Experimental Parasitology 121 (2009) 208–212

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Experimental Parasitology

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Giardia duodenalis: Effects of an ozonized sunflower oil product (Oleozon�)on in vitro trophozoites

Frank Hernández a,*, Damian Hernández a, Zullyt Zamora a, Maritza Díaz a, Odelsa Ancheta a,b,Sandra Rodriguez a,b, Dinorah Torres a,c

a Department of Biomedicine, Ozone Research Center from National Center for Scientific Research, P.O. Box 6412, Havana City 16000, Cubab Laboratory of Electronic Microscopy, Biotechnology Unit, National Center for Scientific Research, P.O. Box 6412, Havana City 16000, Cubac Department of Parasitology, Pedro Kouri Institute, Havana City, Cuba

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 April 2008Received in revised form 9 October 2008Accepted 24 October 2008Available online 5 November 2008

Keywords:Ozonized sunflower oilOzone therapyGiardiasisGiardia duodenalisOleozon

0014-4894/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.exppara.2008.10.009

* Corresponding author.E-mail address: frank.hernandez@cnic.edu.cu (F. H

The ozonized sunflower oil product (Oleozon�) was investigated to explore its cytotoxic activity on Giar-dia duodenalis in vitro cultivated trophozites. Oleozon� produced inactivation of Giardia trophozoites in adose- and cell density-dependent manner. Thirty microliter of Oleozon� with peroxide index value of 500equivalent-mmol of activated oxygen per kilogram were used to achieve a 100% inhibition (<�4.00 logunit) of trophozoites from an initial inoculum of 15 � 104 cells. This potent effect was confirmed by trans-mission electron microscopy where morphological deterioration of superficial structures mainly in theventral disc, and formation of a great number of micro vesicles in the cytoplasm were found. We con-cluded that a direct chemical-oxidation attack by the active substances from Oleozon� is one of thecauses of the parasitocidal effect of this product. We suggest that the dose and cell density-dependenteffect must be taken into account when prescription of this product for giardiasis treatment in humans.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

Giardia duodenalis (Giardia lamblia or Giardia intestinalis), is acommon cause of many gastrointestinal troubles including ende-mic and epidemic diarrhea throughout the world. It is a protozoanparasite which resides in the intestine and the prevalence of infec-tion (giardiasis) may reach 2–7% in Western countries and 20–60%in developing countries (Müller and Gottstein, 1998; Gardner andHill, 2001; Mahmud et al., 1995).

The available drugs for the giardiais treatment are imidazolecompounds. However, patients with infections are already com-mon and increasing in frequency. An alternative natural therapyfor giardiasis has been developed with the use of an ozonized sun-flower oil (Oleozon�) patented by the Ozone Research Center ofCuba. Previous in vitro studies reported good results for antifungaland antibacterial properties of Oleozon� (Lezcano et al., 1996;Lezcano et al., 1998). In a preliminary human study we showedOleozon� had an effectiveness between 70% and 80% in eradicatingthe infection of Giardia duodenalis (Hernández and Menéndez,1997); and in a randomised, open controlled phase III trial its effi-cacy was demonstrated in patients suffering relapsing giardiasis(Amoroto et al., 2002).

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ernández).

However, the mechanism by which Oleozon� produces a cytoto-cic effect on Giardia duodenalis trophozoites is not well understood.

The objective of this work was to ascertain the in vitro efficacyof Oleozon� and its mechanism of action on cultured trophozoitesof Giardia duodenalis.

2. Materials and methods

2.1. Oleozon�

This is a natural product developed by the Ozone Research Cen-ter of Cuba using a stream of ozone gas on a determined amount ofedible commercial sunflower oil, which produced a variety of lipidperoxides by the attack on the unsaturated triglycerides in the oil.The production process has been patented (Molerio et al., 2003)and the product registered as a pharmacological medicament inCuba (Hernández. et al., 2004a). Its potency is mainly indicatedby the peroxide index however, the chemical density and acid in-dex alter with the change in the peroxide index values (Hernándezet al., 2004b). In our study we used Oleozon� with peroxide indicesof 250, 500 and 750 equivalent-mmol/kg with density values of0.9357, 0.9547 and 0.9731 g/mL; and acid index values of 0.22,0.80 and 2.08 mg of KOH/g, respectively. The sunflower oil hadan initial peroxide index value of 7.9 ± 0.13 equivalent-mmol/kg,with a density of 0.9210 ± 0.0001 g/mL, and an acid index valueof 0.12 ± 0.02 mg of KOH/mL.

Table 1Inhibition assay of Giardia duodenalis trophozoites in cultures with initial inoculumsof 45 � 104 cells using Oleozon� of a peroxide index value of 500 equiv-mmol/kg.

Groups Cellular countper tube (�106)

Inhibition(percentage)

Inhibition(log units)

Negat-Cont 18.75 ± 1.98 — —Oil-Cont 30 19.55 ± 0.34 — —Oil-Cont 45 18.75 ± 1.06 — —Oil-Cont 60 19.25 ± 0.43 — —Oleozon 30 1.94 ± 2.66* 91.09 �1.00Oleozon 45 0.25 ± 8.66* 98.67 �1.88Oleozon 60 0.01 ± 0.68* 99.95 �3.28

* Statistical difference respecting its oil-control.

Table 2Inhibition assay of Giardia duodenalis trophozoites in cultures with initial inoculumsof 15 � 104 cells using Oleozon� of Peroxide Index value of 500 equiv-mmol/kg.

Groups Cellular countper tube ( � 106)

Inhibition(percentage)

Inhibition(log units)

Negat-Cont 2.99 ± 0.86 — —Oil-Cont 30 2.65 ± 0.48 — —Oil-Cont 45 2.80 ± 0.43 — —Oil-Cont 60 2.85 ± 0.68 — —Oleozon 30 0 ± 0* 100 <�4.00Oleozon 45 0 ± 0* 100 <�4.00Oleozon 60 0 ± 0* 100 <�4.00

* Statistical difference respecting its oil-control.

F. Hernández et al. / Experimental Parasitology 121 (2009) 208–212 209

The peroxide index represents the number of equivalent-milli-mol of active oxygen that expresses the amount of peroxide con-tained in 1000 g of the oil. Briefly, to obtain this figure, 5 g of asample is mixed with 30 volumes of glacial acetic acid, 20 volumesof chloroform and 0.5 mL of saturated potassium iodide solution.The mixture is shaken for exactly 1 min, mixed with 30 mL ofwater and slowly titrated; shaking continuously, with 0.01 M so-dium thiosulphate until the yellow colour almost disappears. Theperoxide index values are obtained from the expression 10 v/w,where v is the volume of sodium thiosulphate in mL consumedin the titration, and w is the weight in g of substance (British Phar-macopoeia, 2000a).

Acidity is frequently expressed as the acidity index, which is thenumber of mg of potassium hydroxide required to neutralize thefree acids in 1.0 g of the substance (British Pharmacopoeia, 2000b).

2.2. Experimental groups

The following seven groups were included in the study: (1) Neg-ative control [Negat-Cont], culture without adding any oil; (2) Oilcontrol [Oil-Cont] 30, (3) Oil-Cont 45 and, (4) Oil-Cont 60, cultureswith 30, 45 and 60 lL of virgin sunflower oil, respectively; (5)Oleozon 30, (6) Oleozon 45 and (7) Oleozon 60, cultures with 30,45 and 60 lL of Oleozon�, respectively.

2.3. Giardia duodenalis in vitro cultivation

Trophozoites of Giardia duodenalis were isolated from a duode-nal aspirate taken from a pediatric patient following the methodreported previously (Torres et al., 1996). The organisms were rou-tinely maintained in vitro in liquid nitrogen and cultures weremade in Diamond’s TYI-S33 medium enriched with 10% fetal calfserum only after two passes, when the cells were optimally acti-vated with volumes (30, 45 and 60 lL) of Oleozon� or virgin sun-flower oil were added to culture tubes with 5.4 mL of medium,vortexed by 30 s and 0.5 mL of trophozoite suspension with a cor-responding cellular density. The tubes were incubated 48 h at 37 �Cin CO2 atmosphere. Actively growing trophozoites were incubatedon ice for 15 min to detach the parasites from the walls of thetubes. The cells were then sedimented by centrifugation at 600gfor 10 min, washed in phosphate buffered saline (PBS), and resus-pended in a solution of 1% formalin in PBS. Finally, the cell numberwas determined microscopically from 100 lL of each suspensionusing a Neubauer cell-counter chamber. The average of three cul-tures in each group, with duplicate counts for every culture, wereused for obtaining the total number of cells in 1 mL of culture med-ium, or the total number of cells per each tube.

2.4. Calculation of trophozoites inhibition

Growth inhibition was calculated as the percentage of cells inthe cultures with Oleozon� compared to the number of cells inthe control cultures with virgin sunflower oil. Additionally, thegrowth inhibition as log units was also calculated as

log N=N0

where N is the total number of cells in the Oleozon� cultures and N0

the total number of cells in the virgin sunflower oil cultures.

2.5. Electron microscopy examination

Cultures of trophozoites were washed three times in ice-coldPBS and sedimented by centrifugation (see above). They werepre-fixed with 3.2% glutaraldehyde in cacodylate buffer pH 7.4and post-fixed with 2% osmium tetroxide with the same buffer.The cells were then dehydrated with absolute ethanol and embed-

ded in Spurr resin (Spurr, 1969). Sections for transmission electronmicroscopy (TEM) were cut on a LKD Ultratomo III ultramicrotomeand were loaded onto 200-mesh nickel or copper grids. They werestained with uranyl acetate and lead citrate (Reynols, 1963; Smithand Croft, 1991), and the samples were viewed and photographedusing a JEOL JEM 100S electron microscope.

2.6. Statistical analysis

Commercial statistical package NCSS were used to analyse thedata. Results are expressed as mean ± SD. Comparisons were doneby Kruskal-Wallis and Mann-Whitney tests. A p value 6 0.05 wasconsidered statistically significant.

3. Results

3.1. Inhibition of Giardia duodenalis trophozoites

The strong cytotoxic effect of Oleozon� with a peroxide index of500 on in vitro cultivated trophozoites of Giardia duodenalis isdisplayed in Table 1. Virgin sunflower oil added to the culture atthree different volumes (Oil-Cont) had no influence on the growthof the parasites at initial inoculums of 45 � 104 cells, which grewto between 16 and 20 � 106 cells (similar to negative control groupcultures), during 48-h culture under optimal growth conditions.However, Oleozon� added in three different volumes resulted insignificantly reduced recoveries of viable cells with inhibition per-centage ranging from 91.09 to 99.95 (�1.00 to �3.28 log units).

A strong cytotoxic effect of Oleozon� on in vitro cultivated tro-phozoites is illustrated in Table 2 when the initial inoculums were3-fold smaller (15 � 104 cells) than the earlier experiment. In thiscase the three different volumes of Oleozon� were totally effectiveshowing 100 percent (<�4.00 log units) inhibition.

3.2. Dose-response behavior of Oleozon�

To explore the relationship between the inhibitory responseand the potency of Oleozon�, trials with different peroxide indices

210 F. Hernández et al. / Experimental Parasitology 121 (2009) 208–212

were performed using 30lL of the products (Fig. 1). Material withperoxide indices from 250 to 750 with a cellular density of45 � 104 were used. The graph shows a curve representing a clas-sical dose-dependent response.

Another aspect was to evaluate the relationship with cellulardensity (Fig. 2). In this case trophozoite inoculums from 15 � 104

to 45 � 104 trophozoites were treated with an ozonized oil with500 of peroxide index. Fig. 2 shows that the cytotoxicity or inhibi-tion of the trophozoites decreased gradually. As the inoculum den-sity increased.

3.3. Ultrastructural study by electron microscopy

The cell-surface changes on trophozoites that had previouslybeen exposed to Oleozon� was investigated by TEM. The peroxideindex of this ozonized oil was 250 in order to achieve incompletedestruction of the cells and allow to observing the damage in the

Fig. 1. Giardia duodenalis inhibition as a function of peroxide index of Oleozon�

product. Trophozoites cultures were done with inoculums of 45 � 104 cells and30 lL of ozonized oil. Each point is the average of three cultures with duplicatecounts and bars represent the standard deviation.

Fig. 2. Giardia duodenalis inhibition as a function of cellular density. Thirtymicroliter of Oral Oleozon� with Peroxide index of 500 were used in thesetrophozoites cultures. Each point is the average of three cultures with duplicatecounts and bars represent the standard deviation.

cell surface. The potency of this Oleozon� was 3-fold lower thanthe product used in the previous experiments. The normal ultra-structural appearance of untreated trophozoites is illustrated inFig. 3. The parasites have a fine surface membrane, dense cyto-plasm, two nucleuses with basal bodies and well defined microtu-bules. A very dense superficial laminar structure formed bymicrofilaments is also observed in the ventral disc. Oleozon� treat-ment produced marked morphological changes on the surface oftrophozoites (Fig. 4). Careful analysis of thin sections clearly re-vealed loss of dense superficial laminar structures and of cyto-plasm density together with formation of high number of microvesicles in the cytoplasm. The distortion of the ultrastructure ofthe ventral disc with significant loss of microfilaments is notable.The size and shape of the cells were markedly changed showingthe presence of edema.

4. Discussion

In the present study we investigated the effect of Oleozon�

(ozonized sunflower oil) on cultured Giardia duodenalis trophozo-ites obtained from a clinical isolate. Oleozon� with peroxide indexbetween 500 and 750 was highly effective in producing cytotoxic-ity. The results by using ozonized oil with different peroxide indi-ces and trophozoite densities demonstrated a dose- and celldensity-dependent inhibition that seemed to be associated withdisintegration or weakening of cellular structure. The total cyto-toxic effect on 15 � 104 cells was obtained with 30 lL (28.6 mg)of Oleozon�. In previous experiments (not shown), we treatedthe trophozites with smaller amounts (10 and 20 lL) of Oleozon�,finding inhibitions values of 65.4% and 78.8%, respectively. For thatreason in subsequent we used a dose of between 30 and 60 lL ofOleozon�.

Cytotoxicity of Oleozon� is mainly caused by the direct chemi-cal action of the different peroxides in the product, as occurs with

Fig. 3. Transmission electron microscopy of normal cultivated Giardia duodenalistrophozoites Scale bar = 3 lm.

Fig. 4. Transmission electron microscopy of cultivated Giardia duodenalis tropho-zoites treated with an attenuated Oleozon� product (peroxide index of 250). Scalebar = 3 lm.

F. Hernández et al. / Experimental Parasitology 121 (2009) 208–212 211

disinfection of Giardia cysts (which are more resistant than Tro-phozoites) in water by direct treatment with ozone gas (Labatiukand Finch, 1991; Finch et al., 1993).

Active principles of Oleozon� are mainly hydroxyhydroperox-ides, ozonides, hydrogen peroxides and aldehydes (Díaz et al.,2005; Martínez et al., 2005) which have sufficient power to oxidizedifferent biomolecules in the cellular structure. It has been abun-dant demonstrated that Oleozon� has biological activity againstthe majority of microorganisms (Menéndez et al., 1995a,b; Lezcanoet al., 1996; Lezcano et al., 1998; Díaz et al., 2006). The active prin-ciples as separate compounds also have activity (Díaz et al., 1997,2001). Therefore, the main mechanism of action would appear tobe a chemically-mediated attack of the different oxidant com-pound in Oleozon� on the superficial structure of the Giardia duo-denalis trophozoites.

We performed the present study to provide insight into thecytological processes associated with the Oleozon�-mediated par-asite killing. This is likely to be directly responsible for Oleozon�-mediated cytotoxicity and inhibition of cell growth. It was con-firmed by TEM where cell-surface alterations were observed in tro-phozoites previously exposed to attenuated Oleozon�. Incubationof viable cells with cytotoxic amount of this product resulted inmarked damage in the ventral disc; this structure is used by theparasite to attach to the intestine wall. The oxidative injuries pro-duced detachment and agglutination of the cells contributing tothe mechanism involved in intestinal parasite clearance. The for-mation of multiple micro vesicles and loss of cytoplasm densityare other important effects indicate the chemical cytotoxicity onthese cells. Similar morphological changes have been observedon trophozoites treated by a cytotoxic monoclonal antibody andby albendazole, which produced a parasitocide effect (Hemphillet al., 1996).

The effective Oleozon� concentration in the present study ishigher than those used for nitroimidazole products in other studies(Chavez, 1992; Hemphill et al, 1996). However, from the physiolog-ical point of view, we and other authors have used higher concentra-tions of Oleozon� in experimental animal (Remigio et al., 1998;Zamora et al., 2006) and humans (Amoroto et al., 2002; Méndezet al., 2003)) studies without any cytotoxic or side effects in therecipients. Furthermore, we have not found cytotoxic effects onintestinal cells in animal studies for acute and subchronic cytotox-

icity (results not published). As previously stated Oleozon� containsreactive oxygen species which have a potent oxidant activity; there-fore, in terms of toxicity and efficacy, in vivo processes differs fromin vitro conditions because of their by the antioxidant defenses ofthe organism, and lesser oil accessibility to the trophozoites in theintestine. The efficacy of Oleozon� was similar to that of Ornidazolein a phase III clinical trial (Amoroto et al., 2002).

This study demonstrates the cytotoxic effects of the Oleozon�

product on Giardia duodenalis cell cultures; however, host-medi-ated effects should also be considered in human infections. Immu-nological functions of local IgA and IgG systemic antibodies whichpromote antigenic variation of the parasite and are involved incontrol of parasite infection (Stäger and Müller, 1997; Stägeret al, 1997; Müller and Gottstein, 1998), are important aspects toconsider. Clearly, from our in vitro study no conclusion can bedrawn on this. However, exploratory clinical assays have shownthat Oleozon� is useful product to treat Giardia duodenalis infectionin children and adults (Menéndez et al, 1995a,b; Hernández andMenéndez, 1997).

In summary, the data presented in this paper describe the cyto-toxic effect of Oleozon� as an efficient approach for parasite killing.Oleozon� displayed a direct cytotoxic-oxidant effect on Giardiaduodenalis trophozoites in vitro, which is one of the mechanismsof action for a parasitocidal effect of this product. This effect de-pends on dosage and cellular density; aspects that physiciansshould take into account when prescribing this product.

Acknowlegdment

We would like to thank Mrs. Maritza González, Mrs. TaniaValdés and Yahima Harvey for their technical assistance.

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