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Human spermicidal activity of inorganic and organic oxidants
FERTILITY AND STERILITY威 VOL. 76, NO. 1, JULY 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Human spermicidal activity of inorganic and organic oxidants Terrence R. Green, Ph.D.,a Jack H. Fellman, Ph.D.,a and Don P. Wolf, Ph.D.b,c OxiBio, Inc., and Oregon Health Sciences University, Portland, and Oregon Regional Primate Research Center, Beaverton, Oregon
Objective: To identify prospective oxidants that rapidly immobilize human sperm upon contact with human semen. Design: Inorganic, organic, and enzymatically-generated oxidants were mixed with human semen and spermicidal activity was tracked by a modified Sander-Cramer assay. Setting: Commercial and university-based laboratories. Patient(s): Semen samples obtained through a university-based andrology laboratory. Intervention(s): Not applicable. Main Outcome Measures(s): Quantitation of spermicidal activity of test oxidants. Result(s): Sperm lost motility within 20 seconds of exposure to enzymatically generated free iodine (I2). Toluidine blue, phenazine methosulfate, or methylene blue exhibited some, albeit much less, spermicidal activity. Oxidants formed by mixing ascorbic acid with Fe(III)-EDTA, xanthine with xanthine oxidase, or by exposing sperm to the nitric oxide generator, SIN-1 (3-morpholinosydnonimine hydrochloride), were far less potent spermicidal agents. Conclusion(s): Free I2 formed in situ and presented to semen is an extremely potent spermicide. Additional studies on methods of generating de novo I2 may be beneficial in developing a novel new class of nondetergent-based spermicides. (Fertil Steril威 2001;76:157– 62. ©2001 by American Society for Reproductive Medicine.) Key Words: Sperm, spermicidal activity, motility, oxidative damage, free iodine, free radicals, reactive oxygen species
Received September 25, 2000; revised and accepted March 8, 2001. Supported by OxiBio, Inc., Portland, Oregon. Reprint requests: Terrence R. Green, Ph.D., 4640 SW Macadam Avenue, Suite 40, Portland, Oregon 97201 (FAX: 503-274-9906; E-mail: [email protected]). a Research and Development Division, OxiBio, Inc. b Departments of Obstetrics and Gynecology and Physiology and Pharmacology, Oregon Health Sciences University. c Oregon Regional Primate Research Center. 0015-0282/01/$20.00 PII S0015-0282(01)01830-1
Substantial resources have been devoted to the development of effective contraceptive modalities including hormonal approaches, antifertility vaccines, sterilization, the use of abortifacients, intrauterine devices (IUDs), and barrier methods (1). There remains however an unfulfilled need for simple and safe, yet reliable, cost-effective, and culturally acceptable methods that could impact global population growth. Barrier methods used with or without spermicides have often been associated with the additional, hypothetical advantage of protecting the user against sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV). However, recent findings indicate that the most widely used detergent-based spermicide, nonoxynol-9, may actually increase susceptibility to the spread of sexually transmitted HIV (2). This outcome may be linked with the nonspecific disruption of the vaginal epithelium and the creation or enhancement of entry sites for the virus.
Because experimental evidence indicates that intact genital epithelial cells provide a barrier against HIV infection (3), a search for nondetergent-based spermicides, which by their chemical nature would be less likely to disrupt and inflame genital tissues, becomes a high priority. In this regard, we report here varying degrees of susceptibility of human sperm to free radical generators and oxidants, and the discovery that free iodine (I2), formed enzymatically through H2O2 generation and the subsequent peroxidation of inorganic iodide, is an extremely effective spermicidal agent.
MATERIALS AND METHODS Ferricytochrome c, superoxide dismutase (SOD), ascorbic acid, xanthine, xanthine oxidase, glucose oxidase (GO), phenazine methosulfate (PMS), methylene blue (MB) and toluidine blue (TB), and potassium iodide were 157
purchased from Sigma (St. Louis, MO). Ferric chloride and ethylenediaminetetraacetate (EDTA) were purchased from J.T. Baker (Phillipsburg, NJ). SIN-1 was purchased from Molecular Probes, Inc. (Eugene, OR). All other chemicals were of analytical grade. O2 uptake measurements were made using a Clark cell and O2 analyzer as described elsewhere (4). Semen samples were obtained from the Oregon Health Sciences University (OHSU) Andrology Laboratory after diagnostic testing and the removal of all identifying information. Because these studies involved no direct experimentation on humans, no traceable access to patient medical records was possible by the individuals conducting the experiments; because experiments were conducted only on leftover specimens to be discarded, and also because the work was done consistent with human subject guidelines of the Helsinki Declaration of 1975 on human experimentation, institutional review board approval was not sought.
FIGURE 1 Dose and kinetic oxidative effect of PMS vs. xanthine ⫹ xanthine oxidase on sperm motility. Assays initiated with semen additions to test reactants were scored for motility. In the xanthine oxidase experiments, 200 M of xanthine was included in reaction mixtures as substrate, and enzyme was added immediately before the addition of semen. Seminal counts ranged from 37 to 99 ⫻ 106 sperm/mL⫺1. All measurements reflect the average of triplicate determinations per data point. Time zero reflects the control motility that was stable over the duration of the intervals tracked. (Œ), xanthine oxidase, 81 mU mL⫺1; (䊐), xanthine oxidase, 162 mU mL⫺1; (■), PMS, 85 M; (E) PMS, 170 M; and (〫), PMS, 340 M.
Only samples collected from men who had abstained the previous 48 hours from sexual activity were used, and all showed normal seminal characteristics (⬎20 ⫻ 106 spermatozoa mL⫺1, ⬎50% motility, and ⬎50% normal forms). After 30 to 60 minutes liquefaction, samples were diluted threefold in a Tyrode’s albumin lactate pyruvate (TALP) (5) HEPES/1% bovine serum albumin buffer and maintained at 37°C until use. Sperm counts were determined by a hemocytometer that use the average of duplicate counts. Spermicidal activity was determined by a modified Sander-Cramer assay (6). Test mixtures were prepared and within 20 seconds of sperm addition, sperm motility was quantitated in 10-L aliquots under phase contrast microscopy (⫻200) by counting the number of motile and immotile sperm (n ⫽ 100). Test reaction mixtures were prepared fresh in 125 L of 10 mM sodium phosphate, 0.9% NaCl pH 7.4 (PBS), to which sperm in 25 L were then added. Test reactions were maintained at 37°C and the sperm were counted at ambient temperature on glass slides. Sperm showing any movement (twitching and/or nonprogressive or progressive motions) were scored as motile.
Green. Human spermicidal activity. Fertil Steril 2001.
of sperm with glucose ⫹ GO; xanthine ⫹ xanthine oxidase; Fe-EDTA and ascorbate; SIN-1; the organodyes TB, PMS, MB; and combinations of glucose ⫹ GO, used in concert with horseradish peroxidase (HPO) and potassium iodide. The spermicidal activity of each test reaction system was evaluated in terms of the concentration of reactants over time.
Periodically, but at no more than 1-hour intervals, control motility assays were repeated to assess the baseline motility of specific sperm samples used experimentally. Percent residual sperm motility was calculated as the ratio of measured motility of test samples to the measured motility of the same sperm assayed in the absence of the test oxidant or free radical generator, ⫻ 100.
Figure 1 contrasts the kinetic impact on sperm motility of exposing sperm to varying concentrations of PMS or to xanthine ⫹ xanthine oxidase. Except for some evidence of increased motility, sperm were unaffected by exposure to radical products and oxidants of the xanthine oxidase reaction system. Exposure to PMS resulted in a significant loss of motility.
One unit of oxidative activity, expressed in terms of O2 consumed, corresponds to the turnover of 1 mole/minute of substrate or formation of product, respectively.
Similar comparisons were made with several oxidantgenerating systems, the results of which are summarized in Table 1. TB, which exhibited an I50 (the concentration required to cause 50% loss of sperm motility in the modified Sander-Cramer assay) of 26 M, was the most potent among the seven reaction systems listed in Table 1.
RESULTS Radical generators and oxidizing agents tested on sperm samples included redox reactions generated through mixing 158
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Various combinations of the seven radical generators/ Vol. 76, No. 1, July 2001
TABLE 1 Impact of O2 reactive free radical generators on human sperm motility.a Free radical generator/oxidant
Concentration (M)
O2 uptake (mU mL⫺1)b
Control motility (%)
TBc PMSc MBc Xanthine Oxidase SIN-1 Ascorbate-Fe(III)-EDTAd GO
26 ⫾ 1 163 ⫾ 4 2,296 ⫾ 257 (n.a.) 15,083 416 (n.a.)
3⫾0 2⫾1 2⫾1 182 ⫾ 2 150 ⫾ 28 246 ⫾ 10 3,176 ⫾ 103
50 50 50 89 ⫾ 2 109 ⫾ 4 93 ⫾ 4 98 ⫾ 4
a After 3-minute exposure to sperm at 37°C in PBS. Values are the average ⫾1 SD of triplicate assays. Sperm count was 130 ⫻ 106 mL⫺1; control motility ranged from 51% to 64% (CV ⫽ 7%, n ⫽ 42). b Measured rates under experimental conditions in which motility was evaluated. c Reactant concentrations for TB, PMS, and MB reflect the calculated concentration needed to cause 50% loss of sperm motility based upon titrations of sperm motility at varying dye concentrations. d Concentration reflects Fe(III) level chelated with 833 M EDTA in PBS, and to which ascorbate was also added immediately before evaluation to a final concentration of 833 M.
Green. Human spermicidal activity. Fertil Steril 2001.
oxidants listed in Table 1 were also tested on sperm samples with unimpressive results. Combining TB and PMS, or MB with xanthine oxidase, for example, did not enhance sperm immobilization significantly over that of the most potent single reactant tested. On the other hand, combining glucose (11.6 mM) and GO (416 g mL⫺1) with HPO (17 g mL⫺1) and potassium iodide (63 mM) created in situ generation of I2 (evident in the formation of a light yellow coloration in test solutions, absorption maximum ⫽ 350 nm) and resulted in near instantaneous sperm immobilization (20-second exposure). We estimated the steady-state concentration of free I2 to be in the range of approximately 5 to 10 parts per million (ppm) at the time of sperm killing based upon the color intensity of test solutions. Figure 2 summarizes our findings in these experiments. Sperm immobilization was observed to be irreversible because sperm failed to recover motility upon centrifugation and resuspension in fresh isotonic buffer medium. Furthermore, viability assays indicated a strong correlation between the loss of motility and viability (results not shown).
FIGURE 2 Formation of I2-mediated spermicidal activity via GO/HPO enzyme action on glucose and iodide. Semen samples were scored for residual motility at 20 seconds after addition to test mixtures. Control: Semen was added to PBS, pH 7.4, and was assayed for motility as in Figure 1. Results are the average of duplicate determinations, ⫾1 SD, per reaction mixture of a single donor specimen run within approximately 1 to 2 hours of semen collection, but they reflect comparable results obtained on six separate donor specimens obtained on samples of four individual sperm donors. Complete: PBS made up in 11.6 mM glucose, 63 mM KI, 416 g mL⫺1 GO and 17 g mL⫺1 HPO (final concentrations). (Arrow indicates lack of motility in semen samples exposed to the complete reaction mixture.) ⫺GO/HPO: Same as complete, excluding GO and HPO from reaction mixture. ⫺GO: Complete minus GO. ⫺HPO: Complete minus HPO. ⫺I: Complete minus KI.
DISCUSSION In evaluating the efficacy of radical generators and oxidants as prospective spermicides, we chose to use a modified Sander-Cramer assay (7). This assay defines a standardized method of identifying the minimum concentration of a spermicide capable of immobilizing sperm within a 20-second interval. Substances that fail to immobilize sperm within 20 seconds are unacceptable as prospective spermicidal agents acting at the level of the vagina because of the rapidity with which sperm migrate into the cervix and upper reproductive tract. Hence, delayed or incomplete motility loss is unacFERTILITY & STERILITY威
Green. Human spermicidal activity. Fertil Steril 2001.
159
ceptable and all sperm must be immobilized within the 20-second window to ensure complete blockage of the fertilization process. That deliberate exposure of sperm to O2-derived reactive oxygen species and/or other oxidants might prove an effective contraceptive strategy is suggested by the close correlation seen between excess free radical production by sperm and male infertility in significant subpopulations of infertile donors (8, 9), and by the decline in sperm motility, adenosine triphosphate reserves, and the capacity of sperm to undergo the acrosome reaction following in vitro exposure of sperm to exogenous O2-derived reactive oxygen species through the addition of xanthine and xanthine oxidase to semen (10 –13). Additionally, an inverse relationship has been noted between sperm fertility (14) and sperm hypotaurine content, a potent scavenger of hydroxyl radicals (15). Several studies on the vulnerability of sperm to free radical damage have documented aberrant release of O2centered free radicals by sperm (10, 11, 16), and deleterious effects of lipid peroxides on sperm function (17, 18). O2derived reactive species such as O2⫺ and H2O2 have been generated and tested for their inhibitory effects on varying sperm functions required for successful fertilization of the ovum (9, 12, 13). However, prior studies have not documented the kinetic rates of sperm inactivation presented here, especially those seen with the in situ generation of I2 from glucose and GO. In this study, we evaluated O2 reactive generators capable of producing O2⫺, which as a precursor of reactive O2 species (ROS) may be responsible for the oxidative destruction of activities or functions required in the maintenance of normal cellular integrity such as lipid bilayers, thiol residues, or ion channels (19, 20). The organodyes, TB, PMS, and MB, as ROS generators catalyze formation of O2⫺, and through dismutation of O2⫺, the formation of H2O2, by transferring electron equivalents from reducing substances (for instance, as might be found in body fluids) to molecular oxygen (21, 22). In the process, however, they form unstable, highly reactive semiquinone (e.g., organoradical) intermediates (22) which themselves can disrupt essential biochemical processes through both oxidative and polymerization reactions (21–23). Xanthine oxidase ⫹ xanthine, ascorbate-Fe(III)-EDTA and GO ⫹ glucose all catalyze H2O2 formation; and with Fe(III) in the reaction environment, there is formation of hydroxyl radicals (19, 20). When added to an aqueous solution, SIN-1 spontaneously decays through a hydrolytic and oxidative process resulting in production of peroxynitrites, O2⫺, H2O2, and hydroxyl radicals, all powerful oxidizing agents (24 –26). In interpreting the present results, it is important to recognize that the dissolved oxygen content of the reaction environment will limit the rate and quantity of ROS species 160
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produced. The results shown in Table 1 reflect robust rates of ROS formation, especially in the cases of the xanthine oxidase ⫹ xanthine, SIN-1, ascorbate-Fe(III)-EDTA, and GO ⫹ glucose ROS generators. By oxygen uptake analysis, all of these ROS generators caused test solutions to become anaerobic within seconds of their addition to semen samples, reflecting nearly quantitative conversion of the dissolved oxygen in the test solutions into ROS reactants. The modest spermicidal activity of these solutions suggests that sperm must have a remarkably high tolerance to acute fluxes of ROS species. On the other hand, because oxygen is required for these reactions to occur, the consumption of oxygen and subsequent low oxygen tensions create a self-limiting mechanism that may truncate further oxidative damage. For these types of redox reactions to work as effective spermicides in the vagina where the oxygen tension is very low (27, 28), an additional source of oxygen for driving redox reactions would be required. The mixing of semen and vaginal fluid and the concomitant dissolution of oxygen at the air– water interface during sexual intercourse represents one possibility. Sperm have strong antioxidant defenses against ROS products, involving SOD and glutathione peroxidase (29, 30); this could account, in part, for the weak spermicidal activity seen when sperm were exposed to the xanthine ⫹ xanthine oxidase, glucose ⫹ GO, and SIN-1 redox reactions, all of which produced significant fluxes of ROS as evident by the high rates of O2 uptake. On the other hand, TB, PMS, and MB, though showing much lower rates of O2 uptake, were markedly more spermicidal; this suggests that their mechanism is not solely via O2⫺ and ROS formation, but rather by some other set of redox reactions, perhaps involving organoradical intermediates. Using the Sander-Cramer guidelines, the only prospective spermicide of the oxidants tested in this study was I2, or byproducts of its formation (see below) created de novo. In this regard, it is important to recognize that I2 complexed with polyvinylpyrrolidone (e.g., a povidione-iodine formulation) has previously been tested but was found to be far less efficacious as a spermicide (31). This is most certainly due to much of the free I2 being complexed to polyvinylpyrrolidone, rendering the formulation ineffective (32); this lends further credence to the interpretation that spermicidal activity is a property of the free form of I2 and not that of bound I2. Because the free I2 concentration in povidione-iodine is approximately 1 ppm (33), these earlier studies suggest that the critical concentration of free I2 required to affect rapid sperm immobilization must be in excess of 1 ppm. However, in a static formulation such as povidione-iodine wherein free I2 is in equilibrium with bound I2 but total available I2 is fixed, it is also possible that the actual level of free I2 in the povidione-iodine studies dropped below the critical 1 ppm generally recognized to be the cutoff threshold for efficaVol. 76, No. 1, July 2001
cious killing of single-celled organisms (32). This may explain why the enzymatic method of generating free I2 used in our experiments is more efficacious—namely because free I2 is continuously produced, and because any free I2 reduced to inorganic iodide can be reoxidized in the enzymatic formulation to ensure a continuous supply of I2 killing activity. A precise estimate of the amount of free I2 needed to achieve complete killing of sperm within the 20-second Sander-Cramer test window is difficult to establish. This is because the flux of free I2 required depends substantially on the rate of its consumption by reducing equivalents that compete with sperm for free I2. On the other hand, the concentration of reducing equivalents (e.g., hypotaurine, ascorbic acid, reactive thiols, etc.) can vary in semen between men as a consequence of genetic and dietary factors. It is reasonable to expect, however, that the steady-state concentration of free I2 must be at least in excess of 1 ppm, taking into account earlier observations with povidione-iodine formulations which were found, as noted above, to be suboptimal. Although the results of our experiments suggest that formation of free I2 must occur for effective spermicidal activity, alternate species such as I3⫺, or higher oxidation states of iodine such as IO⫺, I2O2, and IO3⫺, may also form in the oxidative environment during conversion of iodide into I2, and these could also be responsible for spermicidal activity. Further research is required before the exact contribution of each of these forms of iodine to the total spermicidal activity of iodine is understood. Because free I2 shows potent broad-spectrum microbicidal and virucidal activities at concentrations in the range of 2 to 5 ppm (32–34), its use in contraceptive applications as a spermicide could additionally provide some protection against the spread of sexually transmitted diseases (STDs). At this concentration range it is nonirritating to tissues (32). This is likely because at these levels free I2 is short-lived. Reducing substances rapidly convert it to inorganic iodide, and hence it can be viewed as a transient oxidant, especially in rich-reducing environments found in vaginal and seminal fluids. One method of controlling production of free I2, and its delivery within the vagina, might entail encapsulation of precursors for its formation within a vaginal insert, relying upon a time-dependent, sustained dissolution of precursors from the delivery device, and subsequent formation of free I2 in situ. Neither the pH of the vagina, nor that of semen, poses problems with regard to the expression of I2-mediated oxidizing activity, because free I2 is an effective oxidizing agent whether in elemental form at low pH, or upon conversion to hypoiodate at more alkaline values above pH 7.0 (33). We envision further studies will be required for the development of free I2– generating delivery devices that can control the rate and duration of its production upon presentation to the vagina. This chemistry might also have applications in conFERTILITY & STERILITY威
doms as an alternative nondetergent-based spermicide used in place of nonoxynol-9. References 1. Senanayake P. Contraception by the end of the 20th century—the role of the voluntary organizations. New Concepts Fertil Control 1994;9: 133– 43. 2. Stephenson J. Widely used spermicide may increase, not decrease, risk of HIV transmission. JAMA 2000;284:949. 3. Greenhead P, Hayes P, Watts PS, Laing KG, Griffin GE, Shattock RJ. Parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides. J Virol 2000;74:5577– 86. 4. Green TR, Wu DE. The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2. J Biol Chem 1986;261: 6010 –15. 5. Bavister BD, Leibfried ML, Lieberman G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983;28:235– 47. 6. Sander FV, Cramer SD. A practical method for testing the spermicidal action of chemical contraceptives. Hum Fertil 1941;6:134 –7. 7. Lee CH. Review: in vitro spermicidal tests. Contraception 1996;54: 131– 47. 8. Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril 1992;57:409 –16. 9. Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 1989; 41:183–97. 10. Gagnon C, Iwasaki A, De Lamirande E, Kovalski N. Reactive oxygen species and human spermatozoa. Ann N Y Acad Sci 1991;637:436 – 44. 11. Riley JC, Behrman HR. Oxygen radicals and reactive oxygen species in reproduction. Proc Soc Exp Biol Med 1991;198:781–91. 12. Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil 1993;97:441–50. 13. Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa. J Reprod Fertil 1995;103:17–26. 14. Holmes RP, Goodman HO, Shihabi ZK, Jarow JP. The taurine and hypotaurine content of human semen. J Androl 1992;13:289 –92. 15. Fellman JH, Green TR, Eicher AL. The oxidation of hypotaurine to taurine: bis-aminoethyl-alpha-disulfone, a metabolic intermediate in mammalian tissue. Adv Exp Med Biol 1987;217:39 – 48. 16. Miesel R, Drzejczak PJ, Kurpisz M. Oxidative stress during the interaction of gametes. Biol Reprod 1993;49:918 –23. 17. Windsor DP, White IG, Selley ML, Swan MA. Effects of the lipid peroxidation product (E)-4-hydroxy-2-nonenal on ram sperm function. J Reprod Fertil 1993;99:359 – 66. 18. Sikka SC, Rajasekaran M, Hellstrom WJ. Role of oxidative stress and antioxidants in male infertility. J Androl 1995;16:464 – 81. 19. Aruoma OI, Kaur H, Halliwell B. Oxygen free radicals and human diseases. J R Soc Health 1991;111:172–7. 20. Marx JL. Oxygen free radicals linked to many diseases. Science 1987; 235:529 –31. 21. Rabinowitch E, Epstein LF. Polymerization of dyestuffs in solution. Thionine and methylene blue. J Biol Chem 1941;63:69 –78. 22. Winterbourn CC, French JK, Claridge RFC. Superoxide dismutase as an inhibitor of reactions of semiquinone radicals. FEBS Lett 1978;94: 269 –272. 23. Green TR, Fellman JH. Effect of photolytically generated riboflavin radicals and oxygen on hypotaurine antioxidant free radical scavenging activity. In: Huxtable R, Michalk DV, eds. Taurine in health and disease. New York: Plenum Press, 1994:19 –29. 24. Hogg N, Darley-Usmar VM, Wilson MT, Moncada S. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem J 1992;281:419 –24. 25. Feelish M. The biochemical pathways of nitric oxide formation from nitrovasodilators: appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions. J Cardiovasc Pharmacol 1991;17(suppl):S25–S33. 26. Brunelli L, Crow JP, Beckman JS. The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch Biochem Biophys 1995;316:327–34. 27. Levin RJ. VIP, vagina, clitoral and periurethral glans—an update on human female sexual arousal. Exp Clin Endocrinol 1991;98:61–9. 28. Levin RJ. The physiology of sexual function in women. Clin Obstet Gynaecol 1980;7:213–52. 29. Kumar PG, Laloraya M, Laloraya MM. Superoxide radical level and superoxide dismutase activity changes in maturing mammalian spermatozoa. Andrologia 1991;23:171–5.
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30. Sinha S, Pradeep KG, Laloraya M, Warikoo D. Over-expression of superoxide dismutase and lack of surface-thiols in spermatozoa: inherent defects in oligospermia. Biochem Biophys Res Commun 1991;174:510 –7. 31. Pfannschmidt N, Nissen HP. Quantitative studies of the interaction of polyvinylpyrrolidone iodine and spermatozoa [in German]. Z Hautkr 1988;63:891– 6. 32. LeVeen HH, LeVeen RF, LeVeen EG. The mythology of povidone-
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iodine and the development of self-sterilizing plastics. Surg Gynecol Obstet 1993;176:183–9. 33. Barabas ES, Brittain HG. Povidone-Iodine. In: Brittain, HG, ed. Analytical profiles of drug substances and excipients. Vol. 25. San Diego, CA: Academic Press, 1998:341– 462. 34. Shikani AH, St. Clair M, Domb A. Polymer-iodine inactivation of the human immunodeficiency virus. J Am College Surg 1996;183:195–200.
Vol. 76, No. 1, July 2001
Human spermicidal activity of inorganic and organic oxidants Terrence R. Green, Ph.D.,a Jack H. Fellman, Ph.D.,a and Don P. Wolf, Ph.D.b,c OxiBio, Inc., and Oregon Health Sciences University, Portland, and Oregon Regional Primate Research Center, Beaverton, Oregon
Objective: To identify prospective oxidants that rapidly immobilize human sperm upon contact with human semen. Design: Inorganic, organic, and enzymatically-generated oxidants were mixed with human semen and spermicidal activity was tracked by a modified Sander-Cramer assay. Setting: Commercial and university-based laboratories. Patient(s): Semen samples obtained through a university-based andrology laboratory. Intervention(s): Not applicable. Main Outcome Measures(s): Quantitation of spermicidal activity of test oxidants. Result(s): Sperm lost motility within 20 seconds of exposure to enzymatically generated free iodine (I2). Toluidine blue, phenazine methosulfate, or methylene blue exhibited some, albeit much less, spermicidal activity. Oxidants formed by mixing ascorbic acid with Fe(III)-EDTA, xanthine with xanthine oxidase, or by exposing sperm to the nitric oxide generator, SIN-1 (3-morpholinosydnonimine hydrochloride), were far less potent spermicidal agents. Conclusion(s): Free I2 formed in situ and presented to semen is an extremely potent spermicide. Additional studies on methods of generating de novo I2 may be beneficial in developing a novel new class of nondetergent-based spermicides. (Fertil Steril威 2001;76:157– 62. ©2001 by American Society for Reproductive Medicine.) Key Words: Sperm, spermicidal activity, motility, oxidative damage, free iodine, free radicals, reactive oxygen species
Received September 25, 2000; revised and accepted March 8, 2001. Supported by OxiBio, Inc., Portland, Oregon. Reprint requests: Terrence R. Green, Ph.D., 4640 SW Macadam Avenue, Suite 40, Portland, Oregon 97201 (FAX: 503-274-9906; E-mail: [email protected]). a Research and Development Division, OxiBio, Inc. b Departments of Obstetrics and Gynecology and Physiology and Pharmacology, Oregon Health Sciences University. c Oregon Regional Primate Research Center. 0015-0282/01/$20.00 PII S0015-0282(01)01830-1
Substantial resources have been devoted to the development of effective contraceptive modalities including hormonal approaches, antifertility vaccines, sterilization, the use of abortifacients, intrauterine devices (IUDs), and barrier methods (1). There remains however an unfulfilled need for simple and safe, yet reliable, cost-effective, and culturally acceptable methods that could impact global population growth. Barrier methods used with or without spermicides have often been associated with the additional, hypothetical advantage of protecting the user against sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV). However, recent findings indicate that the most widely used detergent-based spermicide, nonoxynol-9, may actually increase susceptibility to the spread of sexually transmitted HIV (2). This outcome may be linked with the nonspecific disruption of the vaginal epithelium and the creation or enhancement of entry sites for the virus.
Because experimental evidence indicates that intact genital epithelial cells provide a barrier against HIV infection (3), a search for nondetergent-based spermicides, which by their chemical nature would be less likely to disrupt and inflame genital tissues, becomes a high priority. In this regard, we report here varying degrees of susceptibility of human sperm to free radical generators and oxidants, and the discovery that free iodine (I2), formed enzymatically through H2O2 generation and the subsequent peroxidation of inorganic iodide, is an extremely effective spermicidal agent.
MATERIALS AND METHODS Ferricytochrome c, superoxide dismutase (SOD), ascorbic acid, xanthine, xanthine oxidase, glucose oxidase (GO), phenazine methosulfate (PMS), methylene blue (MB) and toluidine blue (TB), and potassium iodide were 157
purchased from Sigma (St. Louis, MO). Ferric chloride and ethylenediaminetetraacetate (EDTA) were purchased from J.T. Baker (Phillipsburg, NJ). SIN-1 was purchased from Molecular Probes, Inc. (Eugene, OR). All other chemicals were of analytical grade. O2 uptake measurements were made using a Clark cell and O2 analyzer as described elsewhere (4). Semen samples were obtained from the Oregon Health Sciences University (OHSU) Andrology Laboratory after diagnostic testing and the removal of all identifying information. Because these studies involved no direct experimentation on humans, no traceable access to patient medical records was possible by the individuals conducting the experiments; because experiments were conducted only on leftover specimens to be discarded, and also because the work was done consistent with human subject guidelines of the Helsinki Declaration of 1975 on human experimentation, institutional review board approval was not sought.
FIGURE 1 Dose and kinetic oxidative effect of PMS vs. xanthine ⫹ xanthine oxidase on sperm motility. Assays initiated with semen additions to test reactants were scored for motility. In the xanthine oxidase experiments, 200 M of xanthine was included in reaction mixtures as substrate, and enzyme was added immediately before the addition of semen. Seminal counts ranged from 37 to 99 ⫻ 106 sperm/mL⫺1. All measurements reflect the average of triplicate determinations per data point. Time zero reflects the control motility that was stable over the duration of the intervals tracked. (Œ), xanthine oxidase, 81 mU mL⫺1; (䊐), xanthine oxidase, 162 mU mL⫺1; (■), PMS, 85 M; (E) PMS, 170 M; and (〫), PMS, 340 M.
Only samples collected from men who had abstained the previous 48 hours from sexual activity were used, and all showed normal seminal characteristics (⬎20 ⫻ 106 spermatozoa mL⫺1, ⬎50% motility, and ⬎50% normal forms). After 30 to 60 minutes liquefaction, samples were diluted threefold in a Tyrode’s albumin lactate pyruvate (TALP) (5) HEPES/1% bovine serum albumin buffer and maintained at 37°C until use. Sperm counts were determined by a hemocytometer that use the average of duplicate counts. Spermicidal activity was determined by a modified Sander-Cramer assay (6). Test mixtures were prepared and within 20 seconds of sperm addition, sperm motility was quantitated in 10-L aliquots under phase contrast microscopy (⫻200) by counting the number of motile and immotile sperm (n ⫽ 100). Test reaction mixtures were prepared fresh in 125 L of 10 mM sodium phosphate, 0.9% NaCl pH 7.4 (PBS), to which sperm in 25 L were then added. Test reactions were maintained at 37°C and the sperm were counted at ambient temperature on glass slides. Sperm showing any movement (twitching and/or nonprogressive or progressive motions) were scored as motile.
Green. Human spermicidal activity. Fertil Steril 2001.
of sperm with glucose ⫹ GO; xanthine ⫹ xanthine oxidase; Fe-EDTA and ascorbate; SIN-1; the organodyes TB, PMS, MB; and combinations of glucose ⫹ GO, used in concert with horseradish peroxidase (HPO) and potassium iodide. The spermicidal activity of each test reaction system was evaluated in terms of the concentration of reactants over time.
Periodically, but at no more than 1-hour intervals, control motility assays were repeated to assess the baseline motility of specific sperm samples used experimentally. Percent residual sperm motility was calculated as the ratio of measured motility of test samples to the measured motility of the same sperm assayed in the absence of the test oxidant or free radical generator, ⫻ 100.
Figure 1 contrasts the kinetic impact on sperm motility of exposing sperm to varying concentrations of PMS or to xanthine ⫹ xanthine oxidase. Except for some evidence of increased motility, sperm were unaffected by exposure to radical products and oxidants of the xanthine oxidase reaction system. Exposure to PMS resulted in a significant loss of motility.
One unit of oxidative activity, expressed in terms of O2 consumed, corresponds to the turnover of 1 mole/minute of substrate or formation of product, respectively.
Similar comparisons were made with several oxidantgenerating systems, the results of which are summarized in Table 1. TB, which exhibited an I50 (the concentration required to cause 50% loss of sperm motility in the modified Sander-Cramer assay) of 26 M, was the most potent among the seven reaction systems listed in Table 1.
RESULTS Radical generators and oxidizing agents tested on sperm samples included redox reactions generated through mixing 158
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TABLE 1 Impact of O2 reactive free radical generators on human sperm motility.a Free radical generator/oxidant
Concentration (M)
O2 uptake (mU mL⫺1)b
Control motility (%)
TBc PMSc MBc Xanthine Oxidase SIN-1 Ascorbate-Fe(III)-EDTAd GO
26 ⫾ 1 163 ⫾ 4 2,296 ⫾ 257 (n.a.) 15,083 416 (n.a.)
3⫾0 2⫾1 2⫾1 182 ⫾ 2 150 ⫾ 28 246 ⫾ 10 3,176 ⫾ 103
50 50 50 89 ⫾ 2 109 ⫾ 4 93 ⫾ 4 98 ⫾ 4
a After 3-minute exposure to sperm at 37°C in PBS. Values are the average ⫾1 SD of triplicate assays. Sperm count was 130 ⫻ 106 mL⫺1; control motility ranged from 51% to 64% (CV ⫽ 7%, n ⫽ 42). b Measured rates under experimental conditions in which motility was evaluated. c Reactant concentrations for TB, PMS, and MB reflect the calculated concentration needed to cause 50% loss of sperm motility based upon titrations of sperm motility at varying dye concentrations. d Concentration reflects Fe(III) level chelated with 833 M EDTA in PBS, and to which ascorbate was also added immediately before evaluation to a final concentration of 833 M.
Green. Human spermicidal activity. Fertil Steril 2001.
oxidants listed in Table 1 were also tested on sperm samples with unimpressive results. Combining TB and PMS, or MB with xanthine oxidase, for example, did not enhance sperm immobilization significantly over that of the most potent single reactant tested. On the other hand, combining glucose (11.6 mM) and GO (416 g mL⫺1) with HPO (17 g mL⫺1) and potassium iodide (63 mM) created in situ generation of I2 (evident in the formation of a light yellow coloration in test solutions, absorption maximum ⫽ 350 nm) and resulted in near instantaneous sperm immobilization (20-second exposure). We estimated the steady-state concentration of free I2 to be in the range of approximately 5 to 10 parts per million (ppm) at the time of sperm killing based upon the color intensity of test solutions. Figure 2 summarizes our findings in these experiments. Sperm immobilization was observed to be irreversible because sperm failed to recover motility upon centrifugation and resuspension in fresh isotonic buffer medium. Furthermore, viability assays indicated a strong correlation between the loss of motility and viability (results not shown).
FIGURE 2 Formation of I2-mediated spermicidal activity via GO/HPO enzyme action on glucose and iodide. Semen samples were scored for residual motility at 20 seconds after addition to test mixtures. Control: Semen was added to PBS, pH 7.4, and was assayed for motility as in Figure 1. Results are the average of duplicate determinations, ⫾1 SD, per reaction mixture of a single donor specimen run within approximately 1 to 2 hours of semen collection, but they reflect comparable results obtained on six separate donor specimens obtained on samples of four individual sperm donors. Complete: PBS made up in 11.6 mM glucose, 63 mM KI, 416 g mL⫺1 GO and 17 g mL⫺1 HPO (final concentrations). (Arrow indicates lack of motility in semen samples exposed to the complete reaction mixture.) ⫺GO/HPO: Same as complete, excluding GO and HPO from reaction mixture. ⫺GO: Complete minus GO. ⫺HPO: Complete minus HPO. ⫺I: Complete minus KI.
DISCUSSION In evaluating the efficacy of radical generators and oxidants as prospective spermicides, we chose to use a modified Sander-Cramer assay (7). This assay defines a standardized method of identifying the minimum concentration of a spermicide capable of immobilizing sperm within a 20-second interval. Substances that fail to immobilize sperm within 20 seconds are unacceptable as prospective spermicidal agents acting at the level of the vagina because of the rapidity with which sperm migrate into the cervix and upper reproductive tract. Hence, delayed or incomplete motility loss is unacFERTILITY & STERILITY威
Green. Human spermicidal activity. Fertil Steril 2001.
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ceptable and all sperm must be immobilized within the 20-second window to ensure complete blockage of the fertilization process. That deliberate exposure of sperm to O2-derived reactive oxygen species and/or other oxidants might prove an effective contraceptive strategy is suggested by the close correlation seen between excess free radical production by sperm and male infertility in significant subpopulations of infertile donors (8, 9), and by the decline in sperm motility, adenosine triphosphate reserves, and the capacity of sperm to undergo the acrosome reaction following in vitro exposure of sperm to exogenous O2-derived reactive oxygen species through the addition of xanthine and xanthine oxidase to semen (10 –13). Additionally, an inverse relationship has been noted between sperm fertility (14) and sperm hypotaurine content, a potent scavenger of hydroxyl radicals (15). Several studies on the vulnerability of sperm to free radical damage have documented aberrant release of O2centered free radicals by sperm (10, 11, 16), and deleterious effects of lipid peroxides on sperm function (17, 18). O2derived reactive species such as O2⫺ and H2O2 have been generated and tested for their inhibitory effects on varying sperm functions required for successful fertilization of the ovum (9, 12, 13). However, prior studies have not documented the kinetic rates of sperm inactivation presented here, especially those seen with the in situ generation of I2 from glucose and GO. In this study, we evaluated O2 reactive generators capable of producing O2⫺, which as a precursor of reactive O2 species (ROS) may be responsible for the oxidative destruction of activities or functions required in the maintenance of normal cellular integrity such as lipid bilayers, thiol residues, or ion channels (19, 20). The organodyes, TB, PMS, and MB, as ROS generators catalyze formation of O2⫺, and through dismutation of O2⫺, the formation of H2O2, by transferring electron equivalents from reducing substances (for instance, as might be found in body fluids) to molecular oxygen (21, 22). In the process, however, they form unstable, highly reactive semiquinone (e.g., organoradical) intermediates (22) which themselves can disrupt essential biochemical processes through both oxidative and polymerization reactions (21–23). Xanthine oxidase ⫹ xanthine, ascorbate-Fe(III)-EDTA and GO ⫹ glucose all catalyze H2O2 formation; and with Fe(III) in the reaction environment, there is formation of hydroxyl radicals (19, 20). When added to an aqueous solution, SIN-1 spontaneously decays through a hydrolytic and oxidative process resulting in production of peroxynitrites, O2⫺, H2O2, and hydroxyl radicals, all powerful oxidizing agents (24 –26). In interpreting the present results, it is important to recognize that the dissolved oxygen content of the reaction environment will limit the rate and quantity of ROS species 160
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produced. The results shown in Table 1 reflect robust rates of ROS formation, especially in the cases of the xanthine oxidase ⫹ xanthine, SIN-1, ascorbate-Fe(III)-EDTA, and GO ⫹ glucose ROS generators. By oxygen uptake analysis, all of these ROS generators caused test solutions to become anaerobic within seconds of their addition to semen samples, reflecting nearly quantitative conversion of the dissolved oxygen in the test solutions into ROS reactants. The modest spermicidal activity of these solutions suggests that sperm must have a remarkably high tolerance to acute fluxes of ROS species. On the other hand, because oxygen is required for these reactions to occur, the consumption of oxygen and subsequent low oxygen tensions create a self-limiting mechanism that may truncate further oxidative damage. For these types of redox reactions to work as effective spermicides in the vagina where the oxygen tension is very low (27, 28), an additional source of oxygen for driving redox reactions would be required. The mixing of semen and vaginal fluid and the concomitant dissolution of oxygen at the air– water interface during sexual intercourse represents one possibility. Sperm have strong antioxidant defenses against ROS products, involving SOD and glutathione peroxidase (29, 30); this could account, in part, for the weak spermicidal activity seen when sperm were exposed to the xanthine ⫹ xanthine oxidase, glucose ⫹ GO, and SIN-1 redox reactions, all of which produced significant fluxes of ROS as evident by the high rates of O2 uptake. On the other hand, TB, PMS, and MB, though showing much lower rates of O2 uptake, were markedly more spermicidal; this suggests that their mechanism is not solely via O2⫺ and ROS formation, but rather by some other set of redox reactions, perhaps involving organoradical intermediates. Using the Sander-Cramer guidelines, the only prospective spermicide of the oxidants tested in this study was I2, or byproducts of its formation (see below) created de novo. In this regard, it is important to recognize that I2 complexed with polyvinylpyrrolidone (e.g., a povidione-iodine formulation) has previously been tested but was found to be far less efficacious as a spermicide (31). This is most certainly due to much of the free I2 being complexed to polyvinylpyrrolidone, rendering the formulation ineffective (32); this lends further credence to the interpretation that spermicidal activity is a property of the free form of I2 and not that of bound I2. Because the free I2 concentration in povidione-iodine is approximately 1 ppm (33), these earlier studies suggest that the critical concentration of free I2 required to affect rapid sperm immobilization must be in excess of 1 ppm. However, in a static formulation such as povidione-iodine wherein free I2 is in equilibrium with bound I2 but total available I2 is fixed, it is also possible that the actual level of free I2 in the povidione-iodine studies dropped below the critical 1 ppm generally recognized to be the cutoff threshold for efficaVol. 76, No. 1, July 2001
cious killing of single-celled organisms (32). This may explain why the enzymatic method of generating free I2 used in our experiments is more efficacious—namely because free I2 is continuously produced, and because any free I2 reduced to inorganic iodide can be reoxidized in the enzymatic formulation to ensure a continuous supply of I2 killing activity. A precise estimate of the amount of free I2 needed to achieve complete killing of sperm within the 20-second Sander-Cramer test window is difficult to establish. This is because the flux of free I2 required depends substantially on the rate of its consumption by reducing equivalents that compete with sperm for free I2. On the other hand, the concentration of reducing equivalents (e.g., hypotaurine, ascorbic acid, reactive thiols, etc.) can vary in semen between men as a consequence of genetic and dietary factors. It is reasonable to expect, however, that the steady-state concentration of free I2 must be at least in excess of 1 ppm, taking into account earlier observations with povidione-iodine formulations which were found, as noted above, to be suboptimal. Although the results of our experiments suggest that formation of free I2 must occur for effective spermicidal activity, alternate species such as I3⫺, or higher oxidation states of iodine such as IO⫺, I2O2, and IO3⫺, may also form in the oxidative environment during conversion of iodide into I2, and these could also be responsible for spermicidal activity. Further research is required before the exact contribution of each of these forms of iodine to the total spermicidal activity of iodine is understood. Because free I2 shows potent broad-spectrum microbicidal and virucidal activities at concentrations in the range of 2 to 5 ppm (32–34), its use in contraceptive applications as a spermicide could additionally provide some protection against the spread of sexually transmitted diseases (STDs). At this concentration range it is nonirritating to tissues (32). This is likely because at these levels free I2 is short-lived. Reducing substances rapidly convert it to inorganic iodide, and hence it can be viewed as a transient oxidant, especially in rich-reducing environments found in vaginal and seminal fluids. One method of controlling production of free I2, and its delivery within the vagina, might entail encapsulation of precursors for its formation within a vaginal insert, relying upon a time-dependent, sustained dissolution of precursors from the delivery device, and subsequent formation of free I2 in situ. Neither the pH of the vagina, nor that of semen, poses problems with regard to the expression of I2-mediated oxidizing activity, because free I2 is an effective oxidizing agent whether in elemental form at low pH, or upon conversion to hypoiodate at more alkaline values above pH 7.0 (33). We envision further studies will be required for the development of free I2– generating delivery devices that can control the rate and duration of its production upon presentation to the vagina. This chemistry might also have applications in conFERTILITY & STERILITY威
doms as an alternative nondetergent-based spermicide used in place of nonoxynol-9. References 1. Senanayake P. Contraception by the end of the 20th century—the role of the voluntary organizations. New Concepts Fertil Control 1994;9: 133– 43. 2. Stephenson J. Widely used spermicide may increase, not decrease, risk of HIV transmission. JAMA 2000;284:949. 3. Greenhead P, Hayes P, Watts PS, Laing KG, Griffin GE, Shattock RJ. Parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides. J Virol 2000;74:5577– 86. 4. Green TR, Wu DE. The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2. J Biol Chem 1986;261: 6010 –15. 5. Bavister BD, Leibfried ML, Lieberman G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983;28:235– 47. 6. Sander FV, Cramer SD. A practical method for testing the spermicidal action of chemical contraceptives. Hum Fertil 1941;6:134 –7. 7. Lee CH. Review: in vitro spermicidal tests. Contraception 1996;54: 131– 47. 8. Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steril 1992;57:409 –16. 9. Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Reprod 1989; 41:183–97. 10. Gagnon C, Iwasaki A, De Lamirande E, Kovalski N. Reactive oxygen species and human spermatozoa. Ann N Y Acad Sci 1991;637:436 – 44. 11. Riley JC, Behrman HR. Oxygen radicals and reactive oxygen species in reproduction. Proc Soc Exp Biol Med 1991;198:781–91. 12. Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil 1993;97:441–50. 13. Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa. J Reprod Fertil 1995;103:17–26. 14. Holmes RP, Goodman HO, Shihabi ZK, Jarow JP. The taurine and hypotaurine content of human semen. J Androl 1992;13:289 –92. 15. Fellman JH, Green TR, Eicher AL. The oxidation of hypotaurine to taurine: bis-aminoethyl-alpha-disulfone, a metabolic intermediate in mammalian tissue. Adv Exp Med Biol 1987;217:39 – 48. 16. Miesel R, Drzejczak PJ, Kurpisz M. Oxidative stress during the interaction of gametes. Biol Reprod 1993;49:918 –23. 17. Windsor DP, White IG, Selley ML, Swan MA. Effects of the lipid peroxidation product (E)-4-hydroxy-2-nonenal on ram sperm function. J Reprod Fertil 1993;99:359 – 66. 18. Sikka SC, Rajasekaran M, Hellstrom WJ. Role of oxidative stress and antioxidants in male infertility. J Androl 1995;16:464 – 81. 19. Aruoma OI, Kaur H, Halliwell B. Oxygen free radicals and human diseases. J R Soc Health 1991;111:172–7. 20. Marx JL. Oxygen free radicals linked to many diseases. Science 1987; 235:529 –31. 21. Rabinowitch E, Epstein LF. Polymerization of dyestuffs in solution. Thionine and methylene blue. J Biol Chem 1941;63:69 –78. 22. Winterbourn CC, French JK, Claridge RFC. Superoxide dismutase as an inhibitor of reactions of semiquinone radicals. FEBS Lett 1978;94: 269 –272. 23. Green TR, Fellman JH. Effect of photolytically generated riboflavin radicals and oxygen on hypotaurine antioxidant free radical scavenging activity. In: Huxtable R, Michalk DV, eds. Taurine in health and disease. New York: Plenum Press, 1994:19 –29. 24. Hogg N, Darley-Usmar VM, Wilson MT, Moncada S. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem J 1992;281:419 –24. 25. Feelish M. The biochemical pathways of nitric oxide formation from nitrovasodilators: appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions. J Cardiovasc Pharmacol 1991;17(suppl):S25–S33. 26. Brunelli L, Crow JP, Beckman JS. The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch Biochem Biophys 1995;316:327–34. 27. Levin RJ. VIP, vagina, clitoral and periurethral glans—an update on human female sexual arousal. Exp Clin Endocrinol 1991;98:61–9. 28. Levin RJ. The physiology of sexual function in women. Clin Obstet Gynaecol 1980;7:213–52. 29. Kumar PG, Laloraya M, Laloraya MM. Superoxide radical level and superoxide dismutase activity changes in maturing mammalian spermatozoa. Andrologia 1991;23:171–5.
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