Embryotoxicity of magainin-2-amide and its enhancement by cyclodextrin, albumin, hydrogen peroxide and acidification

Ewa T. Mystkowska1, Anna Niemierko1, Aldona Komar1 and Wojciech Sawicki2,3

1 Department of Histology and Embryology and 2 Department of Electron Microscopy, Center of Biostructure, Medical Academy, Warsaw, Poland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The channel-forming antimicrobial peptide, magainin-2-amide, interacts preferentially with negatively charged, non cholesterol-containing membranes, including those of sperm, oocytes and cells of pre-implantation embryos. Cyclodextrin and albumin remove membrane cholesterol and together with hydrogen peroxide (H2O2) are potential enhancers of embryotoxicity. METHODS: Two-cell murine embryos were cultured in vitro with magainin-2-amide at a high effective concentration (250 µg/ml) and at subthreshold concentrations (166 and 200 µg/ml). Embryos treated with sub-threshold concentrations of magainin were additionally treated with cyclodextrin, bovine serum albumin or H2O2 or were cultured under acidified conditions. Cell viability was verified with propidium iodide and fluorescein diacetate. RESULTS: The embryotoxic effect of magainin and H2O2 was dose- and time-dependent. Cyclodextrin, H2O2, acidification of the medium, and to a lesser extent albumin, enhanced the embryotoxicity of magainin at sub-threshold concentrations. CONCLUSION: Magainin on its own is highly embryotoxic. Its embryotoxicity is enhanced by cyclodextrin, albumin, H2O2 and acidification. Thus, magainin which has antibacterial, antifungal and antiprotozoal activity may also have a potential role as a contraceptive agent. The harmful effects of various concentrations of the exogenous H2O2 on 2-cell stage mouse embryos are reported here, to the best of our knowledge, for the first time.

Key words: cyclodextrin/embryotoxicity/hydrogen peroxide/magainine-2-amide/preimplantation mouse embryo


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Magainins represent a family of {alpha}-helix peptides isolated from frog skin (Zasloff, 1987Go). Their ability to kill bacteria and other micro-organisms make them promising antimicrobial agents.

The mode of operation of magainin involves the formation of voltage-dependent channels leading to subsequent cell death as a consequence of ion dissipation. Magainin molecules are first adsorbed to the surface of the membrane from where they intercalate into the membrane forming ion channels (Matsuzaki et al., 1995aGo, 1998Go). Magainins interact preferentially with membranes that are negatively charged, contain acidic phospholipids and have no cholesterol (Matsuzaki et al., 1995bGo; Wieprecht et al., 1997Go). These membrane attributes are characteristic of prokaryotic membranes which is why magainins are good candidates as potential antimicrobial agents.

The cell membrane of preimplantation mouse embryos presents a unique sterol composition (Pratt, 1982Go, 1985Go) that predisposes it to magainin susceptibility.

Magainin at moderate concentration exerts a toxic effect on some tumour cell lines in vitro, melanoma cells in vivo, preimplantation mouse embryos as well as on human spermatozoa (Cruciani et al., 1991Go; Baker et al., 1993Go; Sawicki and Mystkowska, 1999Go; Wójcik et al., 2000Go). At high concentrations it may affect at least some types of normal somatic cells (Chen et al., 1988Go; Matsuzaki, 1998Go). The efficiency in canalization of somatic cell membrane depends on the sequence of amino acids of a peptide antibiotic (Matsuzaki, 1998Go). Moreover, specific modification of the extracellular environment changes the action of magainin and/or susceptibility of the cells (Chen et al., 1988Go; Choi and Toyoda, 1998Go; Lichtenstein et al., 1988aGo; Wójcik et al., 2000Go). Therefore, we tested the effects of albumin, cyclodextrin, hydrogen peroxide (H2O2) and acidification of the medium on the modification of action of magainin.

We reported previously (Sawicki and Mystkowska, 1999Go) that magainin is highly toxic to preimplantation mouse embryos. Therefore, the objective of the present investigation was to confirm magainin embryotoxicity towards murine 2-cell stage embryos, and also to find, and test the chemical and physical factors that enhance this toxicity. It was shown that cyclodextrin, bovine serum albumin (BSA), exogenous H2O2 and acidification of the medium (pH 6.4) enhanced the embryotoxicity of magainin when added at low (subthreshold) concentrations. The effect of magainin in vivo may presumably be enhanced by albumin, H2O2 and by acidification. Our results suggest that magainin can be used as a contraceptive molecule. Additionally, we present for the first time the effects of various concentrations of exogenous H2O2 on preimplantation mouse embryos.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recovery of embryos
Female mice were superovulated with i.p. injection of 10 IU of pregnant mare serum gonadotrophin (PMSG; Folligon, Intervet, Boxmeer, The Netherlands) followed by an injection, 48 h later, of 10 IU of human chorionic gonadotrophin (HCG; Sigma, St Louis, MO, USA) and mated by the same line males. Two-cell embryos were recovered from oviductal ampullae 44–46 h after HCG. A total of 270 female and 50 male mice from a randomly bred colony was used.

Embryo culture and cell viability
The recovered embryos were pooled and 15 embryos transferred to a 5 µl droplet of M2 medium containing 0.4% of either polyvinyl pyrrolidone, PVP (referred to further as M2-PVP medium) or bovine serum albumin, BSA (referred to further as M2-BSA medium). In each experiment at least 7 droplets containing embryos were used. Embryos were then incubated at 37° C in a humidified atmosphere in the air, in droplets of M2 medium under paraffin oil, for 24 h. Visual assessment of cells, as well as tests of their viability, were carried out at various times during the culture.

Cell death was observed by the characteristic structural alterations—enlargement and flattening of blastomeres as well as pronounced cytoplasmic granularity. Additionally, cell death was verified by staining with 50 µg/ml of propidium iodide (Sigma) and 1.5 µmol/l fluorescein diacetate (Sigma) followed by examination under a fluorescence microscope. Propidium iodide, an exclusive vital dye, enters the cell through damaged membranes, while fluorescein diacetate is converted by nonspecific esterases to fluorescein which then penetrates into and stains living cells.

Since we observed that in 2-cell embryos, subjected to various experimental conditions, either one or both blastomeres died, our results are presented as the proportion of dead cells observed divided by the total number of cells used in the experiments.

Chemicals and treatment
Magainin-2-amide (kindly supplied by Dr W.L.Maloy, Magainin Pharmaceuticals, USA and purchased from Sigma) was dissolved in either M2-PVP or M2-BSA medium. We found in the pilot experiments that dramatic embryotoxicity appears at a minimal magainin concentration of 250 µg/ml, at which almost 100% of cells were killed within 30 min. This concentration of magainin was referred to as the threshold concentration. Therefore, in the tests of enhancement of magainin embryotoxicity we applied subthreshold concentrations of the peptide: 200 µg/ml in the tests of cyclodextrin and 166 µg/ml in the tests of H2O2 and acidification. In the subthreshold concentrations of magainin barely 20–30% of cells were killed within 12–24 h. In addition, the M2-BSA medium adjusted to either pH 7.4 or pH 6.4 was prepared and used for culture in medium alone (control) or with subthreshold concentration of 166 µg/ml magainin.

Methyl-ß-cyclodextrine (Sigma) was dissolved in M2-PVP medium at pH 7.4. Preincubation of embryos in 0.7 mmol/l final solution of cyclodextrin lasted for 1.5 h. The embryos were then either tested for viability or transferred to M2-PVP medium alone or to M2-PVP containing 200 µg/ml magainin (subthreshold concentration) and cultured for 24 h.

Concentrations of H2O2 from 10–3 mol/l to 10–8 mol/l were prepared by dilution of 30% H2O2 with water and then with M2-BSA medium at pH 7.4. The effect of various concentrations of H2O2 on the viability of 2-cell embryos was tested. To investigate the effect of H2O2 on the cell response to magainin the embryos were cultured in M2-BSA supplied with 10–7 mol/l H2O2 and a subthreshold concentration of 166 µg/ml of magainin.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Embryo response to magainin, BSA and cyclodextrin
Magainin elicited death of 2-cell embryos in a dose- and time-dependent mode. The dead blastomeres could easily be spotted since soon after contact with magainin, they visibly flattened, increased their size and showed remarkable granulation of cytoplasm. Nevertheless, their viability was carefully checked by staining with either propidium iodide or fluorescein acetate followed by inspection under the fluorescence microscope. At the subthreshold magainin concentration of 166 µg/ml of M2-BSA medium we found 20 and 38% of dead blastomeres after 1 and 24 h of treatment, respectively (Figure 1Go). Magainin at the threshold concentration of 250 µg/ml of M2-BSA medium elicited a dramatic effect on blastomeres of 2-cell embryos: after 30 min, 1 and 24 h of treatment we found 80, 88, and 99% dead blastomeres respectively (Figure 1Go). In contrast, the treatment of 2-cell embryos with 250 µg/ml magainin in M2-PVP medium produced a less pronounced effect than that of the cells cultured in M2-BSA medium (Figure 1Go), although this effect was most evident after 1 h of incubation.



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Figure 1. Responses of 2-cell embryos to a threshold concentration of magainin at 250 µg/ml dissolved in either M2-BSA ({blacksquare}) or M2-PVP medium ({blacklozenge}) and also to a subthreshold concentration of 166 µg/ml magainin in M2-BSA ({triangleup}). Data were obtained from 21 experiments involving 1080 blastomeres.

 
We observed that the blastomeres of 1% of 2-cell embryos treated with 250 µg/ml of magainin fused with each other and produced 1-cell bikaryocytes.

Preincubation of 2-cell embryos for 1.5 h in M2-PVP medium containing cyclodextrin followed by the culture of cells in M2-PVP medium supplied with a subthreshold concentration of 166 µg/ml magainin, enhanced the effect of magainin. Treatment with magainin alone for 30 min or 7 h produced 5.5 and 18.5% of dead blastomeres while cells preincubated with cyclodextrin produced in the corresponding times and with the same concentrations of magainin 48 and 84% of dead blastomeres respectively (Figure 2Go). The blastomeres of 2-cell embryos cultured in M2-PVP medium supplied with cyclodextrin alone remained alive and showed the ability to divide in the course of 24 h (Figure 2Go).



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Figure 2. Responses of 2-cell embryos to a subthreshold concentration of 200 µg/ml magainin alone ({circ}) and to that preceded by 1.5 h preincubation with 0.7 mM cyclodextrin (•). and {blacksquare} denote treatment with cyclodextrin or with medium, alone. Data were obtained from 24 experiments involving 844 blastomeres.

 
Response to hydrogen peroxide
The effect of 10–8–10–3 mol/l H2O2 on the blastomeres of 2-cell embryos was evaluated in M2-BSA medium (Figure 3Go). At a concentration of 10–8 mol/l H2O2 100% of embryos remained alive over the 24 h incubation period. Of those 13% divided producing 4-cell embryos, whereas 37–44% of blastomeres acquired two nuclei in one or both blastomeres of an embryo. We observed that binucleation of blastomeres was the result of the modification of the cell cycle, (i.e. normal karyokinesis but absent cytokinesis) and not cell fusion. In the control, 50–60% of blastomeres divided, producing 4-cell embryos. In M2-BSA medium with added H2O2 at 5x10–8 mol/l and 7x10–8 mol/l concentrations, all embryos survived 24 h in culture and only at a concentration of 8x10–8 mol/l did we note a 24% cell mortality, both after 9 and 24 h of treatment. The augmentation of H2O2 concentration to 10–7–10–6 mol/l increased cell mortality to 26% after 24 h of treatment. Kinetics of embryo mortality at 10–6 mol/l H2O2 concentration is characterized by an acclivity of dead cell number, from 5% at the 3rd hour, to 35% at the 9th hour and to 100% of cell death at 24 h of treatment.



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Figure 3. Responses of 2-cell embryos to various concentrations of H2O2:10–8 mol/l ({circ}), 10–7 mol/l (•), 10–6 mol/l ({square}), 10–5 mol/l ({blacksquare}), 10–4 mol/l ({triangleup}) and 10–3 mol/l ({blacktriangleup}). Data were obtained from 32 experiments involving 3842 blastomeres.

 
The higher concentrations of H2O2, i.e. 10–5, 10–4 and 10–3 mol/l yielded respectively, higher rates of cell death at short treatment times. For example, a 10–3 mol/l concentration of H2O2 killed 100% of cells after the 1st hour of treatment and 3 h of treatment yielded the disappearance of the cells' zona pellucida. As indicated by the kinetics of cell mortality (Figure 3Go) the threshold concentration of H2O2 is 10–6 mol/l and at this or lower concentrations cells are able to adapt. The higher concentrations of H2O2 produced, in addition to cell death, alteration of cell shape, blebbing and cytoplasmic polarization into light and dark domains. The blebbing and polarization of cytoplasm observed with 10–7 and 10–6 mol/l concentrations of H2O2 were not apparent at 10–5 mol/l and above.

Synergism of hydrogen peroxide and magainin
To test the enhancement of the effect of magainin on 2-cell embryos, by H2O2, we used the lowest effective concentration of 10–7 mol/l H2O2 and the subthreshold concentration of magainin (166 µg/ml). While both the magainin and H2O2 acting separately elicited congruent cell death rates of 5% after the 4th, 6–10% after the 9th and 18–24% after 24 h of treatment (Figure 4Go), the simultaneous treatment of both substances at the same time points showed clear synergism: 36% of cells died after 1 h, 58% after 9 h and 65% after 24 h of treatment.



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Figure 4. Response of 2-cell embryos to a subthreshold concentration of 166 µg/ml magainin alone ({blacksquare}), to 10–7 mol/l H2O2 alone (•), or to magainin and H2O2 together ({circ}). Data were obtained from 28 experiments involving 3370 blastomeres.

 
Effect of medium acidification on magainin cytotoxicity
The degree and promptness of the cytotoxic effect of magainin depended on the pH at which cells were cultured. In the M2-BSA medium of pH 7.4 the 30-min treatment with 166 µg/ml magainin alone, yielded 19% dead cells (Figure 5Go) whereas the respective percentage was 45% for the same duration and same magainin concentration in M2-BSA medium of pH 6.4. The highest rate of cell mortality was observed during the first 30 min of treatment. In all variants of the experiment, the progression of cell death proceeded less precipitously thereafter. After 24 h in M2-BSA, at pH 7.4 or 6.4, the proportion of dead cells increased to 27 and 80% respectively.



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Figure 5. Responses of 2-cell embryos to non-acidified, pH 7.4 medium ({square}), acidified medium, pH 6.4 ({blacksquare}), subthreshold concentration of 166 µg/ml magainin at pH 7.4 ({circ}) and 166 µg/ml magainin at pH 6.4 (•). Data were obtained from 20 experiments involving 1714 blastomeres.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Magainin kills embryonic cells in a dose-, stage- and time-dependent manner (Sawicki and Mystkowska, 1999Go). It is thought that the factors which affect the magainin cytotoxicity and its efficiency are connected both to the chemical structure of the cell membrane and to that of magainin (Matsuzaki et al., 1995aGo, bGo) with the former seeming to play the most important role. First, magainin attacks a cell by adsorption of its molecules to the membrane surface. The adsorption depends upon the interaction between acidic lipids of the membrane and the basic peptide of magainin. Then the peptide molecules are inserted into the lipid bilayer and form channel complexes. The cholesterol content of the membrane attenuates to a great extent the cytotoxic effect of magainin (Matsuzaki et al., 1995bGo; Wieprecht et al., 1999Go). This is accompanied by a decrease in membrane fluidity (Wieprecht et al., 1999Go). Moreover, the binding of membrane cholesterol to the magainin glutamine stops the formation of peptide complexes responsible for membrane permeability (Tytler et al., 1995Go).

The cell membrane of preimplantation embryos consists of the same types of phospholipids as those of somatic cells. However, sterol synthesis starts in the 2-cell embryos and proceeds to the lanosterol step only (Pratt, 1982Go, 1985Go). Since lanosterol makes membranes more fluid than cholesterol (Yeagle et al., 1977Go; Yeagle, 1989Go) the blastomere membrane of cleavage embryos could be more susceptible to magainin than that of somatic cells which contain cholesterol. The distribution of lanosterol within the blastomere membrane is noteworthy. It has a mosaic character and is highly asymmetric, being lowest at the surface of attachment to neighbouring cells (Pratt, 1982Go, 1985Go). This feature of blastomere membrane, together with its lanosterol content is presumably responsible for the considerable susceptibility of mouse embryos to magainin. On the other hand, the asymmetric distribution of sterols in blastomere membrane may lead to the fusion of sister blastomeres as observed here.

The present investigation indicates that both cyclodextrin and, to a lesser extent, bovine serum albumin enhanced the cytotoxic effect of magainin. While the magainin alone, at a subthreshold concentration, killed 5–35% of embryonic cells within 0.5–24 h, the supplementation of cyclodextrin, H2O2 or acidification to the medium, increased the rate of the dead cells to 85, 65 and 84% respectively. As observed previously, cyclodextrin removes sterols, including cholesterol, from cell membranes (Haynes et al., 2000Go). Cyclodextrin-treated spermatozoa become highly susceptible to magainin (Kilsdonk et al., 1995Go; Choi and Toyoda, 1998Go; Wójcik et al., 2000Go). Therefore, one might speculate that the elimination of sterols from the membrane by cyclodextrin is the major cause of enhanced susceptibility of blastomeres to magainin found in this study. Like cyclodextrin, serum albumin is thought to be an extracellular acceptor of membrane sterols. However, the molecular mechanisms underlying albumin's effect on membrane cholesterol have not been clearly determined (Haynes et al., 2000Go).

The minimal toxic concentration of magainin for micro- organisms was 10–100 µg/ml whereas the toxicity for tumour cells was 20–200 µg/ml (Zasloff, 1987Go). However, lysis of erythrocytes required as much as 1 mg/ml of magainin (Chen et al., 1988Go; Matsuzaki et al., 1997bGo).

We previously found that mouse oocytes, 1- and 2-cell embryos as well as blastocyst cells were killed in the course of a 1 h treatment with 250 µg/ml magainin (Sawicki and Mystkowska, 1999Go) and that this effect was dose-dependent. The action of magainin is highly sensitive to the lipid composition of the membrane (Matsuzaki, 1998Go; Matsuzaki et al., 1998Go). The efficiency of the binding of magainin to the membrane surface, channel size, density and stability depend on the magainin concentration. At a low concentration of magainin the proportion of peptide to membrane lipids is relatively low and the magainin molecules tend to take an orientation parallel to the lipid bilayer, thus disturbing lipid packing. However, if the proportion of peptide to membrane lipids is relatively high the magainin molecules tend to intercalate into the lipid bilayer and form transmembrane channel complexes composed of three to six molecules each (Ludtke et al., 1996Go; Jo et al., 1998Go; Wenk and Seelig, 1998Go). Experimental models carried out using the membrane of artificial liposomes showed that the complexes are formed in the course of 30 s (Jo et al., 1998Go). The prompt embryotoxic effect of magainin found in this study confirms those findings.

H2O2 is produced both inside and outside cells (Fridovich, 1998Go). Unbalanced production of H2O2 can induce oxidative damage to membrane lipids, proteins and DNA resulting in cellular injury either directly or indirectly through hydroperoxyradicals and lipid hydroperoxides which are precursors of the toxic end product, 4-hydroxy–2–nonenal (HNE) (Uchida et al., 1999Go). At low concentrations exogenous H2O2 induced an adaptative response of cells by promotion of synthesis of antioxidant enzymes and heat shock proteins (Lee and Um, 1999Go). H2O2 can kill cells either by apoptosis or by necrosis (Kim et al., 2000Go). High doses of H2O2 can induce necrosis by impairment of the activity of respiratory enzymes, membrane channels and transporters. This may be done by selectively binding the hydroperoxyl radical to the imidazole moiety, histidine residues and sulphydryl group of cysteine residues (Uchida et al., 1999Go).

For most cells protection against oxidative stress is provided by catalase, glutathione peroxidase and glutathione itself. Production of catalase is initiated in the mouse in the blastocyst stage (Harvey et al., 1995Go); therefore, the critical role in the protection of 2-cell embryos against oxidative stress evoked by exogenous H2O2 may be performed by hypotaurine (Guerin and Menezo, 1995Go), glutathione and glutathione peroxidase. The latter is a major H2O2-consuming enzyme while the former serves as the substrate. Glutathione and its peroxidase are synthesized by oocytes and also by cumulus cells during oogenesis (de Matos et al., 1997Go; El Mouatassim et al., 1999Go). Cleavage mouse embryos cannot synthesize glutathione but can utilize that produced during oogenesis (Gardiner and Reed, 1995Go). Extracellular glutathione has been found in preovular follicular and reproductive tract fluid (Gardiner et al., 1998Go; Józwik et al., 1999Go). Unfavourable embryo culture conditions raise cellular levels of H2O2 and generate the 2-cell block in mouse and rat embryos (Nasr-Esfahani et al., 1990Go, Matsumoto et al., 1998Go).

As indicated by the present investigation, the blastomeres of 2-cell embryos are more sensitive to exogenous H2O2 than somatic cells. For the latter cells cultivated in vitro, and for erythrocytes and leukemic cells, the LD50 was 10–2–10–3mol/l (Lichtenstein et al., 1988bGo), while the blastomeres of 2-cell embryos died at the concentration 10–3–10–5 mol/l H2O2. Our results suggest strongly that H2O2 facilitates the adsorption and binding of magainin to the lipid bilayer. This is in accordance with the fact that the oxidation of fatty acids impairs membrane integrity through elimination of polyunsaturated fatty acids and sterols (Griveau et al., 1995Go) and modification of membrane charge. The results of the present investigation confirm the earlier finding (Lichtenstein et al., 1988bGo) that H2O2 intensified the binding of iodinated neutrophil defensin, another type of antimicrobial peptide, to cell membrane (White et al., 1995Go).

We found that culture of 2-cell embryos in medium acidified to pH 6.4 remarkably enhanced their susceptibility to magainin. The blastomeres of 2-cell embryos cultured in the medium at pH 6.4 survived well with their dividing activity deteriorating only slightly. The blastomeres of cleavage embryos can respond to the low external pH by increasing slightly their internal acidity. An excess of protons outside and within blastomere membranes is buffered by the proteins of proton channels (Baltz et al., 1991Go, 1993Go; Dale et al., 1998Go; Banfi et al., 2000Go; Phillips et al., 2000Go). The ability of embryos to maintain their inner/outer pH emerges at the morula/blastocyst stage when embryos encounter an acidic environment in the uterus (Dale et al., 1998Go).

It is well known that the conformation and self-association of protein molecules, as well as the cytotoxicity and fusogenecity of pore-forming proteins, are pH-dependent (Kagan et al.,1992Go; Hu et al., 2000Go). Acidification enhances the affinity of molecules to bind with the cell surface, and to insert into cell membranes. Acidified media increase the protonation of amino groups of magainin which enhances its helicity, helix stability and maximizes the lytic property of magainin (Matsuzaki et al., 1997aGo). However, the mechanism of the enhancement of embryo sensitivity to magainin continues to be an enigma. It is noteworthy that the bacteria cultured in acidified medium were found to be much more efficiently permeabilized by defensin than those cultured in neutral medium (Sawyer et al., 1988Go). We can, therefore, speculate that acidification can confer on molecules a conformational feature that facilitates the intercalation of magainin into both membrane and channel formation.

Both the antioxidant system of the Graffian follicle (Józwik et al., 1999Go) and pH-stabilization of intrafollicular fluid (Dale et al., 1998Go) are efficient defence mechanisms of the preovulatory oocyte. The ovulated oocyte, zygote and preimplantation embryo are exposed to more changeable conditions in the reproductive tract. However, the physiological pH of oviductal fluid in humans is alkaline (pH 7.7–7.9) although in inflammation the local pH could be reduced to a value below pH 5 (Simmen and Blaser, 1993Go). The sensitivity of preimplantation embryos to peroxidative damage, the devastating effect of magainin, and the synergistic effect of these factors, may be considered as the putative cause of at least some idiopathic infertilities.

The experimental systems magainin/H2O2 and magainin/acidified medium used in the present investigation may mimic, to some extent, the naturally occurring mammalian system, consisting of defensins/cellular environment at inflammation (Lichtenstein et al., 1988bGo). In both systems the enhancement of peptide antibiotic embryotoxicity occurs. Magainin-like immunoreactivity found in human submandibular gland (Hansen et al., 1995Go) renders the potential magainin hazard more probable.

On the other hand, magainin-2-amide can present in-vivo contraceptive ability. It was recently found that 250 and 500 µg of magainin-2-amide vaginally administered exerts anti-nidatory effects in the rhesus monkey (Dhawan et al., 2000Go). We found that cyclodextrin and H2O2 at low concentrations (10–7 mol/l) clearly enhanced the embryotoxicity of magainin-2-amide. Therefore, we propose that a mixture of magainin/cyclodextrin, magainin/H2O2 or magainin/cyclodextrin/H2O2 ought to be a more potent contraceptive than magainin-2-amide alone. Furthermore, peptide antibiotics including magainin have antibacterial, antifungal and antiprotozoal activity, making them promising antimicrobial agents (Hancock, 1997Go). However, if used in vivo as antibiotics, the consequences of their contraceptive side-effects must also be considered.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to Jaroslaw Józwiak and Rafal Bilko for the valuable aid in the graphical presentation of the results.


    Notes
 
3 To whom correspondence should be addressed at: Center of Biostructure, Medical Academy, Chalubinski St. 5, 02–004 Warsaw, Poland. E-mail address: wsawicki{at}ib.amwaw.edu.pl Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 5, 2000; accepted on April 4, 2001.





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