Article Figures & Data
Figures
- FIG 1
Chemical structures of the metal dithiocarbamate complexes: maneb, zineb, and propineb.
- FIG 2
Expression of CA transcripts in L. major and inhibition of LmCA activity by metal dithiocarbamates (DTCs). (A) Total RNA isolated from L. major promastigotes was subjected to RT-PCR using primers specific for the LmCA1 (lane 2) or LmCA2 (lane 5) gene. The respective product sizes (921 and 1,887 bp) are indicated. Lanes 1 and 4 represent the respective negative-control reactions without RT. A DNA ladder was loaded in lane 3. (B) Concentration-dependent inhibition of CA activity (EU/mg) in whole-cell lysates of L. major promastigotes by maneb (◼), zineb (△), and propineb (●). Error bars represent the means ± SD of values from 3 independent experiments. The respective IC50s are given in the index.
- FIG 3
Effect of metal dithiocarbamates on growth and morphology of L. major promastigotes. (A) L. major cells were grown in the absence or presence of increasing concentrations of maneb (◼), zineb (△), or propineb (●), and cells were counted after 96 h of growth. Each data point represents the mean result ± SD from 3 independent experiments. (B) Representative SEM images (at ∼×6,000 magnification) of untreated L. major promastigotes and those treated with 0.625 μM maneb, zineb, or propineb. (C) Bar graph comparing cell length (in μm) of the parasites grown in the absence (untreated) or presence of 0.625 μM maneb, zineb, or propineb. Error bars represent average cell lengths ± SD of values from at least 40 independent measurements. *, Significant difference (P < 0.05) with respect to the untreated control.
- FIG 4
Effect of metal dithiocarbamates (DTCs) on viability of L. major cells. L. major promastigotes were grown in normal medium (pH 7.2) (A) or in acidic medium (pH 5.5) (B) in the absence or presence of the indicated concentrations of maneb (◼), zineb (△), or propineb (●), and the viabilities of the cells were measured by the MTT assay after 96 h of growth. The LD50 values of maneb, zineb, and propineb for Leishmania cells grown in normal or acidic medium are given in the indexes.
- FIG 5
Extent of apoptosis and necrosis in L. major promastigotes treated with metal dithiocarbamates. (A) L. major promastigotes were grown in the absence (untreated) or presence of 0.625 μM maneb, zineb, or propineb for 96 h, and thereafter the cells were stained with Alexa Fluor 488-annexin V conjugate (green), PI (red), and DAPI (blue). Images were captured by an epifluorescence microscope (at ×60 magnification) with appropriate filter sets. (B) Bar graphs represent quantitation of early apoptotic (annexin V+/PI−), late apoptotic (annexin V+/PI+), and necrotic (annexin V−/PI+) cells expressed as a percentage of the total number of DAPI-positive cells for parasites grown in the absence (gray bars) or presence of 0.625 μM maneb (black bars), zineb (dotted bars), or propineb (white bars). At least 200 cells were analyzed for each condition, and the error bars represent the means ± SD of values from triplicate experiments. *, Significant difference (P < 0.05) with respect to the untreated control.
- FIG 6
Effect of metal dithiocarbamates (DTCs) on intracellular amastigotes. L. major-infected J774A.1 macrophages were incubated for 18 h in the absence (0 μM, gray bar) or in the presence of 0.312 μM or 0.625 μM maneb (black bars), zineb (dotted bars), or propineb (white bars), following which the cells were fixed and stained with DAPI. Based on the total number of DAPI-stained nuclei of macrophages and amastigotes in a field, the number of intracellular amastigotes/100 macrophages (parasite load) was calculated from the images captured in an epifluorescence microscope. For each condition, at least 100 macrophages (and the corresponding number of amastigotes) were analyzed. Error bars represent the means ± SD of values from triplicate experiments. *, Significant difference (P < 0.05) with respect to the untreated control.
- FIG 7
Effect of metal dithiocarbamates on viability and CA activity of mammalian cells. (A) J774A.1 (black bars) and NIH 3T3 (white bars) cells were grown in the absence (untreated) or presence of 5 μM maneb, zineb, or propineb, and the viabilities of the cells were measured by the MTT assay after 72 h of growth. (B) J774A.1 (black bars) and NIH 3T3 (white bars) whole-cell lysates were incubated for 15 min in the absence (no inhibitor) or presence of 5 μM maneb, zineb, or propineb, following which the CA specific activities (EU/mg) were measured. Data represent the relative specific activities, i.e., the specific activity in the presence of inhibitor/specific activity in the absence of inhibitor. The error bars in both experiments represent the means ± SD of values from 3 independent experiments.
Tables
- TABLE 1
Intracellular pH of metal dithiocarbamate-treated L. major promastigotes grown in normal (pH 7.2) or acidic (pH 5.5) media
pHea Treatment pHib 7.2 Untreated 6.93 ± 0.02 Manebc 6.68 ± 0.07d Zinebc 6.72 ± 0.03d Propinebc 6.71 ± 0.06d 5.5 Untreated 6.81 ± 0.03 Manebe 6.46 ± 0.05d Zinebe 6.53 ± 0.04d Propinebe 6.49 ± 0.06d ↵a pHe indicates the pH of the growth medium.
↵b pHi values are experimentally determined intracellular pH values of L. major promastigotes grown in the absence (untreated) or presence of metal dithiocarbamates. Means ± SD of values from triplicate experiments are shown.
↵c Treatment with 0.625 μM of the respective metal dithiocarbamate.
↵d Significant difference (P < 0.05) with respect to the untreated control.
↵e Treatment with 0.078 μM of the respective metal dithiocarbamates.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Description of method to culture L. donovani promastigote and data showing that metal dithiocarbamates are effective against L. donovani (Fig. S1).
PDF, 115K
- Supplemental file 1 -
