BIOLOGY OF REPRODUCTION 60, 435–444 (1999)
Spermicidal Activity of Oxovanadium(IV) Complexes of 1,10-Phenanthroline,
2,29-Bipyridyl, 59-Bromo-29-Hydroxyacetophenone and Derivatives in Humans
Osmond J. D’Cruz,1,2,3 Yanhong Dong,2,4 and Fatih M. Uckun2
Drug Discovery Program,2 Departments of Reproductive Biology3 and Chemistry,4 Hughes Institute, St. Paul,
Minnesota 55113
state 14 (IV) function as modulators of cellular redox potential, regulate enzymatic phosphorylation, and exert
pleiotropic effects in multiple biological systems by catalyzing the generation of reactive oxygen species (ROS) [4–
10]. Besides the ability of vanadium metal to assume various oxidation states, its coordination chemistry also plays
a key role in its interactions with various biomolecules. In
particular, organometallic complexes of vanadium(IV) with
bis(cycopentadienyl) moieties or vanadocenes exhibit antitumor properties both in vitro and in vivo, primarily via
oxidative damage [11–13].
Human sperm are exquisitely sensitive to oxidative
stress. This is due to the high content of polyunsaturated
fatty acids in their cell membranes, the low levels of cytoplasmic enzymes for scavenging the ROS, which initiate lipid peroxidation, and the reduced activity of repair
enzymes to recover from oxidative damage [14–17].
ROS such as hydrogen peroxide (H2O2) and hydroxyl
radicals (OH•) affect sperm motility by peroxidation of
membrane lipids and proteins [14, 15, 18, 19]. Oxidative
damage to sperm proteins, carbohydrates, and DNA is an
important pathophysiological mechanism in the onset of
male infertility [18, 20, 21]. Superoxide radicals generated by the action of xanthine oxidase exert a direct, suppressive effect on sperm function, leading to loss of motility, impaired capacitation, and poor sperm-egg interaction [22].
Because of the ability of vanadium(IV)-containing complexes to catalyze the generation of ROS, we synthesized
diverse organovanadium and oxovanadium complexes with
different ligands linked to the central vanadium(IV) atom
by carbon, nitrogen, or oxygen atoms. Our recent studies
on 12 monodentate diacido- and 7 bidentate-coordinated
complexes of bis(cyclopentadienyl)vanadium(IV) demonstrated that these vanadocenes have potent spermicidal and
apoptosis-inducing properties against human sperm [23–
26]. In fact, very short (, 1 min) exposure to vanadocenes
at nanomolar to micromolar concentrations was sufficient
to induce complete sperm motility loss, whereas prolonged
exposure of sperm to millimolar concentrations of inorganic
vanadium (oxidation state IV and V) salts had no effect on
sperm motility [24]. Furthermore, none of the other metallocene dichloro complexes of oxidation state IV containing
titanium, zirconium, molybdenum, or hafnium exhibited
spermicidal activity [24].
Since the redox potential and the stability of metal complexes are greatly affected by the ancillary groups, different
ligands were selected to test their effects on spermicidal
activity and stability. The stability of organometallic complexes with monodentate ligands in aqueous solutions was
found to be improved by chelating effects of certain bidentate ligands, particularly dithiocarbamate and acetylacetonate [25, 26]. Because of the pharmacological and biochemical importance of vanadium compounds, and our novel
finding of vanadocenes as a new class of effective sper-
ABSTRACT
We have recently reported that tetrahedral metallocene
complexes containing vanadium(IV) (vanadocene) have potent
spermicidal activity against human sperm. The spermicidal activity was dependent on vanadium(IV) as the central metal ion
within the bis-cyclopentadienyl (Cp2)-metal complex, but the
variation of diacido groups and/or replacement with bidentate
ligands coordinated to the Cp2-vanadium(IV) moiety also significantly modulated the spermicidal potency. To assess the
structure-activity relationship between vanadocenes and other
coordination complexes of vanadium(IV), a set of 11 oxovanadium(IV) complexes with different geometrical configurations
were synthesized and evaluated for spermicidal activity by
computer-assisted sperm analysis. These complexes included
mono and bis ancillary ligands, 1,10-phenanthroline (phen):
[VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2-phen)2, VO(Clphen), and VO(Cl-phen)2]; 2,29-bipyridyl (bipy): [VO(bipy),
VO(bipy)2, VO(Me2-bipy), and VO(Me2-bipy)2], linked via nitrogen atoms; and 59-bromo-29-hydroxyacetophenone (acph):
[VO(Br,OH-acph)2], linked via oxygen donor atoms. All 11 oxovanadium(IV) complexes elicited concentration-dependent
spermicidal activity at micromolar concentrations (EC50 values:
5.5–118 mM). The bis-phenanthroline complex of oxovanadium(IV), VO(Cl-phen)2, was the most active, and the mono
bipyridyl complex, VO(bipy), was the least active; the order of
efficacy was VO(Cl-phen)2 . VO(phen)2 . VO(Br,OH-acph)2 .
VO(Me2-phen) . VO(bipy)2 . VO(phen) . VO(Cl-phen) .
VO(Me2-phen)2 . VO(Me2-bipy)2 . VO(Me2-bipy) . VO(bipy).
The neutral complex, VO(Br,OH-acph)2, induced rapid sperm
immobilization (T1/2 5 38 sec). The sperm-immobilizing activity of mono- and bis-ligated oxovanadium(IV) complexes was
irreversible, since the treated sperm underwent apoptosis, as
determined by the flow cytometric quantitation of mitochondrial membrane potential, surface Annexin V binding assay, and
in situ DNA nick-end labeling of sperm nuclei. The percentages
of apoptotic sperm quantitated by the flow cytometric assay
correlated well with the spermicidal potency of oxovanadium(IV) complexes. These results provide unprecedented evidence that the spermicidal and apoptosis-inducing activities
of vanadium(IV) complexes are determined by the oxidation
state of vanadium as well as their geometry. Because of its
rapid and potent sperm-immobilizing activity, the bromo-hydroxyacetophenone complex, [VO(Br,OH-acph)2], may be useful as a contraceptive agent.
INTRODUCTION
Vanadium is a physiologically essential element found
in both anionic and cationic forms, with oxidation states
ranging from 21 to 15 (I–V) [1, 2]. This versatility gives
vanadium complexes unique properties [3]. In particular,
the cationic form of vanadium complexes with oxidation
Accepted September 22, 1998.
Received July 15, 1998.
1
Correspondence: Osmond J. D’Cruz, Hughes Institute, 2665 Long
Lake Road, Suite 330, St. Paul, MN 55113. FAX: 651 697 1042;
e-mail: [email protected]
435
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D’CRUZ ET AL.
TABLE 1. Mono- and bis-bidentate oxovanadium(IV) complexes synthesized and tested in this study.
Abbreviation
VO(phen)
VO(phen)2
VO(Me2-phen)
VO(Me2-phen)2
VO(Cl-phen)
VO(Cl-phen)2
VO(bipy)
VO(bipy)2
VO(Me2-bipy)
VO(Me2-bipy)2
VO(Br,OH-acph)2
Oxovanadium(IV) complex
(diaqua)(1,10-phenanthroline)oxovanadium(IV) sulfate
(aqua)bis(1,10-phenanthroline)oxovanadium(IV) sulfate
(diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate
(aqua)bis(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate
(diaqua)(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate
(aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate
(diaqua)(2,29-bipyridyl)oxovanadium(IV) sulfate
(aqua)bis(2,29-bipyridyl)oxovanadium(IV) sulfate
(diaqua)(4,49-dimethyl-2,29-bipyridyl)oxovanadium(IV) sulfate
(aqua)bis(4,49-dimethyl-2,29-bipyridyl)oxovanadium(IV) sulfate
bis(59-bromo-29-hydroxyacetophenone) oxovanadium(IV)
micidal agents [23–26], we have synthesized 11 stable oxovanadium(IV) complexes. These included oxovanadium
complexes with a square pyramidal geometry with the oxoligand in the axial position. The coordination complexes
were stabilized with 5-membered mono- and bis-1,10-phenanthroline (phen): [VO(phen), VO(phen)2, VO(Me2-phen),
VO(Me2-phen)2, VO(Cl-phen), and VO(Cl-phen)2], monoand bis-2,2 9 -bipyridyl (bipy): [VO(bipy), VO(bipy) 2 ,
VO(Me2-bipy), and VO(Me2-bipy)2], and bis-59-bromo-29hydroxyacetophenone (acph): [VO(Br,OH-acph)2], as ancillary ligands linked via nitrogen or oxygen atoms. The
phenanthroline-peroxovanadate complex potentiates the
generation of ROS in cells [27]. Since sperm motility and
function have been shown to be exquisitely susceptible to
ROS, we set out to examine these compounds for spermicidal activity using computer-assisted sperm analysis
(CASA). Our results presented herein provide unprecedented evidence that vanadium IV-bound to phenanthroline,
bipyridyl and acetophenone as ancillary ligands and their
derivatives are potent spermicidal agents and also induce
apoptosis in human sperm.
MATERIALS AND METHODS
Oxovanadium(IV) Complexes Containing 1,10Phenanthroline, 2,29-Bipyridyl, or 59-Bromo-29Hydroxyacetophenone and Derivatives
The names of the 11 oxovanadium complexes synthesized
and tested in this study are listed in Table 1. The chemical
structures of 10 cationic complexes with either mono- or bis1,10-phenanthroline and 2,29-bipyridyl, and one neutral complex, bis-59-bromo-29-hydroxyacetophenone, are depicted in
Figure 1. Nine novel oxovanadium(IV) complexes were synthesized on the basis of the previously published chemistry
of VO(phen) and VO(phen)2 complexes [27]. Briefly, these
complexes were synthesized by reacting an aqueous solution
of vanadyl sulfate with an ethanol solution or a chloroform
solution of the ligands. The complexes purified from chloroform, ether, and/or water were characterized by Fourier transform infrared spectroscopy (FT-Nicolet model Protege 460;
Nicolet Instrument Corp., Madison, WI), UV-visible spectroscopy (DU 7500 spectrophotometer; Beckman Instruments,
Fullerton, CA), mass spectrometry, and elemental analysis
(Atlantic Microlab, Inc., Norcross, GA). The choice of these
three ancillary ligands (phenanthroline, bipyridyl, and acetophenone) was based on the report that the cationic oxovanadium(IV) complex of phenanthroline is superior to cisplatin
(cis-diaminedichloroplatinum([II]) with respect to antitumor
activity [27] and on the structural similarity of bipyridyl ring
to phenanthroline, as well as on the neutral nature of aceto-
phenone complex of oxovanadium(IV). Structural variations
of the ligands included addition of bromo, chloro, or methyl
groups on the phenanthroline, bipyridyl or acetophenone
rings.
Sperm Immobilization Assay (SIA)
To evaluate the spermicidal effects of inorganic complexes of oxovanadium(IV)—VO(phen), VO(phen) 2 ,
VO(Me2-phen), VO(Me2-phen)2, VO(Cl-phen), VO(Clphen)2, VO(bipy), VO(bipy)2, VO(Me2-bipy), VO(Me2bipy)2, and VO(Br,OH-acph)2—a highly motile fraction of
pooled donor sperm (n 5 5) was prepared by discontinuous (90–45%) Percoll gradient (Conception Technologies, San Diego, CA) centrifugation and the swim-up
method as described previously [23, 24, 28]. All donor
specimens were obtained after informed consent and in
compliance with the guidelines of the Hughes Institutional
Review Board. Motile sperm ($ 10 3 106/ml) were suspended in 1 ml of Biggers, Whitten, and Whittingham’s
medium (BWW) containing 0.3% BSA (fraction V; Sigma
Chemical Co., St. Louis, MO) in the presence and absence
of serial 2-fold dilutions of test substance (250–1.9 mM)
in 0.25% dimethyl sulfoxide (DMSO). For each experiment, fresh stock solutions (100 mM) of vanadium compounds were prepared in DMSO. A corresponding volume
of DMSO (0.25%) was added to the control sperm suspensions. After 3 h of incubation at 378C, the percentage
of motile sperm was evaluated by CASA as described previously [24, 25]. The percentages of motilities were compared with those of sham-treated control suspensions of
motile sperm. The spermicidal activity of test compounds
was expressed as the EC50 values (the final concentration
of the compound in medium that decreased the proportion
of motile sperm by 50%).
To test the effect of duration of incubation on SIA in the
presence of the 11 oxovanadium(IV) complexes, a motile
fraction of sperm (107/ml) was incubated at 378C in 1 ml
of BWW-0.3% BSA in the presence of 200 mM each of the
11 complexes or 0.2% DMSO alone. At timed intervals of
2, 5, or 10 min, aliquots (4 ml) were transferred to two 20mm Microcell (Conception Technologies) chambers, and
sperm motility was assessed by CASA.
Sperm Kinematic Parameters
For CASA, 4 ml of each sperm suspension was loaded
into two 20-mm Microcell chambers placed onto a counting
chamber at 378C. At least 5–8 fields per chamber were
scanned for analysis using a Hamilton Thorne Integrated
Visual Optical System (IVOS) version 10 instrument (Ham-
SPERMICIDAL OXOVANADIUM(IV) COMPLEXES
437
FIG. 1. Chemical composition of 11 oxovanadium(IV) (VO) complexes. The oxovanadium complexes have a square pyramidal geometry with the oxo ligand (O22)
in the axial position. These coordination
complexes are stabilized with 5-membered mono or bis-phenanthroline, bipyridyl, and acetophenone-type bidentate ligands with the vanadium atom. Structural
variations of the ligands were made after
addition of bromo, chloro, or methyl
groups on the ancillary ligands of the vanadium(IV) coordination sphere.
ilton Thorne Research Inc., Beverly, MA). Each field was
recorded for 30 sec. The Hamilton Thorne computer calibrations were set at 30 frames at a frame rate of 30 images/
sec. Other settings were as follows: minimum contrast 8;
minimum size 6; low-size gate, 1.0; high-size gate, 2.9;
low-intensity gate, 0.6; high-intensity gate, 1.4; phase-contrast illumination; low path velocity at 10 mm/sec and
threshold straightness at 80%; magnification factor, 1.95.
The performance of the analyzer was periodically checked
using the playback function.
The attributes of sperm kinematic parameters evaluated included numbers of motile (MOT) and progressively (PRG)
motile sperm; curvilinear velocity (VCL; a measure of the
total distance traveled by a given sperm during the acquisition
divided by the time elapsed); average path velocity (VAP; the
spatially averaged path that eliminates the wobble of the
sperm head), straight-line velocity (VSL; the straight-line distance from beginning to end of track divided by time taken),
beat-cross frequency (BCF; frequency of lateral head displacement), amplitude of lateral sperm head displacement (ALH;
the mean width of sperm head oscillation), and the derivatives
straightness (STR 5 VSL divided by VAP 3 100) and linearity (LIN 5 VSL divided by VCL 3 100, departure of sperm
track from a straight line). Data from each individual cell track
were recorded and analyzed. At least 200 sperm were analyzed for each aliquot sampled.
438
D’CRUZ ET AL.
Flow Cytometric Quantitation of Sperm
Acrosome Reaction
In experiments designed to assess the comparative effects of 11 oxovanadium(IV) complexes and a commercial
detergent-based spermicide, N-9, on sperm acrosome reaction, motile fractions of sperm (107/ml) prepared from a
single donor were incubated in 1 ml of BWW-0.3% BSA
in the presence of 100 mM each of the 11 vanadocene complexes—VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2phen)2, VO(Cl-phen), VO(Cl-phen)2, VO(bipy), VO(bipy)2,
VO(Me2-bipy), VO(Me2-bipy)2, and VO(Br,OH-acph)2—in
0.1% DMSO, N-9, or DMSO (0.1%) alone at 378C. After
3 h, 5 mg/ml of purified, phycoerythrin (PE)-conjugated
murine anti-CD46 monoclonal antibody (mAb; clone 122–
2; Research Diagnostics, Flanders, NJ) was added, and the
sperm suspensions were incubated for an additional 30 min.
The suspensions were washed in Tyrode’s salt solution
(Sigma) containing 1% BSA (1% TBSA), and the percentages of CD46-positive sperm were analyzed by flow cytometry using a FACS Vantage flow cytometer (Becton Dickinson, Mountain View, CA), as described previously [29,
30]. Two separate experiments were performed to determine acrosomal loss following exposure of sperm to
oxovanadium(IV) complexes.
Flow Cytometric Assays for Oxovanadium(IV) ComplexInduced Apoptosis
We used three independent flow cytometric apoptotic assays to determine oxovanadium(IV)-mediated quantitative
changes at the mitochondrial, surface membrane, and sperm
nuclear compartments.
Mitochondrial Transmembrane Potential (DCm) Using
JC-1 Dye
The loss of DCm, an early marker for apoptosis, was
quantitated by flow cytometry using the lipophilic cationic
dye, 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolecarbocyanine iodide (JC-1) [31]. This dye accumulates in
the mitochondrial matrix under the influence of the DCm
[32]. The molecule is able to selectively enter into mitochondria, the monomeric form emitting at 527 nm after
excitation at 490 nm. However, depending on the membrane potential, JC-1 is able to form J-aggregates that are
associated with a large shift in emission (590 nm). The
color of the dye changes reversibly from green to greenish
orange as DCm becomes more polarized [33]. To quantitate
changes in sperm DCm following oxovanadium(IV) complex exposure, highly motile fractions of sperm (107/ml) in
duplicate aliquots were incubated at 378C for 3 h in BWW0.3% BSA medium in the presence and absence of 100 mM
each of the 11 oxovanadium complexes. After incubation,
10 mg/ml JC-1 (Molecular Probes, Eugene, OR) was added
from a stock solution in DMSO (1 mg/ml) to the sperm
suspension, which was then incubated for an additional 10
min. At the end of the incubation period, sperm were
washed in Tyrode’s salt solution (Sigma), resuspended in
200 ml of Tyrode’s salt solution, and analyzed by flow cytometry for JC-1-specific fluorescence. The excitation was
at 488 nm; the emissions for green and red/orange fluorescence were 530 nm and 575 nm respectively. JC-1 monomer and aggregated fluorescence were simultaneously measured in oxovanadium(IV) complex-exposed and control
sperm. The percentages of sperm positive for green, orange,
and greenish orange were determined using the cutoff sig-
nals for JC-1-labeled motile sperm. Two separate experiments were performed to assess JC-1 incorporation following exposure of sperm to oxovanadium(IV) complexes.
Sperm Membrane Changes Using Fluorescein
Isothiocyanate (FITC) Annexin V
In order to examine the expression of phosphatidyl serine on the sperm surface after oxovanadium(IV) complex
exposure, we used flow cytometry to evaluate surface binding of FITC-Annexin V [34]. One-milliliter aliquots of
highly motile sperm (107) in triplicate were incubated in
BWW-0.3% BSA at 378C for 12 h with and without 100
mM of each of the 11 oxovanadium(IV) complexes in 0.1%
DMSO. After exposure to these complexes, sperm were
washed with 1% TBSA, and the pellets were resuspended
in the same medium. The sperm suspension was reacted for
30 min at room temperature with 6 mg/ml of FITC-conjugated recombinant human Annexin V (Caltag Laboratories,
San Francisco, CA). After two washes in Tyrode’s salt solution, sperm were resuspended in 1% TBSA containing 1
mg/ml propidium iodide (PI) and analyzed for surfacebound Annexin V and PI permeability by quantitative flow
cytometry using an argon laser for excitation of fluorescence. Annexin V and PI binding were simultaneously measured in oxovanadium(IV) complex-exposed and control
sperm as described previously [25]. The percentages of
sperm positive for Annexin V and PI were determined using the cutoff signals for membrane-intact motile sperm.
Two separate experiments were performed to assess the surface expression of phosphatidyl serine following exposure
of sperm to oxovanadium(IV) complexes.
DNA Fragmentation Using In Situ DNA Nick-End
Labeling by the TUNEL Method
A flow cytometric two-color terminal deoxynucleotidyl
transferase (TdT) assay was employed to detect apoptotic
sperm nuclei by TdT-mediated digoxigenin-uridine triphosphate (dUTP) nick-end labeling (TUNEL [35]). The abilities of 11 oxovanadium(IV) complexes to induce apoptosis
were compared by incubating 1-ml duplicate aliquots of
motile sperm (107/ml) in BWW-0.3% BSA at 378C for 24
h with and without each of the test compounds at a 100mM concentration. Sperm were washed in PBS-1% BSA
and fixed in 4% paraformaldehyde in PBS for 15 min. After
two washes in PBS, they were permeabilized with 0.1%
Triton X-100 in 0.1% sodium citrate for 2 min on ice, and
washed twice with PBS. Labeling of exposed 39-hydroxyl
(39-OH) ends of fragmented sperm nuclear DNA was performed using TdT and detected by FITC-conjugated dUTP
according to the manufacturer’s recommendations (Boehringer-Mannheim, Indianapolis, IN). Sperm aliquots incubated without TdT enzyme served as negative controls.
Non-apoptotic sperm do not incorporate significant
amounts of dUTP because of lack of exposed 39-OH ends,
and consequently have much less fluorescence compared to
apoptotic cells, which have an abundance of 39-OH ends.
Oxovanadium(IV)-induced apoptosis of sperm was shown
by an increase in the number of cells staining with FITCdUTP (M2 gates). The M1 and M2 gates were used to
identify non-apoptotic and apoptotic PI-counterstained
sperm populations, respectively. Two separate experiments
were performed to assess dUTP incorporation following exposure of sperm to oxovanadium(IV) complexes.
439
SPERMICIDAL OXOVANADIUM(IV) COMPLEXES
Confocal Laser Scanning Microscopy
Confocal microscopy of TUNEL-positive and control
sperm was performed using a Bio-Rad MRC 1024 Laser
Scanning Confocal Microscope (Bio-Rad Labs., Richmond,
CA) equipped with an argon-ion laser (excitation at 488
nm and emission at 540 nm) and mounted on a Nikon
Eclipse E800 series upright microscope (Nikon Instruments, Garden City, NY) with high numerical aperture objectives. Confocal images were obtained using a Nikon
3100 (NA 1.4) numerical aperture objective and Kalman
collection filter as described previously [24, 25]. Digital
data were processed using Lasersharp (Bio-Rad), and digitized images were saved on a Jaz disk (Iomega Corp., Roy,
UT) and processed with Adobe Photoshop software (Adobe
Systems, Mountain View, CA). Final images were printed
using a Fuji Pictography 3000 (Fuji Photo Film Co., Tokyo,
Japan) color printer.
Statistical Analysis
Sperm functional parameters are presented as mean 6
SD values. Nonlinear regression analysis was used to find
the EC50 values (i.e., concentrations of compound that result in 50% sperm motility loss) from the concentrationeffect curves using GraphPad PRISM Version 2.0 software
(San Diego, CA). One-way ANOVA followed by Dunnett’s
test was used to obtain statistical significance between control and test results. Linear regression analysis was performed to furnish the correlation coefficient, r2. A p value
of , 0.05 was considered significant.
RESULTS
Oxovanadium(IV) Complexes of 1,10-Phenanthroline,
2,29-Bipyridyl, and 59-Bromo-29-Hydroxyacetophenone
and Derivatives Had Spermicidal Activity
Because of the reported ability of an oxovanadium(IV)
complex of 1,10-phenanthroline to induce hydroxyl radicalmediated cell damage [27, 36], we synthesized a series of
11 oxovanadium(IV) complexes including six phenanthroline (phen)-linked [VO(phen), VO(phen)2, VO(Me2-phen),
VO(Me2-phen)2, VO(Cl-phen), and VO(Cl-phen)2] and four
bipyridyl (bipy)-linked [VO(bipy), VO(bipy)2, VO(Me2bipy), and VO(Me2-bipy)2] via nitrogen atoms, and one
acetophenone (acph)-linked [VO(Br,OH-acph)2] via oxygen
atoms; and tested them for spermicidal activity using
CASA. These complexes were tested side-by-side and at 8
different concentrations ranging from 1.9 mM to 250 mM.
All 11 oxovanadium complexes induced concentrationdependent inhibition of sperm motility assessed after a 3-h
incubation in BWW-0.3% BSA medium. However, marked
differences were noted in their potency. Table 2 shows the
EC50 values calculated from the concentration-response
curves. Among the 6 phenanthroline-linked cationic complexes, the bis-1,10-phenanthroline complex, VO(phen)2,
and its 5-chloro derivative, VO(Cl-phen)2, were the most
potent, with EC50 values of 6.5 mM and 5.5 mM, respectively. Among the 4 bipyridyl-linked cationic complexes,
the bis-2,29-bipyridyl complex, VO(bipy)2, and its 4,7-dimethyl derivative, VO(Me2-bipy)2, were the most active,
with EC50 values of 35 mM and 73 mM, respectively. The
mono-2,29-bipyridal complex, VO(bipy), was the least active (EC50 5 118 mM). The 5-bromo derivative of bis-29hydroxyacetophenone, a neutral complex, was also potent,
with an EC50 value of 13.4 mM. These marked differences
(21-fold) in potency of the spermicidal activity elicited by
FIG. 2. Spermicidal activity of oxovanadium(IV) complexes with mono
or bis 1,10-phenanthroline and 2,29-bipyridyl as ancillary ligands. Concentration-response curves showing the effects of 7 oxovanadium(IV)
complexes on human sperm motility. Highly motile fractions of sperm
were incubated for 3 h with increasing 2-fold concentrations (1.9–250
mM) of oxovanadium(IV) complexes VO(phen), VO(phen)2, VO(Cl-phen)2,
VO(Me2-phen), VO(bipy)2, VO(Me2-bipy), or VO(Me2-bipy)2, or 0.25%
DMSO alone in the assay medium; and the percentages of motile sperm
were evaluated by CASA. Each point represents the mean from two to
four independent experiments. The SD for each drug was , 10% of the
mean values.
the three ancillary heteroligands and their derivatives suggest that the spermicidal potency of oxovanadium(IV)-complexes is modulated by the 5-membered bidentate ligands.
The spermicidal activity of the most potent oxovanadium(IV) complex, VO(Cl-phen)2, was 14-fold more potent
than that of the commercial detergent-based spermicide, N9 (79 mM), when tested under identical experimental conditions.
Figure 2 shows the concentration-response curves of
spermicidal effects of 7 representative oxovanadium(IV)
complexes: VO(phen), VO(phen)2, VO(Cl-phen)2, VO(Me2TABLE 2. Comparative spermicidal activity by CASA and acrosomal loss
by the flow cytometric anti-CD46 mAb binding assay after exposure of
sperm to 11 oxovanadium(IV) complexes containing either mono- and
bis-1,10-phenanthroline, 2,29-bipyidyl, or 59-bromo-29-hydroxyacetophenone and derivatives.
Treatment
DMSO control
VO(phen)
VO(phen)2
VO(Me2-phen)
VO(Me2-phen)2
VO(Cl-phen)
VO(Cl-phen)2
VO(bipy)
VO(bipy)2
VO(Me2-bipy)
VO(Me2-bipy)2
VO(Br,OH-acph)2
N-9
EC50 (mM)a
NAd
37
6.5
20.5
48
39
5.5
118
35
83
73
13.4
79
Anti-CD46 positive
sperm (%)bc
5.2
9.7
9.9
14.0
11.7
11.2
21.5
6.1
4.7
6.4
3.2
24
86
6
6
6
6
6
6
6
6
6
6
6
6
6
0.7
0.7
3.4
2.5e
1.1
0.9
0.5e
1.2
0.3
0.8
0.6
0.6e
2e
a Stock solutions (100 mM) in DMSO were tested in serial 2-fold dilutions
from 250 mM to 1.9 mM.
b Tested at 100 mM.
c Mean 6 SD for two experiments using sperm from different donors.
d NA, not applicable.
e p , 0.05 compared with DMSO control.
440
D’CRUZ ET AL.
FIG. 3. Effect of bis 5-chloro-1,10-phenanthroline oxovanadium(IV) sulfate, VO(Clphen)2, on sperm motion parameters analyzed by CASA. A) Concentration-dependent inhibition of sperm motility parameters. Motile fractions of sperm were
incubated in assay medium in the presence of three increasing concentrations of
VO(Cl-phen)2 (0, 7.8, 15.6, and 31.2 mM)
for 3 h at 378C, and the centroid-derived
motility characteristics were determined
using the Hamilton-Thorne-IVOS version
10 CASA. B) Time-dependent effect on
sperm kinematics. Motile fractions of
sperm were incubated for 5, 10, and 15
min in assay medium in the presence of
200 mM of VO(Cl-phen)2, and the motility
characteristics were determined by CASA
as described in Materials and Methods.
The sperm motion parameters were (left to
right): PRG, progressive motility (%); VCL,
curvilinear velocity (mm/s); VSL, straight
line velocity (mm/s); VAP, average path velocity (mm/s); STR, straightness, VSL/VAP
(%); LIN, linearity, VSL/VCL (%); BCF, beat/
cross frequency (Hz); and ALH, amplitude
of lateral head displacement (mm). Values
are mean 6 SD of two representative experiments. Significant difference ( p ,
0.05) between control and VO(Cl-phen)2treated sperm: progressive motility, VCL,
VAP, and VSL.
phen), VO(bipy)2, VO(Me2-bipy), and VO(Me2-bipy)2. The
spermicidal activity of the oxovanadium(IV) complexes
was strongly dependent on the type of coordinated heteroligands. The oxovanadium(IV) complexes stabilized with
5-membered bis-chelated ligands of phenanthroline, bipyridyl, and acetophenone, with a vanadium(IV) atom conferring a ‘‘butterfly structure,’’ had superior spermicidal activity when compared with diaqua monochelated complexes
(Fig. 1).
Also, in comparison to N-9, the spermicidal activity of
oxovanadium(IV) complexes was not associated with a
concomitant loss of acrosomal membrane or membrane
damage as quantitated by the flow cytometric anti-CD46
mAb binding assay using unfixed sperm suspensions [29,
30]. Despite complete sperm motility loss quantitated after
a 3-h incubation period, 76–97% of the treated sperm remained anti-CD46-negative (acrosome-intact) (Table 2).
The most potent oxovanadium(IV) complexes, VO(Clphen)2 and VO(Br,OH-acph)2, after a 3-h incubation period
induced a 4- to 5-fold increase (21.5% 6 0.5% and 24.3%
6 0.6%, respectively, p , 0.05) in acrosome reactions over
control (5.2% 6 0.7%). However, complete sperm motility
loss with these complexes was achieved within 2 and 10
min of exposure. Thus, the spermicidal activity of oxovanadium(IV) complexes was not concomitantly associated with
disruption of sperm membranes.
Kinetics of Sperm Immobilization by Oxovanadium(IV)
Complexes Was Variable
Interestingly, the kinetics of sperm immobilization by
the 11 oxovanadium(IV) complexes was variable. The corresponding times required for 50% motility loss of progressively motile sperm exposed to these complexes ranged
SPERMICIDAL OXOVANADIUM(IV) COMPLEXES
441
FIG. 4. Flow cytometric quantitation of
oxovanadium(IV) complex-induced apoptotic sperm. Motile sperm were incubated at 378C in either control medium (0.1%
DMSO) or medium supplemented with
100 mM of a representative oxovanadium(IV) complex, VO(Cl-phen)2. The apoptosis-inducing ability of VO(Cl-phen)2
(right panels) in comparison with medium
control (left panels) was tested by three
flow cytometric assays that quantitatively
assess changes of the mitochondrial membrane potential, based on JC-1 staining
(A, B); surface plasma membrane, based
on FITC-Annexin V-staining (C, D); and
sperm nuclear compartment, based on
FITC-dUTP nick-end labeling of fragmented DNA (E, F) after 3, 12, and 24 h, respectively. Note the marked reduction in
JC-1 red fluorescence (aggregates) labeling
with no reduction in green emission
(monomers). In C–F, sperm nuclei were
counter-stained with PI.
from , 1 min to . 60 min. Sperm immobilization by the
neutral complex, VO(Br,OH-acph)2, was the fastest, followed by VO(Cl-phen)2, with T1/2 values of 38 sec and 7.3
min, respectively. The other cationic oxovanadium(IV)
complexes showed a lag period of 30–60 min to bring
about . 50% sperm motility loss. By comparison, sperm
motility in control samples remained stable during the 3-h
monitoring period.
Oxovanadium(IV) Complexes Affected Sperm Kinematics
The observed concentration- and time-dependent decreases in sperm motility after exposure to the 11 oxovanadium(IV) complexes were associated with significant
changes in the centroid-derived movement characteristics
of the surviving sperm, particularly with respect to the track
speed (VCL), straight-line velocity (VSL), and path velocity (VAP). The representative sperm kinematic parameters
observed for VO(Cl-phen)2 versus concentration and time
are shown in Figure 3, A and B, respectively. The decreases
in VCL, VSL, and VAP were similar in magnitude with
increasing concentrations of VO(Cl-phen)2 or exposure
time. However, the linearity (LIN) of the sperm tracks and
the straightness (STR) of the swimming pattern were affected only with increasing concentration of the drug. The
beat-cross frequency (BCF) and the amplitude of lateral
sperm head displacement (ALH) were relatively uniform as
the proportion of motile sperm declined with increasing
concentration (0–15.6 mM) or exposure time (0–10 min).
By contrast, the sperm motion parameters of control sperm
showed insignificant changes during the 3-h exposure.
Oxovanadium(IV) Complexes of 1,10-Phenanthroline,
2,29-Bipyridyl, and 59-Bromo-29-Hydroxyacetophenone
and Derivatives Induced Apoptosis in Human Sperm
Because the phenanthroline complex of vanadium(IV)
has been shown to induce hydroxyl-mediated DNA strand
breaks [27, 36], we tested the effects of the 11 oxovanadium(IV) complexes with phenanthroline, bipyridyl, and
acetophenone as ancillary ligands to induce apoptosis in
human sperm. We used three independent apoptosis assays
to quantitatively assess changes at the mitochondrial, surface membrane, and nuclear level. Analysis by flow cytometry of the mitochondrial membrane potential changes occurring during apoptosis were analyzed with a DCm indicator, JC-1, a carbocyanine cationic dye, by following fluorescence associated with the uptake of JC-1 to evaluate
DCm modifications [33]. Motile sperm exhibited intense
green and red fluorescence of JC-1 (Fig. 4A). It can be seen
that a 3-h treatment with the oxovanadium(IV) complex
VO(Cl-phen)2 resulted in an extinction of the red fluores-
442
D’CRUZ ET AL.
FIG. 5. Confocal laser scanning microscopy images of sperm nuclei undergoing oxovanadium-induced apoptosis. Motile sperm were incubated for
24 h in medium with 100 mM VO(Cl-phen)2, fixed, permeabilized, and visualized for DNA degradation in a TUNEL assay. A) Sperm nuclei counterstained
with PI (red). B) Sperm nuclei visualized for FITC-dUTP incorporation (green). C) Nuclei of VO(Cl-phen)2-treated sperm show dual fluorescence.
Apoptotic nuclei appear yellow because of superimposed labels. Original magnification 31000 (reproduced at 89%).
cence (Fig. 4B), indicating that alteration occurred after
VO(Cl-phen)2 treatment. A 3-h pretreatment of sperm with
7 of the 11 oxovanadium(IV) complexes resulted in variable decreases in DCm-related fluorescence observed as
31–73% reduction (p , 0.05) in JC-1 aggregate (orange/
green) fluorescence without concomitant reduction in JC-1
monomer (green) fluorescence (Table 3). By contrast, .
90% of control sperm were positive for orange/red fluorescence. The most potent spermicidal agents, VO(Cl-phen)2
and VO(phen)2, induced the maximum shift. Therefore,
DCm modifications, evaluated by the uptake of cationic
lipophilic dye, were detected early in the process of apoptosis induced by oxovanadium(IV) complexes.
Changes in the plasma membrane of the cell surface also
appear early in cells undergoing apoptosis [37, 38]. In apoptotic cells, the membrane phospholipid phosphatidyl serine is translocated from the inner to the outer leaflet of the
plasma membrane, thereby exposing phosphatidyl serine to
TABLE 3. Apoptosis-inducing property of spermicidal oxovanadium(IV)
complexes containing mono- and bis-1,10-phanthroline, 2,29-bipyridyl
and 59-bromo-29-hydroxyacetophenone and derivatives.
Treatment
DMSO control
VO(phen)
VO(phen)2
VO(Me2-phen)
VO(Me2-phen)2
VO(Cl-phen)
VO(Cl-phen)2
VO(bipy)
VO(bipy)2
VO(Me2-bipy)
VO(Me2-bipy)2
VO(Br,OH-acph)2
a
JC-1
aggregatepositive
sperm (%)ab
92
35
25
38
63
39
35
95
94
88
62
83
6
6
6
6
6
6
6
6
6
6
6
6
3
12c
1c
2c
2c
4c
1c
1
1
5
5c
3
Annexin
V-positive
sperm (%)ab
7
97
98
96
96
90
99
36
26
47
78
95
6
6
6
6
6
6
6
6
6
6
6
6
3
1c
1c
3c
1c
2c
1c
8c
1c
16c
7c
1c
TUNELpositive
sperm (%)ab
9
98
98
97
94
95
97
43
40
45
63
98
6
6
6
6
6
6
6
6
6
6
6
6
1
1c
1c
1c
1c
1c
1c
16c
22
10c
22c
1c
Motile sperm were incubated at 378C for 3, 12, and 24 h in either
control medium or in medium supplemented with 100 mM each of the
11 oxovanadium(IV) complexes; stained respectively with JC-1, FITC-Annexin V, or FITC-dUTP; and analyzed by flow cytometry.
b Mean 6 SD for two separate experiments using sperm from different
donors.
c p , 0.05 compared with DMSO control.
the external cellular environment [38]. Annexin V binds to
phosphatidyl serine residues that are exposed on the surface
of cells undergoing apoptosis. The apoptosis-dependent surface binding of FITC-labeled recombinant human Annexin
V with 10 of the 11 oxovanadium(IV) complex-treated
sperm showed a dramatic increase in binding of Annexin
V to sperm membrane (Table 3). After 12 h of incubation,
26–99% (p , 0.05) of the treated sperm were apoptotic.
Control sperm exhibited minimal fluorescence (Fig. 4C).
By contrast, 99% of VO(Cl-phen)2-treated sperm were positive for FITC-Annexin V (Fig. 4D), indicating that surface
membrane alteration occurred after prolonged exposure to
oxovanadium(IV) complexes. Control sperm treated with
0.1% DMSO alone showed only 7 6 3% Annexin V positivity at 12 h. The most potent spermicidal complexes,
VO(Cl-phen)2 and VO(phen)2, also induced maximum Annexin V positivity.
Next, TdT-mediated labeling of exposed 39-OH termini
of nuclear DNA with FITC-conjugated dUTP by the in situ
TUNEL method was employed to demonstrate that oxovanadium(IV) complexes induced apoptosis in the sperm
nuclear compartment. Figure 4, E and F, depicts the twocolor flow cytometric contour plots of sperm nuclei of control sperm (E) treated with 0.1% DMSO and test sperm (F),
respectively, treated with 100 mM of VO(Cl-phen)2 in 0.1%
DMSO after staining with FITC-dUTP and counterstaining
with PI. More than 97% of VO(Cl-phen)2-treated sperm
became apoptotic (TUNEL-positive) after 24 h of incubation (Fig. 4F). A 24-h exposure of sperm with any one of
the 11 oxovanadium(IV) complexes evaluated resulted in a
marked increase of TUNEL-positive cells, observed as a
43–98% (p , 0.05) increase in FITC-dUTP fluorescence
(Table 3). By contrast, , 10% of control sperm treated with
0.1% DMSO alone showed apoptotic nuclei after 24 h of
incubation. The percentages of apoptotic sperm quantitated
by the flow cytometric TUNEL assay correlated well with
the potency (EC50 values) of these oxovanadium(IV) complexes in sperm immobilization assays (r2 5 0.557; p ,
0.05). Figure 5 depicts confocal microscopy images of
sperm nuclei treated with 100 mM VO(Cl-phen)2 in 0.1%
DMSO after incubation with TdT and FITC-dUTP with (A
and C) and without (B) PI counterstaining. Confocal images
of TUNEL-positive sperm clearly indicated that the fluo-
SPERMICIDAL OXOVANADIUM(IV) COMPLEXES
rescence was localized to the sperm nuclear region. Nuclei
of VO(Cl-phen)2-treated sperm showed dual fluorescence
(C) consistent with apoptosis.
DISCUSSION
Our results provide unprecedented evidence that oxovanadium(IV) complexes with 1,10-phenanthroline, 2,29-bipyridyl,
or 59-bromo-29-hydroxyacetophenone and their derivatives
linked to vanadium(IV) via nitrogen or oxygen atoms have
potent spermicidal activity against human sperm. The order
of spermicidal efficacy for the 11 oxovanadium(IV) complexes synthesized and evaluated was as follows: VO(Cl-phen)2
. VO(phen)2 . VO(Br,OH-acph)2 . VO(Me2-phen) .
VO(bipy)2 . VO(phen) . VO(Cl-phen) . VO(Me2-phen)2
. VO(Me2-bipy)2 . VO(Me2-bipy) . VO(bipy). Thus, despite the similarities in 5-membered chelating units of phenanthroline and bipyridyl, the phenanthroline complexes of oxovanadium(IV), particularly the bis-phenanthroline complex,
VO(Cl-phen)2, were the most active; and the mono bipyridyl
complex, VO(bipy), was the least active. In addition, the oxovanadium(IV) complexes of the vanadium(IV) atom stabilized with two 5-membered bidentate ligands were 3- to 7fold more potent than the diaqua monochelated complexes of
vanadium(IV).
The kinetics of sperm immobilization by the oxovanadium(IV) complexes was dependent on their net charge.
Structure-activity relationship analyses of 19 vanadocenes
[23–25] and 11 oxovanadium(IV) complexes clearly demonstrated that the spermicidal properties of these complexes
were determined by the oxidation state of the vanadium(IV)
atom. The various ancillary ligands linked by either carbon,
nitrogen, or oxygen atoms to the central vanadium(IV)
atom significantly contributed either to fine tuning of the
spermicidal potency or enhancing the stability of these
complexes in aqueous solution. In addition, similar to our
earlier findings with neutral complexes of vanadocenes
[24], the neutral complex of oxovanadium(IV), VO(Br,OHacph)2, rapidly inactivated sperm in seconds in comparison
to the cationic chelated oxovanadium(IV) complexes or cationic chelated vanadocenes, which required a lag period of
several minutes [25]. Therefore, it appears from our study
that despite the tetrahedral geometry of the ‘‘bent-sandwich’’ structures of vanadocenes or the square pyramidal
geometry/‘‘butterfly’’ structures of oxovanadium(IV) complexes, the rapidity of vanadium(IV)-mediated spermicidal
activity was dependent on the neutrality of these complexes. Because the neutral complex of oxovanadium(IV),
VO(Br,OH-acph)2, was a rapid spermicidal agent, it is likely that this complex of oxovanadium(IV) is rapidly transported across the sperm cell membranes. Because of its
rapidity and potency, VO(Br,OH-acph)2 may be useful as a
contraceptive agent.
The mechanism of sperm motility loss induced by oxovanadium(IV) complexes is yet to be determined. Both the
vanadocenes(IV) and oxovanadium(IV) complexes also
have antitumor activity [11, 12, 27]. The antitumor effects
of vanadium(IV) complexes are thought to be due to their
reaction with the vanadyl-H2O2 system, which results in the
generation of hydroxyl radicals in a Fenton-like reaction
[7, 9, 27]. In particular, the oxovanadium(IV) complex, after dissociation of phenanthroline rings, results in the formation of peroxocompounds that in the presence of H2O2
generate hydroxyl radicals. In support of this hypothesis is
the observation that the vanadyl-phen complex induces hydroxyl radical-dependent DNA cleavage in the presence of
H2O2 [36, 39]. The vanadyl complex [VO(phen)(H2O)2]21
443
has high antitumor activity toward human nasopharyngeal
carcinoma [27]. Hydrogen peroxide is formed in cells by
dismutation of superoxide anions, which are generated in
various systems such as xanthine-oxidase, NADPH oxidase, and NADH-dependent cytochrome P450 and neutrophils [40]. Thus, H2O2 is thought to react with oxovanadium(IV) bound to DNA to generate ROS, resulting in
cleavage of DNA. It is likely that oxovanadium(IV)-induced sperm motility loss and apoptosis are mediated primarily by the ability of these complexes to induce ROSmediated damage to sperm. In sperm, an NADPH-dependent superoxide-generating system has been demonstrated
[41]. In addition, the ability of H2O2 generating Lactobacillus acidophilus, which is present in the vaginas of most
normal women, can further potentiate the spermicidal activity of intravaginally applied oxovanadium(IV) complex.
This is in contrast to the commercial vaginal detergent spermicide, N-9, which is selectively toxic to Lactobacilli [42,
43]. Furthermore, unlike N-9, the spermicidal activity of
the oxovanadium(IV) complexes was not concomitantly associated with membrane disruption.
We ascribe the irreversible nature of the sperm-immobilizing activity of oxovanadium(IV) complexes to their
ability to induce apoptosis. We used three independent
methods that quantitatively assess apoptotic changes in the
mitochondria, surface membrane, and nuclear compartment. Mitochondria are the primary targets for apoptosis,
and alterations in mitochondrial structure and function are
early events of apoptotic cell death [32]. Our studies demonstrated that spermicidal oxovanadium(IV) complexes induced depolarization of sperm mitochondria, an early marker for apoptotic cell death. Prolonged exposure of sperm to
these spermicidal complexes also resulted in increased
FITC-Annexin V binding to sperm surface due to membrane changes during apoptosis, as well as increased dUTP
incorporation in the nuclei of treated sperm. Since vanadium(IV) compounds by themselves do not cleave DNA
[44], the dramatic uptake of dUTP observed in our study
appears to be due to the cleavage of the DNA polymer as
a result of cytotoxicity induced by ROS-mediated effects
of oxovanadium(IV) complexes. The fact that human sperm
are exquisitely sensitive to oxidative stress, and the ability
of oxovanadium(IV) complexes to potentiate these effects
establishes these oxovanadium(IV) complexes as a new
class of gentle contraceptive agents. In addition, our unpublished observations indicate that some of these oxovanadium(IV) complexes selectively inhibit the growth of human testicular cancer cells via the induction of apoptosis.
This activity profile indicates that oxovanadium(IV) complexes could be useful as male antifertility agents as well
as anticancer agents against testicular germ cell tumors.
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