Date Published: May 2, 2019
Publisher: Public Library of Science
Author(s): Cynthia Mmalebna Amisigo, Christine Achiaa Antwi, Jonathan Partt Adjimani, Theresa Manful Gwira, Bin Su.
African trypanosomiasis remains a lethal disease to both humans and livestock. The disease persists due to limited drug availability, toxicity and drug resistance, hence the need for a better understanding of the parasite’s biology and provision of alternative forms of therapy. In this study, the in vitro effects of phenolic acids were assessed for their trypanocidal activities against Trypanosoma brucei brucei. The effect of the phenolic acids on Trypanosoma brucei brucei was determined by the alamarBlue assay. The cell cycle effects were determined by flow cytometry and parasite morphological analysis was done by microscopy. Effect on cell proliferation was determined by growth kinetic analysis. Reverse Transcriptase quantitative Polymerase Chain Reaction was used to determine expression of iron dependent enzymes and iron distribution determined by atomic absorption spectroscopy. Gallic acid gave an IC50 of 14.2±1.5 μM. Deferoxamine, gallic acid and diminazene aceturate showed a dose dependent effect on the cell viability and the mitochondrion membrane integrity. Gallic acid, deferoxamine and diminazene aceturate caused loss of kinetoplast in 22%, 26% and 82% of trypanosomes respectively and less than 10% increase in the number of trypanosomes in S phase was observed. Gallic acid caused a 0.6 fold decrease, 50 fold increase and 7 fold increase in the expression levels of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes respectively while treatment with deferoxamine and diminazene aceturate also showed differential expressions of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes.
African trypanosomiasis (AT) is an infectious disease that affects humans, domestic and wild animals in sub-Saharan Africa and it is transmitted by the tsetse fly . Trypanosoma brucei (T. brucei) is responsible for causing AT in both cattle and humans . The subspecies of T. brucei which includes T. brucei gambiense and T. brucei rhodesiense cause the chronic form of sleeping sickness in West and Central Africa and the acute form of the disease in East and Southern Africa respectively, with about 60 million people being at risk . Trypanosoma brucei brucei is one of the causative agents of Animal African Trypanosomiasis (AAT) or nagana in cattle. About 55 million cattle are at risk with the disease leading to a loss of three million animals annually . Due to the antigenic variation exhibited by the parasites, there is currently no vaccine against trypanosomes hence the mode of treatment is mainly by chemotherapy . Drugs currently in use are toxic, have harmful side effects and are becoming less effective due to resistance. Hence the urgent need for the development of new anti-trypanosomal therapeutics which are safe and efficacious.
Phenolic acids are a class of polyphenols that have been shown to have anti-protozoan, anti-bacterial and anti-cancer properties [17, 18] but only few studies have been done to evaluate their effect on the cell biology of trypanosomes. Of the phenolic acids used in this study, gallic acid showed the highest trypanocidal activity with IC50 comparable to what was obtained by Koide and colleagues . Gallic acid significantly inhibited parasite growth whereas protocatechuic acid, also a hydroxybenzoic acid, did not show any significant activity against the trypanosomes. Factors that can affect the iron chelating properties of these phenolic compounds include the number of the hydroxy groups and the position of the hydroxyl groups on the aromatic ring . The trypanocidal activity of gallic acid could be due to the presence of the extra hydroxyl group and its ability to form reactive oxygen intermediates in the parasite . Although gallic acid showed trypanocidal activity in this study, interestingly, in other studies, gallic acid was inactive against other kinetoplastids such as Leishmania donovani and Trypanosoma cruzi [19, 20] and this could be due to the intracellular nature of these parasites. The amastigote forms of Leishmania and the amastigotes of Trypanosoma cruzi exists intracellularly in mammalian cells. Thus, to effectively kill the intracellular parasites, the iron chelators must be lipophilic to be able to transverse the host cells.
We have shown that phenolic acids with iron chelating properties exhibited good trypanocidal activities with moderate toxicity to mammalian cell lines in vitro compared to the currently used standard animal trypanosomiasis drug. Our data suggests a mechanism of action of deferoxamine, gallic acid and diminazene aceturate to potently inhibit growth of Trypanosoma brucei via perturbation of cell morphology and mitochondrion membrane integrity, and cell cycle arrest while increasing the expression of ribonucleotide reductase and cyclin 2 genes with differential expression of the transferrin receptor. Our data also provides information on the cell biology of T. b. brucei under iron deprivation and provides leads for alternative chemotherapeutic in the treatment of African trypanosomiasis.