Ochratoxin A acts as a photoactivatable DNA cleaving agent

Article abstract of DOI:10.1039/a708275d

The ability of ochratoxin A to photoinduce DNA cleavage is described; in the presence of DNA the photoreaction yields the non-chlorinated derivative, ochratoxin B, while a hydroquinone derivative is produced under anaerobic conditions.

Full text of DOI:10.1039/a708275d

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Ochratoxin A acts as a photoactivatable DNA cleaving agent
Ivan G. Gillman, Jennifer M. Yezek and Richard A. Manderville*
Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486, USA
The ability of ochratoxin A to photoinduce DNA cleavage is
described; in the presence of DNA the photoreaction yields
the non-chlorinated derivative, ochratoxin B, while a
hydroquinone derivative is produced under anaerobic con-
ditions.
However, a marked increase in the extent of photocleavage
-flushed atmosphere, precluding the require-
ment for activated oxygen species. Inhibition of photocleavage
occurred in an N
2
was also provided by copper(ii) ions; a metal that binds 1
18
effectively and quenches its fluorescence spectrum. That both
II
2
Cu and O inhibit DNA cleavage suggests that they quench the
Ochratoxin A (OTA, 1: X = Cl) is a fungal toxin produced by
a species of Aspergillus and Penicillium.1 It contaminates a
wide range of foodstuffs and is implicated in the disease Balkan
endemic nephropathy in which patients suffer from urinary tract
excited state of 1.19
,2
Additional insight into the mode of photocleavage by 1 was
obtained from the finding that the non-chlorinated derivative,
OTB (2: X = H), and the derivative 4,† which lacks the
dihydroisocoumarin (lactone) ring system of 1, failed to
photoinduce DNA strand-scission (Fig. 2). These findings are
remarkably similar to the in vivo toxicity of 1 and indicate a
3
4
tumors. OTA induces single-strand DNA cleavage and DNA
adduction in vivo;5 properties that establish a basis for its
genotoxicity. However, the mechanism of OTA-induced DNA
damage is currently not known.
6
20
requirement for both the chlorine atom and the lactone.
OH
O
Product analysis from the photoreaction of 1 in aqueous
buffered media also provided information regarding the mode
of photocleavage by the toxin. In an ambiently oxygenated
atmosphere, no products were identified in the photoreaction of
CO2H
O
N
H
O
1
alone, even though the toxin was consumed as evidenced by
X
OTA, 1 X = Cl
OTB, 2 X = H
OTOH, 3 X = OH
OH
Cl
CO2H
O
N
H
4
6
Despite this, it is known that the chlorine atom is essential
7
Fig. 1 Cleavage of supercoiled plasmid DNA by 1. Reaction mixtures (20
ml total volume) contained 400 ng of plasmid DNA in 10 mm MOPS buffer,
pH 7.4, and were irradiated on ice through a Pyrex filter with an ILC
Technology 300 W Xenon arc lamp. After 5 min, the reaction mixtures were
analyzed on a 1.2% agarose gel. Lane 1: DNA alone. Lane 2: DNA + 1000
mm 1. Lane 3: irradiated DNA. Lane 4–7: irradiated DNA + 40, 200, 400 and
1000 mm 1, respectively.
and antioxidants inhibit its genotoxic activities. While activa-
tion of OTA appears to be oxidative,3 we have found, using
fluorescence spectroscopy (lext = 380 nm, lem = 441 nm), that
the toxin is particularly susceptible to light and decomposes
over time unless a suitable filter is used to suppress light
–7
intensity. Since certain halogenated compounds have been
shown to efficiently photocleave DNA,8
–10
we hypothesized
that 1 may photoinduce DNA damage. Such activity was also
expected to provide new insight into the toxin’s DNA targeting
activities as photoactivatable agents have proven useful as
probes of DNA structure,11 sequence, and mechanism, both in
terms of DNA strand-scission13 and DNA alkylation.
Table 1 Effect of additives on the extent of photocleavage of supercoiled
DNA (Form 1) by 1
12
Conditionsa
b
c
d
Form I (%)
Form II (%)
Form III (%)
14
Control
89
27
78
66
62
27
27
74
0
11
73
22
34
38
73
73
26
61
0
0
0
0
0
0
0
0
39
Here we describe our preliminary findings on the photo-
nuclease activity of 1. While in vivo activation of 1 may be
+
+
+
+
+
+
1
100 mm NaN
1 ml DMSO
3
15
mediated by cytochrome P450 with subsequent formation of
hydroxyl radicals,1
6,17
our results show that light can also
t
1 ml Bu OH
activate 1 and the products from the photoactivation provide
new insight into species that may participate in its in vivo DNA
targeting activities.
The ability of 1 to facilitate DNA photocleavage was
examined using supercoiled plasmid DNA and agarose gel
electrophoresis. As shown in Fig. 1, no cleavage resulted in the
absence of light (lane 2), highlighting that 1 alone does not
induce DNA cleavage. However, in the presence of light, DNA
cleavage by 1 occurred in a concentration dependent fashion
e
SOD
f
catalase
+10 mm Cu(OAc)2
Anaerobicg
a
Reactions were run at pH 7.4 (10 mm MOPS buffer) using 200 mm 1 and
00 ng of supercoiled plasmid DNA in 20 ml total volume, and were
4
irradiated in ice through a Pyrex filter with an ILC Technology 300 W
Xenon arc lamp for 5 min. Densitometric quantitation of the gels was
6
performed using a Microtek Scanmaker E equipped with PhotoImpact and
(
lanes 4–7). Table 1 shows the effect of various additives on the
b
c
UTHSCSA Image Tool software. Supercoiled DNA. Nicked circular
extent of photocleavage by 1. In an ambiently oxygenated
atmosphere, inhibition was provided by the oxygen radical
scavengers, DMSO, tert-butyl alcohol and sodium azide.
d
e
21
f
DNA. Linear DNA. 1000 units ml super oxide dismutase. 1000 units
ml21 catalase.
g
Reaction with 1 was purged with N2 prior to photo-
cleavage.
Chem. Commun., 1998
647

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work was supported by Grant #IRG from the American Cancer
Society and Wake Forest University.
Notes and References
*
†
E-mail: manderra@wfu.edu
The derivative 4 was prepared in 70% yield from 5-chloro-2-hydroxy-
benzoic acid and l-b-phenylalanine using a DCC coupling procedure; mp
1
7
72–173 °C, d
.92 (m, 1 H), 7.54–7.10 (m, 6 H), 6.94 (d, 1 H, J 8.9), 5.01 (m, 1 H), 3.4–3.1
[(CD CO] 173.0, 169.2, 160.6, 138.3, 134.6, 130.0, 129.2,
127.7, 127.5, 120.3, 116.7, 55.0, 37.7. (Calc. for C16 14ClNO ; C, 60.10; H,
4.41; N, 4.38. Found C, 60.02; H, 4.47; N, 4.39%.)
H 3 2
[(CD ) CO, 200 MHz] 12.3 (br, 1 H), 8.62 (d, 1 H, J 7.9),
Fig. 2 Structure–activity relationships in DNA cleavage of supercoiled
plasmid DNA by 1. Reactions were carried out for 5 min as described in the
caption below Fig. 1. Lane 1: DNA alone. Lane 2: DNA + 200 mm 1. Lane
(m, 2 H); d
C
3 2
)
H
4
3
: irradiated DNA. Lane 4: irradiated DNA + 200 mm 1. Lane 5: irradiated
‡ After 5 min irradiation time, the yield of 2 from the reaction of 100 mm 1
with 100 mm dextrose was ca. 20% based on HPLC analysis. The isolated
sample was identical to authentic 2 purchased from Sigma.
DNA + 200 mm 2. Lane 6: irradiated DNA + 200 mm 4.
§
The hydroquinone derivative 3 was obtained in 80% from the
photoreaction of (100 mm) in N -flushed phosphate buffer (0.1 m, pH 7.4).
]DMSO) 13.08 (s, 1 H), 12.04 (s, 1 H), 9.88 (s, 1 H), 8.95 (d, 1 H,
2
OTB
sugar
2
H 6
d ([ H
J 6.0), 7.72 (s, 1 H), 7.29 (m, 5 H), 4.29 (m, 2 H), 3.12 (m, 3 H), 2.66 (dd,
2
hn
H2O
N2
1 H, J 11.9, 11.6), 1.43 (d, 3 H, J 6.2). d ([ H ]DMSO) 172.5, 169.9, 163.1,
C
6
OTA
OTA*
OTOH, 3
1
5
52.2, 145.8, 136.9, 130.2, 129.3, 128.4, 126.7, 123.4, 118.3, 109.4, 76.2,
3.9, 36.7, 28.1, 20.3. These peaks are identical to a separately prepared
>
290 nm
H2O O2
sodium ascorbate
sample starting from 4-methoxyphenol. Full details will be published
elsewhere.
quinone
Scheme 1 Summary of the photoreaction of 1. Excitation by light ( > 290
nm) to produce 1* is accompanied by production of 2 in the presence of a
1
2
K. J. van der Merwe, P. S. Steyn, L. Fourie, D. B. Scott and J. J. Theron,
Nature, 1965, 205, 1112.
W. van Walbeek, P. M. Scott, F. S. Thatcher, Can. J. Microbiol., 1968,
sugar. Under anaerobic conditions (N
O yields the hydroquinone 3, which is also produced under reducing
conditions in the presence of O
2 2
, H O) substitution of chlorine by
H
2
1
4, 131.
2
.
3
4
R. R. Marquardt and A. A. Frohlich, J. Anim. Sci., 1992, 70, 3968.
J. C. Seegers, L. H. Bohmer, M. C. Kruger, M. L. Lottering and K. M.
de, Toxicol. Appl. Pharmacol., 1994, 129, 1.
5
Y. Grosse, I. Baudrimont, M. Castegnaro, A.-M. Betbeder, E. E.
Creppy, G. Dirheimer and L. A. Pfohl, Chem.-Biol. Interact., 1995, 95,
HPLC. However, in the presence of calf thymus DNA or
dextrose, photoirradiation of 1 yielded the non-chlorinated
derivative 2.‡ Under anaerobic conditions in the absence of a
sugar, photoreaction of 1 produced the hydroquinone deriva-
1
75.
C. Malaveille, G. Brun and H. Bartsch, Mutation Res., 1994, 307,
41.
6
7
1
tive, OTOH (3: X = OH),§ a finding that we attribute to SRN
displacement21 of the chlorine atom by H
O. Interestingly, 3
was also detected in the presence of O when a reducing agent
sodium ascorbate) was added to the photoreaction. This
1
I. Baudrimont, A.-M. Betbeder, A. Gharbi, L. A. Pfohl, G. Dirheimer
and E. E. Creppy, Toxicology, 1994, 89, 101.
2
2
8 T. Matsumoto, Y. Utsumi, Y. Sakai, K. Toyooka and M. Shibuya,
(
Heterocycles, 1992, 34, 1697.
9
J. C. Quada, Jr., M. J. Levy and S. M. Hecht, J. Am. Chem. Soc., 1993,
15, 12 171.
0 Saito, T. Sakurai, T. Kurimoto and M. Takayama, Tetrahedron Lett.,
994, 35, 4797.
observation suggests that the hydroquinone 3 may have
originated from a reactive quinone precursor, in analogy to
photooxidation of halogenated phenols that yield benzoquinone
1
1
1
22,23
derivatives in the presence of O
2
.
The results of these
1
1
1 J. K. Barton, Science, 1986, 233, 727.
2 P. E. Nielsen, C. Hiort, S. H. S o¨ nnichsen, O. Buchardt, O. Dahl and B.
Norden, J. Am. Chem. Soc., 1992, 114, 4967.
studies are summarized in Scheme 1.
In conclusion, the fungal carcinogen 1 does not facilitate
DNA cleavage alone. However, we have demonstrated that light
is one way to activate 1 to induce DNA damage. The finding
that 2 is a product of 1 photoirradiation in the presence of DNA,
suggests that strand-scission is mediated by H-atom abstraction
13 D. Ly, Y. Kan, B. Armitage and G. B. Schuster, J. Am. Chem. Soc.,
1996, 118, 8747.
14 G. B u¨ chi, K. W. Fowler and A. M. Nadzan, J. Am. Chem. Soc., 1982,
1
04, 544.
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996, 51, 207.
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Acta, 1990, 1036, 78.
1
1
1
from deoxyribose sugars through initial C–Cl bond hom-
1
–10
olysis.8 In the absence of a sugar and O
2
, 1 is converted in
high yield to the hydroquinone derivative, 3. This species is also
formed in the presence of O , provided that a suitable reducing
2
7 D. Hoehler, R. R. Marquardt, A. R. McIntosh and H. Xiao, J. Biol.
Chem., 1996, 271, 27 388.
agent is added to the photoreaction. This result suggests
strongly that photooxidation of 1 produces a reactive quinone
18 J. A. Ardus, I. G. Gillman and R. A. Manderville, Can. J. Chem., in the
press.
19 N. J. Turro, Modern Molecular Photochemistry, University Science
Books, Sausalito, CA, 1991, pp. 589–591.
0 D. Hoehler, R. R. Marquardt, A. R. McIntosh and G. M. Hatch, Biochim.
Biophys. Acta, 1997, 1357, 225.
derivative, a species that may be responsible for the toxin’s
DNA adduction properties.5 Presently, experiments are in
progress to identify the cleavage sites and the mechanistic
pathways for photocleavage. We are also studying the oxidation
of 1 to determine the exact nature of oxidized 1, its chemical
reactivity and lifetime in aqueous buffered media and the
potential of such a species to induce DNA adduction.
We thank Dr Fred W. Perrino (Department of Biochemistry,
Wake Forest University) for the sample of plasmid DNA. This
2
2
2
2
1 J. F. Bunnett, Acc. Chem. Res., 1978, 11, 413.
2 K. David-Oudjehani and P. Boule, New J. Chem., 1995, 19, 199.
3 C. Richard and P. Boule, New J. Chem., 1994, 18, 547.
Received in Corvallis, OR, USA, 17th November 1997, 7/08275D
648
Chem. Commun., 1998