Effect of metal ions on the stable adduct formation of 16α- hydroxyestrone with a primary amine via the Heyns rearrangement

Article abstract of DOI:10.1016/S0039-128X(98)00118-4

16α-Hydroxyestrone (16α-OHE₁), one of the major estrogen metabolites in humans that may plays a role in cell transformation, has been found to form stable adducts with nuclear proteins. The mechanism for the formation of a stable covalent adduct of 16α- OHE₁ with protein has been postulated via the Heyns rearrangement after Schiff base formation. The Heyns rearrangement on the steroidal D-ring α-hydroxyimine was investigated using 17-(2- methoxyethylimino)estra-1,3,5(10)-triene-3,16α-diol as a model intermediate. Rates of the Heyns rearrangement and hydrolysis of the steroidal α- hydroxyimine were determined by a high-performance liquid chromatography (HPLC) simultaneously. The Heyns rearrangement was demonstrated to be optimum at pH 6.2 and the reaction rate at physiological pH, 7.3-7.5, was more than 90% of that at the optimum pH. On the other hand, modulator(s) to the reactions were also examined. According to our previous finding of the proton-mediated mechanism of the Heyns rearrangement, the effects of cationic metal ions on the reactions were examined with 29 metal chlorides. Five metal ions, Pt⁴⁺, Cu²⁺, Ni²⁺, Co²⁺, and Mn²⁺, suppressed the formation of Heyns product significantly while Fe²⁺, Y³⁺, Gd³⁺, and Er³⁺ slightly increased it. The suppression rate was synergistically enhanced by the combination of Pt⁴⁺ with Co²⁺, Cu²⁺, or Ni²⁺. These results suggest the five metal ions, Pt⁴⁺, Cu²⁺, Ni²⁺, Co²⁺, and Mn²⁺, reduce the formation of the Heyns product in vivo and, therefore, would be useful tools to clarify the implication of the stable adduct formation of 16α-OHE₁ with protein.

Full text of DOI:10.1016/S0039-128X(98)00118-4

Steroids 64 (1999) 252–258
Effect of metal ions on the stable adduct formation of 16␣-
hydroxyestrone with a primary amine via the Heyns rearrangement
Shinichi Miyairi*, Kaoru Maeda, Tomoyuki Oe, Toyoaki Kato, Akira Naganuma
Department of Molecular and Biochemical Toxicology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
Received 23 September 1998; accepted 11 November 1998
Abstract
16␣-Hydroxyestrone (16␣-OHE1), one of the major estrogen metabolites in humans that may plays a role in cell transformation, has been
found to form stable adducts with nuclear proteins. The mechanism for the formation of a stable covalent adduct of 16␣- OHE1 with protein
has been postulated via the Heyns rearrangement after Schiff base formation. The Heyns rearrangement on the steroidal D-ring ␣-hy-
droxyimine was investigated using 17-(2-methoxyethylimino)estra-1,3,5(10)-triene-3,16␣-diol as a model intermediate. Rates of the Heyns
rearrangement and hydrolysis of the steroidal ␣-hydroxyimine were determined by a high-performance liquid chromatography (HPLC)
simultaneously. The Heyns rearrangement was demonstrated to be optimum at pH 6.2 and the reaction rate at physiological pH, 7.3–7.5,
was more than 90% of that at the optimum pH. On the other hand, modulator(s) to the reactions were also examined. According to our
previous finding of the proton-mediated mechanism of the Heyns rearrangement, the effects of cationic metal ions on the reactions were
examined with 29 metal chlorides. Five metal ions, Pt4ϩ, Cu2ϩ, Ni2ϩ, Co2ϩ, and Mn2ϩ, suppressed the formation of Heyns product
significantly while Fe2ϩ, Y3ϩ, Gd3ϩ, and Er3ϩ slightly increased it. The suppression rate was synergistically enhanced by the combination
of Pt4ϩ with Co2ϩ, Cu2ϩ, or Ni2ϩ. These results suggest the five metal ions, Pt4ϩ, Cu2ϩ, Ni2ϩ, Co2ϩ, and Mn2ϩ, reduce the formation of
the Heyns product in vivo and, therefore, would be useful tools to clarify the implication of the stable adduct formation of 16␣-OHE1 with
protein. © 1999 Elsevier Science Inc. All rights reserved.
Keywords: 16␣-Hydroxyestrone; Stable adduct; Heyns rearrangement; Steroidal D-ring ␣-hydroxyimine; Metal ion effect; Reduction of the adduct formation
Epidemiologic studies have implicated estrogen in the
etiology of breast cancer [1]. The role of major metabo-
lites such as 2- and 16␣-hydroxylated estrogens in this
process are still the subject of great discussion. Although
16␣-hydroxyestrone (16␣-OHE1), which is a principal
16␣-hydroxylated estrogen in humans [2], is suspected to
be a strong risk factor epidemiologically [3,4], the geno-
toxic damage caused by the estrogen metabolite is still
controversial [5,6]. Recently, 4-hydroxylated estrogen, a
minor catechol estrogen, has been found to form geno-
toxic nucleic acid adducts, while its geometric isomer,
2-hydroxylated estrogen, formed non-genotoxic adducts
[7]. On the other hand, a covalent interaction of 16␣-
OHE1 with protein also presents a possible mechanism
for the generation of oncogenic event(s). It has been
revealed that 16␣-OHE1 is an unique long-acting utero-
tropic estrogen, that the affinity to the estrogen receptor
is relatively low [8], and that it forms a stable adduct with
the estrogen receptor [9]. 16␣-OHE1 is supposed to form
a stable covalent adduct at the 17-position with the pri-
mary amino group in protein via the Schiff base (imine)
formation followed by Heyns rearrangement of the D-
ring ␣-hydroxyimine as depicted in Fig. 1 [10]. Previ-
ously, we have demonstrated that an ␣-hydroxyimine of
16␣-OHE1 gave a fairly stable adduct, N-(3-hydroxy-16-
oxoestra-1,3,5(10)-trien-17␤-yl)amine derivative in
aqueous methanol (MeOH) by the Heyns rearrangement.
Although the formation rate of the Heyns product may be
influenced by biologic and environmental factors, these
internal or external factors have not been resolved. Mod-
ulators for adduct formation are urgently needed to clar-
ify the implication of 16␣-OHE1 in carcinogenic
event(s). According to our proton-mediated mechanism
of the Heyns rearrangement proposed previously [11],
metal ions are presumed to interact with the reaction
intermediate as well as the proton. In this study we
* Corresponding author. Tel.: ϩ81-1-22-217-6871; fax: ϩ81-1-22-
217-6871.
E-mail address: miyairi@mail.pharm.tohoku.ac.jp (S. Miyairi)
0039-128X/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: PII SS0039-128X(99)00118-4

S. Miyairi et al. / Steroids 64 (1999) 252–258
253
Fig. 1. Mechanism for the stable adduct formation between 16w␣-OHE1 and protein.
examined the effects of metal cations on the Heyns re-
1.3. Determination of the products of the Heyns
arrangement and the stability of the steroidal ␣-hydroxy-
imine using newly developed HPLC for simultaneous
determination of the Schiff base and products.
rearrangement and hydrolysis of 16␣-OH,17-imine
17-(2-Methoxyethylimino)estra-1,3,5(10)-triene-3,16␣-diol
(16␣-OH,17-imine) was prepared from 16␣-OHE1 and
2-MEA in MeOH as reported previously [11]. The 16␣-
OH,17-imine was determined to be approximately 95% of
the total estrogen in the original preparation by the follow-
1. Experimental
1.1. Materials
Estrone (E1), estradiol (E2), and estriol (E3) were pur-
chased from Steraloids Inc. (Wilton, NH, USA). 2-Me-
thoxyethylamine (2-MEA) was obtained from Tokyo Kasei
Kogyo Ltd. (Tokyo, Japan). 16␣-OHE1 diacetate was pre-
pared from E1 by the method of Leeds et al. with minor
modifications [12]. 3-Hydroxy-17␤-(2-methoxyethylamino)-
esestra-1,3,5(10)-trien-16-one (16-oxo,17␤-amine), 17␤-(2-
methoxyethylamino)estra-1,3,5(10)-triene-3,16␣-diol (16␣-
OH,17␤-amine) hydrochloride and 17␤-(2-methoxyethyl-
amino)estra-1,3,5(10)-triene-3,16␤-diol (16␤-OH,17␤-amine)
hydrochloride were prepared by the methods described previ-
ously (Fig. 2) [11]. 6␣-Hydroxyestradiol (6␣-OHE2) was pre-
pared from E2 by the method of Wintersteiner and Moore [13].
16␤-Hydroxyestradiol (16-epiE3) was produced by sodium
borohydride (NaBH4) treatment of 16-oxoestradiol (16-oxoE2)
which was prepared from 16␣-OHE1 by keto-enol rearrange-
ment under basic condition.
1.2. Apparatus
The apparatus used for HPLC was a LC-10AD solvent
delivery system equipped with a SPD-10A spectrophoto-
meter (at 280 nm) and a CHROMATOPAC C-6A data
processor (Shimadzu, Kyoto, Japan).
Fig. 2. Structures of the major products after reduction with NaBH4 of the
steroidal ␣-hydroxyimine and its rearranged or hydrolyzed products.

254
S. Miyairi et al. / Steroids 64 (1999) 252–258
Table 1
2. Results and discussion
Epimeric ratio of the NaBH4 reduction of carbonyl group at 16- or
17-position
The formation of stable adduct of 16␣-OHE1 to several
proteins has been investigated using a radio-labeled ligand.
The radioactivity was retained preferentially by nuclear
components such as histones [14] and the estrogen receptor
Estrogen
␣-Configuration
␤-Configuration
16-oxo,17␤-amine
16-oxo,17␤-OH
16␣-OHE1
3.1
9.8
93.6
96.9
90.2
6.7
3
[9] after incubation with H-labeled 16␣-OHE1, while E3
failed to show covalent binding [14]. The two-step mecha-
nism for the formation of a covalent adduct of 16␣-OHE1
with protein has been proposed to proceed via Schiff base
(␣-hydroxyimine) formation with an amino group of pro-
teins followed by Heyns rearrangement of the ␣-hydroxy-
imine. Formation of the Schiff base of 16␣-OHE1 at the
17-position with the ⑀-amino group of lysine in aqueous
medium has been revealed in the presence of sodium cya-
noborohydride [15]. Furthermore, we have proved that the
Heyns rearrangement proceeded on the steroidal ␣-hy-
droxyimine with the lysinyl ⑀-amino group in neutral con-
dition and gave N⑀-(3-hydroxy-16-oxoestra-1,3,5(10)-trien-
17␤-yl)-L-lysine, which is considered to be a partial
structure of the stable 16␣-OHE1-protein adduct [16]. How-
ever the reaction condition as well as the effector of the
Heyns rearrangement of the steroidal ␣-hydroxyimine have
not been clarified. As it is difficult to analyze the Heyns
product formed in proteins, a Schiff base from 16␣-OHE1
with 2-MEA, a model amino compound, was utilized to
demonstrate the pH effect and search for the effector related
to the rearrangement.
The ratio was determined by HPLC using the peak areas.
ing HPLC method. 16␣-OH,17-imine (27 mM at the final
concentration) was incubated in a mixture of 50 mM Tris
HCl buffer (various pH condition or pH 6.2) with or without
a metal ion and MeOH (1:1 v/v) for 10 min at 37°C. The
reaction was terminated by the addition of NaBH4. Then the
resulting mixture was loaded onto a Sep-Pak C18 cartridge
after the addition of 6␣-OHE2 (the internal standard (IS) for
HPLC) and water, washed thoroughly with water and eluted
with MeOH (5 ml). The eluate was subjected to HPLC on a
Puresil C18 column (5 ␮m, 150 ϫ 4.6 mm internal diameter;
Millipore-Waters, Milford, MA, USA) using 0.5 (w/v)%
potassium phosphate buffer (pH 6.8)/MeOH (1:1 v/v) as the
eluent.
Fig. 4. Effect of pH on the Heyns rearrangement and the hydrolysis of the
steroidal D-ring ␣-hydroxyimine. 16␣-OH,17-imine was incubated in a
mixture of 50 mM Tris HCl buffer (pH 5.0–8.0) and MeOH (1:1, v/v) for
10 min at 37°C and then treated with NaBH4. The estrogen derivatives in
the solution were subjected to HPLC analysis. f, the stable adduct (16-
oxo-17␤-amine); o, hydrolyzate (16␣-OHE1 ϩ 16-oxoE2).
Fig. 3. Chromatogram of the estrogen derivatives. Mobile phase: 0.5%
potassium phosphate buffer (pH 6.8)/MeOH (1:1, v/v). Flow rate: 1.0
ml/min. UV absorption of the estrogen derivatives were monitored at 280
nm. The numbers on the peaks indicate: 1; 6␣-OHE2 (IS), 2; E3, 3;
16␣-OH,17␤-amine, 4; 16␤-OH,17␤-amine, 5; 16-epiE3.

S. Miyairi et al. / Steroids 64 (1999) 252–258
255
Table 2
(pH 6.8) and MeOH (1:1 v/v) as the eluent (Fig. 3). The
linear relationships of the peak height ratios of the 4 reduced
products to the IS were observed in the range of 0.1:1 to
6.4:1 (data not shown). The extraction of the estrogen de-
rivatives in the reduced mixture using a Sep-Pak C18 car-
tridge was satisfactory.
Metal chloride used for the examination of effects on the Heyns
rearrangement and hydrolysis of 16␣-hydroxyestrone 17-imine
Valency
Divalent
Compound
FeCl2, MnCl2, CoCl2, NiCl2, CuCl2, ZnCl2, BaCl2, MgCl2,
CaCl2, SrCl2
Trivalent
FeCl3, CrCl3, AlCl3, YCl3,
Lanthanides (LaCl3, CeCl3, PrCl3, NdCl3, SmCl3, EuCl3,
GdCl3, TbCl3, DyCl3, HoCl3, ErCl3, YbCl3)
Accordingly, the amounts of 16␣-OH,17-imine, 16-
oxo,17␤-amine, 16␣-OHE1 and 16-oxoE2 in the reaction
mixture were determined from the major reduced products
with a correction based on the epimeric ratios in Table 1. All
the rates (mean Ϯ standard deviation, n ϭ 5) of hydrolysis
and Heyns rearrangement in this paper were expressed as
the percent of a cumulative amount of 16␣-OHE1 and 16-
oxoE2 (observed in a few experiment in the presence of
metal ion(s), but was less than 3% of 16␣-OHE1 formed)
and an amount of 16-oxo,17␤-amine relative to the total
estrogens determined, respectively.
Tetravalent PtCl4, SeCl4
2.1. HPLC system for determination of the products from
the steroidal ␣-hydroxyimine
After incubation of 16␣-OH,17-imine in an aqueous
MeOH, 16-oxo,17␤-amine (the stable adduct), 16␣-OHE1
(the primary hydrolyzed product) and 16-oxoE2 (the sec-
ondary product from 16␣-OHE1 by keto-enol rearrange-
ment) were present in the resulting mixture in addition to
the Schiff base theoretically. Therefore, simultaneous
HPLC analysis of these products and 16␣-OH,17-imine was
performed to evaluate the rates of the Heyns rearrangement
and hydrolysis of 16␣-OH,17-imine. However, the ␣-hy-
droxyimine was too labile to be detected directly by the
system. Hence, the incubation mixture was treated with
NaBH4 to transform it into a stable form, 16␣-OH,17␤-
amine. The other estrogen derivatives that co-existed were
also affected by this treatment. The carbonyl containing
estrogen derivatives, 16-oxo,17␤-amine, 16-oxoE2 and
16␣-OHE1, yielded corresponding hydroxyl products with
␣- and ␤-configurations, while 16␣-OH,17-imine gave 16␣-
OH,17␤-amine quantitatively: the epimeric ratios of the
reduction products are listed in Table 1. The 4 major re-
duced products (16␣-OH, 17␤-amine, 16␤-OH,17␤-amine,
E3 and 16-epiE3) and 6␣-OHE2 (used for IS) were well
separated from each other by HPLC on a Puresil C18 column
using a mixture of 0.5 (w/v)% potassium phosphate buffer
2.2. Effect of pH on the Heyns rearrangement and
hydrolysis of the steroidal ␣-hydroxyimine
The rates of the Heyns rearrangement and hydrolysis of
the steroidal ␣-hydroxyimine were examined in a mixture of
50 mM Tris HCl buffer with various pH (5.0–8.0) and
MeOH (1:1 v/v). As illustrated in Figure 4, the rate of
hydrolysis seemed to be less affected by pH than that of
Heyns rearrangement in the pH range examined. The hy-
drolysis rate appeared to be decreased but was insignificant,
at pH 6.0 and 6.2. Contrarily, the rate of the Heyns rear-
rangement of 16␣-OH,17-imine was significantly increased
in these pH conditions. These results led us to conclude that
the reaction condition with a pH of approximately 6.2 was
optimal for the Heyns rearrangement of the steroidal D-ring
␣-hydroxyimine.
Although the Heyns rearrangement was seemed to be
decreased at the physiological pH according to the amount
of Heyns products at pH 7.3 and 7.5, it was still more than
90% (94% and 92%, respectively) of that at the optimum pH
Fig. 5. Effect of metal ion on the Heyns rearrangement and the hydrolysis of the steroidal D-ring ␣-hydroxyimine. 16␣-OH,17-imine was incubated in a
mixture of 50 mM Tris HCl buffer (pH 6.2) containing metal chloride (5 mM) and MeOH (1:1, v/v) for 10 min at 37°C and then treated with NaBH4. The
estrogen derivatives in the solution were subjected to HPLC analysis. f, the stable adduct (16-oxo-17␤-amine); o, hydrolyzate (16␣-OHE1 ϩ 16-oxoE2).

256
S. Miyairi et al. / Steroids 64 (1999) 252–258
Table 3
Reduction of metal ion promoted Heyns rearrangement and hydrolysis
of the steroidal ␣-hydroxyimine by EDTA
Metal ion
Heyns rearrangement
Hydrolysis
Pt4ϩ
54%
92%
42%
73%
31%
92%
36%
65%
Cu2ϩ
Co2ϩ
Fe2ϩ
Concentrations of metal ion and EDTA were 5 mM.
as shown in Fig. 4. Hence, it is conceivable that the Heyns
rearrangement may proceed on the Schiff base formed from
16␣-OHE1 with protein in vivo, not only under pathologic
conditions but also under physiological conditions. Since
the tissue with inflammation is generally acidic, the forma-
tion rate of stable adduct may be increased in inflamed
(breast) tissue [17].
2.3. Effect of metal ion on the Heyns rearrangement and
hydrolysis of the steroidal ␣-hydroxyimine
Fig. 7. Combination effect of metal ions on the Heyns rearrangement and
the hydrolysis of the steroidal D-ring ␣-hydroxyimine. 16␣-OH,17-imine
was incubated in a mixture of 50 mM Tris HCl buffer (pH 6.2) containing
metal chloride (each 2 mM) and MeOH (1:1, v/v) for 10 min at 37°C and
then treated with NaBH4. The estrogen derivatives in the solution were
subjected to HPLC analysis. f, the stable adduct (16-oxo-17␤-amine); o,
hydrolyzate (16␣-OHE1 ϩ 16-oxoE2).
Twenty-nine metal chlorides, including 13 lanthanides,
were examined for effects on the Heyns rearrangement and
hydrolysis of 16␣-OH,17-imine (Table 2). Tris HCl buffer
was adjusted at pH 6.2 after the addition of metal chloride.
Incubation of the 16␣-OH,17-imine (27 mM at the final
concentration) was performed with a mixture of the buffer
and MeOH (1:1 v/v) for 10 min at 37°C. None of the
precipitate was observed during the incubation. The first
screening was carried out using 5 mM metal ions at the final
Sr2ϩ, Zn2ϩ, Al3ϩ, Cr3ϩ, Fe3ϩ, Se4ϩ, and lighter lanthanide
(atomic number: 57–60) ions) did not show any significant
effect on both reactions (Fig. 5). The rate of the Heyns
rearrangement was modestly enhanced by Fe2ϩ (15%) and
slightly by Y3ϩ (4.1%), Gd3ϩ (5.7%) and Er3ϩ (4.6%). All
concentration. Fe2ϩ, Mn2ϩ, Co2ϩ, Ni2ϩ, Cu2ϩ, Pt4ϩ, Y3ϩ
,
and heavier lanthanide (atomic number: 62–68 and 70) ions
affected the Heyns rearrangement and/or hydrolysis of the
other effective metals, Mn2ϩ, Co2ϩ, Ni2ϩ, Cu2ϩ, and Pt4ϩ
,
suppressed the rearrangement rate significantly (18, 27, 36,
45, and 50%, respectively). On the other hand, the hydro-
16␣-OH,17-imine while 13 metal ions (Ba2ϩ, Ca2ϩ, Mg2ϩ
,
Fig. 6. Effect of metal ion concentration on the Heyns rearrangement and the hydrolysis of the steroidal D-ring ␣-hydroxyimine. 16␣-OH,17-imine was
incubated in a mixture of 50 mM Tris HCl buffer (pH 6.2) containing (A) PtCl4 or (B) FeCl2 and MeOH (1:1, v/v) for 10 min at 37°C and then treated with
NaBH4. The estrogen derivatives in the solution were subjected to HPLC analysis. f, the stable adduct (16-oxo-17␤-amine); o, hydrolyzate (16␣-OHE1 ϩ
16-oxoE2).

S. Miyairi et al. / Steroids 64 (1999) 252–258
257
Fig. 8. Effect of supplementary metal ion concentration on the Heyns rearrangement of the steroidal D-ring ␣-hydroxyimine promoted by Pt4ϩ or Co2ϩ
.
16␣-OH,17-imine was incubated in a mixture of 50 mM Tris HCl buffer (pH 6.2) containing (A) PtCl4 (2 mM) with CoCl2 (0–2 mM) or (B) CoCl2 (2 mM)
with PtCl4 (0–2 mM) and MeOH (1:1, v/v) for 10 min at 37°C and then treated with NaBH4. The estrogen derivatives in the solution were subjected to HPLC
analysis. f, the stable adduct (16-oxo-17␤-amine); o, hydrolyzate (16␣-OHE1 ϩ 16-oxoE2).
lysis rate of the Schiff base was increased by all of the
effective metal ions, for example Pt4ϩ increased it by 26%
(Fig. 5). Both effects promoted by metal ions were reduced
similarly by the addition of a chelating agent, ethylenedia-
minetetraacetic acid (EDTA) (Table 3). The effects of the
rearrangement and hydrolysis of the steroidal D-ring ␣-hy-
droxyimine by Pt4ϩ, Cu2ϩ, Ni2ϩ, Co2ϩ, and Mn2ϩ were in
a dose-dependent manner in a range of 0.5–5 mM. The
effect by Pt4ϩ concentration is depicted in Fig. 6A. On the
other hand, the rate of the Heyns rearrangement increased
by the addition of Fe2ϩ up to 100 ␮M and then became
constant (Fig. 6B).
The combination effect among the five effective metal
ions that suppressed the Heyns rearrangement were exam-
ined at the concentration of 2 mM. As depicted in Fig. 7,
significant synergistic effects on the suppression of the
rearrangement were observed in the combination of Pt4ϩ
with Co2ϩ, Ni2ϩ and Cu2ϩ; 98, 96 and 71%, respectively,
while individual metal ions suppressed it by 21, 8, 11 and
16% at this concentration. Modest synergistic effects were
also observed in the combinations of Cu2ϩ with Ni2ϩ, Mn2ϩ
(alone, 13%) or Co2ϩ; 49, 43, and 41% and Ni2ϩ with Mn2ϩ
or Co2ϩ; both 38%. On the other hand, only the additive
effect on the Heyns rearrangement was observed in the
combination of the metal ions (Fe3ϩ, Y3ϩ, Gd3ϩ, and Er3ϩ),
which individually enhanced the rearrangement by a few
percent (data not shown). The effect of supplementary metal
ion concentration on the Heyns rearrangement and hydro-
lysis of the steroidal ␣-hydroxyimine promoted by Pt4ϩ or
Co2ϩ is shown in Fig. 8. When various concentrations of
Co2ϩ were added to 2 mM of Pt4ϩ, the percent suppression
of the Heyns rearrangement was increased by Co2ϩ in a
dose-dependent manner. However, in the opposite combi-
nation, the synergistic effect was observed only at the con-
centration of 2 mM of Pt4ϩ. These findings suggest that
Pt4ϩ played a predominant role and Co2ϩ worked supple-
mentary in the synergistic effect.
It has been demonstrated that a stable adduct of 16␣-
OHE1 with serum albumin and erythrocyte membrane pro-
tein accumulated in vivo and in vitro [10,18]. The risk of
breast cancer was also revealed to be associated with estro-
gen 16␣-hydroxylation in humans [3] and mice [19]. In
these cases, elevated 16␣-OHE1 levels in certain site(s) may
allow for the accumulation of the stable adduct formed from
16␣-OHE1 and protein(s). The modified protein may play a
role in the genesis of oncogenic event(s) [19,20]. Accord-
ingly, Pt4ϩ, Co2ϩ, Ni2ϩ, and Cu2ϩ would be useful to
clarify an implication of the stable adduct formation of
16␣-OHE1 with protein in the oncogenic event as a sup-
presser of the Heyns rearrangement.
Acknowledgments
This work was supported in part by a grant-in-aid for
Scientific Research from The Ministry of Education, Sci-
ence, Sports and Culture of Japan and by a grant from Kowa
Life Science Foundation.
References
[1] Fishman J, Hershcopf RJ, Bradlow HL. Biochemical epidemiology of
breast cancer. In: Tumor Markers and Their Significance in the
Management of Breast Cancer. New York: Alan R Liss Inc, 1986. pp.
91–103.
[2] Fishman J, Bradlow HL, Gallagher TF. Oxidative metabolism of
estradiol. J Biol Chem 1960;235:3104–7.

258
S. Miyairi et al. / Steroids 64 (1999) 252–258
[3] Schneider J, Kinne D, Fracchia A, Pierce V, Anderson KE, Bradlow
rangement of steroidal D-ring ␣-hydroxyimines. Steroids 1991;56:
361–6.
HL, Fishman J. Abnormal oxidative metabolism of estradiol in
women with breast cancer. Proc Natl Acad Sci USA 1982;70:3047–
51.
[12] Leeds NS, Fukushima DK, Gallagher TF. Studies of steroid ring D
epoxides of enol acetates; a new synthesis of estriol and androstane-
3␤,16␣,17␤-triol. J Am Chem Soc 1954;76:2943–8.
[13] Wintersteiner O, Moor M. The epimeric 6-hydroxy-3,17␤-estradiols.
J Am Chem Soc 1959;81:442–3.
[14] Bucala R, Ulrich PC, Chait BT, Bencsath FA, Cerami A. Structure of
lysine adducts with 16␣-hydroxyestrone and cortisol. J Steroid Bio-
chem 1986;25:127–33.
[15] Yu SC, Fishman J. Interaction of histone with estrogens. Covalent
adduct formation with 16␣-hydroxyestrone. Biochemistry 1985;24:
8017–21.
[16] Miyairi S, Ichikawa T, Nambara T. Preparation of N⑀-(3-hydroxy-
16-oxoestra-1,3,5(10)-trien-17␤-yl)-L-lysine, 16␣-hydroxyestrone-
lysine adduct, via Heyns rearrangement. Tetrahedron Lett 1991;32:
1213–4.
[17] Serrano CV Jr, Fraticelli A, Paniccia R, Teti A, Noble B, Corda S,
Faraggiana T, Ziegelstein RC, Zweier JL, Capogrossi MC. pH de-
pendence of neutrophil-endothelial cell adhesion and adhesion mol-
ecule expression. Am J Physiol 1996;271:C962–70.
[18] Bucala R, Fishman J, Cerami A. The reaction of 16␣-hydroxyestrone
with erythrocytes in vitro and in vivo. Eur J Biochem 1984;140:
593–8.
[19] Bradlow HL, Hershcopf RJ, Martucci CP, Fishman J. Estradiol
16␣-hydroxylation in the mouse correlate with mammary tumor
incidence and presence of murine mammary tumor virus: a pos-
sible model for the hormonal etiology. Proc Natl Acad Sci USA
1985;82:6295–9.
[4] Kabat GC, Chang CJ, Sparano JA, Sepkovic DW, Hu XP, Khalil A,
Rosenblatt R, Bradlow HL. Urinary estrogen metabolites and breast
cancer: a case-control study. Cancer Epidemiol Biomarker Prev 1997;
6:505–9.
[5] Telang NT, Suto A, Wong GY, Osborne MP, Bradlow HL. Induction
by estrogen metabolite 16␣-hydroxyestrone of genotoxic damage and
aberrant proliferation in mouse mammary epithelial cells. J Natl
Cancer Inst 1992;84:634–8.
[6] Newfield L, Bradlow HL, Sepkovic DW, Auborn K. Estrogen me-
tabolism and the malignant potential of human papillomavirus im-
mortalized keratinocytes. Proc Soc Exp Biol Med 1998;217:322–6.
[7] Cavalieri EL, Stack DE, Devanesan PD, Todorovic R, Dwivedy I,
Higginbotham S, Johansson SL, Patil KD, Gross ML, Gooden JK,
Ramanathan R, Cerny RL, Rogan EG. Molecular origin of cancer:
catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc
Natl Acad Sci USA 1997;94:10937–42.
[8] Fishman J, Martucci C. Biological properties of 16␣-hydroxyestrone:
implications in estrogen physiology and pathophysiology. J Clin
Endocrinol Metab 1980;51:611–5.
[9] Swaneck GE, Fishman J. Covalent binding of the endogenous estro-
gen 16␣-hydroxyestrone to estradiol receptor in human breast cancer
cells: characterization and intranuclear localization. Proc Natl Acad
Sci USA 1988;85:7831–5.
[10] Bucala R, Fishman J, Cerami A. Formation of covalent adducts
between cortisol and 16␣-hydroxyestrone and protein:possible role in
the pathogenesis of cortisol toxicity and systemic lupus erythemato-
sus. Proc Natl Acad Sci USA 1982;79:3320–4.
[11] Miyairi S, Ichikawa T, Nambara T. Structure of the adduct of16␣-
hydroxyestrone with a primary amine: evidence for the Heyns rear-
[20] Swaneck GE, Fishman J. Effects of estrogens on MCF-7 cells: pos-
itive or negative regulation by the nature of the ligand-receptor
complex. Biochem Biophys Res Commun 1991;174:276–81.