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Entamoeba histolytica and trichomonas vaginalis: trophozoite growth inhibition by metronidazole electro-transferred water

Contents lists available at Entamoeba histolytica and Trichomonas vaginalis: Trophozoite growth inhibitionby metronidazole electro-transferred water J. Antonio Heredia-Rojas , Antonio Cayetano Torres-Flores , Abraham O. Rodríguez-De la Fuente ,Benito David Mata-Cárdenas Laura E. Rodríguez-Flores , María Porfiria Barrón-González ,Antonio Cayetano Torres-Pantoja Juan M. Alcocer-González a Departamento de Ciencias Exactas y Desarrollo Humano, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Av. Pedro de Alba y Manuel L. Barragán s/n,Cd. Universitaria, C.P. 66451, San Nicolás de los Garza, Nuevo León, Mexicob Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, Av. Pedro de Alba y Manuel L. Barragán s/n, Cd. Universitaria, C.P. 66451, San Nicolás de los Garza, NuevoLeón, Mexicoc Departamento de Patología, Facultad de Medicina, Universidad Autónoma de Nuevo León. Serafín Peña #909 Norte, Monterrey, Nuevo León, C.P. 64000, Mexicod Departamento de Biología Celular y Genética, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Av. Pedro de Alba y Manuel L. Barragán s/n,Cd. Universitaria, C.P. 66451. San Nicolás de los Garza, Nuevo León, Mexicoe Departamento de Microbiología e Inmunología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Av. Pedro de Alba y Manuel L. Barragán s/n,Cd. Universitaria, C.P. 66451, San Nicolás de los Garza, Nuevo León, Mexicof Base Médica, calle Cuatro Ciénegas #175 Col. Mitras Centro, C.P. 64460, Monterrey, Nuevo León, Mexico The influence of low-frequency electromagnetic (LF-EM) waves on microorganisms has been a subject of Received 29 May 2009 experimental investigations for more than two decades and the results are promising. In parallel, an Received in revised form 21 June 2010 interesting procedure known as biophysical-information-therapy or bioresonance therapy (BRT) which Accepted 28 June 2010 in principle is based on LF-EM stimulation, has emerged. BRT was discovered in the late 1980's but it Available online xxxx is still poorly studied. This paper demonstrates that by transferring metronidazole information to watersamples by an electronic amplifier (BRT device), the growth of axenically cultured trophozoites of Ent- amoeba histolytica and Trichomonas vaginalis is significantly inhibited, compared with those cultures trea- ted with non and sham electro-transferred water samples. A positive control of metronidazole, a well- known cytotoxic drug against parasites, was used as a reference.
Ó 2010 Elsevier Inc. All rights reserved.
in the physico-chemical properties of water molecules (For instance, it is known that water possesses several There is a number of alternative and complementary therapeu- paradoxical physical characteristics in comparison with other sub- tics that are unproven or not properly tested. For instance, the use stances and it is extremely sensitive to the impact of physical fac- of electric and magnetic fields, homeopathy, bioresonance therapy, tors such as temperature, magnetic and electric fields, and even and many others. For the last decade, the transferring of bioenergetic mechanical effects; however, the influence of very weak electro- information has been recognized as a novel scientific approach that magnetic energy on water has not yet been investigated, but it is may contribute to the therapy of diverse diseases ().
the subject of intense biophysical research, which concludes that More recently, results of experimental work on the interaction one of the most peculiar features of water is its ability to achieve of water with resonant circuits were presented in an attempt to structural changes and preserve information from specific signals have a physical justification for the changes that are produced in water after this treatment, suggesting permanent modifications There exist a lot of procedures which attempt to inhibit micro- bial and parasitic growth, including the conventional (orthodoxmedicine) use of drugs * Corresponding author. Fax: +81 83762813.
E-mail addresses: (J.A. Heredia-Rojas), ) or by using unconventional approaches such as (A.C. Torres-Flores), electric and magnetic fields. For instance, an early contribution (A.O. Rodríguez-De la Fuente), (B.D. Mata-Cárdenas), from showed an inhibition of infecting (L.E. Rodríguez-Flores), (M.P. Barrón- microorganisms in human wounds by exposure to alternated elec- (A.C. Torres-Pantoja), (J.M. Alcocer-González).
0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi: Please cite this article in press as: Heredia-Rojas, J.A., et al. Entamoeba histolytica and Trichomonas vaginalis: Trophozoite growth inhibition by metronida-zole electro-transferred water. Exp. Parasitol. (2010), J.A. Heredia-Rojas et al. / Experimental Parasitology xxx (2010) xxx–xxx tric fields. Furthermore, reported that processing All chemicals were supplied by Sigma–Aldrich (St. Louis, MO, liquid foods with high-intensity pulsed electric fields inactivated microorganisms with the advantage of only a small increase infood temperature.
2.3. Transmission apparatus Regarding parasite growth inhibition using unconventional pro- cedures, we recently demonstrated an inhibitory growth effect of The equipment used for electronic transmission comprised a 60 Hz sinusoidal magnetic fields at 1.5 and 2.0 mT on Entamoeba bioresonance therapy device, Bicom version 4.4 by Regumed (Reg- invadens trophozoite cultures. In addition, we found that magnetic ulative Medizine Technik GmbH, Germany) serial No. 202057299.
field exposure inhibited the encystation process of this eukaryoticprotozoan 2.4. Metronidazole transmission to water On the other hand, results from reported the activation of human neutrophils by electronically transmitted The source flask containing 0.124 lg/mL or 0.150 lg/mL of met- phorbol-myristate acetate (PMA), suggesting that PMA molecules ronidazole, in a total volume of 10.0 mL of bi-distilled water was emit signals that can be transferred to neutrophils by artificial placed inside the input coil coupled to a bioresonance amplifier, physical means, in a manner that seems specific to the source mol- while in the output coil, a flask containing pure bi-distilled and ster- ecules. Earlier, observed that by transferring acetic ile water was allocated at room temperature. Metronidazole solu- acid information to inorganic salt solutions by means of an elec- tions and bi-distilled water were sterilized by filtration. The tronic amplifier, the pH of the inorganic solutions decreases oscillator was then turned on for the 15 min transmission period.
slightly but significantly. This finding demonstrates that chemical During this procedure, the various parameters such as power, volt- signals can be transferred via an electronic amplifier device. More- age, capacitance and impedance remained constant. Thus, the nat- over, it has been reported that this kind of information appears to ure of the source tube (metronidazole versus vehicle) was the only be coded as a frequency of alternating magnetic vector potential.
variable. According to the bioresonance device manufacturer, a spe- Imprinting a frequency into water may be performed by applying cific program labelled as #196 was used for electronic transmission the field of a permanent magnet or an alternating magnetic field substance to substance. At the end of the transmission period, the at a specific frequency ().
flasks were kept away from light and stored at room temperature In view of this interesting issue involving unorthodox antimi- 1.0 h before being used in bioassays. About 1.0 mL of transferred- crobial strategies and the possibility to transfer drug information water was added to each culture tube for treatment and controls.
to water molecules by means of resonant circuits, we have under-taken the present study to further evaluate the cytotoxic effect of water samples transferred with electronic information of metroni-dazole, a well-known cytotoxic drug against parasites, by using a In order to test the effect of electronically-transferred metroni- bioresonance device on Entamoeba histolytica and Trichomonas vag- dazole, the density of trophozoites was determined in a blind way inalis trophozoite growth.
when both types of parasite cultures reached the end of logarith-mic phase, they were chilled in ice-cold water for 10 min and tro- 2. Materials and methods phozoites were subsequently harvested by centrifugation at 450gand 4 °C, and then washed three times with fresh medium. The resultant pellet was re-suspended in fresh medium and countedin triplicate with a hemocytometer Neubauer chamber.
E. histolytica strain HM1-IMSS was maintained axenically in PEHPS medium ). Briefly, culture tubes 2.6. Statistical analysis containing 11.0 mL of PEHPS were inoculated with 1  104 tropho-zoites/mL, incubated at 37 °C and sub-cultured every 3 days.
The statistical differences were calculated among groups for T. vaginalis GT-13 reference strain was initially isolated by Dr.
trophozoite growth by using analysis of variance for normal distri- Fernando Anaya-Velázquez, Universidad de Guanajuato, México butions. The normality of the data was estimated by means of Kol- and originally maintained in TYI-S-33 medium with 15.5% bovine mogorov–Smirnov test (p < 0.05). All analyses were done using the serum plus the 10 vitamin mixture (Cells SPSS package version 10.0. Differences were considered to be sig- were then sub-cultured in 13  100 mm screw-capped borosilicat- nificant when the probability values were lower than 0.05.
ed culture tubes containing 5.5 mL of PEHPS. The strain wasincubated at 36.5 °C and re-inoculated every 48 h with 1  102 tro- This study evaluated the effect of electronically transmitted 2.2. Experimental design metronidazole to water samples on in vitro E. histolytica and T. vag-inalis trophozoite growth. shows the cell density grouped Two independent experiments using E. histolytica or T. vaginalis mean (for a total of 27 cell counts per group) scored in trophozoites trophozoites were carried out. In each one, the following treatment of E. histolytica after being treated with water samples previously regimen and controls were considered: (a) cells treated with elec- transferred with metronidazole and controls. A statistically signif- tronically-transferred metronidazole water samples, as explained icant decrease in trophozoite growth was observed in cultures below, (b) cells treated with sham electro-transferred water sam- treated with electronically-transferred metronidazole water sam- ples, this is transferring the information from pure water to water, ples compared with non and sham electro-transferred water sam- (c) cells treated with non-transferred water, as a negative control ples. In trophozoites directly exposed to metronidazole (positive and, (d) cells treated with 0.124 lg/mL of metronidazole (IC50) as control), a decrease of about 50% in cell density was found, as a positive control. In the case of T. vaginalis bioassay, an IC100 dos- age of metronidazole (0.150 lg/mL) was used. Nine cultures were Also, the growth of T. vaginalis trophozoites was altered when included for each treatment regimen and controls for a total of exposed to metronidazole electro-transferred water samples.
36 culture tubes for each type of parasite.
shows the arithmetical grouped means of cell densities of Please cite this article in press as: Heredia-Rojas, J.A., et al. Entamoeba histolytica and Trichomonas vaginalis: Trophozoite growth inhibition by metronida-zole electro-transferred water. Exp. Parasitol. (2010), doi: J.A. Heredia-Rojas et al. / Experimental Parasitology xxx (2010) xxx–xxx ering this trend and the lack on consensus on the effectiveness ofthese therapies, it is of considerable interest to examine if there is a significant and measurable biological effect of such unconven-tional procedures. The question has been raised as to whether these unorthodox biomedical techniques can really modify the microbial or parasitic growth.
In the present study, we have observed a statistically significant reduction of both E. histolytica and T. vaginalis trophozoite growth after axenic cultures were treated with water samples processed in a bioresonance device by using a specific procedure called ‘‘sub- stance to substance transference".
These results agreed with previous reports indicating that molecular information can be scanned and transferred by a biore- sonance instrument. Thus, demonstrated thatthe metamorphosis of tadpoles could be greatly slowed down by transferring information from a toxic solution of the hormone thy- transferred water (positive control) roxin to the aquarium water in a number or parallel blind trials. It has also been reported the transfer of the activity of 4-phorbol-12-B-myristate acetate by electronic means on the activation of hu- Fig. 1. Effect of metronidazole electro-transferred water samples on growth ofEntamoeba histolytica trophozoites (HM1-IMSS strain). The amoebas were cultured man neutrophils On the contrary, in PEHPS medium, inoculated with 1  104 trophozoites/mL and incubated at 37 °C.
found no effects from digital signals on the inhibition Nine tubes were used for each treatment regimen and controls, giving a total of 36 of thrombin/fibrinogen coagulation by a digital signal used instead cultures. The cell density was determined in triplicate and in a blind way by using a of the original molecule.
hemocytometer Neubauer chamber.
A full explanation about the possibility of water capacity for storing electronic information is not yet clear, but it is accepted those treatments and controls. The cultures treated with electro- that electromagnetic waves interact with water. One mechanism transferred metronidazole showed a lower cell density when com- that can explains the effect of electromagnetic fields on water is re- pared with sham and non electro-transferred water samples lated to the existence of ‘‘defects" in its molecular structure. These (p < 0.05). Related to the positive control cultures, a significant de- stable structural changes were detected in experiments by the UV creased trophozoite growth was observed in cells exposed to IC luminescence spectrophotometer and they have been related to of metronidazole, as expected.
different water structural defects that include specific centers of In addition, in both bioassays, a slight but statistically signifi- luminescence; the nuclear proton spins were considered to be a cant difference was found in trophozoite growth when comparing primary target of external magnetic fields, since proton lattice of the grouped mean values between non-transferred (negative con- water molecules is unstable and asymmetric ). In this trols) and sham electro-transferred (water to water) water sam- regard, have suggested that the ‘‘anomalous" ples, indicating some induced effect of the bioresonance device states of water are peculiar and unexpected. Their existence may be connected to the occurrence of meta-stable polymorphic statesof water at room temperature; it is therefore important to confirmor to refute the observations of these anomalous states by indepen- dent experimental investigations.
In addition, it has been proposed that the orientation of nuclear There is a trend toward the use of alternative and complemen- proton spins may influence biochemical processes in biological tary therapeutics, mainly in controlling microbial illnesses. Consid- systems, as a result of associations and disintegrations of abovementioned structural defects of water, since ionic structural such defects are chemically active On the other hand, experiments from have proven that exposing water to resonant circuits can permanentlyalter some of its physico-chemical properties. Moreover, they as- sure that water samples after exposure acquire ‘‘biological-like" behavior that lasts for a significant period of time. Based on thesefindings, it is assumed that such behavior will somehow affect any chemical and/or biochemical reactions in which the exposed water may become involved.
The basis for an understanding of the antimicrobial effects of bioresonance techniques is the assumption that the alternating electromagnetic fields, which are detectably emitted by living organisms and molecules and are characterized by intensity (amplitude) and frequency, contain biologically significant infor- Transferred water mation that is used for transmitting a number of signals between transferred water (positive control) cells, tissues, and even molecules Moreover, in the existing literature there are no original articles Fig. 2. Effect of metronidazole electro-transferred water samples on growth of published on E. histolytica nor T. vaginalis or related parasite Trichomonas vaginalis trophozoites (GT-13 strain). The parasites were cultured in growth inhibition by transferring antiparasitic drugs to water sam- PEHPS medium, inoculated with 1  102 trophozoites/mL and incubated at 36.5 °C.
ples. On the other hand, while bioresonance procedures are deni- Nine tubes were used for each treatment regimen and controls for a total of 36 grated by some, research trials conducted primarily in Russia, cultures. The cell density was determined in a hemocytometer in triplicate and in ablind way.
Germany, and Eastern Europe, indicate that electromagnetic waves Please cite this article in press as: Heredia-Rojas, J.A., et al. Entamoeba histolytica and Trichomonas vaginalis: Trophozoite growth inhibition by metronida-zole electro-transferred water. Exp. Parasitol. (2010), J.A. Heredia-Rojas et al. / Experimental Parasitology xxx (2010) xxx–xxx do, indeed, affect biology in single-cell models that can include Castro-Garza, J., Anaya-Velazquez, F., Said-Fernandez, S., González-Garza, M.T., microorganisms ().
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Please cite this article in press as: Heredia-Rojas, J.A., et al. Entamoeba histolytica and Trichomonas vaginalis: Trophozoite growth inhibition by metronida-zole electro-transferred water. Exp. Parasitol. (2010), doi:

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