Ochratoxin A forms a carbon-bonded C8-deoxyguanosine nucleoside adduct: Implications for C8 reactivity by a phenolic radical

Article abstract of DOI:10.1021/ja034221r

The ability of the carcinogenic fungal toxin Ochratoxin A (OTA, 1) to react with deoxyguanosine (dG) has been assessed using electrospray mass spectrometry and NMR. Photoexcitation of OTA (100 μM) in the presence of 50 mol equiv of dG led to the isolation and identification of the C8-deoxyguanosine nucleoside adduct 4. Importantly, the same adduct was formed upon oxidative activation of OTA using horseradish peroxidase (HRP)/H₂O₂ or the transition metals Fe(II) and Cu(II), as evidenced by mass spectrometry. Because the mutagenicity and subsequent carcinogenicity of OTA are believed to stem from oxidative DNA damage (strand scission and oxidative base products) and formation of guanine-specific DNA adducts, the adduct 4 confirms the ability of OTA to react covalently with dG and has important implications for the mechanism of action of OTA and other chlorophenolic toxins that undergo oxidation to yield phenoxyl radicals. The C8 position of dG is susceptible to radical attack, as was amply proven through formation of the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine. The adduct 4 is the first structurally characterized nucleoside adduct of a chlorophenolic toxin, and its formation has important implications for the mutagenicity of phenolic xenobiotics. Copyright

Full text of DOI:10.1021/ja034221r

Published on Web 03/07/2003
Ochratoxin A Forms a Carbon-Bonded C8-Deoxyguanosine Nucleoside
Adduct: Implications for C8 Reactivity by a Phenolic Radical
Jian Dai, Marcus W. Wright, and Richard A. Manderville*
Department of Chemistry, Wake Forest UniVersity, Winston-Salem, North Carolina 27109
Received January 17, 2003; E-mail: manderra@wfu.edu
Ochratoxin A (OTA, 1) is a para-chlorophenolic mycotoxin
produced by strains of Aspergillus and Penicillium1 that is widely
found as a contaminant of improperly stored food products.2 The
toxin is a potent renal carcinogen in rodents3 and is implicated in
human kidney4 and testicular carcinogenesis.5
2 min in 100 mM phosphate buffer (pH 7.4) generated three major
products eluting at 19.4, 23.7, and 24.6 min (see Supporting
Information and Figure S1 for experimental details). Products at
23.7 and 24.6 min had parent ions at m/z 384 and 368 and were
assigned to OTHQ, 3 ([M - H]- ) 384), and OTB, 2 ([M - H]-
) 368).13 The product at 19.4 min also lacked the chlorine isotope
pattern and had a parent ion at [M - H]- ) 633. Its MS/MS
spectrum exhibited a prominent ion at m/z 517 (-deoxyribose (dR)),
which was accompanied by ions at m/z 473 ([M - dR - CO2 -
H]-) and 429 ([M - dR - 2CO2 - H]-), suggesting covalent
attachment of dG to OTA with loss of HCl (i.e., OTA(403) - Cl(35)
+ dG(267) - 1H ) 634).
Semipreparative scale photoreactions yielded the OTA-dG adduct
as an off-white solid; its 1H NMR spectrum in DMSO-d6 is shown
in Figure 1a. Surprisingly, all NH and OH protons of the dG moiety
could be accounted for.17 The sharp singlet at δ 8.09 ppm was either
1
H6 of the OTA moiety or H8 of dG. However, the H-13C long-
range heteronuclear correlation NMR spectrum (HMBC) shown in
Figure 1b established the structure as 4. The sugar H1′ proton
showed a correlation with C8a at 143.3 ppm and C4a at 152.3 ppm,
while the singlet at 8.09 ppm showed correlations with C8a and
the phenolic C8 and amide carbonyl C11. These correlations
confirmed the identity of H6 (8.09 ppm) and that it was the H8a
proton of dG that was lost to yield 4 (NMR assignments of 4 are
given in Figure S2, Supporting Information). It is also worthy to
note that in the NOESY spectrum the sugar H1′ proton showed a
strong cross-peak to the aromatic H6 proton (Figure S3, Supporting
Information), not to the H4 protons, suggesting that the sugar and
the phenylalanine moiety were in close proximity, as depicted for
4.
The authentic sample 4 from photoexcitation of OTA may have
formed via an OTA carbon-centered radical, as carbon radicals form
C8 adducts18 and photolysis of OTA yields the OTA phenolic
radical and solvated electrons (eaq-);16 the latter may initiate
decomposition of the toxin to afford the carbon radical and Cl-.13,16
Thus, it was pertinent to determine whether 4 could be formed under
biologically relevant oxidative conditions where the eaq- would not
be present. Treatment of OTA (100 µM) with 50 equiv of dG in
100 mM phosphate buffer (pH 7.4) at 37 °C in the presence of
either 100 µM Fe(NH4)2(SO4)2, 100 µM Cu(OAc)2, or HRP (Type
VI, 25 units/mL)/1 mM H2O2 gave the extracted ion chromatograms
(EIC) in Figure 2 following 24 h of incubation time. Each oxidation
system converted OTA into 4, but clearly free Fe(II) was the most
efficient (panel b), yielding 4 ca. 5 orders of magnitude greater
than Cu(II) (panel c) and HRP/H2O2 (panel d). These results suggest
that Fe may be an important cofactor in OTA-mediated mutagen-
icity.
In cells, OTA induces oxidative stress6 and oxidative DNA
damage6b,7 through production of reactive oxygen species (ROS).8
From the 32P-postlabeling assay, it appears that OTA facilitates
guanine-specific DNA adducts,9 properties that establish a basis
for its mutagenicity.10 However, recent studies11 suggest that the
spots observed in the 32P-postlabeling assay may be due to OTA
cytotoxicity rather than to direct adduct formation.
Our laboratory has utilized a number of chemical approaches to
assess OTA oxidation.12-14 These studies point to the intermediacy
of the OTA phenoxyl radical and benzoquinone formed from
oxidative dechlorination of OTA. Recently, we presented evidence
that the benzoquinone reacts covalently with reduced glutathione
(GSH).15 Here, the photooxidative properties of the hydroquinone
derivative (OTHQ, 3)13 were utilized to generate an OTA-GSH
conjugate that acted as an analytical standard for biochemical
analyses.15 These results confirmed that OTA undergoes oxidative
dechlorination by liver enzymes, horseradish peroxidase (HRP)/
H2O2, and free Fe(II), which supported our chemical findings.
Presently, we use the photochemistry of OTA13,16 to assess its
reactivity toward deoxyguanosine (dG), the base implicated in OTA-
mediated DNA adduction.9 These studies have led to the isolation
and identification of the carbon-bonded C8-OTA-dG adduct 4 that
is also formed upon oxidation of OTA with HRP/H2O2, free Fe(II),
and Cu(II) ions. These results represent the first definitive evidence
that OTA can react with a DNA-derived nucleophile and suggest
involvement of the OTA phenoxyl radical in reaction with dG.
The photoreactivity of OTA (100 µM) in the presence of 50 mol
equiv of dG was initially investigated using electrospray mass
spectrometry (ES-). Photoirradiation of the OTA/dG mixture for
The results presented in Figure 2 suggest involvement of the
OTA phenoxyl radical in formation of 4. The toxin undergoes a 1e
oxidative process at ∼0.8 V versus SCE (1.04 V vs NHE) to form
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J. AM. CHEM. SOC. 2003, 125, 3716-3717
10.1021/ja034221r CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
formation of ROS with subsequent oxidative DNA damage (strand
breaks and 8-oxoG) and coupling of reactive phenolic radical
intermediates to the C8 position of G to form covalent DNA
adducts.
In summary, the C8-deoxyguanosine nucleoside adduct 4 of the
chlorinated phenolic toxin OTA (1) has been definitively identified
by mass spectrometry and NMR. This represents the first structur-
ally characterized DNA nucleoside adduct of a chlorophenolic toxin
and is an important standard for 32P-postlabeling experiments
dealing with in vivo DNA adduction by OTA. The dG reactions of
other chlorophenolic toxins are being examined to gain mechanistic
insight into this C8 reactivity.
Acknowledgment. Research grant CA 080787 from the Na-
tional Cancer Institute supported this study.
Supporting Information Available: Experimental procedures and
Figures S1-S3 described in the text (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
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Figure 1. (a) 500 MHz 1H NMR spectrum of the isolated OTA-dG adduct
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Figure 2. Extracted ion chromatograms (EIC) of m/z 633 (ES-) for 4 from
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the phenoxyl radical,14 and structure-activity relationships implicate
the OTA phenoxyl radical in OTA-mediated DNA damage.19 The
susceptibility of the C8 position of dG to radical attack has been
amply proven through formation of the hydroxyl radical-derived
DNA lesion, 8-oxodeoxyguanosine (8-oxoG).18 Estrogen radical
anions react at the purine C8 position,20 and most importantly
Ni(salens) that form phenoxyl radicals upon oxidation are speculated
to form C8-dG adducts.21 Thus, the findings reported herein may
help to define the mechanism of mutagenicity for phenolic toxins:
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