Structure-activity relationships imply different mechanisms of action for ochratoxin A-mediated cytotoxicity and genotoxicity

Article abstract of DOI:10.1021/tx200406c

Ochratoxin A (OTA) is a fungal toxin that is classified as a possible human carcinogen based on sufficient evidence for carcinogenicity in animal studies. The toxin is known to promote oxidative DNA damage through production of reactive oxygen species (ROS). The toxin also generates covalent DNA adducts, and it has been difficult to separate the biological effects caused by DNA adduction from that of ROS generation. In the current study, we have derived structure-activity relationships (SAR) for the role of the C5 substituent of OTA (C5-X = Cl) by first comparing the ability of OTA, OTBr (C5-X = Br), OTB (C5-X = H), and OTHQ (C5-X = OH) to photochemically react with GSH and 2′-deoxyguanosine (dG). OTA, OTBr, and OTHQ react covalently with GSH and dG following photoirradiation, while the nonchlorinated OTB does not react photochemically with GSH and dG. These findings correlate with their ability to generate covalent DNA adducts (direct genotoxicity) in human bronchial epithelial cells (WI26) and human kidney (HK2) cells, as evidenced by the ³²P-postlabeling technique. OTB lacks direct genotoxicity, while OTA, OTBr, and OTHQ act as direct genotoxins. In contrast, their cytotoxicity in opossum kidney epithelial cells (OK) and WI26 cells did not show a correlation with photoreactivity. In OK and WI26 cells, OTA, OTBr, and OTB are cytotoxic, while the hydroquinone OTHQ failed to exhibit cytotoxicity. Overall, our data show that the C5-Cl atom of OTA is critical for direct genotoxicity but plays a lesser role in OTA-mediated cytotoxicity. These SARs suggest different mechanisms of action (MOA) for OTA genotoxicity and cytotoxicity and are consistent with recent findings showing OTA mutagenicity to stem from direct genotoxicity, while cytotoxicity is derived from oxidative DNA damage.

Full text of DOI:10.1021/tx200406c

Article
pubs.acs.org/crt
Structure−Activity Relationships Imply Different Mechanisms of
Action for Ochratoxin A-Mediated Cytotoxicity and Genotoxicity
Kheira Hadjeba-Medjdoub,†,§ Mariana Tozlovanu,† Annie Pfohl-Leszkowicz,*,† Christine Frenette,‡,§
Robert J. Paugh,‡ and Richard A. Manderville*,‡
†Laboratory Chemical Engineering, Department Bioprocess & Microbial System, UMR CNRS/INPT/UPS 5503, ENSA Toulouse,
France
‡Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
S
* Supporting Information
ABSTRACT: Ochratoxin A (OTA) is a fungal toxin that is
classified as a possible human carcinogen based on sufficient
evidence for carcinogenicity in animal studies. The toxin is known
to promote oxidative DNA damage through production of
reactive oxygen species (ROS). The toxin also generates covalent
DNA adducts, and it has been difficult to separate the biological
effects caused by DNA adduction from that of ROS generation. In
the current study, we have derived structure−activity relationships
(SAR) for the role of the C5 substituent of OTA (C5−X = Cl) by
first comparing the ability of OTA, OTBr (C5−X = Br), OTB
(C5−X = H), and OTHQ (C5−X = OH) to photochemically
react with GSH and 2′-deoxyguanosine (dG). OTA, OTBr, and OTHQ react covalently with GSH and dG following
photoirradiation, while the nonchlorinated OTB does not react photochemically with GSH and dG. These findings correlate with
their ability to generate covalent DNA adducts (direct genotoxicity) in human bronchial epithelial cells (WI26) and human
kidney (HK2) cells, as evidenced by the 32P-postlabeling technique. OTB lacks direct genotoxicity, while OTA, OTBr, and
OTHQ act as direct genotoxins. In contrast, their cytotoxicity in opossum kidney epithelial cells (OK) and WI26 cells did not
show a correlation with photoreactivity. In OK and WI26 cells, OTA, OTBr, and OTB are cytotoxic, while the hydroquinone
OTHQ failed to exhibit cytotoxicity. Overall, our data show that the C5−Cl atom of OTA is critical for direct genotoxicity but
plays a lesser role in OTA-mediated cytotoxicity. These SARs suggest different mechanisms of action (MOA) for OTA
genotoxicity and cytotoxicity and are consistent with recent findings showing OTA mutagenicity to stem from direct
genotoxicity, while cytotoxicity is derived from oxidative DNA damage.
tumor incidences in humans exposed to much lower doses. For
nongenotoxic chemicals, thresholds based on the induction of
cytotoxicity may be defined, and TDIs can be derived using the
safety factor methodology.9,10 The TDI established for OTA by
the Joint FAO/WHO Expert Committee on Food Additives
uses nephrotoxic effects in pigs and has been set at ∼14.28 ng/
kg bw/day (100 ng/kg bw/week).11 Health Canada has
recently proposed a more stringent TDI of 4 ng/kg bw/day
that considers tumor formation by OTA,5 given that the
characteristics of OTA-induced tumors correspond to those
typically observed for genotoxic chemicals.12,13
INTRODUCTION
■
Ochratoxin A (N-{[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-
3,4-dihydro-1H-isochromen-7-yl]carbonyl}-L-phenylalanine,
OTA, Figure 1) is a mycotoxin produced by several fungi of
Aspergillus and Penicillium species that is a common
contaminant of cereals and agricultural products.1−5 OTA is
nephrotoxic and is a potent renal carcinogen in rats,6 where it
acts as a complete carcinogen (initiator and promoter activity)
rather than as a promoter alone.4,5 OTA is also a potent renal
carcinogen in chicks,7 and the International Agency for
Research on Cancer (IARC) has classified OTA as group 2B
carcinogen (possible human carcinogen) based on sufficient
evidence for carcinogenicity in animal studies.8
Given that OTA is a possible human carcinogen present in
human foodstuffs, government agencies propose tolerable daily
intakes (TDIs) to manage the risk from OTA exposure. Here,
the mechanism of action (MOA) by the toxin has a major
influence on the methods applied for risk assessment.9,10 For
carcinogens whose MOA involves genotoxicity by covalent
DNA adduction (direct mode of genotoxicity), the tumor
incidences observed in animals are used to predict potential
The MOA for OTA has been a controversial issue.14,15 In
mammalian cells, OTA is known to facilitate oxidative DNA
damage16−18 that causes cytotoxicity,17,19,20 and this MOA has
been proposed to play an important role in OTA
carcinogenicity.19,21 This implies that OTA is not a direct
genotoxic carcinogen and that a threshold model can be used
for OTA risk assessment.21 However, using the 32P-postlabeling
Received: September 23, 2011
Published: November 29, 2011
© 2011 American Chemical Society
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diastereomers [3(R/S), Figure 1]. Stock solutions of ochratoxins in
aqueous 50 mM phosphate buffer, pH 7.4, were prepared as outlined
previously.33,34 Glutathione (GSH) was purchased from Sigma-Aldrich
(Oakville, ON), and dG was from ChemGenes (Willmington, MA).
Purchased were the following enzymes: Proteinase K (used as
received), RNase A, RNase T1 (boiled for 10 min at 100 °C to
destroy DNases), and microccocal nuclease (dialyzed against
deionized water) were from Sigma (Saint Quentin Fallavier, France);
spleen phosphodiesterase (centrifuged before use) was from
Calbiochem (VWR, France); and nuclease P1 (NP1) and T4
polynucleotide kinase were from Roche diagnostics (Meylan, France).
[γ 32P-ATP] (444 Tbq/mmol, 6000 Ci/mmol) was from Amersham
(Les Ullis, France); Dulbecco's Eagle's minimum essential medium
(D-EMEM) and Roswell Park Memorial Institute medium (RPMI)
were prepared with Gibco products (Cergy Pontoise, France);
phosphate saline buffer, trypsine, fetal calf serum, streptomycin, and
penicillin were from Life Technologies (Cergy-Pontoise, France);
rotiphenol (phenol saturated with TRIS-HCl, pH 8) was from
Rothsichel (Lauterbourg, France); cellulose MN 301 was from
Macherey Nagel (Duren, Germany); polyethyleneimine (PEI) was
̈
from Corcat (Virginia Chemicals, Portsmouth, VA); Whatman no. 1
paper (ref 6130932) was from VWR (France), and PEI/cellulose TLC
plates used for 32P-postlabeling analyses were prepared in the
laboratory at Toulouse, France. All HPLC solvents were of
chromatography grade. Water used for buffers, and spectroscopic
solutions was obtained from a Milli-Q filtration system (18.2 MΩ).
Methods. Product isolations were carried out using an Agilent
1200 series HPLC equipped with an autosampler, autocollector,
fluorescence, and diode array detector. Separations were performed
using an Agilent C-18 5 μ 150 mm × 4.6 mm column with a flow rate
of 0.75 mL/min using the following mobile phase: 70:30 (0.1% formic
acid in H2O−0.1% formic acid in ACN) to 25:75 in 17 min by a linear
gradient. Mass spectra were obtained at the Biological Mass
Spectrometry Facility (BMSF) at the University of Guelph using an
Agilent 1100 LC-MSD with a single quadrupole detector using
electrospray negative ionization (ESI−) with a capillary voltage of 3500
V. Data were acquired over the m/z range of 50−1000 under normal
scan resolution (13 000 m/z per s). pH measurements were taken at
room temperature with an Accumet 910 pH meter and an Accumet
pH Combination Electrode with stirring. UV−visible absorption
spectra were recorded on a Cary 300-Bio UV−visible spectropho-
tometer equipped with a Peltier block-heating unit and temperature
controller. Standard 1 cm light path quartz glass cells from Hellma
GmbH & Co were used. All UV−vis spectra were recorded with
baseline/background correction.
Photoreactions. Reaction mixtures (2 mL total volume) of 1 mM
ochratoxins (OTA, OTBr, OTB, or OTHQ) in the absence and
presence of 15 molar equiv of GSH or 40 molar equiv of dG were
prepared in 50 mM phosphate buffer, pH 7.4. The reaction mixtures
were irradiated at 350 nm for various lengths of time (2−30 min)
using a Rayonet Chamber Reactor, model RPR-200. The 350 nm light
source provides an irradiance density of 0.01 Mw/cm2/nm in the 325−
375 nm region, which allows selective irradiation of the phenolic ring
system of the ochratoxins. Aliquots (100 μL) were removed and
analyzed by HPLC. For the photodecomposition studies of the
ochratoxins, the starting materials (OTA, OTB, OTBr, and OTHQ)
were used to construct five-point HPLC calibration curves with UV
detection to quantify levels following photoirradiation at 350 nm. For
the diastereomers of OTB, OTBr, and OTHQ, the combined peak
integrals of the two peaks were utilized. Plotted intensity values have
∼5% error determined from three replicate HPLC injections at each
irradiation time point (2−30 min). Peaks of interest were also
collected, concentrated, and characterized by ESI−-MS.
Figure 1. Chemical structures of OTA and OTA analogues.
method, OTA has also been shown to facilitate DNA adduct
formation,22−26 and recent LC-MS/MS data support the
presence of the N-{[(3R,S)-8-hydroxy-3-methyl-1-oxo-3,4-
dihydro-1H-isochromen-7-yl]carbonyl}-L-phenylalanine
(OTB)-2′-deoxyguanosine (dG) adduct (Figure 1) in rat
kidney DNA samples.27 Hibi and co-workers have also
demonstrated the in vivo mutagenicity of OTA in male rat
kidney.28 Deletion mutations were observed in the DNA of the
target tissue (outer medulla) that were not caused by oxidative
DNA damage, suggesting that this indirect MOA does not
contribute to induction of deletion mutations nor renal
carcinogenesis following OTA exposure of rats.28 These new
findings27,28 suggest the possible involvement of a direct
genotoxic mechanism in OTA-mediated renal carcinogenesis.
To further address the MOA for OTA cytotoxicity and
genotoxicity (DNA adduction), we have determined the role of
the C5 substituent by comparing the activity of OTA to its
analogues shown in Figure 1 (C5−X = Br, OTBr; C5−X = H,
OTB; and C5−X = OH, OTHQ). Our data show that the C5−
halogen (Br or Cl) is a structural requirement for OTB-dG
formation29 and that its removal to generate OTB abolishes
DNA adduct formation in mammalian cells but does not inhibit
cytotoxicity. Interestingly, the hydroquinone analogue N-
{[(3R,S)-5,8-dihydroxy-3-methyl-1-oxo-3,4-dihydro-1H-iso-
chromen-7-yl]carbonyl}-L-phenylalanine (OTHQ) has been
found to lack cytotoxicity in mammalian cells, despite being
able to react directly with DNA to generate covalent DNA
adducts.30 The structure−activity relationships (SARs) deter-
mined in this study imply different MOAs for OTA-mediated
cytotoxicity and genotoxicity.
EXPERIMENTAL PROCEDURES
Caution: OTA is toxic and mutagenic and should be handled with
proper safety equipment and precautions.
Cell Culture. Opossum kidney epithelial cells (OK; ATCC CRL-
1840), human bronchial epithelial cells (WI26; ATCC CCL-95.1), and
human kidney cells (HK2; CRL-2190) were provided by ATCC
(American Type Culture Collection, Manassas, VA). WI26 cells were
cultured in RPMI, while OK and HK2 were cultured in D-EMEM
medium containing 44 mM NaHCO3, 5% fetal bovine serum (FBS),
2% vitamins, 2% nonessential amino acids, 1% streptomycin (100 μg/
■
Materials. OTA (benzene free, CAS# 303-47-9) was purchased
from Sigma (Saint Quentin Fallavier, France). Synthetic samples of
bromo-ochratoxin (OTBr),31 OTB,31 and OTHQ32 were available in
the laboratory at Guelph, Canada, and were used as mixtures of
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mL, Gibco), and 1% penicillin (100 U/mL, Gibco) for 48 h at 37 °C
under 5% CO2. After trypsin digestion, the cells were resuspended in
culture medium to obtain 1× 106 cells/mL.
GSH. For OTA (Figure 3A), the new product peaks mainly
stem from substitution of the C5−Cl atom with an H atom
(OTB), OH (OTHQ), or GSH (OTB-GSH). The exception is
OTHQ-GSH (Figure 1) that forms by nucleophilic attachment
of GSH to the unsubstituted position of the quinone
electrophile OTQ that is produced from photoexcitation of
OTA or OTHQ.38 The OTHQ-GSH conjugate has been fully
characterized previously using NMR spectroscopy and
possesses a phenolic absorption at λmax = 352 nm, and analysis
by ESI−-MS shows [M − H]− = 689 with a prominent fragment
ion at m/z 416 from β-elimination of benzoquinol-SH;38 an
established fragmentation of S-peptide-benzoquinone ad-
ducts.39 In this study, its identity was confirmed by UV−vis
and ESI−-MS analysis (Supporting Information).
The major conjugate produced from the photoreaction of
OTA with GSH is OTB-GSH. This species has a phenolic
absorption at λmax = 331 nm and a parent ion at [M − H]− =
673; 16 mass units less than OTHQ-GSH. The conjugate also
undergoes β-elimination of benzoquinol-SH to afford a
fragment ion at m/z 400, which is 32 mass units heavier than
OTB ([M − H]− = 368). These UV−vis and ESI−-MS
characteristics are consistent with the OTB-GSH structure
shown in Figure 1 with replacement of the C5−Cl atom by the
S atom of GSH. The corresponding cysteine (CySH) conjugate
of OTA (OTB-CySH) has been previously characterized, and it
also shows a prominent fragment ion at m/z 400 and a phenolic
absorption at λmax = 330 nm.33
The photoreaction of OTBr with GSH (Figure 3B) showed
the same products as the OTA reaction, despite OTBr being
more photoreactive than OTA and undergoing complete
photodegradation during the 15 min of irradiation time of
the experiment. OTBr was used as a mixture of diastereomers
[3(R/S), Figure 1], and so, two separable peaks were observed
for the photoproducts OTB and OTHQ (Figure 3B). However,
upon attachment of GSH to generate OTB-GSH and OTHQ-
GSH, the stereoisomers were not separable by HPLC using the
conditions for Figure 3. In contrast to OTA and OTBr, OTB
failed to react with GSH upon photoirradiation (Figure 3C),
while OTHQ produced OTHQ-GSH (Figure 3D) from
reaction of GSH with the quinone electrophile OTQ that is
generated from irradiation of OTHQ.38
32P-Postlabeling. WI26 or HK2 cells were treated with 1 μM
OTBr or OTB for 1.5, 6, or 24 h. DNA isolation and extraction were
carried out as fully described in Tozlovanu et al.30 The method used
for 32P-postlabeling was that initially described by Reddy and
Randerath,35 with minor modifications outlined in Faucet et al.26
Cytotoxicity. For cytotoxicity measurements, OK, WI26, or HK2
cells were incubated for 48 h in the presence of 0.5, 1, 2.5, 5, 10, or 25
μM OTA, OTBr, OTB, or OTHQ. Cell viability was assessed using
the MTT assay, as described previously.36 The assay is based on the
capacity of living cells to convert the tetrazolium salt into formazan,
which absorbs at 570 nm and is positively correlated with cell survival.
After one night for the cell adhesion at 37 °C, 10 μL of each
ochratoxin was applied for 48 h at 37 °C. At the end of the incubation,
15 μL of MTT (5 mg/mL, Sigma-Aldrich) in phosphate-buffered
saline (PBS) was added to the 96-well plates for 4 h at 37 °C. The
absorbance at 570 nm was measured with a 96-well plate reader. The
results were measured in six independent experiments and were
expressed as the percentage of viability (%) with respect to the control
(solvent treated cells, 0.1 M HNaCO3, pH 7.4, 1% DMSO), according
to the following formula: [(absorbance treated cells − absorbance
blank)/(absorbance control cells − absorbance blank)] × 100.
Statistical analysis was done using the software SPSS 13.
RESULTS
■
Photoreactivity. We have previously demonstrated that
OTA is photoreactive and that the products from the
photoreaction provide insight into species that may participate
in OTA-mediated DNA damage.34,37 Figure 2 shows the
These differences in photoreactivity for the ochratoxins were
also evident from their reactions with dG.40 Both OTA and
OTBr react with dG to generate the C-linked OTB-dG adduct
(Figure 1) that has been previously characterized by NMR
spectroscopy29 and recently shown to be present in samples of
rat kidney DNA.27 OTB failed to react with dG upon
irradiation, while OTHQ produced a trace amount of an
adduct designated as OTHQ-dG that does not comigrate with
Figure 2. HPLC analysis of the photodegradation of ochratoxins (1
mM) in 50 mM phosphate buffer, pH 7.4, over a span of 30 min of
irradiation at 350 nm. Plotted values have ∼5% error determined from
three replicate HPLC injections at each time point.
relative rates of photodecomposition of the ochratoxins (OTA,
OTBr, OTB, and OTHQ, Figure 1) following irradiation at 350
nm. OTA decomposes relatively slowly upon UV irradiation in
aqueous solutions,34 and following 30 min of irradiation time,
∼50% of the toxin was still present, as evidenced by HPLC
analysis. In contrast, the bromo-analogue OTBr had completely
photodecomposed following ∼10 min of irradiation time, while
OTB and OTHQ (∼80% still present after 30 min) were more
resistant than OTA to photodecomposition.
Further insight into photoreactivity differences for the
ochratoxins was obtained by carrying out the photoreactions
in the presence of excess GSH. Figure 3 shows HPLC
chromatograms for the ochratoxins (1 mM) following photo-
irradiation (15 min) at 350 nm in the presence of 15 equiv of
OTB-dG and exhibits a very different UV spectrum with λmax
=
376 nm.40 On the basis of the mass of the adduct that had a
parent ion at [M − H]− = 631, which is two mass units less
than OTB-dG, OTHQ-dG was ascribed to a benzetheno type
adduct from reaction of dG with the quinone electrophile
OTQ.40 Representative HPLC traces from the photoreaction
(15 min) of the ochratoxins (1 mM) with excess (40 equiv) dG
is shown in Supporting Information (Figure S1). In Table 1 are
given retention times (RT), λmax values, [M − H]− ions, and
fragment ions for the ochratoxins, GSH conjugates, and dG
adducts.
DNA Adduction. Further insight into the role of the C5−X
substituent in OTA reactivity was derived from study of DNA
adduction by OTB and OTBr in human bronchial epithelial
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Figure 3. HPLC chromatograms [70:30 (0.1% formic acid in H2O−0.1% formic acid in ACN) to 25:75 in 17 min by a linear gradient] following 15
min of irradiation (350 nm) of 1 mM (A) OTA, (B) OTBr, (C) OTB, and (D) OTHQ in 50 mM phosphate buffer, pH 7.4, containing 15 equiv of
GSH. OTHQ-GSH has been previously characterized,38 while OTB-GSH is analogous to the previously characterized OTB-CySH conjugate.33.
Table 1. Spectral Data for Photoreactions of Ochratoxins
a
with GSH and dG
b
c
−
species
OTA
RT (min)
13.3
λmax
[M − H]
fragment ions (m/z)
333
332
320
350
331
352
326
376
402
446
368
384
673
689
633
631
358, 314
OTBr
13.6, 14.1
11.6, 11.9
10.9, 11.0
6.8
402, 358
OTB
324, 280
OTHQ
340, 296
OTB-GSH
OTHQ-GSH
OTB-dG
OTHQ-dG
672, 544, 400
688, 560, 416
517, 473, 429
515
6.2
7.5
6.9, 7.0
a
Photoreactions of ochratoxins (1 mM) in the presence of 15 molar
Figure 4. TLC maps of 32P-labeled DNA adducts obtained following
treatment of WI26 with 1 μM OTB or OTBr for 1.5, 6, and 24 h.
equiv of GSH or 40 molar equiv of dG were carried out in 50 mM
phosphate buffer, pH 7.4, for 15 min with irradiation at 350 nm using
a Rayonet Chamber Reactor, model RPR-200. Retention time, HPLC
b
performed using an Agilent C-18 5 μ 150 mm × 4.6 mm column with
a flow rate of 0.75 mL/min using the following mobile phase: 70:30
(0.1% formic acid in H2O−0.1% formic acid in ACN) to 25:75 in 17
c
min by a linear gradient. Phenolic ring absorbance in nm.
(WI26) and human kidney (HK2) cell lines. These cell lines
were used previously to study DNA adduct formation by OTA
and OTHQ.30 For both OTA and OTHQ, low levels of DNA
adducts (8−10 adducts/109 nucleotides) were detected by 32P-
postlabeling following treatment of WI26 and HK2 cells with 1
μM toxin for various lengths of time (2, 7, and 24 h).30 Figure 4
shows the adduct pattern obtained following OTB (1 μM) or
OTBr (1 μM) treatment of WI26 cells, while the corresponding
adduct pattern in HK2 cells is shown in Figure 5. For the
nonchlorinated OTB (C5−X = H, Figure 1) analogue, no
adduct spots were detected in WI26 (Figure 4) or HK2 (Figure
5) cells. In contrast, OTBr was found to form DNA adducts in
WI26 (Figure 4) and HK2 (Figure 5) cells. Faint adduct spots
were detected, especially after 6 h of incubation, as indicated by
the arrows in Figures 4 and 5.
Figure 5. TLC maps of 32P-labeled DNA adducts obtained following
treatment of HK2 with 1 μM OTB or OTBr for 1.5, 6, and 24 h.
exposure (Figure 6). The data presented in Figure 6 show the
natural OTA to be the most cytotoxic of all analogues, showing
cytotoxicity in all cell lines in the high-dose exposure regime
(10, 25 μM). OK cells were the most susceptible to OTA-
mediated cytotoxicity (Figure 6A) followed by WI26 (Figure
6B) and then HK2 (Figure 6C) cells that only showed
cytotoxicity at 10 and 25 μM OTA exposure. As reported
previously, OTB is also cytotoxic to OK cells but not to the
same degree as OTA (Figure 6A).36 In this cell line, OTBr
Cytotoxicity. The MTT assay was performed to determine
the cytotoxicity of the ochratoxins (OTA, OTBr, OTB, and
OTHQ) in OK, WI26, and HK2 cells. For these assays, the
cells were treated with increasing amounts of ochratoxin (0.05−
25 μM), and the survival rate was measured after 48 h of
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4-ClPhOH undergoes photoionization to yield solvated
electrons (eaq) and the phenolic radical.44 For OTA with a
phenolic pKa ∼ 7,45 the transient absorption spectrum
immediately after photoexcitation is dominated by the
absorption spectrum of eaq that reacts with OTA.34 Thus, as
outlined in Scheme 1, this has led to the speculation that the
Scheme 1. Proposed Pathways of OTA Photoreactions
photochemistry of the OTA phenolate leads to reductive
dehalogenation of the toxin with homolytic cleavage of the C−
Cl bond to generate Cl− and an aryl radical in addition to the
phenoxyl radical that is formed in the initial photoionization
process.34 The protonated form of OTA is still expected to
undergo C−Cl bond heterolysis to generate a carbene
intermediate,34 as noted for 4-ClPhOH.43,44
Figure 6. Viability of (A) OK, (B) W126, and (C) HK2 cells after 48 h
of exposure with 0.5, 1, 2.5, 5, 10, and 25 μM OTA, OTBr, OTB, and
OTHQ obtained using the MTT assay. Mean values (SD) were
obtained in six independent experiments.
shows similar cytotoxicity to OTA at high doses, while the
hydroquinone OTHQ failed to show cytotoxicity. In W126
cells, high doses (25 μM) of OTB and OTBr were as cytotoxic
as OTA (Figure 6B), while again OTHQ failed to show
cytotoxicity. In HK2 cells (Figure 6C), only OTA showed
measurable cytotoxicity.
The SARs for the photodecomposition of the ochratoxins
presented in Figure 2 are consistent with the hypothesis that
cleavage of the C5−X bond initiates the photodecomposition
process.34 The energy required for homolytic bond cleavage for
aromatic compounds (C6H5−X) bearing different X substitu-
ents has been tabulated by Blanksby and Ellison in kcal mol−1:
C6H5−H (112.9 0.5), C6H5−OH (112.4 0.6), C6H5−Cl
(97.1 0.6), and C6H5−Br (84 1).46 Clearly, the C−Br bond
is the weakest, followed by C−Cl and then C−OH and C−H,
which are of equal stability. Both OTB and OTHQ were
resistant to photodecomposition (Figure 2) because they lack a
suitable leaving group to undergo C5−X bond cleavage either
through heterolysis or homolytic cleavage. In contrast, OTBr is
more photoreactive than OTA because the C5−Br bond is
more labile than the corresponding C5−Cl bond in OTA.
Cleavage of the C5−X bond in OTA also provides a rationale
for the products generated in the presence of GSH (Figure 3)
or dG (Figure S1). For OTA, irradiation of the protonated
species is expected to generate the carbene intermediate.
Reaction of the carbene with an H-donor generates OTB, while
reaction with O2 gives the benzoquinone OTQ and reaction
with H2O generates the hydroquinone OTHQ (Scheme 1).34,37
DISCUSSION
■
To determine SAR for the ochratoxins shown in Figure 1, we
first assessed their photoreactivity in the absence (Figure 2) and
presence of GSH (Figure 3) or dG (Figure S1). Our choice of
GSH and dG as representative biological nucleophiles stemmed
from our previous studies showing that the parent OTA reacts
covalently with GSH to generate OTHQ-GSH.38 Experiments
carried out by Obrecht-Pflumio and Dirheimer also suggested
that OTA forms guanine-specific DNA adducts,41,42 and we
have shown that OTA reacts photochemically with dG to form
OTB-dG.29 Thus, the impact of the C5 substituent on reactivity
toward GSH and dG could be determined. Our previous
studies have also shown that the product distribution from
OTA photochemistry is analogous to the photoproducts of 4-
chlorophenol (4-ClPhOH).34,37 Heterolysis of the C−Cl bond
to form a carbene intermediate is well documented for the
protonated form of 4-ClPhOH.43,44 However, the phenolate of
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The stable OTB and OTHQ products were detected (Figures 3
and S1), while OTQ is too reactive for detection in aqueous
media at physiological pH.32 The quinone electrophile OTQ is
also generated from photoirradiation of OTHQ,38 and it reacts
covalently with GSH to generate OTHQ-GSH (Figure 3)38
and with dG to form OTHQ-dG (Figure S1 and Table 1).40
Direct reaction of the OTA-carbene with GSH would give rise
to OTB-GSH, while reaction with dG would furnish OTB-dG.
These species may also be generated from the aryl radical
intermediate formed by reduction of OTA by eaq.34 Aryl radicals
are known to react covalently with dG to generate C8-dG
W126 and HK2 cells. These results suggest that OTB lacks
direct genotoxicity, while OTA, OTBr, and OTHQ can be
viewed as direct genotoxins, ochratoxins capable of forming
covalent DNA adducts.
For induction of OTA cytotoxicity, recent studies have
shown a close correlation between the onset of oxidative DNA
damage.17,19,20 These studies have employed the comet assay
(single cell gel electrophoresis) to indirectly detect oxidative
DNA damage mediated by OTA with the aid of the repair
enzyme formamido-pyrimidine-DNA-glycosylase (Fpg). The
enzyme Fpg cleaves DNA at a variety of oxidized guanine
adducts, producing DNA fragmentation. The onset of OTA-
induced cytotoxicity in cell cultures is highly correlated with the
induction of enhanced DNA breakage by the comet assay
following Fpg treatment.17,19,20 This suggests that oxidative
DNA damage mediated by ROS is responsible for the observed
cytotoxicity, given that OTA has been shown to stimulate ROS
production in bacteria, rat liver microsomes, mitochondria, and
rat hepatocytes.49,50 In HK2 cells, ROS generated by OTA
treatment were determined by using the fluorescent probe
dihydrodichlorofluorescein diacetate (H2DCF-DA).19 Follow-
ing 6 h of OTA exposure, an increased level of ROS was
observed. Pretreatment of the cells with N-acetylcysteine
(NAC) showed a significant decrease in ROS levels, resulting
in a significant increase in cell viability at all concentrations of
OTA tested.19 These studies have firmly established that
oxidative DNA damage causes OTA-mediated cytotoxic-
ity.17,19,20
Previous studies carried out in cultured cells have also shown
cells to display a range of sensitivities to OTA-mediated
cytotoxicity.19,20 In our experiments, the viability of OK cells
was strongly decreased by OTA concentrations >2.5 μM,
whereas WI26 and HK2 cells were more resistant (Figure 6). In
OK cells, the decrease in cell number mediated by OTA is due
to apoptosis and necrosis.51 OK cells lack sulfotransferase
enzymes.36 In contrast, WI26 and HK2 cells are of human
origin and contain all metabolic capacity. Thus, the heightened
sensitivity of OK cells to OTA-mediated cytotoxicity may be
due to the lack of OTA sulfoconjugation.
Interestingly, a correlation between photoreactivity and
cytotoxicity was not observed. In OK and WI26 cells, OTBr
and OTB displayed cytotoxicity at a level similar to OTA at 25
μM, while the hydroquinone OTHQ failed to exhibit
cytotoxicity (Figure 6). In HK2 cells, all of the synthetic
samples (OTBr, OTB, and OTHQ) failed to exhibit
cytotoxicity under the conditions employed. One factor that
can play a role in cytotoxicity is stereochemistry, and the
synthetic samples of OTBr, OTB, and OTHQ were used as
mixtures of diastereomers [3(R/S), Figure 1], while the natural
product OTA was a stereochemically pure sample. However,
recent results show that the L-configuration of the phenyl-
alanine moiety of OTA is important for cytotoxicity, while the
stereocenter at the dihydroisocoumarine structure is of less
importance,52 suggesting that use of the diastereomer mixture
for OTHQ did not play a dramatic role in its inability to induce
cytotoxicity.
Oxidative DNA damage and hence cytotoxicity mediated by
OTA are expected to result from direct redox cycling reactions
involving OTA, coupled to indirect mechanisms resulting from
reduction of cellular antioxidant defenses, which is expected to
amplify the oxidative-mediated effects of OTA.21 OTA contains
a phenolic ring system, and a potential mechanism for oxidative
stress mediated by phenols has been described as futile thiol
adducts,47 while nucleophilic attachment of GS− by an SRN
process48 would yield OTB-GSH.
1
Given that product formation from photoirradiation of OTA
stems from cleavage of the C5−Cl bond (Scheme 1), it is not
surprising that the same products were generated from the
more reactive bromo-analogue OTBr (Figures 3 and S1).
However, the nonhalogenated analogue OTB failed to react
with GSH and dG following photoirradiation. For simple
phenols, photoionization to generate phenoxyl radicals and eaq
is the primary photoprocess, especially when the phenols are
deprotonated.43 The C5 substituent of the ochratoxins
influences the phenolic pKa, and a pKa value ∼8 has been
estimated for OTB.45 Thus, at pH 7.4 (conditions of the
photoreactions), sufficient OTB phenolate is present that the
photoreaction is expected to generate the OTB phenoxyl
radical and eaq. Because OTB lacks a leaving group, it cannot
interact with eaq to generate an aryl radical, nor will it undergo
C5−H heterolysis to yield a carbene intermediate. This leaves
the OTB phenoxyl radical as the reactive species, and it is
expected to be quenched by GSH through an H-atom
abstraction process to regenerate OTB and produce the thiyl
radical of GSH (GS•).15 The OTB phenoxyl radical is also
unreactive toward covalent attachment to dG, given the lack of
product formation from the photoreaction (Figure S1C). For
the hydroquinone derivative OTHQ, which also has a phenolic
pKa ∼ 8,32 the photoreaction will generate a semiquinone
radical that can undergo a further oxidative process to generate
the quinone electrophile OTQ.32 The quinone electrophile
OTQ reacts covalently with GSH to generate OTHQ-GSH
(Figure 3)38 and with dG (Figure S1D)40 and DNA in cell
cultures.30
The photochemistry of the ochratoxins highlights the impact
of the C5 substituent. Photoirradiation of OTA generates
electrophiles capable of reacting covalently with GSH (Figure
3)38 and dG (Figure S1).29 Not surprisingly, the C5−Cl atom
of OTA can be replaced with a Br atom (OTBr) without
change in chemical reactivity. However, replacement of the
C5−Cl with an OH group (OTHQ) alters the photochemistry
to one now dominated by the quinone electrophile OTQ where
the photoproducts stem from Michael addition instead of
attachment to the C5 position. Complete removal of the C5−
Cl atom to yield OTB removes the production of electrophiles
capable of reacting covalently with GSH (Figure 3C) or dG
(Figure S1C).
A correlation between the photochemistry of the ochratoxins
and their ability to form DNA adducts in cell cultures was
observed. The analogues that can react with dG following
photoirradiation (OTA, OTBr, and OTHQ, Figure S1) also
react with DNA in cell cultures to generate covalent DNA
adducts, as evidenced by 32P-postlabeling. OTB fails to show
DNA adduct formation under conditions where OTA,30 OTBr
(Figures 4 and 5), and OTHQ30 generate adduct spots in both
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Chemical Research in Toxicology
Article
pumping.53,54 This generates ROS, thiol oxidation, and
antioxidant depletion, and OTA is known to reduce GSH
levels in mammalian cell lines17 and stimulate ROS
production.19,49,50 Such direct redox cycling pathways are
initiated by production of the phenoxyl radical,53,54 and
electrochemical measurements show that OTA undergoes a
1e oxidative process in aqueous media to form the phenoxyl
radical at E1/2 = 1.04 V vs NHE.55 Peroxidase enzymes are
deemed important for OTA bioactivation,56,57 and peroxidases
are well-known to catalyze the conversion of phenols into
phenoxyl radicals.58,59
formation from that of ROS generation.64 An accepted
mechanism for quinone-mediated oxidative stress involves
reduction of the quinone by reductases to a semiquinone
radical, which reduces oxygen to O2•− and reforms the
quinone.61−63 This futile redox cycling forms cytotoxic levels
of H2O2, and GSSG is retained by the cell and causes cytotoxic
mixed protein disulfide formation.63 Our studies on the
OTHQ/OTQ redox couple show OTQ to be an arylating
quinone with the C6 position available for Michael adduct
formation.38 We have also found OTQ to be highly reactive,
undergoing decomposition in aqueous media to form a polar
diacid derivative.32 Thus, as outlined in Scheme 2B, it is
expected that once OTQ is formed it will react immediately
with H2O and biological nucleophiles, making its formation
from the semiquinone radical essentially irreversible. This
concept has been demonstrated for other arylating quinones;
that is, quinones with higher chemical reactivity display lower
cytotoxicity due to more rapid detoxification by Michael
addition reactions.64 This concept provides a rationale for lack
of cytotoxicity displayed by OTHQ (Figure 6).
The pathways in Scheme 2 permit a rationale for the role of
the C5−X substituent in OTA-mediated cytotoxicity (Figure
Scheme 2. Proposed Pathways for (A) ROS Generation by
OTA, OTBr, and OTB and (B) Lack of ROS Generation by
OTHQ
The C5−X substituent of OTA also plays a role in OTA
metabolism.65,66 OTA is cleaved by carboxypeptidase A to the
nontoxic ochratoxin α (OTα) and phenylalanine (Phe), leading
to detoxification. OTB is cleaved much quicker by this
enzyme,65,66 while OTBr undergoes hydrolysis at a rate
comparable to OTA.66 That OTB undergoes hydrolysis by
carboxypeptidase at a quicker rate than OTA has been evoked
as a rationale for its lower toxicity.67 In these studies,
administration of OTB to male rats failed to induce major
histopathological features in the kidneys (karyomegaly and
apoptosis of tubular epithelial cells in the outer stripe of the
outer medulla) that is characteristic of OTA-mediated
nephrotoxicity.6,28 Because differences in toxicokinetics be-
tween OTA and OTB were viewed to be responsible for their
different potential to induce nephrotoxicity, the cytotoxicities of
OTA and OTB were compared in a cell system where
toxicokinetics play a minor role.67 The cytotoxicity of OTA was
more pronounced only at low concentrations; at higher
concentrations, both OTA and OTB induced a similar
reduction in cell viability (i.e., Figure 6B), suggesting that
these two analogues are equally cytotoxic.67 Additional
experiments carried out in rats also showed that OTB generates
a similar degree of DNA breakage at the same dose of OTA,68
and that the C5−Cl atom of OTA is not a structural
requirement for OTA-mediated cytotoxicity.67,68
Our findings show that the C5−Cl atom is critical for direct
genotoxicity mediated by OTA but plays a lesser role in OTA-
mediated cytotoxicity. Thus, cytotoxicity is not a true measure
of OTA reactivity and overall toxicity to cells. The new findings
on OTA mutagenicity favor direct genotoxicity and rule out
oxidative DNA damage as a contributor to induction of
deletion mutations or renal carcinogenesis.28 However,
oxidative DNA damage is regarded as the contributor to the
induction of OTA-mediated cytotoxicity.17,19,20 Our SAR for
OTA photoreactivity, DNA adduction, and cytotoxicity in
mammalian cells is consistent with different MOA for OTA-
mediated cytotoxicity and direct genotoxicity. These data
support the hypothesis that toxicity and carcinogenicity
induced by OTA are driven by two different MOAs.69
6). Replacement of the C5−Cl atom with Br or H will not
dramatically impact the reactivity of the phenoxyl radical, given
that the phenolates of PhOH, 4-ClPhOH, and 4-BrPhOH have
almost identical 1e oxidative potentials (E° ∼ 0.85 V vs
NHE).60 Thus, for OTA, OTBr, and OTB the redox cycling
pathway in Scheme 2A should take place, and these ochratoxins
should be cytotoxic, as observed in OK and WI26 cells (Figure
6A,B). In contrast, replacement of the C5−Cl atom with OH to
generate OTHQ does have a dramatic impact on the reactivity
of the phenoxyl radical. OTHQ undergoes a 1e oxidative
process in aqueous media to form the phenoxyl radical
(semiquinone radical) at E1/2 = 0.58 V vs NHE.55 Thus, the
semiquinone radical of OTHQ is a much weaker oxidant than
the phenoxyl radicals of OTA, OTBr, and OTB and is less
likely to be reduced by GSH (Scheme 2B). Instead, a second 1e
oxidative process occurs at E1/2 = 0.89 V vs NHE that is
ascribed to oxidation of the semiquinone radical into the
quinone electrophile OTQ (Scheme 2B).55
In general, hydroquinone/quinone redox couples are well-
known to stimulate cytotoxicity,61−63 although it is difficult to
separate the biological effects caused by Michael adduct
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Chemical Research in Toxicology
Article
(8) IARC (1993) Monographs on the Evaluation of Carcinogenic Risks
to Humans. Some Naturally Occurring Substances: Food Items and
Constituents, Heterocyclic Aromatic Amines and Mycotoxins, No. 56,
Ochratoxin A, pp 489−521, International Agency for Research on
Cancer, Lyon.
(9) Bolt, H. M., Foth, H., Hengstler, J. G., and Degen, G. H. (2004)
Carcinogenicity categorization of chemicals-new aspects to be
considered in a European perspective. Toxicol. Lett. 151, 29−41.
(10) Bolt, H. M., and Degen, G. H. (2004) Human carcinogenic risk
evaluation, part II: Contributions of the EUROTOX specialty section
for carcinogenesis. Toxicol. Sci. 81, 3−6.
ASSOCIATED CONTENT
■
S
* Supporting Information
ESI−-MS spectra of GSH and dG adducts derived from
photoirradiation of the ochratoxins and Figure S1 described in
the text. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: leszkowicz@ensat.fr (A.P.-L.) or rmanderv@uoguelph.
ca (R.A.M.).
■
(11) JECFA (2001) Safety Evaluation of Certain Mycotoxins in Food,
Fifty-Sixth Meeting of the Joint FAO/WHO Expert Committee on Food
Additives (JECFA), WHO Food Additives Series, Vol. 47, pp 281−415,
World Health Organization, Geneva.
Author Contributions
§These authors contributed equally to this work.
(12) Kuiper-Goodman, T. (1996) Risk assessment of ochratoxin A:
an update. Food Addit. Contam. 13 (Suppl.), 53−57.
Funding
Support for this research was provided by the European Union
(“Ochratoxin A-risk assessment” QLK1-2001-01614), the
Region Midi-Pyren
(13) Lock, E. A., and Hard, G. C. (2004) Chemically induced renal
tubule tumors in the laboratory rat and mouse: Review of the NCI/
NTP database and categorization of renal carcinogens based on
mechanistic information. Crit. Rev. Toxicol. 34, 211−299.
(14) Turesky, R. J. (2005) Perspective: Ochratoxin A is not a
genotoxic carcinogen. Chem. Res. Toxicol. 18, 1082−1090.
(15) Manderville, R. A. (2005) A case for the genotoxicity of
ochratoxin A by bioactivation and covalent DNA adduction. Chem. Res.
Toxicol. 18, 1091−1097.
́
ee
́
s, the French Ministry of Research, the
center cancer (ARC), Natural Sciences
Association recherche
́
and Engineering Research Council of Canada (NSERC), the
Canadian Foundation for Innovation (CFI), and the Ontario
Innovation Trust Fund (OIT).
ACKNOWLEDGMENTS
We thank Dr. Wojciech Gabryelski (University of Guelph) for
assistance with the mass spectral analysis.
■
(16) Gautier, J.-C., Holzhaeuser, D., Markovic, J., Gremaud, E.,
Schilter, B., and Turesky, R. J. (2001) Oxidative damage and stress
response from ochratoxin A exposure in rats. Free Radical Biol. Med.
30, 1089−1098.
ABBREVIATIONS
■
(17) Kamp, H. G., Eisenbrand, G., Schlatter, J., Wurth, K., and
̈
OTA, N-[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihy-
dro-1H-isochromen-7-yl]carbonyl}-L-phenylalanine; OTBr, N-
{[(3R,S)-5-bromo-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-
isochromen-7-yl]carbonyl}-L-phenylalanine; OTB, N-{[(3R,S)-
8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-isochromen-7-yl]-
carbonyl}-L-phenylalanine; OTHQ, N-{[(3R,S)-5,8-dihydroxy-
3-methyl-1-oxo-3,4-dihydro-1H-isochromen-7-yl]carbonyl}-L-
phenylalanine; SAR, structure−activity relationships; MOA,
mechanism of action; dG, 2′-deoxyguanosine; 4-ClPhOH, 4-
chlorophenol; OK, opossum kidney epithelial cells; WI26,
human bronchial epithelial cells; HK2, human kidney cells; D-
EMEM, Dulbecco's Eagle's minimum essential medium; RPMI,
Roswell Park Memorial Institute medium.
Janzowski, C. (2005) Ochratoxin A: Induction of (oxidative) DNA
damage, cytotoxicity and apoptosis in mammalian cell lines and
primary cells. Toxicology 206, 413−425.
(18) Kamp, H. G., Eisenbrand, G., Janzowski, C., Kiossev, J.,
Latendresse, J. R., Schlatter, J., and Turesky, R. J. (2005) Ochratoxin A
induces oxidative DNA damage in liver and kidney after oral dosing to
rats. Mol. Nutr. Food Res. 49, 1160−1167.
(19) Arbillaga, L., Azqueta, A., Ezpeleta, O., and Lopez de Cerain, A.
(2007) Oxidative DNA damage induced by Ochratoxin A in the HK-2
human kidney cell line: evidence of the relationship with cytotoxicity.
Mutagenesis 22, 35−42.
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A., Khana, Q. M., and Heflich, R. H. (2011) Comparative analysis of
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