PLoS One. 2016 Aug 18;11(8):e0161207.
Blood Stage Plasmodium falciparum Exhibits Biological Responses to Direct Current Electric Fields
Lorena M. Coronado, Stephania Montealegre, Zumara Chaverra, Luis Mojica, Carlos Espinosa, Alejandro Almanza, Ricardo Correa, José A. Stoute, Rolando A. Gittens, Carmenza Spadafora.
Electromagnetic radiation can interact with living cells, including pathogens. In this study, we evaluated the in vitro effect of direct current (DC) capacitive coupling electrical stimulation on the biology and viability of Plasmodium falciparum. Hence, a device was designed to expose infected erythrocytes to different capacitively coupled electric fields in order to evaluate their effect on P. falciparum. After exposure, different parameters which determine the fate of the parasite were measured. The effect of DC on growth of the parasite, replication of DNA, mitochondrial membrane potential and level of reactive oxygen species after exposure demonstrated that the parasite responds to electric fields. This response involves several calcium signaling pathways. To our surprise, after late trophozoites were exposed to DC electric fields, the population of infected erythrocytes increased in comparison with unexposed controls. The increase in parasitemia could be related to higher DNA replication levels due to the generation of more merozoites (infecting stages) inside each infected erythrocyte.
The mode of action of the applied DC electric fields in this study seems to be related to the reduction of ROS, hyperpolarization of the mitochondrial membrane, and an increase in the synthesis of DNA, which translates into higher proliferation rates and an increase in the number of parasites.
- PMID:27537497; DOI:10.1371/journal.pone.0161207
Electrical stimulation effects have been widely studied in multiple cell types such as neurons, osteoblasts, epithelial cells, human keratinocytes, and human mesenchymal stem cells (1-3), but there is no report on the effects of electrical stimulation on Plasmodium parasites. We hypothesized that Plasmodium falciparum is able to respond to electricalstimulation. With the need to find new alternatives against the malaria-causing agent, our group set out to study the effects of different forms of energy on the malaria parasite to find parameters that could help kill or decrease the infecting capability of the parasite.
Fig 1. Capacitive coupling electrical stimulation system.
For this purpose, two stainless steel plates were designed to cover all the wells of a 96-well plate without interfering with their stacking capability. Then, a small protruding strip on each plate was used for electrical connections (Fig 1). The parasitemia of P. falciparum in infected erythrocytes was affected after exposure to DC electric fields at 1 and 100 V. An evaluation of the parasite fold change showed that the groups stimulated with 1 and 100 V exhibited significantly higher growth than the controls after 24 hours. (Fig 2)
Fig 2. Evaluation of the fold change of growth of different experimental groups.
We then had to identify the mechanism by which this happens. We knew that electric stimulation in other biological systems has been related to calcium signaling. So, we began to evaluate different biological parameters to explore the mechanisms of action of electrical fields on malaria parasite growth by using a set of inhibitors of the most important pathways. First, the increase in P. falciparum parasitemia caused by 1 and 100 V electrical stimulation was significantly inhibited by TMB8, which blocks the release of Ca2+ from intracellular storage. In addition, Other inhibitors known as W7 (calmodulin inhibition), bromophenacyl bromide and indomethacin (cell membrane-related pathways disruption) as well as verapamil (L-type voltage gated channels inhibition) were used to evaluate the role of these Ca2+ signaling pathways. The only inhibitor that presented a difference between the two different voltage-stimulated groups was neomycin, which blocks the inositol trisphosphate pathway, which rescued the effect caused on the parasites by the 100 V treatment. (Fig 3). When parasites were assessed after 48 h, the effect of the stimuli had almost disappeared and parasites grew slightly more in comparison with unexposed controls, with the inhibitors not making a difference in this situation.
Fig 3. Evaluation of the fold change of growth of the different electrically stimulated groups with the use of different signal transduction inhibitors.
We then tested two parameters that are directly related to the proliferation of cells: mitochondrial polarization and reactive oxygen species. To test if the proliferative response of the parasites is related to hyperpolarization of the mitochondrial membrane, we used flow cytometry to measure the mitochondrial membrane potential (ΔΨm) of P. falciparum. In the electrically-stimulated groups, both 1 V and 100 V treatments revealed a hyperpolarization of the mitochondrial membrane, by an increase of 13.9% and 16.3%, respectively.
Reactive oxygen species (ROS) were also involved in the growth of the cells. P. falciparum-infected erythrocytes exhibited reduced levels of reactive oxygen species (ROS) after exposure to DC capacitive stimulation.
The pathways targeted by the electrical stimulation seem to be related to the Ca2+ signaling cascade. Signal transduction in both the 1 V and 100 V stimuli points to activation of intracellular Ca2+ activated channels that flood the cytosol with the ion and promote the activation of calmodulin. The mode of action of the DC electrical fields also seems to be that of reducing the production of ROS, hyperpolarizing the mitochondrial membrane and increasing the synthesis of DNA, all of which translates into higher proliferation rates and an increase in the number of parasites.
Importance of the study: The most important finding of this study is that P. falciparum is able to respond to direct current electric fields. In this study we have demonstrated that there are biological effects associated with exposure to this type of energetic stimulation. Our results seem to suggest that the mechanism of action of these effects involves specific signaling pathways related to calcium and directly involved in the increase of mitochondrial membrane potential polarization.
We have found that specific DC electric fields help the malaria parasite to proliferate, which was not our initial expected outcome. Nonetheless, showing that the parasite is capable of reacting when confronted with electrical stimulation opens up the possibility of finding appropriate energy parameters with which to manipulate their response.
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Funding: This work was supported by the Bill & Melinda Gates Foundation, Seattle, WA, www.gatesfoundation.org [grant number 51797] to CS and JAS; the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panamá, www.senacyt.gob.pa [Ph.D. scholarships] to LMC and RC; and National System of Investigators, Panama, www.senacyt.gob.pa [grant numbers 38-2014, 91-2015] to CS and RG.
The authors would like to thank all the Spadafora team for help in the laboratory, and the Centro Nacional de Metrología de Panamá CENAMEP for help in providing the electrical stimulation setup.
Affiliation Center of Cellular and Molecular Biology of Diseases (CBCMe), Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP), City of Knowledge, Panama, Republic of Panama