17beta-Estradiol nitration by peroxidase/H2O2/NO2-: a chemical assessment.

Article abstract of DOI:10.1016/j.bmc.2004.03.036

Nitration of 17beta-estradiol by H(2)O(2) and nitrite in the presence of various peroxidases, viz. horseradish peroxidase, lactoperoxidase, and peroxidase-containing homogenates from bovine uteri, was systematically investigated to assess on a chemical basis its potential relevance to the mechanisms of impairment of estrogen functions under oxidative/nitrosative stress conditions. In the presence of excess nitrite 17beta-estradiol reacted smoothly to give 2-nitroestradiol (1), 4-nitroestradiol (2), and 2,4-dinitroestradiol (3). With 10-300 microM estradiol, formation yields of 1-3 were 12-55%, but dropped to 1% or less at lower estrogen concentration, for example, 1 microM, or in plasma as the reaction medium. Time course analysis showed that 2 is the prevalent nitration product under conditions of slow generation of nitrating species, suggesting some regioselectivity for estradiol nitration at C-4, whereas 1 prevails with bolus addition of reagents, due to faster degradation of 2. Competition experiments carried out with (15)NO(2)- showed that 2 is about twice more susceptible to nitration than 1 as determined by (15)N NMR analysis of the resulting 3. The biological effects of 1 and 2 were preliminarily tested on in vitro bovine embryo cultures. When 1 and 2 were substituted to the standard 17beta-estradiol in the oocyte maturation, a significant decrease in both cleavage and blastocyst efficiency was observed in the case of 1 but not 2. Overall, these results suggest that estradiol nitration is a potential pathway of hormonal dysfunction and toxicity but would require elevated estrogen levels of questionable physiological relevance.

Full text of DOI:10.1016/j.bmc.2004.03.036

Bioorganic & Medicinal Chemistry 12 (2004) 2927–2936
ꢀ
17b-Estradiol nitration by peroxidase/H2O2/NO2 : a chemical
assessment
Alessandro Pezzella,a Paola Manini,a Paola Di Donato,b Raffaele Boni,c
Alessandra Napolitano,a Anna Palumbob and Marco d’Ischiaa,*
aDepartment of Organic Chemistry and Biochemistry, University of Naples ‘‘Federico II’’, Complesso Universitario Monte S.Angelo,
Via Cinthia 4, I-80126 Naples, Italy
bLaboratory of Biochemistry and Molecular Biology, Zoological Station ‘‘Anton Dohrn’’, Villa Comunale I-80121 Naples, Italy
cDepartment of Animal Science, University of Basilicata, Campus Macchia Romana, 85100 Potenza, Italy
Received 21January 2004; revised 5 March 2004; accepted 16 March 2004
Available online 22 April 2004
Abstract—Nitration of 17b-estradiol by H2O2 and nitrite in the presence of various peroxidases, viz. horseradish peroxidase, lac-
toperoxidase, and peroxidase-containing homogenates from bovine uteri, was systematically investigated to assess on a chemical
basis its potential relevance to the mechanisms of impairment of estrogen functions under oxidative/nitrosative stress conditions. In
the presence of excess nitrite 17b-estradiol reacted smoothly to give 2-nitroestradiol (1), 4-nitroestradiol (2), and 2,4-dinitroestradiol
(3). With 10–300 lM estradiol, formation yields of 1–3 were 12–55%, but dropped to 1% or less at lower estrogen concentration, for
example, 1 lM, or in plasma as the reaction medium. Time course analysis showed that 2 is the prevalent nitration product under
conditions of slow generation of nitrating species, suggesting some regioselectivity for estradiol nitration at C-4, whereas 1 prevails
with bolus addition of reagents, due to faster degradation of 2. Competition experiments carried out with 15NO2 showed that 2 is
ꢀ
about twice more susceptible to nitration than 1 as determined by 15N NMR analysis of the resulting 3. The biological effects of 1
and 2 were preliminarily tested on in vitro bovine embryo cultures. When 1 and 2 were substituted to the standard 17b-estradiol in
the oocyte maturation, a significant decrease in both cleavage and blastocyst efficiency was observed in the case of 1 but not 2.
Overall, these results suggest that estradiol nitration is a potential pathway of hormonal dysfunction and toxicity but would require
elevated estrogen levels of questionable physiological relevance.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
regarded as the main nitrating agent in vivo, but
increasing evidence indicates that nitrite ions, the main
physiologic metabolite of NO, may also be involved.
Nitration reactions targeted to cellular constituents
contribute to the broad range of geno- and cytotoxic
mechanisms in oxidative stress-based disease states.1 At
loci of inflammation, macrophages, neutrophils, and
other cells of the immune system become activated to
secrete several mediators of the inflammatory cascade
including abnormally high fluxes of nitric oxide (NO).
Under oxidative stress conditions, NO is partly diverted
from its physiological roles via interaction with reactive
oxygen species (e.g., superoxide), leading to aberrant
production of peroxynitrite, nitrogen dioxide, and other
reactive nitrogen species purportedly responsible for
nitration of biomolecules.2–4 Peroxynitrite is commonly
ꢀ
Relatively high levels of NO2 are found in physiolog-
ical fluids (e.g., 0.5–3.6 lM in plasma and up to 210 lM
in saliva)5 increasing dramatically under pathological
conditions.6 Following oxidation, for example, by per-
oxidases such as horseradish peroxidase, lactoperoxi-
dase, myeloperoxidase, and eosinophil peroxidase, or by
Fenton-like reagents nitrite may be converted to the
nitrating agent NO2.3;7–9
The most typical target of nitrating species is protein
tyrosine, giving 3-nitrotyrosine.1;3;7;8 Although tyrosine
nitration may occur in healthy subjects with no apparent
dysfunction in nitrated proteins,10 it has been reported to
alter the functional properties of a variety of enzymes11
and has been implicated in the pathogenesis of a range of
diseases.12 3-Nitrotyrosine per se appears to be involved
in tubulin degradation13;14 and in neurodegeneration.15
Keywords: Nitroestradiols; Nitrite; Peroxidases; Peroxynitrite.
* Corresponding author. Tel.: +39-081674132; fax: +39-081674393;
e-mail: dischia@cds.unina.it
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.036

2928
A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
Catecholamines are another class of phenolic substrates
that are susceptible tꢀo nitration, for example, by the
peroxidase/H2O2/NO2 system, leading to regioselective
conversion to 6-nitrocatecholamines.16;17
The widespread observation of tyrosine nitration by
NO-derived nitrating systems indicates that nitration of
other phenolic biomolecules, such as estrogens, would
be likely in biological systems. Estrogens are responsible
for the development of most secondary sex characteris-
tics in females, and appear also to play important roles
in the nervous system,18 whereby they are ranked among
the neurosteroids. NO synthase (NOS) occur in female
reproductive tract19 and in the central nervous system.20
Elevated levels of NOSs are found in human breast
tumors21 and a correlation has been reported between
tumor grade progression and increased NOS activity in
many cancers,22 although the actual relationship
remains to be assessed.
Figure 1. HPLC elution profileꢀof the products formed by reaction of
estradiol with HRP/H2O2/NO2 system. Estradiol (300 lM) in 0.1M
ꢀ
phosphate buffer pH 7.4 was treated with HRP (1U/mL), NO
(3.0 mM) and H2O2 (600 lM) portionwise over 40 min and the mixture
analyzed at 50 min reaction time (eluant II).
2
Recently, preliminary data on the nitration of 17
b-estradiol by different systems has been reported, as
part of a study on the nitrosation and nitration of the
selective estrogen receptor modulator ꢀraloxifene.23 In
this, horseradish peroxidase/H2O2/NO2 was shown to
be the most effective nitrating system for estradiol. We
report herein a systematic study of the nitration of
estradiol promoted by horseradish and mammalian
peroxidases with a view to assessing the chemical feasi-
bility of the process in vitro and the underlying reaction
pathways. The effects of nitroestradiols on the in vitro
maturation of bovine oocytes were also explored.
identification of the product as 2,4-dinitroestradiol (3).
The structural identity of the product was secured by
comparison with a synthetic sample obtained by HNO3
nitration of estradiol,24;25 as well as by its formation by
ꢀ
separate nitration of 1 or 2 by HRP/H2O2/NO2
.
OH
OH
O2N
HO
HO
OH
2. Results and discussion
NO2
1
2
2.1. Estradiol nitration: isolation of reaction products
Preliminary experiments were aimed at isolating and
characterizing the products formed by nitration of
estradiol. To this aim, estradiol (300 lM) was exposed to
O2N
HO
ꢀ
the horseradish peroxidase (HRP)/H2O2/NO2 system,3
NO2
3
using 600 lM H2O2 and a 10-fold excess nitrite, as well
as to peroxynitrite, an established phenol nitrating agent
in vitro and in vivo.8 In both cases, HPLC analysis
showed the generation of three main products (Fig. 1).
2.2. Kinetic experiments
The products eluted under peaks A and C were identi-
fied as the known 2-nitroestradiol (1) and 4-nitroestra-
diol (2), respectively, by straightforward spectral
analysis and comparison of the chromatographic
behavior with those of synthetic samples.24 The same
products were recently described in preliminary experi-
ments with the HRP/H2O2/NO2ꢀ system as well as in the
reactions of estradiol (0.2 mM) with SIN-1in 40% ace-
tonitrile–phosphate buffer, pH 7.4, and with spermine-
NONOate.23 The third peak (marked as B) gave a
product with a molecular ion peak at m=z 362, sug-
gesting a dinitro derivative. The 1H NMR spectrum
displayed only a single proton in the aromatic region at
In a subsequent series of experiments, the kinetics of
estradiol decay and nitroestradiol formation induced by
various peroxidases were investigated with the substrate
at 300 lM concentration and a 10-fold excess nitrite.
Figure 2 compares the results obtained with a bolus
addition of 600 lM H2O2 (graph A) and using 560 lM
glucose/glucose oxidase as a physiologically relevant
source of H2O2 (graph B) with HRP in the presence of
NO2ꢀ. In both cases, the three main nitration products
were clearly detectable.
ꢀ
In the reaction with the HRP/H2O2/NO2 system,
estradiol consumption was about 85% at 1h reaction
time with an overall yield of nitration products of about
12%. Apparently, the low mass balance was accounted
for by a complex mixture of products (possibly oxida-
1
d 8.11 giving a cross peak in the H, 13C HMQC spec-
trum with a carbon signal at d 123.45. 2D homo- and
heterocorrelated NMR analysis eventually allowed

A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
2929
ꢀ
Table 1. Estradiol and tyrosine nitration by HRP/H2O2/NO2 and
peroxynitrite
300
200
100
25.0
20.0
15.0
10.0
5.0
A
Product yield (lM)b
Substratea
Product
ꢀ
Peroxynitrite HRP/H2O2/NO2
Tyrosine
Estradiol
3-Nitrotyrosine
6.1 1.2
10.4 1.5
1
2
3
.5 2<.40.121
<0.3
3.1 0.5
6.3 0.7
9.0 0.8
a At 300 lM concentration.
0.0
0
b Determined at 60 min reaction time by HPLC analysis as detailed
under Section 4. Data shown are the means of three experi-
ments SD.
0
20 40 60 80 100 120
min
Figure 3 reports data obtained for estradiol nitration
using lactoperoxidase (LPO)/hydrogen peroxide and
tandem glucose/glucose oxidase/LPO as oxidizing sys-
tems under the above conditions.
10.0
8.0
6.0
4.0
2.0
0.0
300
200
100
0
B
As in the case of the HRP-catalyzed reaction, 2 was the
prevailing product under conditions of slow generation
of nitrating species (Fig. 3B), whereas the situation was
reversed with bolus addition of reagents (Fig. 3A).
Figure 4 shows formation of 1 and 2 from 10 lM
estradiol with LPO (1.0 U/mL) and H2O2 (20 lM), as a
function of nitrite concentration. The data, determined
in the early stages of the reaction when nitroestradiol
consumption was not significant, indicated the prefer-
ential formation of 2 over the whole range of nitrite
concentrations. The profile of estradiol consumption
0
100
200
300
min
Figure 2. Time course of ꢀestradiol nitration and formation of 1–3 by
the peroxidase/H2O2/NO2 system. Estradiol (300 lM) was incubated
with HRP (1.0 U/mL), NO2 (3.0 mM) and H2O2 (600 lM) or glucose
(560 lM), glucose oxidase (0.1U/mL) as detailed in Section 4. Data are
the means of three experiments, and SD <5%. Plot A: left axis (dashed
line), consumption of estradiol (ꢁ), and right axis (solid line), forma-
tion of 1 (m), 2 (r), and 3 (j). Plot B: as in Plot A with generation of
H2O2 by the glucose/glucose oxidase system.
ꢀ
300
200
100
0
60
50
40
30
20
10
0
A
tion products) all in very low amounts, which could not
be identified. At 50% substrate ꢀconsumption, 1 prevailed
over 2 in the HRP/H2O2/NO2 reaction, whereas with
the glucose/glucose oxidase system the products were
formed in comparable amounts.
ꢀ
0
20 40 60 80 100 120
The reaction with the HRP/H2O2/NO2 system was also
min
run with the substrate at 1.0 lM concentration and all
other reagents maintained at the same molar ratios. At
2 h reaction time estradiol consumption was about 60%
and 1 and 3 were formed in about 0.5% yield, whereas 2
was not detectable.
300
200
100
0
10.0
B
8.0
6.0
4.0
2.0
0.0
To assess the feasibility of estradiol nitration in a
physiological fluid, the estrogen was reacted at 300 lM
concentration with the HRP/H2O2/NO2 system using
human plasma as the medium. At 2 h reaction time
about 50% substrate consumption was observed and
both 1 and 3 were formed at ca 0.3 and 0.4 lM con-
centration, respectively, whereas 2 was below detection
limits.
ꢀ
0
100
200
300
min
Figure 3. Time courꢀse of estradiol nitration and formation of 1–3 by
the LPO/H2O2/NO2 system. Estradiol nitration was carried out as
described in the legend to Figure 2. Data are the means of three
experiments, and SD <5%. Plot A: left axis (dashed line), consumption
of estradiol (ꢁ), and right axis (solid line), formation of 1 (m), 2 (r)
and 3 (j). Plot B: as in Plot A with generation of H2O2 by the glucose/
glucose oxidase system.
The relative susceptibility of estradiol and tyrosine to
ꢀ
nitration by HRP/H2O2/NO2 and peroxynitrite was
compared under the reported conditions.3 The results in
Table 1indicated that estradiol is at least as susceptible
to nitration as tyrosine.

2930
A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
30.0
25.0
20.0
15.0
10.0
5.0
5.0
4.0
3.0
2.0
1.0
0.0
0.0
1
2
3
0
20
40
60
µ
80
100
-
[NO2 ] ( M)
Figure 5. Yields of nitroestradiols obtained from estradiol by action of
different peroxidase-containing fractions from bovine uterus. Estradiol
(300 lM) was incubated with SF (gray bars) or MF (open bars) frac-
tions in the presence of 32 mM CaCl2 and in the presence of hydrogen
peroxide (600 lM) and nitrite (3.0 mM) in 0.05 M phosphate buffer
(pH 7.2). Shown are the mean + SD values for three separate experi-
ments.
Figure 4. Formation of 1 and 2 and estradiol consumption as a
function of nitrite concentration. Estradiol (10 lM) was incubated with
LPO (1U/mL) and H2O2 (20 lM) and the mixture was analyzed at 4 h
reaction time as detailed in Section 4. Data are the means of three
experiments, and SD <10%. Solid line: 1 (m), 2 (r); dashed line:
estradiol (ꢁ).
and mass balance data suggest that estradiol oxidation is
somewhat inhibited at the higher nitrite concentrations,
whereas at low nitrite concentrations other reaction
pathways are operative in addition to nitration. In the
presence of 10-fold excess nitrite, an about 60% con-
sumption of estradiol is associated with formation of
nitroestradiols 1 and 2 in around 80% overall yield
based on reacted material. On the other hand, at lower
nitrite to substrate ratios, for example, in the presence of
20 lM nitrite, estradiol is completely consumed with
formation of nitroestradiols in less than 20% yield.
no detectable nitroestradiol formation. Under the reac-
tion conditions investigated nitroestradiol yields
obtained with the MF fraction were significantly higher
than those of the SF fraction. Product 1 invariably pre-
vailed over the isomer 2 while dinitroestradiol 3 was
formed to a lower extent.
2.3. Mechanistic issues
Ample experimental evidence indicates that the active
forms of HRP as well as of mammalian peroxidase
lactoperoxidase and myeloperoxidase are competent in
The data in Figures 2–4 were suggestive of a different
susceptibility to decomposition of 1 and 2. A competi-
tion experiment was therefore performed, in which an
equimolar mixture of 1 and 2 (10 lM each) was reacted
ꢀ
bringing about oxidation of NO2 to NO2.3;26 The
ability of peroxidase including HRP and uterine per-
oxidase to effect oxidation of estradiol is also docu-
mented27 and it can therefore be expected that estradiol
may compete with nitrite for oxidation by peroxidase
compound I and II under the reaction conditions
examined. Such a competition may account for the
observed decrease in estradiol consumption with increasing
nitrite/estradiol molar ratios (Fig. 4). Nitroestradiols 1
and 2 would then result from coupling of the semiqui-
none radical I arising from one electron oxidation of
estradiol, with NO2 (Scheme 1). Such a mechanism is
similar to that proposed for the haem-peroxidase gen-
eration of 3-nitrotyrosine by cooperative oxidation of
nitrite and tyrosine.1 Given the susceptibility of mono-
phenolic compounds to undergo radical nitration by
NO2,28 generation of I from estradiol brought about by
NO2 followed by radical coupling should be also con-
sidered a feasible route to nitroestradiols 1–3. In the case
of tyrosine nitration, the higher rate of reaction of
ꢀ
with the LPO/H2O2/NO2 system. The results showed
that 2 was far more reactive to oxidation than 1. At
15 min reaction time, the consumption of 1 and 2 was
about 40% and 80%, respectively.
In another series of experiments, estradiol nitration was
investigated in crude tissue homogenates from bovine
uteri. Two cellular fractions were obtained, which con-
tained peroxidase activity: a soluble fraction (SF) and a
microsomal fraction (MF) (see Section 4). The results in
Figure 5 indicated that both fractions contain significant
levels of peroxidase capable of mediating conversion of
estradiol to nitroestradiols 1–3 in the presence of H2O2
and nitrite. For comparative purposes, in these experi-
ments substrate and reagent concentrations were iden-
tical with those adopted in the case of the HRP and
LPO-induced reactions.
tyrosine wꢀit1h ꢀ1peroxidase compound II at pH 7
29
(1.6 · 104 M
s
)
)
with respect to that of nitrite
suggests that the enzyme can bring
Omission of nitrite in the incubation mixture resulted in
substrate consumption without appreciable formation of
nitroestradiols. In the absence of Ca2þ the peroxidase
containing fractions proved unable to mediate nitroestra-
diol formation, supporting the role of peroxidase in the
nitrite-dependent nitration. Omission of H2O2 resulted in
30
(5.5 · 102 Mꢀ1 sꢀ1
about oxidation of both substrates. The lack of kinetic
data for the reaction of estradiol with peroxidase does
not allow to extend this conclusion to the case of
estradiol nitration.

A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
2931
OH
OH
OH
Peroxidase
17β-estradiol
.
.
O
O
O
.
I
O
NO2
-
NO2
FeIV
-
NO2
OH
Peroxidase
OH
compound II
NO2
O2N
O
.
+
+
FeIV
FeIII
H
Peroxidase
Peroxidase
native form
O
O
NO2
compound I
H
2
1
H2O
H2O2
ꢀ
Scheme 1. Proposed mechanism of formation of nitroestradiols 1 and 2 by the peroxidase/H2O2/NO2 system.
Data in Figures 2–4 indicate that 2 prevails under con-
ditions of slow generation of the oxidizing species, for
example, with the tandem glucose/glucose oxidase–per-
oxidase system, at relatively low substrate concentration
or in the early stages of the reaction, whereas 1 is the
main product with bolus addition of reagents or when-
ever the oxidizing/nitrating species are in large excess or
are generated at rapid rate. These lines of evidence
indicate that 2 is the prevalent nitration product but is
oxidized to a greater extent than 1 under forcing reac-
tion conditions.
Calculation of Mulliken atomic spin densities for the
geometry optimized structure of estradiol phenoxyl
radical in vacuo and with the polarizable continuum
Figure 6. Energy minimized structures of nitroketones formed by
coupling of estradiol phenoxy radical with NO2 at C-2 and C-4
position.32
model,31 to keep into account possible solvent effects,
gave values of 0.340 and 0.300 at C-2 and of 0.237 and
0.296 at C-4, respectively, which were not compatible
with the apparent regioselectivity at C-4 for estradiol
nitration. Likewise, inspection of SOMO isosurfaces31
did not reveal appreciable differences between the 2- and
4-positions. Interestingly, however, a comparison of the
energy minimized conformations32 for each of the a and
b stereoisomers of the 2- and 4-nitroketone arising by
free radical coupling of estradiol phenoxyl radical with
NO2 (see Scheme 1) revealed that the stereoisomers of
the 4-nitroketone were more stable than those of the
2-nitroketone. In particular, the most stable conformer
of the 4-nitro series, viz. the 4b-nitro conformer having
the O–N–C–H dihedral angle of 0.3ꢁ, displayed an
energy 1.03 kcal/mol lower than that of the most stable
conformer of the 2-nitro series, viz. the 2a-nitro con-
former having the O–N–C–H dihedral angle of )26ꢁ
(Fig. 6). Whether this has any bearing on the regio-
chemical outcome of estradiol nitration is difficult to
assess at the present level of analysis.
tion, in the presence of nitrite (10 M equiv), HRP (2.0 U/
mL) and H2O2 (2 M equiv), and at 30 min reaction time,
2 was consumed to a higher extent than 1 (30% vs 20%)
leading to 3 in 16% yield versus 10% yield in the case of
1. Furthermore, a competition experiment was per-
formed in which an equimolar mixture of the nitro-
estradiols was oxidized by peroxidase/H2O2 in the
presence of an excess of 15NO2ꢀ. The reaction was hal-
ted at 50 min, and the resulting 3 was isolated and
1
subjected to NMR analysis (Scheme 2). The H, 15N
HMBC spectrum (Fig. 7) indicated two distinct 15N
signals at d 369.5 and 369.8, the former due to labeled
nitro group at C-2 (well discernible J correlation with
3
the aromatic proton resonance at d 8.11 (H-1)) and the
latter to the labeled nitro group at C-4. The assignment
was confirmed by separate preparation and analysis of
15
the mono N-labeled derivatives of 3. The integrated
The relative susceptibility of 1 and 2 to undergo further
nitration to give 3 was then investigated. Under identical
conditions, that is with substrate at 300 lM concentra-
areas of the nitrogen signals, under conditions ensuring
complete relaxation of the nuclei, were in the approxi-
mate ratio of 2:1, denoting a prevalent location of the

2932
A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
15
O2N
80
*
*
60
HRP/H2O2
Na15NO2
HO
NO2
1 +2
40
+
O2N
HO
**
20
**
15NO2
0
1
2
Control
3
Figure 8. Effect of nitroestradiols 1 and 2 in comparison with estradiol
(control) on the efficiency of cleavage and blastocyst production during
the in vitro maturation of cattle oocytes. Shown are the means of three
replicates + SD. Gray bars: cleavage; open bars: blastocysts. Con-
centration of estradiols was 1 lg/mL. COC used for the experiments
with 1 or 2 or 17b-estradiol were 76, 94, and 85 in the order. * P<0.05;
** P<0.01.
Scheme 2. Formation of 15N labeled 3 by reaction of 1 and 2 with
peroxidase/H2O2 in the presence of 15NO2
.
ꢀ
embryonic development. 17b-Estradiol is a component
naturally present in the pre-ovulatory follicle and is
involved in the oocyte meiotic competence.34 As shown
in Figure 8, substitution of 17b-estradiol with 1 (1 lg/
mL) resulted in significant inhibition of embryo devel-
opment, as evaluated by cleavage and blastocyst pro-
duction.
Addition of 17b-estradiol (1 lg/mL) to incubation mix-
tures containing 1 lg/mL of 1 partially counteracted the
inhibitory effects of 1 (data not shown). In all cases
nitroestradiols 1 and 2 were recovered apparently
unchanged from the incubation media.
Figure 7. Selected region of the 15N NMR spectrum of 3 obtained by
reaction of equimolar 1 and 2 with HRP/H2O2 in the presence of
15NO2ꢀ. The integrated areas of 15N resonances are reported below the
scale. Inset: shown is the correlation peak between the proton reso-
3. Conclusions and biological relevance
Aim of the present study was to address whether aro-
matic nitration may be a potential conversion route of
estradiol under oxidative and nitrosative stress condi-
tions. Based on these results and previous23 lines of
evidence, peroxidase/H2O2/nitrite may be a candidate
system to induce estradiol nitration. In female repro-
ductive apparatus, peroxidase is present in the uterus,35
and it is relevant that the uterine peroxidase has been
found to be competent to bring about estradiol nitration
in crude homogenates.27 The observed formation of a
dinitroderivative, along with two nitroderivatives,
denoted a chemical susceptibility to nitration at least
comparable to that of tyrosine, the nitratable biological
target par excellence.
nance at 8.11 ppm and the 15N resonance at 369.49 ppm in the 1H, 15
N
HMBC spectrum.
label on the 2-position due to the faster formation of the
phenoxyl radical from 2.
A useful interpretative basis of the higher reactivity
exhibited by 2 with respect to 1 is provided by the
structural properties of the compounds.33 The crystal
structure of 1 indicated that the nitro-oxygens are
coplanar to the aromatic ring, thus ensuring efficient
electron conjugation. In contrast, in 2 the nitro group is
significantly twisted out of the plane of the aromatic ring
and is thus nonconjugating. This entails a decreased
influence of the nitro group on the electronic properties
of the phenol ring making the reactivity of 2 somewhat
closer to that of the parent estrogen.
This notwithstanding, the balance of available data fails
to meet expectations of substantial estradiol nitration
in vivo, mainly because the reaction becomes significant
at substrate concentrations in the lM range, that is far
from physiological levels. Moreover, tyrosine, both free
and protein bound, may be a more abundant target for
nitrating species in vivo. However, the possibility that
some nitration may occur under acute oxidative/nitro-
sative stress conditions accompanying inflammation and
with abnormally high estrogen levels, for example, after
administration for therapeutic purposes, should not be
overlooked.
2.4. Effects of nitroestradiols on in vitro oocyte matura-
tion
In a final set of experiments, the effect of 1 and 2 on
bovine oocyte maturation in vitro was investigated, to
determine the possible influence of nitroestradiols on

A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
2933
In addition to the chemical elucidation of reaction
4.3. Methods
pathways, the results of the present study have provided
preliminary evidence for a potential inhibitory effect of 1
and, to a lesser extent, 2 on oocyte cleavage and
embryonic development. Differential biological effects of
isomeric nitroestradiols have already been noted and are
confirmed in the present study. For example, they dis-
play different receptor binding affinities:33 1 bound to
estrogen receptors with 1/1000th the affinity of estradiol
and was inefficient in gene stimulation, whereas 2 dis-
played a relative binding affinity 40-fold greater than
that of the 2-nitro derivative. Thus nitroestradiols,
especially 1, appear to be valuable candidate substances
to modulate estradiol actions. Product 1 moreover is an
efficient inhibitor of steroid alcohol sulfotransferase, an
enzyme catalyzing metabolically important sulfation of
steroids.36 The toxicity data presented here point to
nitroestradiols as potentially useful compounds for
investigative purposes and could stimulate their search
as novel endogenous modulators of receptor functions.
A better understanding of their biological properties
may open up new avenues for the therapy of malignant
diseases, infertility, and CNS disorders.
UV and IR spectra were performed using a diode array
1
and spectrophotometer. H, 13C spectra were recorded
1
at 400.1and 100.6 MHz, respectively. 1H, H COSY,
1
1
1H, 13C HMQC, H, 13C HMBC, and H, 15N HMBC
experiments were run at 400.1MHz using standard pulse
programs from the Bruker library. For 15N NMR
experiments delay values up to 10 s were used. 15N
chemical shifts are relative to NH3 (liquid, 298 K,
0.0 ppm). For EIMS spectra samples were ionized with a
70 eV beam, and the source was taken at 230 ꢁC. Main
fragmentation peaks are reported with their relative
intensities (percent values are in brackets). Analytical
and preparative TLC analyses were performed on F254
0.25 and 0.5 mm silica gel plates or high performance
TLC (HPTLC) using cyclohexane/ethyl acetate 60:40,
eluant A. Column chromatography was performed
using 60–230 mesh silica gel. Analytical and preparative
HPLC was carried out with an instrument equipped
with a UV detector set at 254 or 280 nm. Octadecylsilane
coated columns, 4.6 · 250 mm (5 lM particle size)
or 22 · 250 mm (10 lM particle size) were used for
analytical or preparative runs at a flow rate of 1.3 or
15 mL/min, respectively. Elution conditions: 1% acetic
acid/acetonitrile 40:60 (eluant I); 1% acetic acid/aceto-
nitrile 90:10 (solvent A), acetonitrile (solvent B),
from 20% to 55% solvent B gradient, 30 min, 55%
solvent B, 10 min (eluant II); 0.05 M phosphate
buffer, pH 3.0/methanol 90:10 (eluant III); 0.5%
acetic acid/acetonitrile 60:40 (solvent A), acetonitrile
(solvent B); 0% solvent B, 20 min, from 0% to
25% solvent B gradient, 10 min, 25% solvent B, 20 min
(eluant IV).
4. Experimental
4.1. Materials
17b-Estradiol, tyrosine, 3-nitrotyrosine,
D-glucose,
potassium thiocyanate, ferrous sulfate heptahydrate,
nonstabilized hydrogen peroxide (35% solution in
water), [15N]NaNO2 (>99% isotopic enrichment) were
from Aldrich Chemie. Horseradish peroxidase (HRP)
(donor: H2O2 oxidoreductase, EC 1.11.1.7) type II (167
pyrogallol U/mg, RZ E430=E275 ¼ 2:0), lactoperoxidase
(LPO) from bovine milk (80 pyrogallol U/mg RZ
Production of H2O2 by glucose oxidase was quantitated
by oxidation of Fe (II) and formation of Fe (III)-thio-
cyanate complex.3 Nitrite was determined spectropho-
tometrically at 538 nm using the Griess reagent (1%
sulfanylamide, 0.1% naphthylethylenediamine in 2.5%
phosphoric acid).38
E
412=E280 ¼ 0:76), glucose oxidase from Aspergillus niger
(b- -glucose: oxygen 1-oxidoreductase EC 1.1.3.4) type
D
II (20,000 U/mg) were obtained from Sigma and used
without further purification. Peroxynitrite was prepared
from sodium nitrite and hydrogen peroxide in acid using
a quenched flow reactor as described,37 and its concen-
tration
was
determined
spectrophotometrically
4.4. Synthesis of nitroestradiols 1–3
(e ¼ 1:67 mMꢀ1 cmꢀ1 at 302 nm). Plasma samples from
freshly drawn heparinized blood was deep frozen at
)70 ꢁC until use.
The reaction was carried out by procedures previously
reported24;25 with modifications. In brief, to a solution of
1 (200 mg, 0.74 mmol) in glacial acetic acid (20 mL)
taken at 70 ꢁC in a water bath concentrated nitric acid
(40 lL) was added under stirring. The mixture was
allowed to stand for 18 h at rt, diluted with H2O and
extracted with ethyl acetate (3 · 10 mL). The organic
layers dried over sodium sulfate were taken to dryness
and the residue fractionated by silica gel chromato-
graphy (1.0 · 50 cm column) using cyclohexane/ethyl
acetate (70:30 to 40:60 gradient mixtures) as the
eluant to afford pure 2-nitroestradiol (1) (70 mg, 30%
yield), 4-nitroestradiol (2) (59 mg, 25% yield). When
nitration reaction was carried out as above but
using higher amounts of HNO3 (160 lL) work up and
column chromatography of the reaction mixture affor-
ded as the main product 2,4-dinitroestradiol (3) (30 mg,
12% yield).
4.2. Animals
The uterus of 30 cycling 14–18 months old heifers were
collected at abattoir at different times within a three
month period (Sept–Dec) and promptly stored into a
thermal bag at 4 ꢁC. At the arrival to the laboratory,
within 3 h from collection, the organs were layered on
crushed ice and washed with saline solution at 4 ꢁC. The
uterine bodies and horns were opened along their length
to remove endometrium slices. Pools of endometrium
slices were stored at )80 ꢁC. Ovaries from slaughtered
cows were collected from the abattoir and transported in
a thermal bag at 27–30 ꢁC to the laboratory within 3–4 h
of collection. The laboratory temperature was 30 ꢁC.

2934
A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
4.5. 2-Nitroestradiol (1)33
bands, which were found to consist of pure 1 (12 mg,
10% yield), 2 (5 mg, 4% yield) and 3 (10 mg, 8% yield). In
separate experiments 1 or 2 were exposed to the HRP/
H2O2/NO2 system under the above conditions and the
reaction course was monitored by HPLC analysis (elu-
ant I or II).
(Rf 0.45, eluant A; tR 39 min, eluant I); dH (acetone-d6)
0.74 (3H, s), 1.11–1.52 (7H, m), 1.65 (1H, m), 1.85–1.96
(3H, m), 2.12 (1H, m), 2.29 (1H, m), 2.86 (2H, m), 3.64
(1H, m), 6.83 (1H, s), 7.90 (1H, s), 10.21 (1H, s); dC
(acetone-d6) 11.69 (CH3), 23.88 (CH2), 27.08 (CH2),
27.50 (CH2), 31.12 (CH2), 31.78 (CH2), 37.56 (CH2),
39.33 (CH), 44.11 (C), 44.42 (CH), 51.07 (CH),
81.84 (CH), 119.86 (CH), 122.22 (CH), 133.06 (C),
134.85 (C), 150.11 (C), 153.37 (C). EIMS m=z 317
(Mþ, 97), 287 (15), 271 (10), 258 (100). HREIMS
calcd mass for C18H23NO4 317.1627 Da, found m=z
317.1618 Da.
ꢀ
4.9. Oxidation of 1 and 2 in the presence of nitrite ions
A solution of the appropriate nitroestradiol (5 mg,
0.016 mmol) in methanol (1 mL) was added to 0.1 M
phosphate buffer, pH 7.4 (52 mL) and treated with
nitrite ions (11 mg, 0.16 mmol) and HRP (2.0 U/mL)
under vigorous stirring. Hydrogen peroxide was added
in 4 aliquots (0.008 mmol each) over 10 min. Aliquots of
the reaction mixture were periodically withdrawn,
extracted with ethyl acetate and analyzed by HPLC
using eluant I. In either case 2,4-dinitroestradiol (3) was
the only detectable reaction product. Identification and
quantitation of reaction products was carried out by
comparing retention times and integrated peak areas
with external calibration curves for authentic samples.
At 30 min reaction time consumption of 2 (tR 6 min,
eluant I) or 1 (tR 17 min, eluant I) was 30% and 20%,
respectively, and formation yields of 3 (tR 12 min, eluant
I) were 16% and 10%, respectively.
4.6. 4-Nitroestradiol (2)33
(Rf 0.22, eluant A; tR 28 min, eluant I); dH (acetone-d6)
0.81 (3H, s), 1.20–1.52 (7H, m), 1.70 (1H, m), 1.80–2.20
(3H, m), 2.23 (1H, m), 2.32 (1H, m), 2.74 (2H, m), 3.67
(1H, m), 6.94 (1H, d, J ¼ 8:8 Hz), 7.35 (1H, d,
J ¼ 8:8 Hz), 9.30 (1H, br s); dC (acetone-d6) 11.69 (CH3),
23.73 (CH2), 24.94 (CH2), 27.01(CH 2), 27.23 (CH2),
30.98 (CH2), 37.60 (CH2), 39.06 (CH), 44.01(C), 44.74
(CH), 50.67 (CH), 81.79 (CH), 115.32 (CH), 128.89
(CH), 129.44 (C), 133.41 (C), 144.22 (C), 147.42 (C).
EIMS m=z 317 (Mþ, 100), 300 (73), 287 (20), 258 (79).
HREIMS calcd mass for C18H23NO4 317.1627 Da,
found m=z 317.1599 Da.
4.10. 15N labeling experiments
An equimolar mixture of 1 and 2 (50 mg each,
0.15 mmol) dissolved in methanol (10 mL) was added to
0.1M phosphate buffer, pH 7.4 (500 mL) and the solu-
tion was treated with [15N] NaNO2 (1.5 mmol) and
peroxidase (1.0 U/mL). Hydrogen peroxide was added
portionwise up to 0.30 mmol final concentration within
40 min. The reaction course was followed by HPLC
(eluant II). At 50 min reaction time, when HPLC anal-
ysis revealed formation of 3 but with residual starting
materials, the mixture was extracted with ethyl acetate
(3 · 200 mL) and the combined organic layers were dried
over sodium sulfate and taken to dryness. The residue
was fractionated by PLC (eluant A) to afford labeled 3
(14 mg) dN (acetone-d6) 369.49, 369.82; EIMS: m=z 363
(Mþ, 30), 317 (Mþ–15NO2, 46), 258 (Mþ–15NO2–C3H7O,
100). In other experiments 1 or 2 were treated separately
with [15N] NaNO2 and labeled 3 was isolated by frac-
tionation of the reaction mixture as above.
4.7. 2,4-Dinitroestradiol (3)25
(Rf 0.28 eluant A; tR 36 min eluant I); dH (acetone-d6)
0.80 (3H, s), 1.21–1.45 (7H, m), 1.70 (1H, m), 1.93–2.04
(3H, m), 2.25 (1H, m), 2.36 (1H, m), 2.78 (2H, m), 3.70
(1H, m), 8.11 (1H, s); dC (acetone-d6) 11.36 (CH3), 23.51
(CH2), 25.19 (CH2), 26.37 (CH2), 26.83 (CH2), 30.86
(CH2), 37.19 (CH2), 38.52 (CH), 43.81(C), 44.02 (CH),
50.44 (CH), 81.47 (CH), 123.45 (CH), 133.70 (C), 133.81
(C), 138.72 (C), 145.82 (C), 148.81 (C). EIMS m=z 362
(Mþ, 6), 332 (3), 317 (62), 258 (100). HREIMS calcd
mass for C18H22N2O6 362.1478 Da, found m=z
362.1455 Da.
4.8. Reaction of estradiol with HRP/H2O2/NaNO2:
isolation of 1–3
A solution of estradiol (100 mg, 0.37 mmol) in methanol
(20 mL) was added to 0.1M phosphate buffer pH 7.4
(1.2 L). The mixture was treated under vigorous stirring
at room temperature with sodium nitrite (255 mg,
3.7 mmol) and HRP (1U/mL). Hydrogen peroxide
(10%) was added in 8 aliquots (0.09 mmol each) over
40 min. The reaction course was followed by HPLC
analysis (eluant I or II). After 50 min the reaction was
halted by addition of Na2S2O5 (70 mg, 0.37 mmol) and
the mixture was extracted with ethyl acetate
(3 · 500 mL). The combined organic layers were dried
over sodium sulfate and taken to dryness. The residue
was fractionated by PLC (eluant A) to afford three main
4.11. Kinetic experiments
To solutions of estradiol (300 lM) in 0.1M phosphate
buffer pH 7.4 in a water bath thermostated at 37 ꢁC,
sodium nitrite (3.0 mM) and HRP or LPO (1.0 U/mL
final concentration) were added under vigorous stirring.
Hydrogen peroxide was added portionwise over 10 min
up to 600 lM final concentration. Aliquots of the reac-
tion mixture were periodically withdrawn, treated with a
solution of Na2S2O5 in water (600 lM final concentra-
tion) to terminate oxidation, extracted with ethyl ace-
tate, and analyzed by HPLC using eluant I or II. Similar

A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
2935
experiments were performed in which: (i) the substrate
was at 1 lM; (ii) H2O2 was generated in situ using
up in methanol for HPLC analysis (eluant IV). Identi-
fication and quantitation of nitroestradiols were carried
out as described above.
D
-glucose (560 lM) and glucose oxidase (0.1U/mL, final
concentration); (iii) the substrate was at 10 lM with
nitrite varying in the range 5–100 lM. In other experi-
ments peroxynitrite was added to estradiol (300 lM) in
0.1M phosphate buffer pH 7.4 portionwise at 5 min
intervals up to 300 lM concentration. Aliquots of the
reaction mixture were analyzed by HPLC (eluant I)
following work up as above. When estradiol nitration
was carried out in plasma as the medium, a solution of
estradiol in methanol (20 lL) was added to plasma
(2 mL) up to 300 lM concentration, followed by sodium
nitrite (3.0 mM) and HRP (1.0 U/mL) and the mixture
was taken at 37 ꢁC. Hydrogen peroxide was added
portionwise over 10 min up to 1.20 mM final concen-
tration. At 2 h reaction time the mixture was worked up
as above. In all experiments, identification and quanti-
tation of reaction products were carried out by com-
paring retention times and integrated peak areas with
external calibration curves for authentic samples. All
experiments were run at least in triplicate.
4.13. Tyrosine nitration
A solution of tyrosine (300 lM) in 0.1M phosphate
buffer pH 7.4 was treated sequentially with HRP (1U/
mL), sodium nitrite (3.0 mM) and hydrogen peroxide
(600 lM) as described above for estradiol or with
peroxynitrite portionwise up to 300 lM concen-
tration. Aliquots of the reaction mixture were periodi-
cally withdrawn, treated with Na2S2O5 (300 lM
final concentratiꢀon) in the case of the reaction with
HRP/H2O2/NO2 system and analyzed by HPLC (elu-
ant III) for identification and quantitation of 3-
nitrotyrosine.
4.14. Effect of 1 and 2 on in vitro maturation of cattle
oocytes
In vitro production of cattle embryos was performed by
the procedure previously described.40 Immature oocytes
were collected from 2 to 8 mm follicles by an 18-G
needle under controlled pressure (50–70 mm Hg).
Cumulus-oocyte complexes (COC) were isolated from
the follicular fluid and washed three times with tissue
culture medium (TCM199) supplemented with 0.05%
polyvinylalcohol and 10 mM HEPES. The COC were
transferred into the maturation medium within 4-well
plates (Nunclon, Nunc, DK) (30 lL/COC) and left in
an incubator at 39.0 ꢁC in 5% CO2 humidified air for
24 h.
4.12. Uterine peroxidase
Crude uterine peroxidases were extracted as previously
reported.39 Briefly, endometrium from uterine horns was
finely minced, frozen on dry ice–acetone, reduced to a
fine powder, and homogenized in ice cold 0.01M Tris–
HCl pH 7.2 (20% w/v), using a glass/glass homogenizer.
Homogenates were centrifuged (Beckman, Model L8-
70M) at 40,000ꢁg for 30 min, at 4 ꢁC. The supernatants
represented the soluble fraction (SF). Pellets were
homogenized again in ice cold 0.01M Tris–HCl pH 7.2
(2 mL) containing 0.5 M CaCl2 and centrifuged at
100,000ꢁg for 30 min at 4 ꢁC; the supernatants collected
at this stage represented the calcium-extractable mi-
crosomial fraction (MF).
Maturation medium was TCM199 supplemented with
10% fetal calf serum and 10 IU/mL luteinizing hormone
and 0.1IU/mL follicle stimulating hormone. This
medium was further supplemented with either 17b-
estradiol (1 lg/mL) or 1 (1 lg/mL) or 2 (1 lg/mL).
Peroxidase activity of either SF and MF fractions was
determined by the o-phenylenediamine assay as follows.
The incubation mixture contained 154 mM sodium cit-
rate buffer, pH 5 (650 lL), 75 mM o-phenylenediamine
(250 lL), 100 mM H2O2 (50 lL) and a suitable amount
of the enzyme. SF and MF were adjusted to 0.25 M
CaCl2 before assay. The reaction course was followed
spectrophotometrically by recording the absorbance at
496 nm at 37 ꢁC. One unit was defined as the amount of
enzyme required to produce one unit increase of the
absorbance under the assay conditions. SF fraction,
diluted 1:3 (v/v) with 0.05 M sodium phosphate buffer
(pH 7.2), was incubated at 37 ꢁC with estradiol (300 lM)
in the presence of 32 mM CaCl2. The reaction was
started by addition of H2O2 (600 lM) and sodium nitrite
(3.0 mM). Incubation with MF fraction diluted 1:3 (v/v)
with 0.05 M sodium phosphate (pH 7.2) was carried out
under the same conditions. Other experiments were
carried out as above without addition of CaCl2 or
nitrite. In all cases, after 2 h, reaction mixtures were
extracted three times with ethyl acetate, and the com-
bined organic layers, dried over anhydrous sodium sul-
fate, were evaporated to dryness. The residue was taken
After maturation, COCs were freed from the cumulus
cells by vortexing for 3 min and parthenogenetically
activated by 5 min exposure to 7.5 lM Ca-ionophore,
A23187, in Fert-TALP medium. The oocytes were then
transferred in culture dishes (Nunclon, Nunc, DK)
containing Fert-TALP medium supplemented with
2.5 mM 6-dimethylaminopurine and incubated for 3.5 h.
Finally, the oocytes were transferred in synthetic ovi-
ductal fluid supplemented with essential and nonessen-
tial amino acids and bovine serum albumin
(SOFaaBSA), covered with embryo-tested oil and cul-
tured under controlled gaseous environment (5% CO2,
7% O2, 88% N2) for 8 days post-activation for blastocyst
development. Culture plates were changed every 2 days.
The recovery of 1 or 2 in the incubation media was
determined by ethyl acetate extraction followed by
HPLC analysis (eluant I).
The developmental potential of cattle oocytes exposed
either to 1 or 2 or control (17b-estradiol), measured
as cleavage and blastocyst rates, was compared by
ANOVA after arcsine transformation.

2936
A.Pezzella et al./ Bioorg.Med.Chem.12 (2004) 2927–2936
Palumbo, A.; Napolitano, A.; Barone, P.; d’Ischia, M.
Chem.Res.Toxicol. 1999, 12, 1213.
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This study was carried out in the frame of the MIUR
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the Centro Interdipartimentale di Metodologie Chimico
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