Fig 1.
Combination effect of vitamin K3 and vitamin C.
The isobolograms illustrate the effect of the VK3 + VC combination against epimastigote forms (A), trypomastigote forms (B), and intracellular amastigote forms (C). The dotted lines correspond to an additive effect. Points below the line indicate a synergistic effect. Points above the line indicate an antagonistic effect. The points show median values.
Fig 2.
Morphological and ultrastructural alterations in epimastigote forms of Trypanosoma cruzi that were treated with vitamin K3 and vitamin C alone and combined at 72 h.
SEM images: (A) Untreated epimastigote forms. (B) Epimastigote forms that were treated with 1.90 μM VK3. (C) Epimastigote forms that were treated with 0.61 mM VC. (D) Epimastigote forms that were treated with 1.90 μM VK3 + 0.61 mM VC. TEM images: (E) Untreated epimastigote forms. (F) Epimastigote forms that were treated with 1.90 μM VK3. (G) Epimastigote forms that were treated with 0.61 mM VC. (H) Epimastigote forms that were treated with 1.90 μM VK3 + 0.61 mM VC. Star, cytoplasmic vacuoles; asterisk, lipid bodies; white arrowhead, myelin-like structure; f, flagellum; g, Golgi complex; k, kinetoplast; m, mitochondrion; n, nucleus. Scale bars = 2 μm in A-D and 0.2 μm in E-H.
Fig 3.
Morphological and ultrastructural alterations in trypomastigote forms of Trypanosoma cruzi that were treated with vitamin K3 and vitamin C alone and combined at 24 h.
SEM images: (A) Untreated trypomastigote forms. (B) Trypomastigote forms that were treated with 0.35 μM VK3. (C) Trypomastigote forms that were treated with 0.20 mM VC. (D) Trypomastigote forms that were treated with 0.35 μM VK3 + 0.20 mM VC. TEM images: (E) Untreated trypomastigote forms. (F) Trypomastigote forms that were treated with 0.35 μM VK3. (G) Trypomastigote forms that were treated with 0.20 mM VC. (H) Trypomastigote forms that were treated with 0.35 μM VK3 + 0.20 mM VC. Star, cytoplasmic vacuoles; f, flagellum; g, Golgi complex; k, kinetoplast; m, mitochondrion. Scale bars = 1 μm in A-D and 0.2 μm in E-H.
Fig 4.
Morphological and ultrastructural alterations in intracellular amastigote forms of Trypanosoma cruzi that were treated with vitamin K3 and vitamin C alone and combined at 24 h.
SEM images: (A) Untreated intracellular amastigote forms. (B) Intracellular amastigote forms that were treated with 0.30 μM VK3. (C) Intracellular amastigote forms that were treated with 0.18 mM VC. (D) Intracellular amastigote forms that were treated with 0.30 μM VK3 + 0.18 mM VC. TEM images: (E) Untreated intracellular amastigote forms. (F) Intracellular amastigote forms that were treated with 0.30 μM VK3. (G) Intracellular amastigote forms that were treated with 0.18 mM VC. (H) Intracellular amastigote forms that were treated with 0.30 μM VK3 + 0.18 mM VC. Star, cytoplasmic vacuoles; arrow, blebs in plasma membrane; f, flagellum; k, kinetoplast; m, mitochondrion; n, nucleus. Scale bars = 1 μm in A-D and 0.2 μm in E-H.
Fig 5.
Total ROS production in parasitic forms of Trypanosoma cruzi that were treated with vitamin K3 and vitamin C, alone and combined, for 24 h using H2DCFDA labeling.
(A) Epimastigote forms that were treated with 1.90 μM VK3 and 0.61 mM VC, alone and combined. (B) Trypomastigote forms that were treated with 0.35 μM VK3 and 0.20 mM VC, alone and combined. (C) Amastigote forms that were treated with 0.30 μM VK3 and 0.18 mM VC, alone and combined. H2O2 used as a positive control is also shown. Total ROS were measured as an increase in fluorescence that is caused by the conversion of nonfluorescent dye to fluorescent DCF. The results are expressed as the mean fluorescence (in arbitrary units [A.U.] ± SE) of at least three independent experiments. * Indicate significant differences compared with the control group (untreated cells; p ≤ 0.05).
Fig 6.
Nitric oxide production in parasitic forms of Trypanosoma cruzi that were treated with vitamin K3 and vitamin C, alone and combined, for 24 h using DAF-FM diacetate labeling.
(A) Epimastigote forms that were treated with 1.90 μM VK3 and 0.61 mM VC, alone and combined. (B) Trypomastigote forms that were treated with 0.35 μM VK3 and 0.20 mM VC, alone and combined. (C) Amastigote forms that were treated with 0.30 μM VK3 and 0.18 mM VC, alone and combined. The NO was measured as an increase in fluorescence that is caused by the conversion of DAF-FM to form fluorescent benzotriazole. The results are expressed as the mean fluorescence (in arbitrary units [A.U.] ± SE) of at least three independent experiments. * Indicate significant differences compared with the control group (untreated cells; p ≤ 0.05).
Fig 7.
Reduced thiol levels in parasitic forms of Trypanosoma cruzi that were treated with vitamin K3 and C, alone and combined, for 3, 24, and 48 h using DTNB.
(A) Epimastigote forms that were treated with 1.90 μM VK3 and 0.61 mM VC, alone and combined. (B) Trypomastigote forms that were treated with 0.35 μM VK3 and 0.20 mM VC, alone and combined. (C) Amastigote forms that were treated with 0.30 μM VK3 and 0.18 mM VC, alone and combined. The results are expressed as the mean percentage (± SE) of at least three independent experiments. * Indicate significant differences compared with the control group (untreated cells; p ≤ 0.05).
Fig 8.
Lipid peroxidation in parasitic forms of Trypanosoma cruzi that were treated with vitamin K3 and vitamin C, alone and combined, for 24 h.
(A-C) Lipid peroxidation, determined by DPPP labeling. The results are expressed as the mean fluorescence (in arbitrary units [A.U.] ± SE) of at least three independent experiments. (D-F) Lipid peroxidation, determined as the amount of TBARS in terms of MDA levels. The results are expressed as the mean MDA nmol/mg protein (± SE) of at least three independent experiments. (A, D) Epimastigote forms that were treated with 1.90 μM VK3 and 0.61 mM VC, alone and combined. (B, E) Trypomastigote forms that were treated with 0.35 μM VK3 and 0.20 mM VC, alone and combined. (C, F) Amastigote forms that were treated with 0.30 μM VK3 and 0.18 mM VC, alone and combined. * Indicate significant differences compared with the control group (untreated cells; p ≤ 0.05).
Fig 9.
Cell cycle in epimastigote forms of Trypanosoma cruzi that were treated with 1.90 μM vitamin K3 (VK3) and vitamin C (VC), alone and combined, for 24 h, evaluated by flow cytometry.
The results are expressed as the mean percentage of cells in each stage of the cell cycle (± SE) of at least three independent experiments. * Indicate significant differences compared with the control group (untreated cells; p ≤ 0.05).
Fig 10.
Autophagic vacuoles in parasitic forms of Trypanosoma cruzi that were treated with vitamin K3 and vitamin C, alone and combined, for 24 h using MDC labeling.
(A) MDC fluorescence microscopy images: (a, a’) Untreated epimastigote forms. (b, b’) Epimastigote forms that were treated with 1.90 μM VK3. (c, c’) Epimastigote forms that were treated with 0.61 mM VC. (d, d’) Epimastigote forms that were treated with 1.90 μM VK3 + 0.61 mM VC. (e, e’) Untreated trypomastigote forms. (f, f’) Trypomastigote forms that were treated with 0.35 μM VK3. (g, g’) Trypomastigote forms that were treated with 0.20 mM VC. (h, h’) Trypomastigote forms that were treated with 0.35 μM VK3 + 0.20 mM VC. (i, i’) Untreated amastigote forms. (j, j’) Amastigote forms that were treated with 0.30 μM VK3. (k, k’) Amastigote forms that were treated with 0.18 mM VC. (l, l’) Amastigote forms that were treated with 0.30 μM VK3 + 0.18 mM VC. Arrows, stained autophagic vacuoles; MDC, monodansylcadaverine; MDC + WTM: monodansylcadaverine + wortmannin. Scale bars: 20 μm. (B-D) MDC fluorescence obtained by ImageJ: (B) Epimastigote forms. (C) Trypomastigote forms. (D) Amastigote forms. * Indicate significant differences compared with the control group (untreated cells; p ≤ 0.05) and # indicate significant difference compared with the vitamins in combination without WTM (p ≤ 0.05).