N,N'–Dithiobisphthalimide, a disulfide aromatic compound, is a potent spermicide agent in humans

Abstract

Several studies have shown that users of vaginal preparations containing nonoxynol-9 (N-9) are at a high risk for sexually transmitted diseases, including HIV. Therefore, there is a great interest in identifying compounds that can specifically inhibit sperm without damaging the vaginal lining, possess a powerful spermicide activity, and can be used in contraceptive vaginal preparations to replace N-9. In this work, we studied the spermostatic and/or spermicidal activity of five non-detergent, disulfide compounds on human sperm, HeLa cells, and Lactobacillus acidophilus. The motility and viability of human sperm in semen and culture medium was evaluated after treatment with different concentrations of the disulfide compounds (2.5 – 100 µM). In addition, we evaluated the cytotoxic effect on HeLa cells and L. acidophilus. We identified compound 101, N,N'-dithiobisphthalimide (No. CAS 7764-30-9), as the most effective molecule. It has a half maximal effective concentration (EC₅₀) of 8 µM and a minimum effective concentration (defined as the concentration that immobilizes 100 percent of the sperm in 20 sec) of 24 µM. At these concentrations, compound 101 does not affect the viability of the sperm, HeLa cells, or L. acidophilus. Our results indicate that dithiobisphthalimide has a potent spermostatic, irreversible effect with no toxic effects on HeLa cells and L. acidophilus.

Keywords:

• contraception
• fertility
• human sperm
• spermostatic agent

Introduction

The world's population continues to grow at an alarming speed, threatening the ecological balance of the planet [http://www.census.gov/ipc/www/popclockworld.html]. In developing countries, where resources are scarce, the population grows even faster, presenting an obstacle to improving living conditions. Family planning is one relevant aspect of this problem and benefits people and countries in many ways. If unintended pregnancies could be avoided, then approximately one-quarter of maternal deaths could be prevented [WHO 2006].

Contraception allows women to limit pregnancies to the years during which they experience good health and fertility. Furthermore, contraception allows women to avoid giving birth more often than is desirable for their health. Spacing pregnancies at least two years apart helps women have healthier children and contributes to improving child survival by 50% [WHO 2006]. In addition, family planning encourages the adoption of less risky sexual practices and thereby helps to prevent the transmission of sexually transmitted diseases (STDs), including HIV/AIDS [McMahon et al. 2004] and helps to prevent abortions from occurring under unsafe conditions. Fertilization and STDs have a common mode of transmission (sexual), relevant anatomic environment (the female reproductive tract), and transport vehicle (semen and cervical fluid) [Doncel 2005]. These three common features justify the development of agents that can block the fertilization and simultaneously the transmission of STDs. Given that the sperm and the causative agents of STDs both travel in the semen and come into contact with their targets after vaginal penetration and ejaculation, it seems reasonable that a formulation that could be applied to the cervical cavity before intercourse could have both a contraceptive and microbicide effect [Dwivedi et al. 2007; Jain et al. 2007]. Regardless of their specific molecular targets, contraceptive microbicides mainly interact with the sperm and the causative agents of STDs in the vagina. The spermicide that is most widely employed in medicated barrier methods (condoms, caps, and diaphragms) or in stand-alone spermicidal foams and creams is the detergent nonoxynol-9 (N-9). N-9 has been available over the counter in the United States for almost 50 years. Although N-9 was only approved for use as a contraceptive, some studies have suggested that it can be a potential microbicide. However, the use of N-9 as a contraceptive agent in humans has also been associated with genital tract inflammation and an increased risk of contracting HIV, chlamydia, gonorrhea, and other STDs [Fichorova et al. 2001; Van Damme et al. 2002]. In light of these data, there is clearly an urgent need to identify a powerful, non-detergent spermicide to replace N-9.

Spermicides that are capable of killing 100% of human sperm almost instantaneously at physiological concentrations in vitro are likely to provide adequate pregnancy protection in vivo. However, the molecules that are designed for such an activity often target other cells in the vagina, such as the cervico-vaginal epithelia and Lactobacilli that are crucial for maintaining a natural barrier to pathogen invasion, especially HIV. N-9 is one such molecule that kills sperm, bacteria (including lactobacillus), viruses, and cervico-vaginal cells in vitro [Beer et al. 2006; Dover et al. 2007; Ojha et al. 2003] because of its strong surfactant action. Hence, there is a need for molecules that specifically target the sperm cells in the vagina, while remaining practically inert to other cell types at spermicidal concentrations.

Recently, a new series of non-detergent, disulfide esters of carbothioic acid were discovered and found to include several very promising spermicidal molecules. Some of these molecules could, in theory, replace N-9 in contraceptive preparations [Dwivedi et al. 2007; Jain et al. 2007; Jain et al. 2009; Jain et al. 2010]. In this work, we report the effect of five aromatic compounds containing a disulfide bond in their structure on human sperm. We also evaluate the effect of these molecules on HeLa cells and L. acidophilus.

Results

Evaluation of the effect of the disulfide molecules on human sperm in semen

The spermicidal/spermostatic effect of the five disulfide molecules described in Tables 1 and 2 was evaluated at different concentrations using human semen containing sperm. The results of the Sander-Cramer test showed that four of the five compounds have a potent sperm immobilizing effect (Fig. 1A). The calculated minimum effective concentrations (MECs) were 24 µM, 41 µM, 82 µM, and 88 µM for compounds 101, S2, 116, and J2, respectively. The reported MEC for N-9 is 811 µM [Jain et al. 2007; Lakshmi et al. 2008]. Compound 269 does not have any significant effect on human sperm movement (Fig. 1A). Sperm viability is not affected by any of the disulfide compounds at any concentration tested (Fig. 1B). This result indicates that the molecules have a spermostatic, rather than a spermicidal, effect, at the concentrations tested. Compound 101 was the most potent of the molecules tested. It immobilized 100% of the sperm within 20 sec at a concentration of 24 µM without causing any toxic effects (86 ± 2% (mean ± sem) viable sperm at all concentrations tested) (Fig. 1B).

Figure 1.  The effect of disulfide compounds on sperm immobilization (A) and viability (B) in human semen samples evaluated by the method of Sander and Cramer (1941). A) Semen was mixed with different concentrations of the disulfide compounds and gently vortexed for 10 sec. Then, the percentage of motile sperm was evaluated under a phase contrast microscope at 37°C. B) Sperm viability was evaluated using eosin Y staining according to the protocol outlined by the World Health Organization [WHO 2010]. The mean ± sem of five experiments performed with different donors is shown.

Table 1. Disulfide symmetric compounds.

Name	Formula	Purity	Identification No.	Molecular Weight	Chemical Structure
2,2′-Dithiobis (5-nitropyridine) No. CAS: 2127-10-8	C10H6N4O4S2	96%	116	310.31
5,5′-Dithiobis(1-phenyl-1H-tetrazole) No. CAS: 5117-07-1	C14H10N8S2	95%	269	354.42
N,N'-Dithiobisphthalimide No. CAS: 7764-30-9	C16H8N2O4S2	95%	101	356.38

Table 2. Disulfide asymmetric compounds.

Name	Identification No.	Molecular Weight	Symmetrical Compounds	Composition	Chemical Structure
5-(5-Nitropiridin-2-ildithio)-1-phenyl-1H-tetrazole	S2	332.37	116 + 269	One 5 Nitropyridine ring and one 1-phenyl-1H-tetrazole, with a disulfide bond
(5-5-nitropyridine-2-ildithio)- phthalimide	J2	333.35	116 + 101	One 5 Nitropyridine ring and one phthalimide group, with a disulfide bond

We also found that the effect of the compounds on the sperm is irreversible. When sperm were treated with the compound in semen and then thrice washed in culture medium and incubated for a further 2 h, the percentage of immotile sperm remained significantly high (compare black and white bars in Fig. 2).

Figure 2.  The irreversible effect of the disulfide compounds on human sperm motility. Semen (black bars) was mixed with different concentrations of the disulfide compounds, and the motility recorded immediately thereafter. Control samples received 0.1% DMSO (0 µM concentration bars). The sperm was then washed with Tyrode medium (white bars) and incubated for 2 h. Motility was recorded again. The mean ± sem of three experiments performed with different donors is shown. *P < 0.001 comparing all treatments.

Effect of the disulfide molecules on human sperm in culture medium

The effect of the disulfide molecules on human sperm separated by a percoll gradient and then resuspended in culture medium was found to be very similar to its effect on sperm in neat semen (Fig. 3). Again, the most potent compound evaluated was compound 101; compound 269 has no significant effect on sperm movement. We next tested the effect of each compound on the viability of human sperm in culture medium (Fig. 4). Sperm aliquots were incubated with each molecule for 30 min, and then the viability was evaluated. At the MEC for each compound, sperm viability was found to be unaffected (Fig. 4), with the exception of compound J2, which was found to cause a significant decrease in sperm viability at 25 µM. In contrast, compound 269 does not affect sperm viability at any of the concentrations tested (data not shown).

Figure 3.  The effect of the disulfide compounds on sperm immobilization in samples resuspended in culture medium. Motile spermatozoa were selected by centrifugation through a two-step Percoll gradient, resuspended in culture medium and treated with different concentrations of the disulfide compounds for 30 min Then, the percentage of motile sperm was measured under a phase contrast microscope. The mean ± sem of eight experiments performed with different donors is shown.

Figure 4.  The effect of the disulfide compounds on sperm viability and immobilization in human samples resuspended in culture medium. Motile spermatozoa were selected by centrifugation through a two-step Percoll gradient, resuspended in culture medium and treated with different concentrations of the disulfide compounds for 30 min. Control samples received 0.1% DMSO (0 µM concentration bars). Then, the percentage of viable and immotile sperm was evaluated. The mean ± sem of eight experiments performed with different donors is shown. *P < 0.01 compared with the control.

The effect of the assayed disulfide molecules on HeLa cells

HeLa cells were exposed to different concentrations of each compound for 24 h (Fig. 5). Viability significantly decreased after treatment with compounds S2 and 116 at concentrations above 15 µM (68% and 64% viable cells, respectively). At the highest concentration tested (100 µM), cell viability was found to be 43% and 53% for compounds S2 and 116, respectively. Treatment with compound 101 did not decrease HeLa cell viability at concentrations below 75 µM (59% viable cells at 75 µM and 42% viable cells at 100 µM) (Fig. 5). Thus, compound 101 is the least cytotoxic to HeLa cells. As with sperm HeLa cells were unaffected by compound 269 (data not shown).

Figure 5.  The cytotoxic effect of the disulfide compounds on the human cervical (HeLa) cell line. HeLa cells were incubated for 24 h with different concentrations of the disulfide compounds. Control samples received 0.1% DMSO (0 µM concentration bars). Then, cell viability was evaluated using a Cell Proliferation Assay from Promega (see Materials and Methods). The results represent the mean ± sem of five experiments. *P < 0.01 compared with the control.

We next performed a series of dose-response experiments to measure the MEC and the half maximal effective concentration (EC₅₀) of compound 101 for human sperm and HeLa cells (Table 3). We determined that the sperm MEC of compound 101 is 24 µM and that the EC₅₀ is 8 µM. For HeLa cells, we determined that the MEC is 475 µM and that the EC₅₀ is 86 µM. We observed no cytotoxicity for compound 101 in HeLa cells at the sperm MEC (24 µM) (Fig. 5). The security index of compound 101 is 3.853 (the quotient of the cytotoxic EC₅₀ and spermostatic EC₅₀).

Table 3. Effect of compound N,N'-dithiobisphthalimide (101) on sperm motility and HeLa cell viability.

Concentration (μM)	% Motile sperm (mean ± sem)	% Viable HeLa cells (mean ± sem)
0	80 ± 4.1	100 ± 0
15	23 ± 3	99 ± 1.6
20	9 ± 1	98 ± 1.2
25	0 ± 0	100 ± 1.4
50	0 ± 0	96 ± 1.1
75	0 ± 0	59 ± 2.0
100	0 ± 0	42 ± 2.4
200	0 ± 0	18 ± 2.7

The effect of the disulfide molecules on Lactobacillus acidophilus

Compound 101 was further assayed for its effect on L. acidophilus (Fig. 6). This compound did not affect the growth of L. acidophilus at the spermostatic MEC of 24 µM. The MEC and half maximal inhibitory concentration (IC₅₀) of this compound on L. acidophilus were determined to be 2951 µM and 530 µM, respectively (Fig. 6). It has been reported that N-9 significantly inhibits the growth of Lactobacillus colonies by approximately 84% at the MEC and 100% at 2 x MEC [Jain et al. 2007; Lakshmi et al. 2008].

Figure 6.  The cytotoxic effect of disulfide compound 101 on the growth and survival of Lactobacillus acidophilus colonies in vitro. L. acidophilus were incubated for 24 h with different concentrations of the disulfide compound 101. Control samples received 0.1% DMSO (0 µM concentration bars). Then, the number and size of colonies were recorded. The results represent the mean ± sem of five experiments. Data are expressed as a percentage of the control, which was considered to be 100%. *P < 0.01 compared with the control.

Discussion

Considerable effort in the last few years has been devoted to testing compounds that lack the side effects of N-9. N-9 and other surfactants exhibit detergent-type cytotoxicity for vaginal cells, thereby increasing the risk of transmitting an STD/HIV [Roddy et al. 2002; Stephenson 2000; Van Damme et al. 2002]. In addition, N-9 is known to inhibit the growth of Lactobacilli, further increasing the likelihood of transmitting an STD/HIV [Martin et al. 1999; Myer et al. 2005; Taha et al. 1998]. Commensal bacteria populate the vaginal mucosa of healthy women of a childbearing age and help to prevent urogenital infections [Redondo-Lopez et al. 1990]. Lactobacilli are key members of the vaginal microflora that help to maintain a sterile environment by producing hydrogen peroxide and an acidic environment. The depletion of vaginal Lactobacilli is associated with establishing opportunistic infections and an increased risk of acquiring HIV and herpes simplex virus type 2 [Cherpes et al. 2005; Cohn et al. 2001]. The principal Lactobacillus species that is isolated from the vaginal mucosa of healthy women is L. acidophilus. This species was therefore chosen for the evaluation of the safety of the disulfide molecules tested. Here, we have provided evidence to indicate that several disulfide bond containing aromatic molecules have a potent immobilizing effect on human sperm, either in neat semen or upon resuspension in culture medium. This effect was found to be irreversible and rather specific to sperm cells, as the molecules do not affect HeLa cells or L. acidophilus at the same effective concentration. The results show that the disulfide molecules specifically affect the movement of the sperm, rendering them immotile after 20 sec but otherwise alive. These compounds do not affect the viability of the sperm, HeLa cells, or L. acidophilus. Compound 101 was the least cytotoxic to HeLa cells and L. acidophilus. Compound 269 had no effect on these cells. Of the five compounds tested, 116, 101, S2, and J2 decreased sperm motility but not viability. Compounds116 and S2 were cytotoxic to HeLa cells. Thus, compound 101 has a potent spermostatic effect and a low cytotoxic effect on human sperm and HeLa cells.

The role of sulfhydryl (SH) and disulfide (SS) groups in maintaining sperm membrane fluidity and integrity [Nivsarkar et al. 1998; Yelinova et al. 1996], and consequently normal sperm function [Nivsarkar, et al. 1998; Sinha et al. 1993] is well documented. Others have suggested that the SH groups change during sperm maturation and capacitation [Calvin and Bedford 1971] in the head and tail [Bedford and Calvin 1974; Calvin and Bedford 1971; Huang et al. 1984]. Loss of these groups causes inhibition of superoxide dismutase activity, both in human and rat caudal sperm, resulting in enhanced production of superoxide radicals and ultimately a decrease in motility [Kumar et al. 1990]. Reactive oxygen species (ROS) enhances membrane fluidity via lipid peroxidation [Nivsarkar et al. 1998], whereas the membrane SH groups play an important role in modulating the sperm membrane by reducing membrane fluidity. Thus, molecules that interact with these groups are likely to be detrimental to sperm, as has been reported for several sulfhydryl-binding agents, such as N-ethylmaleimide [Solanki et al. 2005], SSRI antidepressants [Kiran Kumar et al. 2006b], benzenepropanamine analogues [Kiran Kumar et al. 2006a], and sodium dialkyl dithiocarbamates [Holzaepfel et al. 1959; Tripathi et al. 1996]. SH groups can be used as a marker to assess infertility in unexplained male infertility and provide targets for contraceptive research [Nivsarkar et al. 1998]. The interconversion of thiol groups to disulfides is an important biochemical event for the survival of sperm under oxidative stress [Storey 1997]. Several antimicrobial peptides [Andreu and Rivas 1998] with intramolecular disulfide linkages exert their activity against pathogens by forming channels in the cell membranes; their reduced (thiol) form causes acute membrane permeabilization. Thus, sulfhydryl-binding compounds with disulfide groups may exhibit spermicidal and antimicrobial activity.

As presented above, we found that four of the five aromatic disulfide compounds studied have spermicidal activity of varying levels (116, 101, S2, and J2). Not one of these compounds is a detergent, unlike N-9, which is a detergent surfactant that can affect the cell plasma membrane. N-9 has been shown to be irritating in animal models and in humans [WHO/CONRAD 2001]. The aromatic disulfide compounds that were studied here do not have that feature and, therefore, should not have these non-specific effects. Compound 101 was found to have a powerful spermostatic activity without being spermicidal. In addition, this compound does not damage other cell types, such as HeLa cells and L. acidophilus, making it a very promising molecule to replace N-9 in vaginal contraceptive preparations.

In addition to the side effects caused by the surfactant nature of N-9, this molecule is also required in high concentrations to achieve its spermicidal effect in vaginal preparations. Thus, compound 101 is 37 times safer than N-9, requiring much lower concentrations than N-9 in preparations of vaginal contraceptives. The MEC and EC₅₀ of compound 101 is 24 µM and 8 µM for sperm and 475 µM and 86 µM for HeLa cells, respectively. These are much higher than the MEC and EC50 reported for N-9 (811 µM and 129 µM for sperm and an EC50 of 13.5 µM for HeLa cells) and thus compound 101 should be 37 times safer. It is worth mentioning that the EC₅₀ values of compound 101 for inducing toxicity in HeLa cells and L. acidophilus are much lower than those reported for N-9 [Jain et al. 2007; Lakshmi et al. 2008]. N-9 is known to inhibit the growth of Lactobacillus [Krebs et al. 2002; Ojha et al. 2003] and to irritate the cervical-vaginal epithelium. This effect perhaps causes the observed increased susceptibility to HIV infection by providing a direct subcutaneous entry for the virus [Catalon et al. 2005].

In summary, we have identified a compound that immobilizes 100% of human sperm in 20 sec with a MEC of 24 µM and an EC₅₀ of 8 µM. In addition, this compound does not prevent the growth of HeLa cells or L. acidophilus.

Materials and Methods

Semen Samples

Semen samples were obtained from normal donors after two to three days of sexual abstinence. Prior permission was obtained from the ethics committee of the University of Antofagasta. All samples had normal semen parameters outlined by the WHO [2010] guidelines. The specimens were allowed to liquefy for 30 to 60 min at 37°C in a slide warmer before the effect of the disulfide compounds were tested. To obtain a population of highly motile sperm in the culture medium, they were selected by centrifugation through a two-step Percoll gradient, as described previously [Morales et al. 2003]. Briefly, it consisted of a lower layer of 80% Percoll and an upper layer of 40% Percoll. Aliquots of semen were layered over the top layer of the Percoll gradient and centrifuged for 20 min at 300 g. The pellet was diluted in 10 ml of modified Tyrode medium consisting of 117.5 mM NaCl, 0.3 mM NaH₂PO₄, 8.6 mM KCl, 25 mM NaHCO₃, 2.5 mM CaCl₂, 0.5 mM MgCl₂, 2 mM glucose, 0.25 mM Na pyruvate, 19 mM Na lactate, 70 µg/ml of both streptomycin and penicillin, phenol red, and 0.3% bovine serum albumin (BSA). The sample was then centrifuged again at 300 g for 10 min and resuspended in the same medium but with 2.6% BSA. The sperm concentration was adjusted to10x10⁶ cells/ml, and the cells were incubated at 37°C and 5% CO₂.

Disulfide compounds

Five disulfide compounds were used. Three were purchased from Sigma-Aldrich (St. Louis, MO. USA) and are the symmetrical compounds 116, 2,2′-dithiobis(5-nitropyridine) (Nº CAS 2127-10-8); 269, 5,5′-dithiobis(1-phenyl-1H-tetrazole) (Nº CAS 5117-07-1); and 101, N,N'-dithiobisphthalimide (Nº CAS 7764-30-9) (Table 1). The other two compounds, S2 and J2, are asymmetrical disulfides and were synthesized in the Inorganic Chemical Research Laboratory at the University of Antofagasta (Table 2). Details of their synthesis are given below. All compounds were dissolved in dimethyl sulfoxide (DMSO). Stock solutions of 10 mg/ml were prepared by diluting each compound in saline. Before use, serial dilutions were made in saline. The concentration of DMSO in the sperm aliquots was never more than 0.1%.

Synthesis of the disulfide compounds

The 5-(5-nitropyridin-2-yldithio)-1-phenyl-1H-tetrazole (S2) and 5-(5-nitropyridin-2-yldithio)-phthalimide (J2) were synthesized as follows. All reactions were carried out under an atmosphere of purified nitrogen. The solvents used were dried and distilled prior to use. Both compounds were obtained as yellow block-shaped crystals according to the method described by Tanaka and Ajiki [2004]. Briefly, the following was placed into a 20 ml, three-necked flask equipped with an overhead stirrer: for S2, 5,5′-dithiobis(1-phenyl-1H-tetrazole) (269) (177.2 mg, 4 mmol) and 2,2'-dithio-bis(5-nitropyridine) (116) (31.03 mg, 1 mmol); for J2, N,N'-dithiobisphthalimide (101) (86 mg, 1 mmol) and 2,2'-dithio-bis(5-nitropyridine) (116) (31.03 mg, 1 mmol) in CH₂Cl₂ (5 ml). Once the components were mixed, bis(1,5-cyclooctadiene)rhodium(I) tetrafluoroborate, (Rh(cod)2)BF4 (6 mg, 0.03 mmol) was added, and the resulting mixture was stirred for 3 h at room temperature. The resulting solution was maintained at 298 K for 1.5 h under air. The solution was concentrated and purified by silica-gel chromatography (hexane-ethyl acetate = 20:1 v/v). Yellow block-shaped crystals of S2 suitable for X-ray analysis were grown from a solution in hexane:EtOAc (1:1 v/v) at 298 K over a period of a few days in air. The structure was determined by x-ray diffraction methods [Brito et al. 2007b]. The compound J2 was characterized by classical spectroscopic methods.

The compounds 2,2'-dithio-bis(5-nitropyridine) (116; Nº CAS 2127-10-8), 5,5'-dithiobis(1-phenyl-1H-tetrazole) (269; Nº CAS 5117-07-1), and N,N'-Dithiobisphthalimide (101; Nº CAS 7764-30-9) were purchased from Sigma-Aldrich. Crystals suitable for single-crystal x-ray diffraction analysis were grown by slow evaporation of a solution in ethyl acetate. The structures of both compounds were determined by x-ray diffraction methods [Brito et al. 2007a; Brito et al. 2007c].

The effect of the disulfide compounds on human sperm in semen

A spermicidal test was performed with each dilution, ranging in concentration from 10 µM to the minimum effective concentration (MEC), following the modified method of Sander and Cramer [1941]. This test measures the concentration of the spermicidal agent that is required to immobilize 100% of the sperm in less than 20 sec. Briefly, 0.05 ml of human semen was added to 0.25 ml of the spermicidal compound solution and vortexed (at very low speed) for 10 sec. The controls were aliquots of semen treated with saline plus 0.1% DMSO. A drop of the mixture was immediately placed on a microscope slide, covered with a cover glass and examined under a phase contrast microscope (Olympus, Japan) at 37°C. The sample was scored positive if 100% of the spermatozoa became completely immotile within 20 sec and remained immotile for another 60 min, even after a dilution with excess buffer. Semen samples were maintained at 37°C throughout the experiments. The MEC and the concentration immobilizing ∼50% of sperm (EC₅₀) were determined. EC₅₀ values for the spermicides were determined by mixing semen with spermicide solutions (serially diluted from MEC) and analyzed immediately for percentage motility using phase-contrast optics and a 37°C heated stage microscope. A concentration-versus-percentage motility curve was plotted to determine the approximate EC₅₀ [Jain et al. 2007]. Each analysis was conducted in triplicate using semen from several donors.

Evaluation of sperm viability

Eosin staining was performed to assess sperm viability according to the protocol of the World Health Organization [WHO 2010]. Immediately after performing the Sander and Cramer test, one drop of semen was mixed with two drops of 1% eosin Y between a cover slide and a glass slide. The prepared slide was immediately examined with a phase-contrast microscope at 37°C. At least 100 sperm were recorded.

Evaluation of the irreversibility of the effect of the disulfide compounds (Sperm revival test)

To determine whether the effect of the disulfide compounds was irreversible, 100 µl aliquots of semen were mixed with 20 µl of different dilutions of each compound as described above. Before each incubation, sperm motility and viability were recorded. Subsequently, the sperm were thrice washed with Tyrode medium, resuspended in 100 µl of the same medium and incubated for an additional 2 h at 37 °C and 5% CO₂. Motility and viability were recorded as described [WHO 2010].

The effect of the disulfide compounds on human sperm in culture medium

In addition to assessing the effect of the disulfide compounds on sperm in semen, we also tested the effect of these compounds on sperm resuspended in culture medium. For this experiment, we used a modification of the original method of Sander and Cramer [1941] described above. Briefly, 0.05 ml aliquots of motile human sperm resuspended in modified Tyrode's medium at 10x10⁶ cells/ml were mixed with 0.25 ml of spermicidal compound solution. These sperm were exposed to each compound for 30 min. Then, each sample was gently vortexed for 10 sec. A drop was placed on a microscope slide, covered with cover glass, and examined under a phase contrast microscope (Olympus, Japan) at 37°C. The MEC and EC₅₀ were determined as described above. Sperm viability was determined using the eosin staining, as described above.

The effect of the disulfide compounds on human cervical (HeLa) cells in culture

The CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay from Promega (Promega Corporation, Madison, WI, USA) was used according to the manufacturers protocol to evaluate the cytotoxicity of the disulfide compounds against HeLa cells (a gift from Dr. Ivan Neira). HeLa cells, derived from a human cervical carcinoma cell line, have been extensively used in toxicity assays, including the search for novel spermicides [Beer et al. 2006; Dwivedi et al. 2007; Hughes et al. 2009; Jain et al. 2010; Jain et al. 2009; Lakshmi et al. 2008]. In brief, cells seeded at a density of 2x10⁴ cells/well in a 96-well plate were incubated in Dulbecco's Modified Eagle Medium (DMEM) for 24 h at 37°C in a 5% CO₂ air atmosphere, pH 7.4. After 24 h, the culture medium was replaced with fresh medium containing a dilution of disulfide compounds (from 15 to 100 µM). Control wells contained 0.1% DMSO. After a 24 h incubation, 20 µl of the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) tetrazolium compound was added to each well. After an additional 2 h incubation, the OD was read at 490 nm in a microplate reader (BioRad Laboratories Inc., Hercules, CA, USA). During the assay, the MTS tetrazolium compound is converted to a soluble formazan product. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.

The effect of disulfide compounds on Lactobacillus acidophilus

Lactobacilli acidophilus were cultured in MRS (de Man, Rogosa, and Sharpe) broth, pH 6.5, for 24 h at 37°C in a microaerophilic atmosphere (5 -10% oxygen). After this period, 500 µl aliquots of a suspension containing ∼100 bacteria/ml were placed in eppendorf tubes and incubated with different concentrations of the disulfide molecules at 37°C in a microaerophilic atmosphere. After 24 h, the bacteria were serially diluted to ∼100 bacteria/ml. MRS agar was prepared, and 100 µl aliquots were seeded and incubated for an additional 24 h as above. The number and size of the colonies were recorded at the end of the experiment.

Abbreviations

N-9:	=	nonoxynol-9
EC50:	=	half maximal effective concentration
MECs:	=	minimum effective concentrations
STDs:	=	sexually transmitted diseases
SH:	=	sulfhydryl
SS:	=	disulfide
BSA:	=	bovine serum albumin
ROS:	=	reactive oxygen species
DMSO:	=	dimethyl sulfoxide.

Declaration of Interest: This work was supported by Fondo Nacional de Ciencia y Tecnología de Chile, FONDECYT 1080028 (P.M.) and Fundación Minera Escondida. M.F. thanks the fellowship from the Antofagasta Institute. All authors declare that there are no conflicts of interest.