Oxidation of ochratoxin A by an Fe-porphyrin system: Model for enzymatic activation and DNA cleavage

Article abstract of DOI:10.1021/tx9901074

Ochratoxin A (OTA, 1) is a fungal toxin that facilitates single-strand DNA cleavage, DNA adduction, and lipid peroxidation when metabolically activated. To model the enzymatic activation of OTA, we have employed the water-soluble iron(III) meso-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS) oxidation system. In its presence, OTA has been found to facilitate single- strand cleavage of supercoiled plasmid DNA through production of reactive oxygen species (ROS) (i.e., the hydroxyl radical, HO·). The reaction of OTA with the FeTPPS oxidation system also generated three hydroxylated products (chlorine atom still attached), which was taken as evidence for production of the known hydroxylated metabolites (2-4) of OTA. This result suggested that the FeTPPS system served as a reasonable model for the enzymatic activation of OTA. When the reaction of OTA with FeTPPS was carried out in the presence of excess hydrogen peroxide (H₂O₂) and sodium ascorbate, a hydroquinone species (OTHQ, 5) was detected in which an OH group has replaced the chlorine atom of OTA. The production of OTHQ (5) was dependent on the presence of the reducing agent, sodium ascorbate, which suggested that the oxidation catalyst furnished the quinone derivative OTQ (6) that was subsequently reduced to OTHQ (5) by ascorbate. Utilizing a synthetic sample of OTHQ (5), the hydroquinone was found to undergo autoxidation with a t( 1/2 )' of 11.1 h at pH 7.4, and to possess a pK(a) value of 8.03 for the phenolic oxygen ortho to the carbonyl groups. Our findings imply that the hydroquinone (OTHQ) and quinone (OTQ) metabolites of OTA have the ability to cause alkylation/redox damage and have allowed us to propose a viable pathway for oxidative damage by OTA.

Full text of DOI:10.1021/tx9901074

1066
Chem. Res. Toxicol. 1999, 12, 1066-1076
Oxid a tion of Och r a toxin A by a n F e-P or p h yr in System :
Mod el for En zym a tic Activa tion a n d DNA Clea va ge
Ivan G. Gillman, T. Nicole Clark, and Richard A. Manderville*
Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486
Received J une 16, 1999
Ochratoxin A (OTA, 1) is a fungal toxin that facilitates single-strand DNA cleavage, DNA
adduction, and lipid peroxidation when metabolically activated. To model the enzymatic
activation of OTA, we have employed the water-soluble iron(III) meso-tetrakis(4-sulfonato-
phenyl)porphyrin (FeTPPS) oxidation system. In its presence, OTA has been found to facilitate
single-strand cleavage of supercoiled plasmid DNA through production of reactive oxygen
species (ROS) (i.e., the hydroxyl radical, HO•). The reaction of OTA with the FeTPPS oxidation
system also generated three hydroxylated products (chlorine atom still attached), which was
taken as evidence for production of the known hydroxylated metabolites (2-4) of OTA. This
result suggested that the FeTPPS system served as a reasonable model for the enzymatic
activation of OTA. When the reaction of OTA with FeTPPS was carried out in the presence of
excess hydrogen peroxide (H2O2) and sodium ascorbate, a hydroquinone species (OTHQ, 5)
was detected in which an OH group has replaced the chlorine atom of OTA. The production of
OTHQ (5) was dependent on the presence of the reducing agent, sodium ascorbate, which
suggested that the oxidation catalyst furnished the quinone derivative OTQ (6) that was
subsequently reduced to OTHQ (5) by ascorbate. Utilizing a synthetic sample of OTHQ (5),
the hydroquinone was found to undergo autoxidation with a t1/2 of 11.1 h at pH 7.4, and to
possess a pKa value of 8.03 for the phenolic oxygen ortho to the carbonyl groups. Our findings
imply that the hydroquinone (OTHQ) and quinone (OTQ) metabolites of OTA have the ability
to cause alkylation/redox damage and have allowed us to propose a viable pathway for oxidative
damage by OTA.
In tr od u ction
Ochratoxin A (OTA,1 1, Figure 1) is a mycotoxin
produced by several species of Aspergillus and Penicil-
lium fungi (1). It colonizes a wide range of foodstuffs and
has been implicated in numerous mycotoxicoses in farm
animals, especially in pigs (2, 3). Of greatest concern to
humans is its implication in a fatal kidney disease called
Balkan endemic nephropathy in which patients suffer
from high incidences of kidney, pelvic, ureter, and urinary
bladder carcinomas (4, 5). In terms of OTA genotoxicity,
the toxin promotes single-strand DNA cleavage in mouse
spleen cells in vitro and in the kidneys, liver, and spleen
of mice in vivo (6, 7). Using the 32P-postlabeling method
(8), OTA has been shown to induce DNA adduct forma-
tion in the kidneys, testicles, liver, spleen (9, 10), and
urinary bladder (11) of mice. Although these results
establish a basis for its genotoxicity, the mechanisms of
OTA-induced DNA damage and carcinogenicity are cur-
rently not known.
* To whom correspondence should be addressed: Department of
Chemistry, Wake Forest University, Winston-Salem, NC 27109-7486.
Telephone: (336) 758-5513. Fax: (336) 758-4656. E-mail:
manderra@wfu.edu.
1
Abbreviations: OTA, ochratoxin A; OTB, ochratoxin B; OTHQ,
ochratoxin hydroquinone; OTQ, ochratoxin quinone; SOD, superoxide
dismutase; TCP, 2,4,6-trichlorophenol; PCP, pentachlorophenol; FeT-
PPS, iron(III) meso-tetrakis(4-sulfonatophenyl)porphyrin; TPPS,
5,10,15,20-tetraphenyl-21H,23H-porphine-P,P′,P′′,P′′′-tetrasulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid; CHES, 2-(cyclohexylamino)-
ethanesulfonic acid; TMEDA, N,N,N′,N′-tetramethylethylenediamine;
LDA, lithium diisopropylamide; DPPA, diphenylphosphoryl azide; TEA,
triethylamine; MeCN, acetonitrile; SCE, saturated calomel electrode.
F igu r e 1. Structure of ochratoxin A (OTA, 1) and its analogues.
Despite this, structure-activity studies on OTA have
shown that the chlorine atom is essential for its geno-
toxicity, as the nonchlorinated derivative ochratoxin B
10.1021/tx9901074 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/01/1999

Model for Oxidative Damage by OTA
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1067
drate (FeSO4‚7H2O), 4-morpholinepropanesulfonic acid (MOPS),
2-(cyclohexylamino)ethanesulfonic acid (CHES), N,N,N′,N′-tet-
ramethylethylenediamine (TMEDA), sec-butyllithium (sec-
BuLi), lithium diisopropylamide (LDA), diethylcarbamyl chlo-
ride, iodomethane, diphenylphosphoryl azide (DPPA), triethyl-
amine (TEA), and nitrosonium tetrafluoroborate (NOBF4) (all
from Aldrich Chemical Co.), L-phenylalanine tert-butyl ester
hydrochloride (ICN), and catalase (Sigma). Other inorganic
reagents and solvents were obtained from Fisher Scientific.
(OTB) is not genotoxic (12). DNA adduct formation is also
effectively inhibited by antioxidants (11), i.e., R-tocoph-
erol (vitamin E) and ascorbic acid, and by the enzymes
catalase and superoxide dismutase (SOD) (13). These
findings suggest that an oxidative pathway, which may
be activated by cytochrome P450 (14) and/or enzymes
with peroxidase activity (11), is responsible for OTA-
induced DNA damage and carcinogenicity. However,
studies aimed at elucidating the nature of OTA oxidation
(15, 16) have shown that OTA is metabolized into
hydroxylated products 2-4 (Figure 1), which are not toxic
(17).
While literature data on the oxidation of OTA are
lacking, we speculated that its deleterious activities could
be rationalized by oxidation of its para chlorophenolic
group to a quinone species. This hypothesis stemmed
from our recent finding that photoirradiation of the
molecule generates the hydroquinone derivative OTHQ
(5, Figure 1) in the presence of sodium ascorbate (18).
This observation suggested that OTHQ may have origi-
nated from the quinone precursor OTQ (6, Figure 1) in
analogy to the photooxidation of other halogenated phe-
nols that yield benzoquinone derivatives (19). Chlorinated
phenols, such as 2,4,6-trichlorophenol (TCP) and pen-
tachlorophenol (PCP), have also been shown to undergo
bioactivation to benzoquinones (20-23). Because qui-
nones can redox cycle, creating oxidative stress, and form
covalent adducts with a variety of cellular macromol-
ecules (24), the oxidation of OTA to OTQ would provide
a rationale for its in vivo genotoxicity and would be
consistent with the importance of the chlorine atom
derived from structure-activity relationships (12).
Meth od s. Elemental analyses were carried out by Atlantic
Microlab Inc. High-resolution mass spectral analyses were
carried out by either the Nebraska Center for Mass Spectrom-
etry (Lincoln, NE) or Mass Consortium (San Diego, CA). 1H
NMR spectra were recorded on either a Varian VXR-200 (200
MHz) or a Bruker AVANCE 300DMX (300 MHz) spectrometer.
Chemical shifts are given in parts per million relative to
tetramethylsilane (TMS), and coupling constants (J ) are re-
ported in hertz. 13C NMR spectra were recorded on a VXR-200
spectrometer operating at 50.3 MHz. Spectra were referenced
to the residual solvent peak. UV spectra were obtained on a
Hewlett-Packard 8452A diode array spectrometer equipped with
a temperature-controlled cell. IR spectra were recorded on a
Mattson 4020 Galaxy FT-IR system. Thin-layer chromatography
was carried out on Analtech 250 µM layer, UV254 silica gel
plates with glass backing, while silica gel chromatography was
performed using Kieselgel 60, 230-400 mesh. Distilled, deion-
ized water from a Milli-Q system was used for all aqueous
solutions and manipulations. Other solvents were purified and
dried according to standard procedures. FeTPPS was prepared
from the reaction of FeSO4‚7H2O with 1 equiv of TPPS (26).
The concentration of the FeTPPS solution was quantified by
UV-vis and was ca. 80% FeIII(H2O)(TPPS)3- (ꢀ393 ) 1.5 × 105
M-1 cm-1). Agarose gel electrophoresis was carried out in 40
mM Tris-acetate buffer (pH 8.0), containing 5 mM EDTA.
Agarose gel loading buffer consisted of 40 mM Tris-acetate (pH
8.0), 5 mM EDTA, 40% glycerol, and 0.3% bromophenol blue.
To model enzymatic activation of OTA, we have
employed the water-soluble iron(III) meso-tetrakis(4-
sulfonatophenyl)porphyrin (FeTPPS) complex, which was
recently used by Meunier to study the oxidation of TCP
(22). In its presence, OTA has been found to facilitate
single-strand cleavage of supercoiled plasmid DNA
through production of reactive oxygen species (ROS) (i.e.,
the hydroxyl radical, HO•). Our studies also demonstrate,
for the first time, that an Fe-porphyrin oxidation system
converts OTA into OTHQ (5) in the presence of a suitable
reducing agent (sodium ascorbate). This observation
suggested strongly that the oxidation of OTA generated
the quinone derivative, OTQ (6). This finding, together
with the DNA cleavage results, has allowed us to propose
a model for oxidative damage by OTA. Our model is
consistent with the in vivo results of Malaveille and co-
workers (12) that implicated the chlorine atom as an
important determinant of OTA genotoxicity.
HP LC. Reverse-phase HPLC was performed using a Hitachi
7400 pump and a Waters 991 photodiode array detector set to
acquire data from 220 to 800 nm. Analytical separations were
carried out using a Waters symmetry shield C-18 column (3.9
mm × 150 mm) at a flow rate of 1 mL/min using the following
mobile phase: 20/80 methanol/50 mM phosphate buffer (pH 7.0)
followed by a linear gradient to 80/20 methanol/50 mM phos-
phate buffer (pH 7.0) over the course of 30 min.
LC/MS. Electrospray mass spectra were acquired using a
Micromass Quattro II instrument operating in the negative ion
spray mode (ES-). The system acquired signals over a m/z range
of 200-1000 at a rate of 8 s/scan. Separations were carried out
using a Waters symmetry shield C-18 column (2.1 mm × 150
mm) at a flow rate of 0.1 mL/min using the following mobile
phase: 20/80 methanol/10 mM ammonium bicarbonate followed
by a linear gradient to 80/20 methanol/10 mM ammonium
bicarbonate over the course of 30 min. A postcolumn split was
used which generated a 10 µL flow to the electrospray source
with 90 µL diverted to a Hewlett-Packard photodiode array.
F eTP P S Oxid a tion of OTA. Under one set of conditions,
100 µM OTA in 10 mM phosphate buffer (pH 7.0) was allowed
to react with 0.1% FeTPPS and 6 molar equiv of H2O2 and
sodium ascorbate. The reaction mixture (1 mL total volume) was
incubated at 37 °C for varying lengths of time, and then 20 µL
was injected on an analytical scale reverse-phase HPLC system.
In separate experiments, 100 µM OTA solutions in 10 mM
phosphate buffer (pH 7.0) were reacted with 0.1% FeTPPS and
6 molar equiv of H2O2. After the lengths of time had been varied,
the reactions were quenched by addition of 10 µL of 0.2 M
sodium ascorbate (final concentration of 2 mM). The quenched
solutions were then analyzed by reverse-phase HPLC.
Syn th esis of OTHQ (5). The synthesis of OTHQ (5) was
carried out starting from the N,N-diethylcarbamate 7 (Scheme
1), which was prepared from 4-methoxyphenol (27).
N,N-Dieth yl-2,5-d im eth oxy-3-(d ieth ylca r ba m oyl)ben z-
a m id e (8). To a stirred solution of 7 (2.71 g, 12.14 mmol) in
Exp er im en ta l P r oced u r es
Ca u tion : The work described herein involves the synthesis
and handling of hazardous agents and was therefore conducted
in accordance with the NIH Guidelines for the Laboratory Use
of Chemical Carcinogens.
Ma ter ia ls. Ochratoxin A (OTA, 1, Figure 1) was purchased
from R. R. Marquardt (Department of Animal Sciences, Uni-
versity of Manitoba, Winnipeg, MB) and was used without
further purification. Supercoiled plasmid DNA (form I) was a
gift from F. W. Perrino (Department of Biochemistry, Wake
Forest University). The plasmid was a derivative of pOXO4
containing the dnaQ gene (25). The following enzymes and
reagents were obtained commercially and used without fur-
ther purification: 5,10,15,20-tetraphenyl-21H,23H-porphine-
P,P′,P′′,P′′′-tetrasulfonic acid (TPPS), ferrous sulfate heptahy-

1068 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Sch em e 1. Syn th esis of OTHQ (5), w ith Isola ted Yield s in P a r en th eses
Gillman et al.
tetrahydrofuran (THF, 200 mL) at -78 °C under argon were
added via syringe TMEDA (1.99 mL, 14.57 mmol) and then sec-
BuLi (11.2 mL, 14.57 mmol from a 1.3 M solution in cyclohex-
anes) over the course of 10 min. The resulting solution was
stirred for 1 h at -78 °C, and then via syringe was added
diethylcarbamyl chloride (1.65 g, 12.14 mmol). The resulting
solution was allowed to warm to room temperature and then
stirred for an additional 20 h, at which point the solution was
again cooled to -78 °C and treatment with TMEDA and sec-
BuLi was repeated. After 20 h at room temperature, the
resulting black solution was neutralized with 100 mL of brine
followed by 1 M HCl. The solution was concentrated under
reduced pressure and extracted with CH2Cl2 (3 × 100 mL).
Following the standard workup, concentration of the CH2Cl2
solution afforded a dark yellow oil. The crude product was
dissolved in 200 mL of acetone, and then excess K2CO3 followed
by iodomethane (10 g, 70.45 mmol) was added and the resulting
suspension heated at reflux for 24 h. The suspension was then
concentrated under reduced pressure and extracted with CH2-
Cl2. The organic layer was separated, dried over MgSO4, and
then concentrated under reduced pressure, followed by purifica-
tion by silica gel chromatography (ethyl acetate) to afford 8 as
a light yellow oil: Rf ) 0.27; yield 2.11 g (52%); 1H NMR (CDCl3)
δ 6.68 (s, 2H), 3.70 (s, 6H), 3.6-3.0 (br, 6H), 1.17 (t, 3H, J )
7.14 Hz), 0.982 (t, 3H, J ) 7.15 Hz); 13C NMR (DMSO-d6) δ
169.0, 165.2, 156.0, 146.8, 135.1, 114.8, 111.7, 74.5, 56.1, 28.6,
20.3; IR 2976, 2940, 1634, 1477, 1234, 1052 cm-1. Anal. Calcd
for C18H28O4N2: C, 64.26; H, 8.39; N, 8.33. Found: C, 64.18; H,
8.48; N, 8.37.
at -78 °C under argon was added via syringe LDA (5.10 mL,
10.21 mmol from a 2.0 M solution). The reaction mixture was
stirred for 1 h, and then acetaldehyde (1 mL, 17.9 mmol) was
added via syringe. The solution was allowed to warm to room
temperature, and after 20 h, the mixture was neutralized with
50 mL of brine and 1 M HCl. The solution was concentrated
under reduced pressure and then extracted with CH2Cl2 (3 ×
50 mL). Following the standard workup and concentration under
reduced pressure, the resulting crude oil was dissolved in 100
mL of N2-purged 6 N HCl and the reaction mixture was refluxed
under argon for 6 days. The mixture was cooled and filtered to
yield a yellow powder. Recrystallization from CH2Cl2/MeOH
afforded 10 as a white powder: yield 0.795 g (49%); 1H NMR
(DMSO-d6) δ 9.80 (s, 1H), 7.56 (s, 1H), 4.63 (m, 2H), 3.10 (dd,
2H, J ) 17, 3 Hz), 2.60 (dd, 1H, J ) 11.6, 5.6 Hz), 1.40 (d, 3H,
J ) 6.2 Hz); 13C NMR (DMSO-d6) δ 168.6, 165.8, 155.0, 144.9,
132.8, 122.3, 115.1, 111.1, 74.7, 28.8, 20.4; IR 3284, 3121, 1699,
1618, 1454, 1402, 1221 cm-1. Anal. Calcd for C11H10O6(H2O)1/4
C, 54.44; H, 4.36. Found: C, 54.47; H, 4.32.
:
OTHQ ter t-Bu tyl Ester (11). To a stirred solution of 10 (100
mg, 0.420 mmol) in 10 mL of dry DMSO were added under argon
2.2 equiv of TEA (0.093 g, 0.924 mmol) and DPPA (254 mg, 0.924
mmol) followed by 1 equiv of L-phenylalanine tert-butyl ester
hydrochloride (108.3 mg, 0.420 mmol). The solution was stirred
for 12 h and then concentrated under reduced pressure. The
resulting residue was resuspended in 30 mL of CH2Cl2 and
washed with 1 M phosphate buffer (pH 7.0). The organic layer
was dried over MgSO4, filtered, and concentrated under reduced
pressure to yield an oil that was purified by silica gel chroma-
tography (80/20 CH2Cl2/ethyl acetate) to afford 11 as a light
N ,N -Die t h yl-2,5-d im e t h oxy-3-(d ie t h ylca r b a m oyl)-6-
m eth ylben za m id e (9). To a stirred solution of 8 (2.00 g, 5.95
mmol) in THF at -78 °C under argon were added via syringe
TMEDA (0.97 mL, 7.13 mmol) and then sec-BuLi (5.5 mL, 7.13
mmol from a 1.3 M solution in cyclohexanes) over the course of
10 min. The resulting solution was allowed to stir at -78 °C
for 1 h, and then iodomethane (5 g, 35.23 mmol) was added via
syringe. The solution was allowed to warm to room temperature,
and after 20 h, the reaction mixture was neutralized with 100
mL of brine followed by 1 M HCl. The solution was concentrated
under reduced pressure and extracted with CH2Cl2 (3 × 75 mL).
Following the standard workup and concentration under re-
duced pressure, the resulting oil was purified by silica gel
chromatography (ethyl acetate) to afford 9 as a light yellow oil:
1
yellow oil: Rf ) 0.57; yield 70.7 mg (40%); H NMR (CDCl3) δ
12.03 (s, 1H), 9.80 (s, 1H), 8.51 (d, 1H, J ) 4.0 Hz), 7.71 (s, 1H),
7.24 (m, 5H), 4.80 (m, 1H), 4.68 (d, 1H, J ) 6.9 Hz), 3.14 (m,
2H), 2.73 (m, 2H), 1.36 (br, 12H); 13C NMR (CDCl3) δ 170.4,
170.3, 152.6, 146.1, 137.0, 130.7, 130.5, 129.6, 128.6, 127.1,
123.8, 118.7, 109.7, 81.6, 76.5, 54.7, 37.4, 28.4, 27.9, 20.6; IR
3468, 1735, 1660, 1637, 1595, 1534, 1367, 1158 cm-1; MS
(HRFAB) m/z 442.1878 (MH+) (C24H27NO7 requires 441.1866).
OTHQ (5). To a round-bottom flask containing 11 (50 mg,
0.113 mmol) was added 10 mL of dry HCl (40 mmol in dioxane)
under argon. The solution was allowed to stir at room temper-
ature for 12 h, and then the solvent was removed under reduced
pressure. To the flask was then added 20 mL of water, and the
suspension was filtered to give an off-white solid. The material
was dissolved in 10 mL of saturated sodium carbonate and
precipitated with 1 N HCl. This was filtered to give pure 5: yield
34.7 mg (80%); 1H NMR (DMSO-d6) δ 13.08 (s, 1H, COOH),
12.04 (s, 1H, OH), 9.88 (s, 1H, OH), 8.95 (d, 1H, J ) 6.0 Hz,
NH), 7.72 (s, 1H), 7.29 (m, 5H), 4.29 (m, 2H), 3.12 (m, 3H), 2.66
(dd, 1H, J ) 11.9, 11.6 Hz), 1.43 (d, 3H, J ) 6.2 Hz); 13C NMR
(DMSO-d6) δ 172.5, 169.9, 613.1, 155.2, 145.8, 136.9, 130.2,
129.3, 128.4, 126.7, 123.4, 118.3, 109.4, 76.2, 53.9, 36.7, 28.1,
1
Rf ) 0.24; yield 1.60 g (77%); H NMR (CDCl3) δ 6.65 (s, 1H),
3.78 (s, 3H), 3.73 (s, 3H), 3.6-3.0 (br, 6H), 2.08 (s, 3H), 1.23 (t,
3H, J ) 7.20 Hz), 1.03 (m, 3H); 13C NMR (CDCl3) δ 169.5, 167.0,
151.0, 147.8, 127.9, 124.8, 117.4, 109.0, 76.1, 56.2, 42.9, 39.1,
28.2, 20.8, 14.1, 12.8; IR 2981, 2940, 2878, 1638, 1472, 1436,
1275, 1088 cm-1. Anal. Calcd for C19H30O4N2(H2O)1/4: C, 64.29;
H, 8.66; N, 7.89. Found: C, 64.53; H, 8.60; N, 7.74.
3-Met h yl-5,8-d ih yd r oxy-7-ca r b oxy-2,3-d ih yd r oisocou -
m a r in (10). To a stirred solution of 9 (2.3 g, 6.81 mmol) in THF

Model for Oxidative Damage by OTA
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1069
OTA (25 µM) in the presence of FeTPPS (5 µM) was able
to effect single-strand breaks (lane 4) and convert form
I into nicked circular DNA (form II). These results were
in direct contrast to our DNA cleavage studies with free
Fe, where no role for the toxin could be established (25).
The oxidative nature of the strand scission was con-
firmed by the finding that the enzyme catalase, which
lowers solution concentrations of H2O2, almost completely
inhibited cleavage (lane 7). Hydroxyl radical scavengers,
Me2SO (lane 5) and tert-butyl alcohol (t-BuOH, lane 6),
also provided partial protection against strand scission.
Additional experiments showed that the cleavage was
insensitive to the ionic strength of the medium (10-500
mM NaCl, data not shown). Taken together, these results
strongly suggested that the neutral HO• mediated the
DNA cleavage by the FeTPPS/OTA mixture (29). The
results also suggested that OTA provided the reducing
equivalents to convert Fe(III)TPPS to Fe(II)TPPS. HPLC
combined with electrospray mass spectrometry was then
utilized to examine the reaction of OTA with FeTPPS and
H2O2 in more detail.
Rea ction of OTA w ith F eTP P S/H2O2/Sod iu m As-
cor ba te. As described by Meunier (22), the FeTPPS/H2O2
system efficiently catalyzes the oxidation of chlorinated
phenols (ortho- or para-substituted) to their respective
benzoquinones. Our goal was to utilize the FeTPPS/H2O2
system to carry out the oxidation of OTA in the presence
of a reducing agent (sodium ascorbate), which would
reduce the anticipated quinone, OTQ (6), into OTHQ (5),
as outlined in eq 1
F igu r e 2. DNA cleavage by OTA (25 µM) in the presence of
Fe(III)TPPS (5 µM). Cleavage was carried out at 37 °C for 30
min in 10 mM MOPS (pH 7.4) buffer solution: lane 1, DNA
alone; lane 2, DNA and Fe(III)TPPS; lane 3, DNA and OTA;
lane 4, DNA and OTA and Fe(III)TPPS; lane 5, DNA and 1 µL
of DMSO; lane 6, DNA and 1 µL of t-BuOH; and lane 7, DNA
and 1000 units/mL catalase.
20.3; IR 2927, 2854, 1731, 1582, 1442, 1148, 1021 cm-1; MS
(HRFAB) m/z 386.1251 (MH+) (C20H19NO7 requires 385.1161).
Au toxid a tion of OTHQ (5). Autoxidation reactions for
OTHQ (5) were followed spectrophotometrically using a Hewlett-
Packard (HP-8452) UV-vis spectrometer at 350 or 396 nm.
Reactions were initiated by adding 10 µL of a stock solution of
5 (2.6 mM) in MeOH to a 3 mL cuvette containing 2 mL of buffer
(10 mM) and 100 mM NaCl equilibrated at 37 °C. Mixing could
be achieved within 2 or 3 s, and the data, which were analyzed
using the ENZFITTER program, gave good first-order rate
constants for at least two half-lives.
P r oton Affin ity. The pH measurements were obtained at
25 °C on a Fisher Scientific Accumet 910 pH meter using
standard glass electrodes. Calibration was carried out using
commercial buffers (BDH, pH 4.00, 7.00, and 10.00, all (0.01).
Over the pH range of 6.6-9.5, the proton affinity of 5 was
determined spectrophotometrically (28) at 25 °C using a 1 mL
cuvette containing 1 mL of a 10 mM solution of buffer with 100
mM NaCl. To the sample cell was added 10 µL of a 2.6 mM
stock solution of 5 in MeOH. UV-vis spectra were recorded by
the overlay method in the wavelength range of 250-500 nm.
Oxid a tion of OTHQ (5) by Nitr oson iu m Tetr a flu or obo-
r a te. Reactions of OTHQ (5) with nitrosonium tetrafluoroborate
(NOBF4) were followed by UV-vis using a Hewlett-Packard
(HP-8452) spectrometer set to acquire data over the range of
220-600 nm. The oxidation was carried out in dry distilled
acetonitrile (MeCN) and oven-dried quartz cuvettes. A stock
solution of OTHQ (13 mM) was prepared, and 10 µL was added
to a 1 mL cuvette containing 990 µL of MeCN. To this solution
was added 10 molar equiv of NOBF4. The resulting yellow
solution was passed through a strong ion exchange cartridge to
remove excess NOBF4.
OTA (1) FeTPPS8 OTQ (6) sodium ascorbate8 OTHQ (5) (1)
H2O2
In this way, we could monitor for the presence of OTHQ
through comparison to our authentic sample, which was
generated from the photoreaction of OTA in N2-flushed
phosphate buffer (18).
Under one set of conditions, 100 µM OTA in 10 mM
phosphate buffer (pH 7.0) was allowed to react with 0.1%
FeTPPS and 6 molar equiv of both H2O2 and sodium
ascorbate. The reaction mixture was incubated at 37 °C
for varying lengths of time and then analyzed by reverse-
phase HPLC (detection at 330 nm). Representative HPLC
results are depicted in Figure 3. The HPLC profile
exhibited seven products (a -g); UV spectra (250-500
nm) for the four major products (d -g), including OTA,
are also shown. Note that OTA shows two absorbances
at ca. 330 and 380 nm. The phenolic pKa of OTA is 7.0
(25), and so both the protonated (λmax ) 333 nm) and
deprotonated (λmax ) 380 nm) forms of the toxin are
present. Compounds d -f exhibited UV spectra similar
to that of OTA, while compound g exhibited a single
absorbance at 350 nm.
Insight into the nature of products d -g was achieved
using electrospray mass spectrometry with negative
ionization (ES-). As shown in Figure 4, products d -f had
identical masses with an [M - H]- ion at 418 (Figure
4B), which is 16 mass units heavier than OTA (Figure
4A, [M - H]- ion at 402). The ES- spectrum of these
products also exhibited the characteristic chlorine isotope
splitting pattern (Figure 4B). While we lack authentic
standards to unambiguously characterize d -f, the UV-
vis characteristics and the ES- data were consistent with
formation of the three hydroxylated metabolites of OTA
(2-4, Figure 1) that have been previously characterized
using liver microsomes to oxidize the toxin (16). In
Rela xa tion of Su p er coiled P la sm id DNA by OTA/F eT-
P P S. Reaction mixtures (20 µL total volume) contained 400 ng
of supercoiled plasmid DNA (form I), 10 mM MOPS (pH 7.4),
25 µM OTA, and 5 µM FeTPPS. Reaction mixtures were
incubated at 37 °C for 30 min, and then reactions were quenched
by the addition of 4 µL of loading buffer. Samples were loaded
onto a 1% agarose gel containing ethidium bromide (1 µg/mL).
The gel was run at 110 V for 2 h and visualized by UV
illumination. In separate experiments, the effects of catalase
(1000 units/mL), Me2SO (1 µL), tert-butyl alcohol (1 µL), and
NaCl (10-500 mM) were examined.
Resu lts
DNA Clea va ge by OTA. The ability of OTA to effect
DNA cleavage in the presence of FeTPPS was determined
using supercoiled plasmid DNA (form I) and agarose gel
electrophoresis. As shown in Figure 2, cleavage by OTA
did not result in the absence of FeTPPS (lane 3), nor could
FeTPPS effect strand scission alone (lane 2). However,

1070 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Gillman et al.
F igu r e 3. HPLC elution profile and accompanying UV-vis spectra of peaks d -g and OTA from the reaction of OTA with FeTPPS/
H2O2/sodium ascorbate at 37 °C and pH 7.0.
contrast, product g had an [M - H]- ion at 384 with the
loss of the chlorine isotope pattern (Figure 4C), which
was consistent with OTHQ (5). Product g also comigrated
with our authentic sample of OTHQ (verified by sample
spiking) and had an identical UV spectrum at pH 7.0
(λmax ) 350 nm). These results firmly established product
g as the hydroquinone of OTA, i.e., OTHQ (5).
coupling reagent, diphenylphosphoryl azide (DPPA) (31)
was used to attach the L-phenylalanine tert-butyl ester
moiety to afford 11, which was subsequently treated with
acid (dry HCl/dioxane) to yield a sample of OTHQ (5).
The sample of 5 was obtained as a mixture of diastere-
omers. Examination by reverse-phase HPLC indicated
the presence of two equivalent peaks eluting at 17.6 and
18.7 min (data not shown). Confirmation that the peak
eluting at 18.7 min was the 3R-epimer was established
through comparison to our sample of 5 generated from
the photoreaction of OTA (18), which generates the 3R-
epimer as the photoreaction does not alter the stereo-
chemistry of the dihydroisocoumarin.
Aqu eou s Rea ctivity of OTHQ/OTQ. Experiments
were performed to determine the rate of OTHQ autoxi-
dation at 37 °C and at different pH values. Representa-
tive UV-vis data for the decomposition of OTHQ are
shown in Figure 6. At pH 8.0, the autoxidation was
accompanied by a decrease and “blue” shift of the
absorbance at ca. 350-400 nm (at pH 8.0, the phenolate
of OTHQ is also present, with a pKa of 8.03; see below),
along with an increase in absorbance at 270 nm. The fact
that the decomposition was due to autoxidation was
confirmed by the finding that an N2-purged solution of
OTHQ remained relatively stable at pH 8.0 (monitored
over a 12 h period). First-order rate constants for the
autoxidation of OTHQ are given in Table 1, which shows
that the autoxidation was pH-dependent between pH 7.0
and 9.0. As noted for other hydroquinone systems, this
observation indicates that proton removal is involved in
the rate-determining step, which is consistent with
semiquinone and subsequently quinone formation (32).
In the presence of sodium ascorbate, the hydroquinone
5 was generated in roughly 5% yield from the reaction
of OTA with FeTPPS/H2O2. Also, only ca. 15% of the
starting OTA was consumed over a 14 h period (data not
shown). In a separate experiment, OTA was incubated
with the FeTPPS/H2O2 mixture under physiological
conditions and excess sodium ascorbate was added at
various times. Figure 5 shows that under these conditions
the % concentration of OTA was reduced to ca. 30% after
30 min. The hydroquinone OTHQ (5) and the hydroxy-
lated products could still be detected, but their concen-
tration remained at ca. 5%. In the absence of sodium
ascorbate, the hydroxylated products were noted, but no
OTHQ could be detected in the HPLC traces from the
reaction of OTA with FeTPPS/H2O2 (data not shown).
These results were consistent with our hypothesis that
OTA oxidation generates the quinone OTQ (6) that was
reduced to OTHQ in the presence of sodium ascorbate
(eq 1).
Syn th esis of OTHQ (5). To study the characteristics
of the hydroquinone OTHQ (5) in more detail, a sample
was prepared using total synthesis. Scheme 1 outlines
the procedures used for the preparation of 5 that were
based on strategies described by Snieckus and co-workers
for the synthesis of OTA (30). From 10, the peptide

Model for Oxidative Damage by OTA
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1071
F igu r e 5. Time course of the loss of OTA in the reaction with
0.1% Fe(III)TPPS and 6 molar equiv of H2O2. Aliquots were
combined with 2.0 mM sodium ascorbate at various times and
analyzed by HPLC.
F igu r e 6. UV-vis data for the autoxidation of OTHQ (5) in
10 mM MOPS buffer (pH 8.0) containing 100 mM NaCl at 37
°C. Scans were taken every 100 min.
Ta ble 1. Su m m a r y of th e Ra te Con sta n ts for th e
Au toxid a tion of OTHQ (5)a
b
pH
kobs × 105 (s-1
)
t1/2 (h)
7.0
7.4
8.0
8.5
9.0
1.13
1.74
3.50
4.72
8.92
16.98
11.09
5.49
4.08
2.16
a
All experiments were conducted in 10 mM buffer and 100 mM
b
NaCl at 37 °C. Estimated error in rate constants was (5%.
for this phenolic group of OTHQ (5), which is ca. 1.0 pH
unit above the corresponding pKa of OTA (25).
Further insight into the aqueous decomposition of
OTHQ (5) was obtained through MS analyses. Figure 7
shows the ES- spectrum obtained from the reaction of
OTHQ with aqueous NH4HCO3 (10 mM, pH 8.0). Neither
the starting hydroquinone ([M - H]- ) 384) nor the
anticipated quinone product OTQ (6) ([M - H]- ) 382)
was detected. Instead, a series of peaks were noted with
major species at 387, 432, and 449. Collision-induced
dissociation indicated that peaks at 449 and 387 were
derived from the peak at 432. On the basis of the known
chemistry of quinone systems (22, 34), we speculated that
the decomposition product with an [M - H]- ion at 432,
which is 48 mass units (three oxygen atoms) heavier than
OTHQ, may have originated from the reaction of OTQ
(6) with nucleophilic species (H2O/HO- or HOO-) in
solution (22, 34). To test this hypothesis, the oxidation
of OTHQ using excess nitrosonium tetrafluoroborate
(NOBF4) in a non-nucleophilic “dry” acetonitrile (MeCN)
solution was examined, and the subsequent product
ascribed to OTQ (6) was reacted with water (NH4HCO3,
pH 8.0). Our choice to utilize NOBF4 as the oxidant
stemmed from the fact that the formal potential of NO+
F igu r e 4. Negative ion electrospray mass spectra (ES-) of
peaks d -g and OTA from Figure 3. (A) OTA, with an [M - H]-
ion at 402. (B) d -f, with an [M - H]- ion at 418. (C) g, with an
[M - H]- ion at 384 [OTHQ (5)].
This work demonstrated that OTHQ is oxidized by O2
alone (t1/2 ) 11.1 h at pH 7.4; Table 1), which is not the
case for OTA (33).
In the pH range of 7.0-9.0, OTHQ also underwent a
change in UV-vis characteristics exhibiting a red shift
in λmax from 350 nm at pH 7.0 to 396 nm at pH 9.0. This
change was consistent with deprotonation of the phenolic
oxygen ortho to the carbonyl groups. At 25 °C, a pKa value
of 8.03 ( 0.06 (95% confidence interval) was established

1072 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Gillman et al.
ated DNA damage (8-12) and lipid peroxidation (32), the
exact pathway by which OTA initiates oxidative damage
has remained elusive. One scenario put forward is that
OTA forms a complex with Fe(III) and the resulting Fe-
(III)‚OTA chelate is reduced by NADPH-cytochrome
P450 reductase to an Fe(II)‚OTA complex, which acti-
vates molecular O2 to initiate oxidative damage (36).
However, Hoehler has recently shown that the phenoxy
O-methylated derivative of OTA, which does not chelate
Fe(III) effectively, facilitates HO• production in hepato-
cytes, mitrochondria, and microsomes from rats just as
efficiently as OTA (37). Our recent studies also show that
OTA facilitates DNA cleavage by the Cu(II) complex of
1,10-phenanthroline [Cu(OP)2], a prototypical Cu-medi-
ated nuclease that requires an external reducing agent
for activation (25). In the presence of free Fe(III) or Cu-
(II) with an equimolar amount of or excess OTA, no role
for the toxin could be established. Like Hoehler’s results
(37), these studies also implied that oxidative damage
by OTA can be attributed to its redox properties, instead
of to its ability to coordinate redox active transition
metals (25).
F igu r e 7. Negative ion electrospray mass spectrum (ES-)
taken following the aqueous decomposition of OTHQ (5) from
the reaction in NH4HCO3 (10 mM, pH 8.0).
The studies presented here on the ability of OTA to
facilitate DNA cleavage in the presence of FeTPPS also
support this notion. Iron-porphyrin systems have been
shown to cause DNA cleavage in the presence of O2 and
a reducing agent, usually a thiol such as dithiothreitol
or 2-mercaptoethanol (38, 39). A mechanism for DNA
cleavage by these systems involves production of HO•
through a Fenton type of reaction between Fe(II) and
H2O2 (39). The ability of the FeTPPS system to facilitate
strand scission in the presence of OTA (Figure 2) sug-
gested that OTA provides the reducing equivalents to
convert Fe(III)TPPS to Fe(II)TPPS. The fact that the
enzyme catalase inhibited the cleavage provided a direct
role for H2O2. The cleavage was also found to be insensi-
tive to the ionic strength of the medium, and the hydroxyl
radical scavengers, Me2SO and tert-butyl alcohol, provide
protection (Figure 2). Taken together, these results
strongly suggested that HO• initiated cleavage (29). This
hypothesis was also consistent with the demonstrated
ability of the toxin to facilitate HO• production in cellular
systems (33, 40).
Once it was established that the FeTPPS/OTA system
facilitates DNA cleavage, the reaction of OTA with
FeTPPS in the presence of H2O2 was investigated. We
anticipated that FeTPPS/H2O2 would oxidize OTA to the
quinone derivative OTQ (6), in analogy to the oxidation
of other chlorinated phenols (20-23). This prediction was
also based on our recent findings regarding the ability
of OTA to act as a photoactivatable DNA cleaving agent
(18). Here, the photocleavage was found to stem from
initial C-Cl bond homolysis, and product analysis of the
photoreaction showed a direct correlation with the known
photochemistry of p-chlorophenol (19). For example,
photoirradiation of OTA under anaerobic conditions
produced the hydroquinone derivative, OTHQ (5). Inter-
estingly, OTHQ was also detected in the presence of O2
when a reducing agent (sodium ascorbate) was added to
the photoreaction mixture. This observation suggested
that OTHQ originated from its oxidized precursor OTQ
(6), in analogy to photooxidation of halogenated phenols
in the presence of O2 (19). Given the fact that the
photochemical behavior of OTA mimicked that estab-
lished for p-chlorophenol, we speculated that the oxida-
tion of OTA would similarly involve the quinone deriva-
F igu r e 8. UV-vis spectra of OTHQ (5) in the absence
(spectrum a) and presence of 10 equiv of NOBF4 (spectrum b)
in acetonitrile.
is 1.27 V versus SCE in MeCN (35), which is more than
sufficient to oxidize OTHQ.2
Figure 8 shows the UV-vis changes in OTHQ (spec-
trum a) upon addition of 10 equiv of NOBF4 (spectrum
b). The OTHQ solution immediately turned yellow, and
its absorption maxima at 350 nm (ꢀ350 ) 3400 M-1 cm-1
)
and 240 nm were replaced by a broad absorbance band
in the visible region along with an intense absorbance
at 260 nm. While attempts to isolate the oxidized product
failed, these spectral changes were in good agreement
with those reported for the oxidation of TCP to its
respective benzoquinone (23). Addition of 10 mM NH4-
HCO3 (100 µL) to the yellow solution ascribed to OTQ
(6) (Figure 7) led to the immediate disappearance of the
intense absorbance at 260 nm. Inspection of the solution
by ES- again showed the presence of the species in Figure
7. A possible reaction sequence for the aqueous decom-
position of OTHQ (5) is outlined in the Discussion.
Discu ssion
The ability of OTA to induce single-strand DNA
cleavage (6, 7) and DNA adduction (8-11) in vivo, coupled
with its implication in human kidney carcinogenesis (4,
5), has stimulated numerous investigations into its mode
of action (12-17). While these studies have established
that an oxidative mechanism is involved in OTA-medi-
2
In MeCN, the electrochemical oxidation of OTA exhibits behavior
almost identical with that of p-chlorophenol. The half-wave potential
of OTA is 1.45 V versus SCE, while the half-wave potential of OTHQ
is 0.92 V in MeCN. Upon successive scans of OTA, a secondary
oxidation peak with a half-wave potential of 0.92 V versus SCE was
observed which corresponds to OTHQ (M. W. Calcutt, R. E. Noftle,
and R. A. Manderville, unpublished results).

Model for Oxidative Damage by OTA
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1073
Sch em e 2. Mod el P a th w a y for th e Oxid a tion of OTA to OTQ (6)
tive OTQ (6). To provide evidence for the intermediacy
of OTQ in OTA oxidation, we carried out the oxidation
with FeTPPS/H2O2 in the presence of sodium ascorbate,
which was used as a trapping agent to convert OTQ (6)
to its reduced form OTHQ (5). As predicted, the hydro-
quinone OTHQ (5) was only detected when sodium
ascorbate was added to the reaction mixture, which was
consistent with direct formation of OTQ (6) with subse-
quent reduction to OTHQ.
The reaction of OTA with the FeTPPS system also
generated three hydroxylated products (chlorine atom
still attached, Figure 4B), which was taken as evidence
of the production of 2-4 (Figure 1), i.e., the known
hydroxylated metabolites of OTA (16). This finding
suggested that the FeTPPS system served as a reason-
able model for the bioactivation of OTA. Further insight
into the nature of OTA oxidation was derived from the
finding that production of the hydroxylated products did
not depend on the presence of sodium ascorbate. This
result was in direct contrast to the production of OTHQ
(5), and suggested that like the quinone OTQ (6), the
hydroxylated products are the direct result of OTA
oxidation. This finding allows one to speculate that the
inability of others to detect OTHQ (5) in reactions of OTA
with microsomal/sodium ascorbate mixtures could be that
OTQ (6) reacts with adventitious protein prior to reduc-
tion by sodium ascorbate (41). This factor, coupled with
the lack of authentic standards, could make detection of
OTHQ in a microsomal assay problematic.
Scheme 2 outlines a possible pathway for the oxidation
of OTA by the FeTPPS/H2O2 system. The first step
involves production of the OTA phenoxyl radical (12).
This species may result from electron abstraction by a
high-valent Fe-oxoporphyrin generated from the reac-
tion of FeTPPS with H2O2 (22), or by electron transfer to
Fe(III)TPPS to produce Fe(II)TPPS, as suggested in the
DNA cleavage results. Further oxidation of the phenoxy
radical by a second molecule of FeTPPS or molecular O2
would then yield the phenoxonium cation. The presence
of the chlorine leaving group provides a viable pathway
for OTQ (6) production through nucleophilic attachment
of H2O with the loss of HCl (22). The fact that we detect
low levels of OTHQ in the oxidation of OTA by FeTPPS/
H2O2 in the presence of sodium ascorbate is attributed
to two factors. First, sodium ascorbate would be expected
to reduce the OTA phenoxy radical to the OTA phenoxy
anion (42). This would make the oxidation process
inefficient, as was noted when sodium ascorbate was
added to the reaction mixture (long incubation times and
a slow rate of turnover of the toxin). This observation is
also consistent with the ability of sodium ascorbate to
quench the genotoxicity of OTA (13). Second, the antici-
pated electrophilicity of the quinone OTQ would make
trapping by sodium ascorbate difficult. For example,
when the incubation of OTA with FeTPPS/H2O2 was
carried out in the absence of sodium ascorbate, the toxin
was consumed over the course of approximately 50 min
(Figure 5). Upon quenching the reaction with sodium
ascorbate, we could still only detect ca. 5% OTHQ. We
attribute this to the fact that the quinone product OTQ
reacts with nucleophilic species in solution [H2O/HO- or
HOO- (34)] to form decomposition products (Figure 7)
prior to being reduced by sodium ascorbate. This factor
makes it difficult to determine the yield of OTQ from the
oxidation.
The aqueous reactivity of OTHQ (5) also suggests that
if it were formed in vivo, then it would make a significant
contribution to the toxicity of OTA. Like other hydro-
quinones (32, 43), OTHQ undergoes autoxidation (Figure
6 and Table 1), a process that generates superoxide and
subsequently H2O2 (32). This information demonstrates
that OTHQ is more reactive than OTA, which is not
oxidized by O2 alone (33). Autoxidation of OTHQ was also
anticipated to yield the quinone OTQ (6). While we did
not detect OTQ, we did find evidence for formation of a
species with an [M - H]- ion at 432, which was ac-
companied by other species with [M - H]- ions at 449
and 387 (Figure 7). While we currently lack unambiguous
evidence regarding the identity of these decomposition
products, a possible decomposition pathway for OTHQ
is outlined in Scheme 3. The proposed pathway is based
on the available literature information on the chemistry
of quinone systems and is consistent with the MS data.
Thus, autoxidation of OTHQ is believed to generate OTQ
(6) that subsequently reacts with H2O/HO- to yield the
o-hydroquinone that can undergo oxidation to the cat-
echol. As described by Foote (34), catechols undergo C-C
cleavages through reaction with HOO- via an acyclic
Baeyer-Villiger type mechanism. This type of reaction
for the catechol would yield the diacid derivative (both
tautomers are shown), which has an [M - H]- ion at 432
(Figure 7). Treatment of the diacid with NH4HCO3 would
yield the ammonium salt with an [M - H]- ion at 449,
while decarboxylation would generate a species with an

1074 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Sch em e 3. P r op osed P a th w a y for th e Aqu eou s Decom p osition of OTHQ (5)
Gillman et al.
Sch em e 4. Mod el for th e En zym a tic Activa tion of OTA
[M - H]- ion at 388 and an [M - 2H]2- ion at 387. The
fact that the chemical oxidation of OTHQ by NOBF4 in
dry acetonitrile (Figure 8) also generated the same
species upon reaction with aqueous NH4HCO3 provides
additional evidence that the decomposition products
originated from the reaction of OTQ (6) with nucleophilic
species in solution. Hence, the anticipated reactivity of
the quinone OTQ makes it a likely candidate for covalent
attachment to biopolymers (24, 41) and provides a
rationale for the ability of OTA to generate DNA adducts
in vivo.
the quinone OTQ may react with DNA-derived nucleo-
philes to produce DNA adducts.
In addition to the pathways outlined in Scheme 4, we
have also demonstrated that OTA can activate DNA
cleavage by the Cu(OP)2 complex (25). New evidence from
our laboratory3 also shows that the Cu(II) complex of
OTA (25, 44) can facilitate DNA cleavage by Cu(II) bound
to the DNA surface. Since copper activation represents
a nonenzymatic pathway by which certain phenolic
compounds (45-47), including hydroquinone metabolites
of PCP (47), facilitate DNA damage, we also expect that
the presence of Cu(II)/Cu(I) will contribute to the geno-
toxicity of OTA. In fact, copper activation may be more
efficient than the enzymatic pathway proposed in Scheme
4, as copper coordination by OTA facilitates DNA binding,
since by itself OTA is electrostatically repelled by the
DNA helix.
On the basis of the results from these studies, we
propose the model outlined in Scheme 4 for the enzymatic
activation of OTA. The reaction of OTA with an Fe-
porphyrin system, such as cytochrome P450 (14, 15), is
envisioned to follow two pathways. The CH-oxidation
pathway generates hydroxylated metabolites 2-4 (16).
These species are readily eliminated in the bile of rats
(possibly through glucuronide formation) and conse-
quently are far less toxic than OTA (17). Thus, the CH-
oxidation route is regarded as a detoxification pathway.
The phenol-oxidation route converts OTA into the qui-
none OTQ (6). This pathway generates reactive oxygen
species (ROS) (H2O2, HO•, or O2•-) that can lead to DNA
cleavage (6, 7) and lipid peroxidation (36). The quinone
OTQ can undergo either a two-electron reduction to form
OTHQ or a one-electron reduction to yield a semiquinone
anion radical. These events establish futile redox cycles
in which formation of ROS is amplified (32, 41). Finally,
Current efforts in our laboratory are focused on deter-
mining whether the quinone OTQ, or its electrophilic
precursors depicted in Scheme 2, react with DNA bases,
as anticipated.
Ack n ow led gm en t. We thank Dr. Fred W. Perrino
(Department of Biochemistry, Wake Forest University
School of Medicine) for the sample of plasmid DNA. This
work was partially supported by Grant IRG from the
American Cancer Society and Wake Forest University.
NMR spectra were recorded on instruments purchased
3
I. G. Gillman and R. A. Manderville, unpublished results.

Model for Oxidative Damage by OTA
Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1075
(21) Waidyanatha, S., Po-Hsiung, L., and Rappaport, S. M. (1996)
Characterization of chlorinated adducts of hemoglobin and albu-
min following administration of pentachlorophenol to rats. Chem.
Res. Toxicol. 9, 647-653.
with the partial support of the NSF (CHE-9708077) and
NCBC (9703-IDG-1007).
(22) Labat, G., Seris, J .-L., and Meunier, B. (1990) Oxidative degrada-
tion of aromatic pollutants by chemical models of ligninase based
on porphyrin complexes. Angew. Chem., Int. Ed. 29, 1471-1473.
(23) Wiese, F. W., Chang, H. C., Lloyd, R. V., Freeman, J . P., and
Samokyszyn, V. M. (1998) Peroxidase-catalyzed oxidation of 2,4,6-
trichlorophenol. Arch. Environ. Contam. Toxicol. 34, 217-222.
(24) Peter, M. G. (1989) Chemical modifications of biopolymers by
quinones and quinone methides. Angew. Chem. 101, 572-587.
(25) Ardus, J . A., Gillman, I. G., and Manderville, R. A. (1998) On
the role of copper and iron in DNA cleavage by ochratoxin A.
Structure-activity relationships in metal binding and copper-
mediated DNA cleavage. Can. J . Chem. 76, 907-918.
(26) Barley, M. H., Takeuchi, K. J ., and Meyer, T. J . (1986) Electro-
catalytic reduction of nitrite to ammonia based on a water-soluble
porphyrin. J . Am. Chem. Soc. 108, 5876-5885.
(27) Lustig, E., Benson, W. R., and Duy, N. (1967) Hindered rotation
in N,N-dimethyl-carbamates. J . Org. Chem. 32, 851-852.
(28) Albert, A., and Sergeant, E. P. (1962) The determination of
ionization constants, Chapman & Hall, London.
(29) Pogozelski, W. K., and Tullius, T. D. (1998) Oxidative strand
scission of nucleic acids: routes initiated by hydrogen abstraction
from the sugar moiety. Chem. Rev. 98, 1089-1107.
(30) Sibi, M. P., Chattopadhyay, S., Dankwardt, J . W., and Snieckus,
V. (1985) Combinational O-aryl carbamate and benzamide direct
ortho metalation reactions. Synthesis of ochratoxin A and och-
ratoxin B. J . Am. Chem. Soc. 107, 6312-6315.
(31) Shioiri, T., Ninomiya, K., and Yamada, S.-I. (1972) Diphenylphos-
phoryl azide. A new convenient reagent for a modified Curtius
reaction and for the peptide synthesis. J . Am. Chem. Soc. 94,
6203-6205.
(32) Kwok, E. S. C., and Eastmond, D. A. (1997) Effects of pH on
nonenzymatic oxidation of phenylhydroquinone: potential role in
urinary bladder carcinogenesis induced by o-phenylphenol in
Fischer 344 rats. Chem. Res. Toxicol. 10, 742-749.
(33) Xiao, H., Madhyastha, S., Marquardt, R. R., Li, S., Vodela, J . K.,
Frohlich, A. A., and Kemppainen, B. W. (1996) Toxicity of
ochratoxin A, its opened lactone form and several of its analogs:
structure-activity relationships. Toxicol. Appl. Pharmacol. 137,
182-192.
(34) Sawaki, Y., and Foote, C. S. (1983) Mechanism of C-C cleavage
of cyclic 1,2-diketones with alkaline hydrogen peroxide. The
acyclic mechanism and its application to the basic autoxidation
of pyrogallol. J . Am. Chem. Soc. 105, 5035-5040.
(35) Connelly, N. G., and Geiger, W. E. (1996) Chemical redox agents
for organometallic chemistry. Chem. Rev. 96, 877-910.
(36) Omar, R. F., Hasinoff, B. B., Mejilla, F., and Rahimtula, A. D.
(1990) Mechanism of ochratoxin A stimulated lipid peroxidation.
Biochem. Pharmacol. 40, 1183-1191.
(37) Hoehler, D., Marquardt, R. R., McIntosh, A. R., and Hatch, G.
M. (1997) Induction of free radicals in hepatocytes, mitochondria
and microsomes of rats by ochratoxin A and its analogs. Biochim.
Biophys. Acta 1357, 225-233.
(38) Ward, B., Skorobogaty, A., and Dabrowiak, J . C. (1986) DNA
cleavage specificity of a group of cationic metalloporphyrins.
Biochemistry 25, 6875-6883.
(39) Meunier, B. (1992) Metalloporphyrins as versatile catalysts for
oxidative reactions and oxidative DNA cleavage. Chem. Rev. 92,
1411-1456.
(40) Hoehler, D., Marquardt, R. R., McIntosh, A. R., and Xiao, H.
(1996) Free radical generation as induced by ochratoxin A and
its analogs in bacteria (Bacillus brevis). J . Biol. Chem. 271,
27388-27394.
(41) Bolton, J . L., Pisha, E., Zhang, F., and Qiu, S. (1998) Role of
quinoids in estrogen carcinogenesis. Chem. Res. Toxicol. 11,
1113-1127.
(42) Sturgeon, B. E., Sipe, H. J ., J r., Barr, D. P., Corbett, J . T.,
Martinez, J . G., and Mason, R. P. (1998) The fate of the oxidizing
tyrosyl radical in the presence of glutathione and ascorbate. J .
Biol. Chem. 273, 30116-30121.
Refer en ces
(1) van der Merwe, K. J ., Steyn, P. S., Fourie, L., Scott, D. B., and
Theron, J . J . (1965) Ochratoxin A, a toxic metabolite produced
by Aspergillus ochraceus Wilh. Nature 205, 1112-1113.
(2) Pohland, A. E., Nesheim, S., and Friedman, L. (1992) Ochratoxin
A: A review. Pure Appl. Chem. 64, 1029-1046.
(3) Kuiper-Goodman, T., and Scott, P. M. (1989) Risk assessment of
the mycotoxin ochratoxin A. Biomed. Environ. Sci. 2, 179-248.
(4) Castegnaro, M., Bartsch, H., and Chernozemsky, I. (1987) En-
demic nephropathy and urinary tumors in the balkans. Cancer
Res. 47, 3608-3609.
(5) Tatu, C. A., Orem, W. H., Finkelman, R. B., and Feder, G. L.
(1998) The etiology of balkan endemic nephropathy: still more
questions than answers. Environ. Health Perspect. 106, 689-700.
(6) Creppy, E. E., Kane, A., Dirheimer, G., Lafarge-Frayssinet, C.,
Mousset, S., and Frayssinet, C. (1985) Genotoxicity of ochratoxin
A in mice: DNA single-strand break evaluation in spleen, liver
and kidney. Toxicol. Lett. 28, 29-35.
(7) Kane, A., Creppy, E. E., Roth, A., Ro¨schenthaler, R., and Dir-
heimer, G. (1986) Distribution of the [3H]-label from low doses of
radioactive ochratoxin A ingested by rats, and evidence for DNA
single-strand breaks caused in the liver and kidneys. Arch.
Toxicol. 58, 219-224.
(8) Grosse, Y., Baudrimont, I., Castegnaro, M., Betbeder, A.-M.,
Creppy, E. E., Dirheimer, G., and Pfohl-Leszkowicz, A. (1995)
Formation of ochratoxin A metabolites and DNA-adducts in
monkey kidney cells. Chem.-Biol. Interact. 95, 175-187.
(9) Pfohl-Leszkowicz, A., Chakor, K., Creppy, E. E., and Dirheimer,
G. (1991) DNA adduct formation in mice treated with ochratoxin
A. In Mycotoxins, endemic nephropathy and urinary tract tumors
(Castegnaro, M., Plestina, R., Dirheimer, G., Chernozemsky, I.
N., and Bartsch, H., Eds.) pp 245-253, IARC Scientific Publica-
tion 115, International Agency for Research on Cancer, Lyon,
France.
(10) Pfohl-Leszkowicz, A., Grosse, Y., Kane, A., Creppy, E. E., and
Dirheimer, G. (1993) Differential DNA adduct formation and
disappearance in three mice tissues after treatment by the
mycotoxin ochratoxin A. Mutat. Res. 289, 265-273.
(11) Obrecht-Pflumio, S., Grosse, Y., Pfohl-Leszkowicz, A., and Dir-
heimer, G. (1996) Protection by indomethacin and aspirin against
genotoxicity of ochratoxin A, particularly in the urinary bladder
and kidney. Arch. Toxicol. 70, 244-248.
(12) Malaveille, C., Brun, G., and Bartsch, H. (1994) Structure-
activity studies in E. coli strains on ochratoxin A (OTA) and its
analogues implicate a genotoxic free radical and a cytotoxic thiol
derivative as reactive metabolites. Mutat. Res. 307, 141-147.
(13) Baudrimont, I., Betbeder, A.-M., Gharbi, A., Pfohl-Leszkowicz,
A., Dirheimer, G., and Creppy, E. E. (1994) Effect of superoxide
dismutase and catalase on the nephrotoxicity induced by sub-
chronical administration of ochratoxin A in rats. Toxicology 89,
101-111.
(14) De Groene, E. M., Hassing, I. G. A. M., Blom, M. J ., Seinen, W.,
Fink-Gremmels, J ., and Horbach, G. J . (1996) Development of
human cytochrome P450-expressing cell lines: application in
mutagenicity testing of ochratoxin A. Cancer Res. 56, 299-304.
(15) Omar, R. F., Gelboin, H. V., and Rahimtula, A. D. (1996) Effect
of cytochrome P450 induction on the metabolism and toxicity of
ochratoxin A. Biochem. Pharmacol. 51, 207-216.
(16) Stømer, F. C., Støren, O., Hansen, C. E., Pedersen, J . I., and
Assen, A. J . (1983) Formation of (4R)- and (4S)-hydroxyochratoxin
A and 10-hydroxyochratoxin A from ochratoxin A by rabbit liver
microsomes. Appl. Environ. Microbiol. 45, 1183-1187.
(17) Li, S., Marquardt, R. R., Frohlich, A. A., Vitti, T. G., and Crow,
G. (1997) Pharmacokinetics of ochratoxin A and its metabolites
in rats. Toxicol. Appl. Pharmacol. 145, 82-90.
(18) Gillman, I. G., Yezek, J . M., and Manderville, R. A. (1998)
Ochratoxin A acts as a photoactivatable DNA cleaving agent.
Chem. Commun., 647-648.
(43) Zhang, F., Chen, Y., Pisha, E., Shen, L., Xiong, Y., van Breemen,
R. B., and Bolton, J . L. (1999) The major metabolite of equilin,
4-hydroxyequilin, autoxidizes to an o-quinone which isomerizes
to the potent cytotoxin 4-hydroxyequilenin-o-quinone. Chem. Res.
Toxicol. 12, 204-213.
(19) Grabner, G., Richard, C., and Ko¨hler, G. (1994) Formation and
reactivity of 4-oxocyclohexa-2,5-dienylidene in the photolysis of
4-chlorophenol in aqueous solution at ambient temperature. J .
Am. Chem. Soc. 116, 11470-11480.
(44) Gillman, I. G., Day, C. S., and Manderville, R. A. (1998) Stepwise
formation of a nonsymmetric dinuclear copper complex of ochra-
toxin A. Inorg. Chem. 37, 6385-6388.
(45) Singh, U. S., Scannell, R. T., An, H., Carter, B. J ., and Hecht, S.
M. (1995) DNA cleavage by di- and trihydroxyalkylbenzenes.
(20) Den Besten, C., Smink, M. C. C., De Vries, E. J ., and Van
Bladeren, P. J . (1991) Metabolic activation of 1,2,4-trichloroben-
zene and pentachlorobenzene by rat liver microsomes: a major
role for quinone metabolites. Toxicol. Appl. Pharmacol. 108, 223-
233.

1076 Chem. Res. Toxicol., Vol. 12, No. 11, 1999
Gillman et al.
Characterization of products and the roles of O2, Cu(II), and
alkali. J . Am. Chem. Soc. 117, 12691-12699.
(46) Li, Y., Trush, M. A., and Yager, J . D. (1994) DNA damage caused
by reactive oxygen species originating from a copper-dependent
oxidation of the 2-hydroxy catechol of estradiol. Carcinogenesis
15, 1421-1427.
(47) Naito, S., Ono, Y., Somiya, I., Inoue, S., Ito, K., Yamamoto, K.,
and Kawanishi, S. (1994) Role of active oxygen species in DNA
damage by pentachlorophenol metabolites. Mutat. Res. 310,
79-88.
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