. Whereas, at higher exposure concentrations, it
appeared that the cells reprogrammed their metabolism to maintain the cell homeostasis via
activation of pentose phosphate pathway (PPP).
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Table of content
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INTRODUCTION
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Exposure to environmental and occupational contaminants has long been known to be
attributed to different kinds of lifeꢀthreatening diseases. Bladder cancer is a classic example of
such diseases, with studies reporting occupational exposure as the primary cause of
approximately 5 to 25% of different kinds of bladder tumours.1 Smoking and exposures to
aromatic amines are wellꢀknown risk factors, however, different studies have also reported the
potential of combustion products as a possible contributor to bladder cancer development.2–7
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3ꢀnitrobenzanthrone (3ꢀnitroꢀ7Hꢀbenz[de]anthracenꢀ7ꢀone, 3ꢀNBA), a nitroꢀpolycyclic
aromatic hydrocarbon, has attracted a lot of attention due to its high genotoxic and mutagenic
potential.8–13 Although present only in minute quantities in diesel exhaust particles (0.647 –
6.61 µg/g), it induced the highest number of revertants per nanomole in the Ames test, that
has been reported to date.14 Moreover, the mechanistic data on the effects of 3ꢀNBA lead to
its classification as a possible human carcinogen (Group 2B) by the International Agency for
Research on Cancer.15
Bound to diesel exhaust particles, the primary route of exposure to 3ꢀNBA is via the
respiratory tract. However, formation of DNA adducts after 3ꢀNBA exposure in secondary
organs of rodents with a particularly increased mutant frequency in urinary bladder have also
been reported.12 Biomonitoring studies of the urine of mine workers occupationally exposed
to diesel emissions also demonstrated the presence of a minute quantity of
3ꢀaminobenzanthrone (3ꢀABA), the main metabolite of 3ꢀNBA.16
Keeping in mind that 3ꢀNBA exerts a mutagenic effect in the bladder and the appearance of a
urinary metabolite that can be oxidised to a reactive intermediate, the objective of the current
study was to elucidate the mechanism of 3ꢀNBAꢀinduced bladder toxicity. Human bladder
cancer epithelial cells (RT4 cell line) were exposed to environmentally relevant doses of 3ꢀ
NBA, inter alia 0.0003 µM, the similar concentration of 3ꢀABA found in the 24 h urine of
exposed mine workers.16
To elucidate the doseꢀdependent response of 3ꢀNBA in the bladder, we studied its uptake and
bioactivation, cytotoxicity, and finally its potential to alter the cellular metabolism RT4 cells.
With the present work, we shed light on the cellular and metabolic response, highlighting the
transition from an adaptive, respectively controlled state, to a repair and protection mode
(maintaining homeostasis), to adverse effects that the cells undergo when exposed to 3ꢀNBA.
MATERIALS AND METHODS
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Chemicals and Reagents
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Standard chemicals for cell culture (culture medium, foetal calf serum, supplements) were
purchased from c.c.pro (Oberdorla, Germany). Chemicals for the synthesis of 3ꢀNBA were
purchased from SigmaꢀAldrich (Steinheim, Germany), Merck Millipore (Darmstadt,
Germany) and Alfa Aesar (Karlsruhe, Germany). The required chemicals for the metabolic
assays were purchased from Alfa Aesar (Karlsruhe, Germany), SigmaꢀAldrich (Steinheim,
Germany) and Carl Roth (Karlsruhe, Germany). Fluorescence probes and chemicals for the
toxicity assays were purchased from Life Technologies (Darmstadt, Germany), Promega
(Mannheim, Germany), Santa Cruz Biotechnologie (Heidelberg, Germany) and Alfa Aesar
(Karlsruhe, Germany). Solvents for extraction of intermediates of energy metabolism and
required derivatisation agents were purchased from Merck Millipore (Darmstadt, Germany)
and SigmaꢀAldrich (Steinheim, Germany). All chemicals used were of analytical grade.
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Cell culture
The human bladder cancer cell line RT4 (obtained from ATCC, Cat. No. ATCC® HTBꢀ2™)
was cultured until confluence in McCoy’s 5A medium, supplemented with 10 % foetal bovine
serum, 7.4 mg/mL Lꢀglutamine (0.75 %), 100 units/mL penicillin, and 100 mg/mL
streptomycin at 37°C in an atmosphere of 95% air and 5% CO2.
The assays were performed in 96ꢀwell plates two days after seeding of 20,000 cells per well.
The exposure was performed by incubation with culture medium containing 3ꢀNBA (the
concentration ranged from 0.3 nM to 10 µM) dissolved in DMSO (<0.1 % of the final
concentration) for 24 hours. The same percentage of DMSO was used to expose control cells
in all experiments. All experiments were performed at least five times in independent runs
unless otherwise stated.
Synthesis of 3-NBA
3ꢀNBA was synthesised by the nitration of benzanthrone. The first step of the synthesis was to
dissolve benzanthrone (2.5 g, 10.8 mM) in nitrobenzene (30 mL). Then, concentrated nitric
acid (4.0 mL) was added and the mixture was stirred at 40ꢀ50°C for 3h. Crude 3ꢀNBA
precipitated as a yellow solid from the cold reaction mixture and was recrystallised from
glacial acetic acid. The purity of the product (98.8%) was determined by GCꢀMS.
Metabolic assays
Uptake and metabolic activation of 3ꢀNBA to 3ꢀABA
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The uptake and metabolic conversion of the nonꢀfluorescent 3ꢀNBA were monitored based on
the fluorescence of 3ꢀABA.17,18 3ꢀABA emitted fluorescence was measured at a wavelength of
620 nm when excited at 470 nm. Fluorescence was monitored over a time span of 30 minutes
to 72 h. Cells treated with normal cell culture medium were used to correct for autoꢀ
fluorescence of the cells. In addition, spontaneous formation of 3ꢀABA was monitored in a
cellꢀfree environment.
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Determination of the catalytic activities of phase I and II biotransformation enzymes.
Catalytic activities of the following enzymes were measured: cytochrome P450 (CYP450),
glutathione Sꢀtransferase (GST), two Nꢀacetyltransferase isozymes (NAT1 & NAT2,
sulfotransferase 1A1 (SULT1A), xanthine oxidase (XO), cytochrome P450 oxidoreductase
(POR), and NAD(P)H:quinone oxidoreductase (NQO1). Detailed protocols of the assays for
each enzyme are available in the Supporting Information.
Briefly, cells seeded in 96ꢀwell plates were exposed against 0.0003 to 80 µM 3ꢀNBA for 24h.
At the end of the exposure period, the medium was removed and the cells were washed three
times with PBS before storage at ꢀ70°C. At the day of the experiment, the 96ꢀwell plates were
thawed for 10 minutes before usage. Afterward, 100µL reaction mixture containing PBS or
Tris as buffer, triton xꢀ100 for cell lysis, and required cofactors and detection reagent for the
corresponding enzyme, were added. The corresponding enzyme product was monitored by
fluorescence or absorption measurement.
Determination of toxicological endpoints
Formation of reactive oxygen species (ROS)
ROS were detected by their reaction with the fluorescent indicator dye 2′,7′ꢀdichloroꢀ
dihydrofluorescein diacetate (H2DCFꢀDA; Life Technologies). Briefly, cells treated with
normal cell culture medium were used to normalise between the independent experiments
(n=5), while 50 ꢁM of tertꢀbutyl hydroperoxide served as positive control. After 24h of
exposure, cells were washed three times with phosphateꢀbuffered saline (37°C) and incubated
with 10 ꢁM H2DCFꢀDA in PBS for 30 min at 37°C. Afterward, the cells were washed three
times and the fluorescence was measured in PBS immediately at λ(ex/em) 485/535 nm.
Cell proliferation
Cell proliferation was monitored by incorporation of the thymidine analogue 5ꢀethynylꢀ2'ꢀ
deoxyuridine (EdU, Santa Cruz Biotechnology) and detection with fluoresceinꢀ5ꢀazide (FAMꢀ
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5ꢀazide, Lumiprobe). 3.5 h before the end of the exposure period, 20 µL EdU labelling
solution (10 µM/well) was added to each well and the exposure was continued for the
remaining time. Afterwards, the culture medium was removed and the cells were washed
twice with PBS followed by fixation for 15 minutes with 4% formaldehyde, 0.1% triton xꢀ100
in PBS. To detect the incorporated EdU, the fixed cells were washed three times with PBS
and incubated in a freshly prepared fluorescence labelling solution of 100 mM TrisꢀHCl (pH
8.0), 100 mM ascorbic acid, 1 mM CuSO4 and 10 µM FAMꢀ5ꢀazide for 30 minutes. The
fluorescence was monitored after three washing steps with PBS at λ(ex/em) 485/535 nm in PBS.
Blank wells were used for background readꢀout, while unexposed cells were used to
normalise the results between the independent experiments (n=5).
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Mitochondrial membrane potential (MMP, ꢁψm)
ꢂψm was analysed by using the fluorescent mitochondrial membrane potential indicator
rhodamin 123 (Rh123, Life Technologies). Cells treated with normal cell culture medium
were used as negative controls while valinomycin (25µM) served as positive control. After
exposure, the cells were washed three times with PBS (37°C) and incubated with 10 µM
Rh123 for 30 min at 37°C. At the end of the incubation period, the dye solution was removed
and the cells were washed twice with PBS (37°C) before measurement of the fluorescence
intensity at λ(ex/em) 485/535 nm in PBS. Control cells were used to normalise the results
between the independent experiments (n=5).
The cellular viability (MTT assay)
The viability respectively metabolic activity was determined by using the tetrazolium dye
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT). Two hours before the
end of the exposure period, 20 µL of a freshly prepared and sterile filtered MTT solution
(5 mg/mL) was added to each well and mixed gently. Upon completion of the incubation, the
culture medium was removed and the cells were washed twice with PBS. Cell lysis was
performed with a mixture of 0.6 % glacial acetic acid, and 10 % SDS in DMSO. The mixture
was added to each well (100 µL), and the plate was incubated for 5 min at RT, which was
continued for 5 min on an orbital shaker. The cellular viability was determined by
measurement of the absorbance at 492 nm to the reference wavelength of 620 nm. Blank
wells were used for background readꢀout. Control cells were used to normalise the results
between the independent experiments (n=5).
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Glutathione quantification
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Cellular glutathione (GSH) levels were measured by means of 5,5'ꢀdithiobisꢀ(2ꢀnitrobenzoic
acid) (DTNB, Ellman's reagent).19,20 After exposure, the cells were washed three times with
PBS before stored at ꢀ70°C. At the day of the experiment, the cells were thawed for 10
minutes at RT. Afterward, 100 µL of a reaction mixture (5 mM EDTA, 1 mM DTNB, 1 U/mL
GSSG reductase, 0.6% sulfosalicylic acid, 0.2% Triton Xꢀ100 in PBS) was added and the
plates were incubated for 5 minutes at RT. To initiate the reaction NADPH was added to each
well (0.25 mM final conc. per well) and the absorbance were measured immediately at
415 nm for five minutes in one minute intervals.
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To determine the cellular GSSG (glutathione disulphide) content, 1ꢀmethylꢀ2ꢀvinylpyridinium
triflate (M2VP, 20 µL/well of 3 mM solution in 0.2% Triton Xꢀ100) was added 2 minutes
before the reaction mixture was added.
Calibration standards, GSH and GSSG, respectively, were treated similarly to the samples.
The assay was performed four times (n=4) and the results were normalised to 106 cells.
Metabolomic profiling
Extraction of carbohydrate and lipid metabolites from cell lysates
The intermediates of energy metabolism were extracted by a methyl tertꢀbutyl ether/methanol
(MTBE/MeOH)ꢀbased technique.21 Cells grown in a 75 cm2 flask were exposed to five
different concentrations of 3ꢀNBA (0.3 nM, 10 nM, 80 nM, 10 µM and 80 µM) at 80 %
confluence for 24h. At the end of the exposure, the cells were harvested according to standard
procedures. The resulting cell pellet was washed three times with normal saline solution
(isotonic saline solution, 0.9% NaCl) to remove remaining culture medium. During the last
washing step, the cells were transferred to 2 mL reaction tubes for metabolite extraction. Cell
lysis was performed by incubating the cells in a mixture of MTBE/MeOH (10:3, 400µL) by
vortexing with 45 mg glassꢀbeads (acid washed) for 2 minutes. The lysed cells were then
centrifuged to separate the cell debris from the metabolites at 20,000 xg for 5 minutes at 4°C.
The supernatant was removed and collected in 1.5 mL reaction tubes. The remaining pellet
was reꢀextracted with 400 µL MTBE/MeOH (10:3) and the supernatants were combined. The
separation of hydrophilic and lipophilic metabolites phase was induced by adding 200 µL
water. After vortexing, the samples were centrifuged at 20,000 xg for 5 minutes at 4°C and
the upper (organic) phase was transferred into a new 2 mL tube. The remaining aqueous phase
was washed once with
a
waterꢀsaturated MTBE/MeOH solution (10:3:2.5;
MTBE:MeOH:H2O) and the organic phases were combined after centrifugation at 20,000 xg
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for 5 minutes at 4°C. The aqueous phase was transferred into a GCꢀvial. Both metabolic
fractions were dried in a vacuum centrifuge at RT.
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Derivatisation of the metabolites
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Aqueous fraction
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50 µL of a 20 mg/mL solution of methoxyamine hydrochloride in pyridine was pipetted into
the vial of the aqueous fraction and incubated at RT overnight (> 16h). Afterwards, 30 µL
MSTFA was added and the fraction was further incubated for 30 minutes at 80°C
Organic fraction
100 µL toluene was added to the dried organic fraction, followed by 750 µL MeOH and
150 µL of an 8 % HCl solution in methanol. After each addition the samples were mixed.
Afterwards, the samples were incubated overnight (> 16h) at RT to derivatised fatty acids to
their corresponding methyl esters. At the end of incubation, the organic compounds were
extracted by adding 150 µL hexane and 150 µL water. After centrifugation at 20,000 xg for
5 minutes at 4°C, the (upper) organic phase was transferred to a new vial and the aqueous
phase was reꢀextracted with 150 µL. The organic phases were combined and dried in a
vacuum centrifuge at RT. Finally, the dried sample was suspended in 50 µL pyridine and
derivatised with 30 µL MSTFA for 30 min at 80°C.
GC/MS setup
The isolated and derivatised metabolites were identified and quantified by GC/MS analysis
using an HP 6890/5973 GC/MS system (Agilent Technologies, Waldbronn, Germany). The
instrument was equipped with a 30 m x 320 µm (i.d.) Optima 5 column coated with a 5%
phenyl/95% methylpolysiloxane crossꢀlinked stationary phase (0.25 ꢁm film thickness;
MACHEREYꢀNAGEL, Düren, Germany). Helium was used as carrier gas (flow rate of
1.5 mL/min). The analytes (1 µL each) were injected in the splitless mode with a solvent cutꢀ
off time of 6 minutes. The injector temperature was maintained at 280°C. The oven
temperature was kept at 70°C for 1 min and then linearly increased at a rate of 1°C/min up to
76°C and from there at a rate of 6°C/min up to 300°C, where it was maintained for 5 min. The
MS was operated in the electron impact ionisation mode at 70 eV with the quadrupole
temperature set at 150°C and the source temperature at 230°C. Full scans were acquired by
repetitively scanning over the mass range from 50 to 550 Da at a scan rate of 500 msec/scan.
GC/MS spectrum analysis
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The resulting GC/MS spectra were deconvoluted with AMDIS (Automated Mass spectral
Deconvolution and Identification System) and analysed with SpecConnect.22 The resulting
integrated signal matrix was used to calculate the metabolite ratios between the various
groups and for statistical analysis using the KNIME Analytics Platform (ANOVA). The peak
identification was performed with the GOLM metabolome database (Ver. 20111121),23 and
massbank.24 The metabolite profiling was performed in four independent experiments (n=4).
The resulting metabolites were annotated with ChEBI identifiers and submitted for
enrichment analysis using MetaboAnalyst.25 The STITCH v5.0 was used to investigate
metaboliteꢀprotein interactions. The list of regulated metabolites was uploaded and the
metabolic network was studied using STITCH v5.0. The network was computed under the
assumption of a very high confidence interaction, > 0.9.26
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Statistical analysis and multivariate analysis
All experiments were performed at least five times in independent runs unless otherwise
stated. Microplate reader readouts of fluorescence or absorbance measurements were
normalised to control wells. Control values were set to 100 %. Each analysis comprises three
technical replicates. The level of statistical significance relative to control was calculated by
using the analysis of variance (ANOVA) with Tukey’s postꢀhoc test as a statistical method.
The significance is represented in the graphs by asterisks (*, p ≤ 0.05; **, p ≤ 0.01;
***, p ≤ 0.001).
Principal component analysis of the combined toxicological assays (activity and cytotoxicity
assays) and GCꢀMS data supplemented with data from the combined toxicological assays
(conc. 0.0003, 0.01, 0.08, 10 and 80 µM) was performed using the Multibase package
(Numerical Dynamics, Japan).
RESULTS
Biotransformation of 3-NBA
Based on the intrinsic fluorescence of 3ꢀABA, one of the main metabolites of 3ꢀNBA, the
uptake, metabolic activation and its progress over 72 h were monitored. Exposure of RT4
cells to 17 different concentrations ranging from 0.0003 µM to 80 µM revealed a timeꢀ and
doseꢀdependent increase in the cellular fluorescence intensity of 3ꢀABA compared to nonꢀ
exposed cells. Elevated cellular fluorescence levels were detected for concentrations starting
from 0.01 µM after 30 minutes of exposure. During the observation phase no saturation of
fluorescence was detected (Fig. 1A). We assume that the trend of the reduced slope of the
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fluorescence intensity after a longer exposure time reflects the depletion of 3ꢀNBA in the
culture medium, thus leading to a steady state of 3ꢀABA fluorescence.
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The microscopic analysis of the cellular distribution of formed 3ꢀABA revealed a primary
cytosolic localization of 3ꢀABA (Supporting Information, Fig. S1).
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The analyses of the activity of phaseꢀI and II enzymes revealed a nonꢀmonotonic doseꢀ
response which was mostly divided into three parts ranging from 0.0003 µM to 0.005 µM,
0.01 µM to 0.6 µM and 1.25 µM to 80 µM. The most pronounced activity alterations, in
general, were observed for NQO1 (up to 122%), for XO (up to 105%) and a decrease for
NATs to at least 88% (NAT1) respectively 90% (NAT2) (Fig. 1B).
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The other activity assays, POR, CYP450, GST, and SULT1A, showed only minor alterations
except for GST and SULT1A (Fig. 1B). They showed a concentrationꢀdependent increase in
activity at the highest exposure concentrations of 116% (SULT1A) and 118% (GST)
compared to control.
Determination of toxicological endpoints
Various cellular endpoints were analysed to elucidate the cellular doseꢀresponse and to
identify the beginning of disturbance in cellular homeostasis. The analysed toxicological
endpoints showed a nonꢀlinear response to 3ꢀNBA with partitions comparable to the shape of
the doseꢀresponse curve of the metabolising enzymes.
ROS production and cell proliferation
The analysis of the cellular ROS levels revealed that at low exposure concentrations a slight
decrease in ROS formation was observed, while at higher exposure concentrations, the
cellular ROS increased up to 132 % when cells were treated with 0.01 µM to 10 µM. The
maximal ROS level of 172 % was observed for 80 µM (Fig. 2A).
The proliferation rate showed a significant decrease, at the highest concentrations, of 14% as
compared to control. However, at lower exposure concentrations no alterations were observed
(Fig. 2B).
Mitochondrial membrane potential and cell viability
A significant increase in ꢂψm was observed only for the highest exposure concentrations with
levels from 110% (5 µM) to 118% (80 µM, Fig. 2D) as compared to the control.
The MTT assay as an indicator of cell viability exhibited a nonꢀmonotonic response in the
exposure range from 0.0003 µM to 0.3 µM followed by a concentrationꢀdependent decrease at
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high 3ꢀNBA dosages. The minimal cell viability was observed for 80 µM where the viability
decreased to 64%, compared to the control levels (Fig. 2C).
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Glutathione quantification
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The determination of cellular glutathione levels revealed a concentration dependent
significant increase in the total cellular content of glutathione from 0.147±0.014 mM in
control cells to 0.215±0.018 mM when cells were exposed to 80 µM of 3ꢀNBA. A similar
result was obtained for oxidised glutathione which showed a concentration dependent
significant increase from 4.4±2.6 µM in control to 25.4±2.6 µM when cells were exposed to
80 µM. The ratio of GSH to GSSG decreased from 32.33 in control to 7.47 in the cells
exposed to 80 µM 3ꢀNBA (Fig. 3).
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Metabolomic profiling
Based on the results of the described assays, five different concentrations (0.0003 µM,
0.01 µM, 0.08 µM, 10 µM and 80 µM) were selected to observe changes in the cellular
metabolism. The exposure to 3ꢀNBA revealed various significantly altered cellular
metabolites, 15 for cells exposed to 0.0003 µM (10 identified), 33 for 0.01 µM (28 identified),
183 for 0.08 µM (62 identified), 109 for 10 µM (36 identified) and 81 metabolites (43
identified) after the treatment with 80 µM. These metabolites fulfilled our criteria of an
altered metabolite when compared to control (volcano plots of the metabolites are shown in
Supporting Information, Fig. S4). A metabolite was considered altered when its relative
abundance has changed by a factor not less than ± 1.5. The significantly altered and identified
metabolites are listed in Supporting Information (Tabl. S1ꢀ5). The metabolites with altered
concentrations include, among others, fatty acids (e.g. palmitic acid, oleic acid, linolenic
acid), amino acids (e.g. glycine, serine, phenylalanine), substrates of glycolysis and TCA
cycle (e.g. fructoseꢀ6ꢀphosphate, citric acid) and antioxidant (hypotaurine, αꢀtocopherol).
Enrichment analysis of the altered metabolites based on their ChEBi identifiers linked them to
various metabolic pathways, among them pentose phosphate pathway, glutathione
metabolism, glycolysis, gluconeogenesis, betaꢀoxidation of very long chain fatty acids, alphaꢀ
linolenic acid and linoleic acid metabolism (Fig. 4; Supporting Information Fig. S5ꢀS9). The
STITCH tool was used to provide a visualisation of predicted interactions of chemicals and
proteins after exposure to 0.08 µM 3ꢀNBA. The interaction network revealed that a large
number of metabolites are linked to NADPH and 10ꢀformylꢀtetrahydrofolate (Fig. 5).
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The multivariate analysis of the obtained toxicological data including the data of the activity
assays revealed three clusters for both principal component analyses. The software clustered
the concentrations according to the cellular effect. The resulting groups consist of the
concentrations 0.0003 to 0.02 µM (including control cells) cluster 1 and 0.04 to 10 µM cluster
2 and 80 µM cluster 3 (Fig. 6).
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DISCUSSION
Found in diesel engine exhaust, which significantly contributes to air pollution, the nitrated
polycyclic aromatic hydrocarbon 3ꢀNBA is considered a suspected human carcinogen,27
probably affecting the lungs. Studies have reported the presence of 3ꢀABA, the main
metabolite of 3ꢀNBA in urine samples of salt mine workers occupationally exposed to diesel
engine exhaust.16 Given that the bladder is the main organ where urine is stored, it is plausible
to expect toxic effects on bladder epithelial cells, too. Therefore, the objective of the present
study was to describe the doseꢀresponse behaviour of 3ꢀNBA in vitro, and to elucidate the
underlying cellular mechanisms. To reflect environmental condition, concentrations as low as
0.0003 µM 3ꢀNBA, similar to 143 ng/24 h (1.5 L; 0.0004 µM 3ꢀABA) urine observed for
3ꢀABA,16 were tested in the utilised cell model. In the high doses range, up to 10 µM was
tested equivalent to ~ 3 mg/24 h urine. Due to the absence of strong toxic effects, a critical
dose of 80 µM was tested although this concentration cannot be reached under environmental
conditions.
The resulting data pointed to a nonꢀlinear effect with modest deviations from control
conditions at low doses of 3ꢀNBA and pronounced toxic effects at higher doses. At an
intermediate level of exposure (0.04 – 10 µM 3ꢀNBA, obtained by principal component
analysis, additional processes were activated which became increasingly less effective in
maintaining a controlled cellular state, as is evident for the obtained ROS results. Finally,
pronounced toxic effects occurred at a concentration of 80 µM
Uptake and metabolic activation of 3-NBA into its toxic metabolite 3-ABA
An important determinant of the toxicity of a xenobiotic is the ability of a specific tissue to
metabolise it. A suitable candidate for monitoring is 3ꢀABA, one of the main metabolites of 3ꢀ
NBA, due to its autoꢀfluorescence. We could demonstrate that these cells were capable of
metabolising 3ꢀNBA into its toxic metabolite.17,18,30,31 In our experimental setting, 3ꢀABA was
detected as early as 30 minutes even in the cells exposed to low concentrations such as
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0.01 µM 3ꢀNBA. During the 72 h exposure, we observed a timeꢀdependent accumulation of
3ꢀABA with no evidence of approaching saturation (Fig. 1A). Microscopic evaluation of the
distribution within the cells revealed that 3ꢀABA was mainly found in the cytosol where the
enzymes involved in the metabolism of 3ꢀNBA are located.
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So far, the bioactivation of 3ꢀNBA has not been studied in detail in bladder cells. Therefore,
the activity of a panel of enzymes ꢀ involved in the metabolism of 3ꢀNBA ꢀ was measured.
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The analysis revealed a high activity of NQO1 indicating that this enzyme might be the
main contributor in metabolic conversion of 3ꢀNBA in the utilized cell model, which is in
agreement with previous studies (Fig. 1B).36,37 The other enzymes, POR, XO, and CYP450
had an only negligible influence on bioactivation of 3ꢀNBA. In addition, we also measured
the activity of the conjugation enzymes NAT1/2 and SULT1A, which was reported to
enhance the 3ꢀNBAꢀderived DNA adduct formation.38,33 However, treatment with 3ꢀNBA
only moderately influenced NAT1/2 by reducing their activity while SULT1A was only
affected at the highest concentrations. The additionally performed test for GSTs, a key group
of enzymes for cellular detoxification, showed an equal result. Interestingly, most described
enzymes are regulated by the aryl hydrocarbon receptor (AhR) and polycyclic aromatic
hydrocarbons are potent ligands for AhR. Nevertheless, the nitroꢀpolycyclic aromatic
hydrocarbon 3ꢀNBA was not shown to induce these enzymes via AhR.32–34
Cytotoxicity of 3-NBA
After analysing the bioactivation of 3ꢀNBA, we analysed the cytotoxic potential of 3ꢀNBA
within the wide concentration range used in this study (Fig. 2). To avoid interferences of 3ꢀ
ABA autoꢀfluorescence at λem 550 to 700 nm, the fluorescence based toxicological assays
were performed at excitation and emission wavelength λex 488 nm and λem 535 nm.
ROS plays an important part in cellular stress response; in comparable studies a significant
increase in ROS production was demonstrated, beginning at 0.01 µM 3ꢀNBA in human
alveolar cells (A549), and particularly 10 µM in RT4 cells, with both cell lines being exposed
for 30 minutes.37,39 In our model, treatment with 3ꢀNBA induced a doseꢀdependent ROS
production at a concentration of 0.01 µM. Nevertheless, compared to the aboveꢀmentioned
studies, the increase in ROS production seemed to be less pronounced in our experimental
setting, which might be due to the duration of the exposure. We assumed that within 30
minutes of exposure the cellular oxidative defence is not able to restore the cellular
homeostasis as effectively as after an exposure of 24 h. After an increase of ROS the cells try
to maintain a steady state, probably by activating specific pathways to face the increasing
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oxidative stress, while their compensatory limit was exceeded when high concentrations of 80
µM was applied. This assumption is supported by our metabolomic data, described in detail in
the “metabolomic profiling” section.
Furthermore, oxidative stress is closely intertwined with energy metabolism and, in particular,
with mitochondrial activity. Measurement of cellular ATP levels (Supporting Information,
Fig. S3) showed no perturbation over a wide concentration range, which was further
confirmed by supportive ꢂψm measurements. Alterations of both endpoints decreased ATP
levels and hyperpolarisation of ꢂψm, were only observed for the higher dosages tested.
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Metabolomic evaluation
Due to the complexity of this topic, we selected five concentrations based on the principal
component analysis to compare different cellular states. This analysis grouped the
concentrations according to the observed cellular effects i.e. 0.0003 to 0.02 µM (including
control cells) as cluster 1, 0.04 to 10 µM as cluster 2 and 80 µM as cluster 3 (Fig. 6), out of
which 0.0003, 0.01, 0.08, 10, and 80 µM were further selected for metabolomic analysis.
At lower concentrations of 3ꢀNBA (0.0003 and 0.01 µM), several metabolites of pathways
were altered which were associated with the antioxidant defence such as the
taurine/hypotaurine, glutathione or vitamin B6 metabolism (Fig. 4).40,41
Exposure to 0.08 µM 3ꢀNBA led to the regulation of the highest number of pathways
indicating that the maintenance of the cellular homeostasis became increasingly challenging.
Thus, 3ꢀNBA impact on the protein biosynthesis, the amino sugar metabolisms or the pentose
phosphate pathway might be a sign of the increasing cellular effort to provide energy and to
face the disturbance of the redox state. Exposing the cells to 10 and 80 µM intensified the
shift in energy metabolism, particularly towards the pentose phosphate pathway. The
preference of the pentose phosphate pathway was also reported after the exposure to other
xenobiotics such as iron, 4ꢀmonochlorobiphenyl or TCDD.42–44
A common toxic effect of these xenobiotics is an induction of oxidative stress, which can be
counterbalanced by the glutathione system, which was also observed in our cell model (Fig.
3). Regeneration of oxidised glutathione is dependent on NADPH, which in turn is
substantially produced in the oxidative pentose phosphate pathway. That and the additional
generation of ribose 5ꢀphosphate for the nucleotide synthesis make it perfectly understandable
and obvious that the pentose phosphate pathway plays an important role in the redox defence
and in the repair of DNA damage. In addition, the serineꢀdriven oneꢀcarbon metabolism
contributes markedly to the NADPH production. By the conversion of serine to glycine, the
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methyl group is transferred to tetrahydrofolate, followed by oxidation to 10ꢀformylꢀ
tetrahydrofolate which is coupled to the reduction of NADP+ to NADPH.45 Accordingly,
serine and glycine were increased after the exposure to 0.08 µM 3ꢀNBA. The association
network (Fig. 5), generated with STITCH, further highlights the connection between the
altered metabolites, NADPH and 10ꢀformylꢀtetrahydrofolate.
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Furthermore, 3ꢀNBA also altered the fatty acid metabolism. Exposure to the lowest
concentration already increased the levels of saturated fatty acids (palmitic acid, arachidic
acid, behenic acid, lignoceric acid), which could be oxidised in the peroxisomes and
mitochondria to fuel respiration. In addition, also polyunsaturated fatty acids (PUFAs) were
mobilised. In this case, however, differences between the individual concentrations became
apparent. Thus, levels of members of the nꢀ6 family (γꢀlinolenic acid, arachidonic acid (AA))
were elevated in response to 0.01 and 0.08 µM 3ꢀNBA while the nꢀ3 fatty acids (αꢀlinolenic
acid, docosahexaenoic acid (DHA)) increased after treatment with 10 µM. In addition, AA
and DHA serve as substrates for the downstream synthesis of lipid mediators, which modulate
inter alia inflammation: those derived from nꢀ6 PUFAs (AA) enhance, whereas nꢀ3 PUFAs
(DHA) derived mediators suppress this process.46 Also in context with exposure to polycyclic
aromatic hydrocarbons, the protective effect of nꢀ3 PUFAs was recently reported.47 In
addition, PUFAs are a major determinant of the cellular membrane and therefore have a deep
impact on mitochondrial function.48 Thus, alterations of DHA or AA modulate ROS
generation and interfere with the respiratory chain complex. Nevertheless, the direction of
these modulations depends on many factors and requires further investigation.
In conclusion, the present study highlights the doseꢀresponse relationship of 3ꢀNBA in
bladder cancer cells regarding cytotoxicity, metabolising enzymes and the metabolomic state.
Starting from a relevant environmental dose of 0.0003 µM up to 80 µM, we described the
driving mechanism behind the different cellular states. Up to a concentration of 0.02 µM, 3ꢀ
NBA exerted only lowꢀdose effects given a functioning oxidative stress defence. In an
intermediate exposure range from 0.04 µM to 10 µM, the cell increasingly utilised processes
to protect the cells from the consequences of exposure. Higher doses of 3ꢀNBA drove the
cells irreversibly to a toxic state. The metabolomic data suggested that the pentose phosphate
pathway and the folate metabolism play a leading role in the transition from an adaptive to an
adverse cellular state. From the perspective of risk assessment, one might come to the
cautious conclusion that the urinary concentration of exposed workers seemed to exert a lowꢀ
dose effect. Nevertheless, this has to be further validated.
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ASSOCIATED CONTENT
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SUPPORTING INFORMATION
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Methods and corresponding references of applied assays to determine enzyme activities of
NQO1, CYP450, XO, POR, GST, SULT1A, NAT1 and NAT2; microscopic images of the
cellular distribution of 3ꢀABA; bar graphs representation of the enzyme activity results and
observed cellular ATP levels; volcano plot as graphical representation of the observed cellular
metabolites by GCꢀMS; results of the enrichment analysis of significantly altered metabolites
observed after exposure to 0.0003, 0.01, 0.08, 10 or 80 µM 3ꢀNBA, by MetaboAnalyst; tables
of significantly altered metabolites observed after exposure to 0.0003, 0.01, 0.08, 10 or 80 µM
3ꢀNBA.
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The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: mario.pink@fau.de. Telephone: (+49) 9131ꢀ85ꢀ26102. Fax: (+49) 9131 ꢀ85ꢀ22317.
ORCID
Mario Pink: 0000ꢀ0002ꢀ5148ꢀ5138
Nisha Verma: 0000ꢀ0000ꢀ7558ꢀ167X
Funding
The authors acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG) under
Grant No. SCHM1207/5ꢀ1.
Conflict of interest
The authors declare no conflicts of interest.
ABBREVIATIONS
AA, arachidonic acid; 3ꢀABA, 3ꢀaminobenzanthrone (3ꢀaminoꢀ7Hꢀbenzo[d,e]anthracenꢀ7ꢀ
one); AMDIS, Automated Mass spectral Deconvolution and Identification System; ChEBI,
Chemical Entities of Biological Interest; CuSO4, copper sulphate; CYP450, cytochrome P450;
DHA, docosahexaenoic acid; DMSO, dimethyl sulphoxide; EdU, 5ꢀethynylꢀ2'ꢀdeoxyuridine;
FAMꢀ5ꢀazide, fluoresceinꢀ5ꢀazide; GCꢀMS, gas chromatographyꢀmass spectrometry; GSH,
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glutathione; GSSG, glutathione disulphide; GST, glutathione Sꢀtransferase; H2DCFꢀDA, 2′,7′ꢀ
dichloroꢀdihydrofluorescein diacetate; MeOH, methanol; MMP, ꢂψm, mitochondrial
membrane potential; MSTFA, NꢀmethylꢀNꢀ(trimethylsilyl)trifluoroacetamide; MTBE, methyl
tertꢀbutyl ether; MTT, tetrazolium dye, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium
bromide; NADP+, nicotinamide adenine dinucleotide phosphate, oxidised; NADPH,
nicotinamide adenine dinucleotide phosphate, reduced; NAT, Nꢀacetyltransferase isozyme;
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3ꢀNBA,
3ꢀnitrobenzanthrone
(3ꢀnitroꢀ7
H
ꢀbenzo[d,e]anthracenꢀ7ꢀone);
NQO1,
NAD(P)H:quinone oxidoreductase, PBS, phosphate buffered saline; POR, cytochrome P450
oxidoreductase; PUFA, polyunsaturated fatty acids, PPP, pentose phosphate pathway; Rh123,
rhodamine 123; ROS, reactive oxygen species; RT, room temperature; SDS, sodium dodecyl
sulphate; STITCH, Search Tool for Interactions of Chemicals; SULT1A1, sulfotransferase
1A1; TCA cycle, tricarboxylic acid cycle; XO, xanthine oxidase
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Figure 1: Determination of 3-ABA accumulation and analysis of enzyme activities. (A)
Determination of the metabolic activation of 3ꢀNBA to 3ꢀABA over 72 h. The intrinsic
fluorescence of 3ꢀABA (λex 485 nm, λem 595 nm) was used to monitor the metabolic
activation and its progress. Elevated fluorescence levels were observed starting from 0.1 µM
and 30 minutes of exposure. At lower exposure concentrations no increase in fluorescence
could be measured. During the measurement, no saturation in 3ꢀABA fluorescence was
observed.
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(B) Heat map illustration of the measured enzyme activities. The activity of enzymes reported
to be involved in metabolic activation of 3 NBA, NQO1, POR, XO and CYP450 and
conjugation, NAT1 and 2, SULT1A and GST as an important enzyme in detoxification, were
tested after exposure to 3ꢀNBA for 24 h. The represented data in the heat map are average
values of 7 independent experiments; bar graphs to each tested enzyme were shown in
Supporting Information (Fig. S2).
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Figure 2: Determination of 3-NBA cytotoxicity. Cytotoxic effects caused by 3ꢀNBA after
24 h exposure was monitored by measuring alterations of cellular ROS levels (A) and
proliferation (EdU, B), cell viability (MTT, C) and mitochondrial membrane potential (MMP,
D). All measured toxicological endpoints exerted a nonꢀmonotonic response to 3ꢀNBA with
modest alteration up 0.001 µM, an intermediate range of response between 0.004 and 10 µM
and a toxic response after treatment with 80 µM. The obtained values were normalised to
controls (n = 5; *, p<0.05).
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Figure 3: Glutathione quantification. After 24 h of exposure with 3ꢀNBA, the GSH content
was quantified by means of DTNB (Ellman’s reagent). The measurements were performed by
using a microplate reader (OD412nm). The data were presented as mean ± SEM of four
independent experiments. The level of significance relative to control was determined by
using the tꢀtest (*, p ≤ 0.05; **, p ≤ 0.01).
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Figure 4: Cumulative representation of the enrichment analysis. The graphs illustrate
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signalling pathways that are enriched on the basis of the metabolites passing the filtering
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criteria. The corresponding metabolites are listed in Supporting Information (Tabl. S1ꢀ5). The
bars indicate the degree of enrichment of the specific pathway. The analysis highlighted the
shift in cellular metabolism when exposed to higher dosages of 3ꢀNBA. Starting at an
exposure to 0.08 µM 3ꢀNBA, the cells reꢀschedule the cellular metabolism towards protein
biosynthesis, the amino sugar metabolisms or the pentose phosphate pathway. This metabolic
shift might be a sign of the increasing cellular effort to maintain homeostasis by the
generation of redox equivalents and to provide substances for cell repair, lipids, and
backbones for nucleotides.
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Figure 5: Stitch annotation network based on the altered metabolites observed after
exposure to 0.08 µM 3ꢀNBA. The network computed under the assumption of very high
confidences interactions, > 0.9, with 1 being the highest possible confidence. The network
shows strong interactions between the observed metabolites, 10ꢀformylꢀtetrahydrofolate and
NADPH. Functional enrichments of the network assigned them to oneꢀcarbon metabolic
process and tetrahydrofolate interconversion/metabolism.
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Figure 6: Principal component analysis of the obtained data. The analysis was performed
to discriminate for concentrationꢀdependent differences in the cellular behaviour. Two
analysis were performed, once based on the GCꢀMS data supplemented with data from the
monitored toxicological endpoints and activity assays (A, conc. 0.0003, 0.01, 0.08, 10 and
80 µM) and a second time for all tested concentrations based on toxicological endpoint and
activity assay results (B). The analysis discriminates in both analyses three clusters of
concentrations based on their cellular behaviour. Categorised in the first cluster are (cluster 1)
concentrations ranging from 0.0003 µM to 0.02 µM including control, categorised in the
second cluster are concentrations ranging from 0.04 µM to 10 µM (cluster 2) and in the third
cluster 80 µM (cluster 3).
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Table of Content
272x96mm (150 x 150 DPI)
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Figure 1
246x137mm (150 x 150 DPI)
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