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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. References

Sapphyrins and a series of related porphyrinoid macrocycles have been investigated as potential agents for the treatment of leishmaniasis. The effectiveness of the compounds was evaluated in vitro upon incubation with Leishmania tarentolae or L. panamensis amastigotes and promastigotes. Their effectiveness was also assessed against intracellular L. panamensis. The cytotoxicity of the compounds was evaluated in vitro using the U937 human promonocyte cell line. Effectiveness and cytotoxicity were assessed in the presence and absence of visible light to assess the photodynamic activity of the compounds. Sapphyrin and two related heterosapphyrins were shown to be particularly effective as inhibitors of Leishmania. A photodynamic effect was observed, which may be attributed to the formation of reactive oxygen species. Yields of singlet oxygen (1O2) produced were determined in ethanol solutions by direct measurement of 1O2 phosphorescence. Confocal microscopy demonstrated that sapphyrin and related macrocycles were taken up by the Leishmania cells and that their presence induces the formation of mitochondrial superoxide. Sapphyrins have been widely investigated as anticancer agents and we here show activity against the Leishmania parasites.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. References

Sapphyrins, the first examples of expanded porphyrin systems, were first investigated in the 1960s and have been the subject of considerable synthetic interest (1–5). These conjugated pentapyrrolic macrocycles have been shown to have considerable promise in medicinal applications, including cancer treatment and photodynamic therapy (6–9). In addition, sapphyrins have been shown to be effective phosphate receptors (10) and can act as catalysts for phosphate hydrolysis (11) and in nucleotide transport (12). Furthermore, sapphyrins have been used for the photodynamic inactivation of the herpes simplex virus (13). We have been investigating the use of porphyrin analogues for their ability to inhibit Leishmania (14,15) and considered sapphyrins to be potentially useful in this type of application. Leishmania are obligate intracellular parasites that lead to leishmaniasis diseases, which affect more than 12 million people worldwide, but current treatments are unsatisfactory. Treatments for leishmaniasis diseases with fewer side effects and a lower cost than present drugs are needed because current treatments have severe side effects including joint pain, fatigue, gastrointestinal upset and anemia. Additionally, the drugs currently in use are toxic, expensive and can cause refractory infection (16). Syntheses of sapphyrins, heterosapphyrins (oxasapphyrins and thiasapphyrins), acenaphtho and phenanthrosapphyrins and benzocarbasapphyrin (Fig. 1) have been developed (3,17,18). In addition, a novel synthesis of an expanded sapphyrin-like system, pentaphyrin (3.1.0.1.3), has been reported (19). The objective of this study was to evaluate the effect of these expanded porphyrins on the cell viability of Leishmania parasites both under axenic and intracellular conditions. Evaluation included the effectiveness of the compounds in two species of Leishmania (Leishmania tarentolae and L. panamensis) and their cytotoxicity toward human promonocytic cells (U937 line). Effects of light exposure on viability and cytotoxicity were also evaluated, as were levels of induced intracellular superoxide formation.

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Figure 1.  Structures of sapphyrins and related expanded porphyrins (pentaphyrins).

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Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. References

Culture methods.  The initial promastigote culture of L. tarentolae was obtained from the American Tissue Culture Collection (ATCC #30143). The promastigotes were cultured (at 26°C) in the dark in heat-sterilized brain heart infusion medium (BHI; Becton-Dickinson) with 20 μm hemin and 100 U mL−1 penicillin and 0.1 mg mL−1 streptomycin (Sigma-Aldrich). Stock cultures were grown in 25 mL Corning flasks (Fisher Scientific) containing 10 mL of medium and transferred every 4 days to fresh medium. An additional L. tarentolae strain, LEM-125, was a generous gift of Dr. Larry Simpson (Howard Hughes Medical Institute, University of California, Los Angeles, CA) and cultured as indicated below. LEM-125 promastigotes were cultured at 26°C as indicated above, but with addition of 10% fetal bovine serum (FBS, Invitrogen). In some cases, however, the LEM-125 parasites were cultured in Schneider medium (Sigma) at pH 6.9 supplemented with 10% FBS, 100 IU mL−1 penicillin, 0.1 mg mL−1 streptomycin and 2 mm l-glutamine (Sigma). Medium was changed every 3 days. In order to obtain axenic amastigotes from the LEM-125 strain, the method of Taylor et al. (20) was followed. Promastigotes were cultured for 4–5 days as described above. The parasite density was then adjusted to 2 × 105 parasites mL−1 in Schneider medium at pH 5.4 enriched with 10% FBS, 100 U mL−1 penicillin, 0.1 mg mL−1 streptomycin and 2 mm L-glutamine. All the parasite cultures were incubated at 28°C for 3 days. This process was then repeated with a stepped temperature increase to 30°C, then 32°C. Once the amastigote growth was established, temperature was maintained at 32°C. The medium was changed every 2–3 days keeping the cell density at 2 × 105 mL−1.

L. panamensis (MHOM/CO/87/UA140) were isolated from Colombian patients infected with cutaneous leishmaniasis. Parasites in the promastigote stage were cultured at 26°C in Schneider medium at pH 6.9 supplemented with 10% FBS, 100 U mL−1 penicillin, 0.1 mg mL−1 streptomycin and 2 mm L-glutamine. Medium was changed every 3 days and parasite count was maintained at 10 × 106 parasites mL−1. In order to obtain axenic amastigotes, promastigotes were cultured for 4–5 days as described above. The parasite density was then adjusted to 2 × 106 parasites mL−1 in Schneider medium at pH 5.4 enriched with 20% FBS, 100 U mL−1 penicillin, 0.1 mg mL−1 streptomycin and 2 mm l-glutamine. The parasite culture was incubated at 28°C for 3 days. This process was then repeated with a stepped temperature increase to 30, 31 and 32°C. Once amastigote growth was established, the temperature was maintained at 32°C. The medium was changed every 3–4 days and the cell density was maintained at 5 × 106 parasites mL−1.

Human promonocyte U937 cells (ATCC No. CRL-1593.2) were used. U937 cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% FBS, 100 U mL−1 penicillin, 0.1 mg mL−1 streptomycin and 2 mm l-glutamine, pH 7.2 at 37°C in an atmosphere of 5% CO2. The medium was changed every 2–3 days, keeping the cell density at 2 × 105 cells mL−1.

Compound effectiveness.  The expanded porphyrinoid macrocycles used in this study (Fig. 1) were synthesized following the procedures of Lash and coworkers (3,17–19). These conjugated macrocycles were dissolved in absolute ethanol to obtain stock solutions that were stored at −20°C and in the dark to prevent light inactivation. For each L. tarentolae strain, a 1 mL aliquot from a 1 day old culture was placed in each well of a Falcon 24 well plate (Becton-Dickinson). After overnight incubation, the parasites were inoculated with expanded porphyrin (each well receiving one of the seven compounds studied) to obtain a final concentration between 0.1 and 10 μm compound and 1% ethanol. Control cells were given ethanol only. Each compound was incubated with either strain of L. tarentolae in the presence or absence of visible light from a fluorescent lamp (350–750 nm, 750 Lux) for 3 h. After 3 h incubation, light microscopy, confocal microscopy and the MTT viability assay (21), as reported by Morgenthaler et al. (14), were conducted. In some cases, cell viability of cultures was evaluated every day for 4 days with n = 3 replicates and reported as percent of day 1 controls. For the EC50 determinations, viability was assessed only after 48 h of incubation with the compounds. For the LEM-125 axenic amastigotes and the human promonocyte cells, the same procedure as above was followed.

The effectiveness of the compounds against L. panamensis axenic amastigotes was established by the MTT viability colorimetric method (21). Cultures of axenic amastigotes were set in the wells of a 96 well plate at a density of 2 × 106 parasites mL−1 in Schneider medium at pH 5.4 supplemented with 20% FBS and in the presence of different test compound concentrations (six serial double dilutions) at 32°C for 72 h. Parasites maintained under the same conditions in the absence of test compounds served as a positive control for viability. The effectiveness of the compound was compared to the traditional medicines meglumine antimoniate (Glucantime®) and Amphotericin B (Fungizone®). The viability determination was carried out in duplicate. Results are expressed as effective concentrations (EC) corresponding to 50% parasite death (EC50) and calculated using the statistical probit analysis (22). The photodynamic effect was measured upon exposing cultures to 2 h per day of visible light provided by a fluorescent lamp (20 W) positioned at 12 cm from the culture plates providing 750 Lux of illumination.

The effect of the compounds against intracellular amastigotes of green fluorescent protein (GFP)-transfected L. panamensis strain (MHOM/CO/87/UA140epir GFP) was evaluated by flow cytometry of infected phorbol myristate acetate (PMA)-induced U937 cells incubated in the presence or absence of each drug as reported by Varela et al. (23). Briefly, U937 cells were dispensed in 24 well plates at a concentration of 3 × 105 cells per well, which were treated with 1 μm of PMA for 48 h at 37°C, after which they were infected with promastigotes of L. panamensis in stationary growth phase (day 5) in modified NNN medium in a proportion of 1:25 cell/parasite. After 3 h of incubation at 34°C in 5% CO2, noninternalized parasites were removed by washing and infected macrophages were incubated again at 34°C and 5% CO2 to allow differentiation to the amastigotes form. After 24 h of incubation, the compounds with the appropriate dilution, without exceeding the LC50 were added. Infected and treated cells were maintained at 34°C and 5% CO2 for 72 h. The leishmanicidal effect was measured in a flow cytometer at 488 nm of excitation and 525 nm of emission. The results are expressed as the EC50 calculated by the probit statistical method (22). The data are the average of three independent experiments conducted in duplicate. Additionally, infected cells exposed to amphotericin B and meglumine antimoniate were used as indicators of leishmanicidal activity.

Infected cells cultivated in the absence of the drug served as the control. The percentage of infection was calculated as previously described (24). The data reported are the average of three independent experiments conducted in duplicate. The results obtained both for axenic and intracellular amastigotes are expressed as 50% of the EC50 calculated by the probit method (22).

Relative fluorescence and singlet oxygen yields in ethanol solution.  Fluorescence spectra of the compounds in ethanol solution were determined using a luminescence spectrophotometer (Perkin Elmer LS55) set at an excitation wavelength of 477 nm (for sapphyrin and oxasapphyrin) or 488 nm (for all other heterosapphyrins). Fluorescence quantum yields were determined relative to a solution of acridine yellow in ethanol (φf = 0.47, 25) matched to have the same absorbance of the sample solution at the excitation wavelength. Instrument settings were identical for reference and sample. Singlet oxygen (1O2) yields of the compounds dissolved in ethanol were determined from the direct measurement of the phosphorescence emitted by the Δg state at 1270 nm. Yields were relative to a rose bengal solution in ethanol (φΔ = 0.68, 26). 1O2 was produced upon excitation of the sample with a frequency modulated (800 Hz) beam from an argon ion laser (Lexel 95) tuned at either 477 or 488 nm. Emitted light was collected at 90° relative to the excitation beam and passed through a monochromator (PTI technologies) equipped with a near infrared grating (1250 nm blaze). The output from the monochromator was filtered using a 1000 nm long pass glass filter before reaching a germanium detector (Judson Technologies) cooled with liquid nitrogen. The detector signal was amplified (×105) with a transimpedance preamplifier (Judson Technologies) and fed to a lock-in amplifier (Stanford Research Systems). The intensity of the phosphorescence signal was linearly dependent on the laser power in the 50–900 mW range. Relative quantum oxygen yields were obtained by comparison of the slopes of the power-dependence plots obtained from ethanol solutions of sample and references that were saturated with oxygen and matched to have similar absorbances (27).

Microscopy.  The induced production of superoxide was tested using the MitoSox™ fluorescent imaging probe (Invitrogen) according to the manufacturer’s recommendations. Before imaging, 200 μL of L. tarentolae parasites (either strain) containing one of the seven different sapphyrin or heterosapphyrin compounds were incubated with 400 μL of a 5 μm MitoSox working solution for 10 min. Confocal fluorescence imaging of the probe was carried out using a confocal microscope (Leica DMIRE2) with an argon ion laser set at 514 nm for excitation and detecting the emission in the 550–660 nm range.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. References

MTT viability assay data presented in Fig. 2 indicate that the porphyrinoid macrocycles can affect the L. tarentolae promastigotes either positively or negatively.

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Figure 2.  Viability assay results for Leishmania tarentolae promastigotes upon incubation with 1% ethanol (control) or 10 μm of pentaphyrin compound in ethanol solution (1%). (a) ATCC 30143 strain after light treatment, (b) ATCC 30143 strain treated in the dark, (c) LEM125 strain after light treatment and (d) LEM125 strain treated in the dark.

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Aqueous ethanol (1% vol/vol) was used as a solvent control and data are reported as percent of day 1 of these control cells. Values larger than 100% indicate that parasites’ populations increase relative to day 1 control or initial viability determination. Preliminary studies conducted by Morgenthaler et al. indicated that 1% (vol/vol) ethanol was minimally inhibiting to Leishmania promastigotes of the ATCC 30413 strain (14), although this study indicates that LEM125 parasites are more sensitive to 1% ethanol. Sapphyrin, oxasapphyrin or thiasapphyrin (at 10 μm) showed a substantial detrimental effect on the growth of the ATCC 30143 strain of L. tarentolae promastigotes either following exposure to visible light (Fig. 2a) or in the dark (Fig. 2b), with the other porphyrinoid analogues showing far smaller negative effects. A 3 h exposure to visible light in the presence of sapphyrin, oxasapphyrin or thiasapphyrin reduced cell viability by more than 91% on the day following additions and the growth of parasites seemed to only moderately recover 3 days after the addition of compound (Fig. 2b). The dark companion cultures (Fig. 2b) for these three compounds still exhibited a 42% to 82% decrease of cell viability with sapphyrin, oxasapphyrin or thiasapphyrin but not with the others tested. The promastigotes of the LEM-125 strain of parasites (Figs. 2c,d) appears to be as sensitive to the compounds as the ATCC strain. For instance, sapphyrin, oxasapphyrin and thiasapphyrin also affected them the most, although the LEM125 promastigotes exhibited a smaller photodynamic effect after 3 days of testing (Fig. 2c). The other compounds tested either had no effect or even showed a modest increase in the viability of the both strains of promastigotes. The LEM125 parasites seem to recover over a period of 72 h from exposure to sapphyrin in contrast to what is observed for the ATCC 30143 parasites. This suggests that the LEM125 parasites have a mechanism by which sapphyrin is inactivated, which may not be present in the other strain.

It can be seen from the MTT viability assay data presented in Fig. 3 that sapphyrin, oxasapphyrin and thiasapphyrin at 1 μm had a large negative effect on axenic L. tarentolae amastigotes.

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Figure 3.  Viability results for Leishmania tarentolae LEM-125 axenic amastigotes cells incubated and tested 24 and 48 h after addition of 1 μm of each expanded porphyrin. (a) Cultures exposed for 3 h per day of visible light and (b) cultures tested in the dark.

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The effect was greater than the effect seen with the promastigote cultures (Fig. 2). The experiments with axenic amastigotes also showed a difference between those amastigotes incubated and exposed to visible light and those kept in the dark indicating, in some cases, a photodynamic effect (Fig. 3a,b). Axenic amastigotes in the dark appeared to be even more sensitive than the LEM-125 strain promastigotes in the dark.

The EC of the compounds (EC50) was evaluated in both axenic and intracellular amastigotes of L. panamensis, which causes cutaneous leishmaniasis in humans. Table 1 shows EC50 values and indicates that the trend observed for nonpathogenic L. tarentolae is also evident in the human pathogen, L. panamensis. Sapphyrin, oxasapphyrin and thiasapphyrin are the most effective compounds both in the dark and when parasites were exposed to visible light.

Table 1.   Heterosapphyrin effectiveness (EC50) against axenic and intracellular Leishmania panamensis and cytotoxicity (LC50) toward human promonocyte cell line U937.
CompoundAxenic amastigotesIntracellular amastigotesCytotoxicity (U937)
EC50m) darkEC50m) lightEC50m) darkEC50m) lightLC50m) darkLC50m) light
Sapphyrin17.7 ± 1.37.8 ± 1.39.8 ± 0.010.6 ± 0.0118.0 ± 1.92.33 ± 0.03
Oxasapphyrin11.4 ± 0.57.7 ± 1.124.1 ± 0.62.9 ± 0.0833.0 ± 3.75.4 ± 0.20
Thiasapphyrin34.0 ± 0.217.1 ± 0.8N.D.5.5 ± 0.0587.9 ± 4.49.1 ± 0.9
Benzocarbasapphyrin168 ± 1113.7 ± 0.2166.7 ± 1.69.7 ± 0.3173 ± 1612.2 ± 0.9
Phenanthrosapphyrin255 ± 5145 ± 5N.D.121 ± 5305 ± 9182 ± 6
Acenaphthosapphyrin101 ± 4101 ± 12N.D.153 ± 8953 ± 188174 ± 3
Glucantime®929 ± 7N.D.19 ± 3N.D.3500 ± 110N.D.
Amphotericin B0.05 ± 0.002N.D.0.05 ± 0.001N.D.22.63 ± 0.02N.D.

All compounds are more effective when cultures were exposed to visible light indicating a photodynamic mechanism of action. The effectiveness is enhanced 2–17 times and the photodynamic effect is enhanced in the intracellular parasites. Interestingly, benzocarbasapphyrin is the compound that exhibits the largest photodynamic enhancement in both axenic and intracellular parasites. In general, compounds are more effective against intracellular amastigotes that were exposed to visible light for 2 h daily. For instance, sapphyrin is the most effective compound against intracellular amastigotes with an EC50 of 0.6 μm, followed by oxasapphyrin and thiasapphyrin with EC50 equal to 2.9 μm and 5.5 μm, respectively. Both acenaphthosapphyrin and phenanthrosapphyrin have EC50 above 120 μm upon light exposure of intracellular parasites. The effectiveness of sapphyrin, oxasapphyrin and thiasapphyrin in combination with light exposure against intracellular parasites (Table 1) is better than that obtained with the conventional medicine glucantime (EC50 = 19 μm), although it does not surpass the efficacy shown by amphotericin B (EC50 = 0.05 μm).

In general, the compounds tested seem to be toxic to the human promonocyte U937 cell line (see LC50 values in Table 1). Trends in cytotoxicity follow those of effectiveness, with sapphyrin being the most toxic to promonocytes both in the dark and when exposed to visible light. However, when comparing the EC50 to LC50 values, the compounds seem to be somewhat more selective toward intracellular parasites than the human promonocytic cells. It would be relevant to investigate encapsulation techniques targeted to improve the selectivity of the compounds.

Confocal fluorescence microscopy was used to evaluate if the expanded porphyrins were accumulated by the parasites. The confocal fluorescence microscope excitation laser was set at 458 nm in order to excite the macrocycles while emission was detected at in the 500–750 nm range. The compounds are, however, not very fluorescent (Table 2) in ethanol solution, with fluorescence quantum yields in the 0.002–0.023 range, which makes its intracellular detection difficult.

Table 2.   Relative fluorescence (ϕf) and singlet oxygen (ϕΔ) quantum yields of heterosapphyrins in ethanol solution.
 SAPOSAPTSAPBCSAPPSAPANSAP
  1. *Relative to acridine yellow in ethanol (ϕf = 0.47), percent error is 5%.

  2. †Relative to rose bengal in ethanol (ϕΔ = 0.68).

  3. SAP = sapphyrin; OSAP = oxasapphyrin; TSAP = thiasapphyrin; BCSAP = benzocarbasapphyrin; PSAP = phenanthrosapphyrin; ANSAP = acenaphthosapphyrin.

ϕf*0.0130.0120.0020.0050.0040.023
ϕΔ0.21 ± 0.030.30 ± 0.040.09 ± 0.010.10 ± 0.010.08 ± 0.020.57 ± 0.01

The cultures, both those exposed to light and those grown in the dark were evaluated by confocal fluorescence microscopy on the second day of incubation after additions. Figure 4 shows that cells exhibit fluorescence following addition of those heterosapphyrins that are fluorescent enough to be detected inside the parasites (such as sapphyrin and oxasapphyrin).

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Figure 4.  Confocal microscopy images of Leishmania tarentola LEM-125 promastigotes. (a) Transmission image of parasites incubated in the dark with 10 μm thiasapphyrin. (b) Fluorescence image of same cells. (c) Overlay of transmission and fluorescence images of parasites incubated with 10 μm sapphyrin after being exposed to visible light. (d) Control parasites that have not been exposed to a compound do not exhibit any intrinsic fluorescence. Scale bars are 10 μm.

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The images indicate the ability of cells to take up these compounds. Fluorescence was not detected in the flagellar region of the parasites. The uptake of sapphyrin, oxasapphyrin or thiasapphyrin by the parasites is correlated with a decrease in cell viability. Cells incubated with ethanol only did not exhibit fluorescence (Fig. 4d).

Confocal microscopy showed that the cell shape changed in the presence of the sapphyrin, thiasapphryin or oxasapphyrin. The promastigote cells changed from the motile, elongated shape (Fig. 4d) to a more rounded, nonmotile shape (Fig. 4a,c). The shape and motility of the two L. tarentolae strains appeared to be affected in the same manner as evaluated by microscopy.

The mechanism of photodynamic action invokes the formation of an excited state that is able to activate molecular oxygen into reactive oxygen species (ROS) that ultimately induce cellular death (28). Maiya et al. (7) demonstrated that sapphyrin is able to induce the formation of 1O2 in solution with quantum yields of 0.1–0.3 depending on the solvent. All the sapphyrin derivatives studied here also photosensitize the production of 1O2 when dissolved in ethanol. The 1O2 quantum yields are listed in Table 2. Acenaphthosapphyrin has the largest 1O2 yield in ethanol solution, followed by oxasapphyrin and sapphyrin. There is no direct correlation between 1O2 yields in ethanol and the in vitro efficacies and cytotoxicities upon light exposure, although this is expected because in vitro performance is highly influenced by the ability of the compound to localize in the parasites or cells. Also, 1O2 yields may be dependent on the local environment around the photosensitizer (7). For instance, the in vitro results (Table 1) suggest that acenaphthosapphyrin is not internalized efficiently by the parasites, which explains the lack of activity of this compound in comparison to other compounds which may be less efficient at producing 1O2via a photodynamic mechanism. Interestingly, the presence of a fused ring to one of the pyrrole units (acenaphthosapphyrin, benzocarbasapphyrin and phenanthrosapphyrin) seems to decrease the in vitro activity relative to sapphyrin and the analog heterosapphyrins. This fact suggests that the presence of such a fused ring perturbs the penetration of the compound into the parasite and human cells. The performance of the benzocarbasapphyrin is particularly interesting because it shows the best enhancement of activity in parasites upon exposure to light. This suggests that once the compound enters the parasite it localizes in a region which is quite susceptible to oxidation by ROS produced via a photodynamic mechanism.

A fluorescent probe (MitoSox) was used to detect superoxide formation inside the cells and to evaluate a possible mechanism of cell inhibition by the heterosapphyrins. Wang et al. reported that sapphyrin kills tumor cells by the formation of ROS (9), although they did not employ any light treatment in their studies. It can be seen in Fig. 5 that the presence of sapphyrins caused the cells to produce superoxide. Control cells exposed to just ethanol did not exhibit a positive superoxide response (data not shown).

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Figure 5.  Confocal fluorescence and transmission images of L. tarentolae promastigotes incubated with the superoxide probe MitoSox™ and exposed to light. (a1) and (a2) in the presence of 10 μm benzocarbasapphyrin (scale bars are 10 μm). (b1) and (b2) in the presence of 10 μm sapphyrin (scale bars are 25 μm).

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The use of the MitoSox probe showed that one mechanism of parasite death involves the formation of superoxide. Turrens reviewed the link between one mode of mitochondrial formation of superoxide and the respiratory chain of aerobes (29). Although superoxide is not a strong oxidant, it is well established to be a precursor of other ROS and thus oxidative damage in cells. Previous studies demonstrate that the activation of cell apoptosis is clearly associated with production of superoxide in the mitochondria (30). It has also been reported (31,32) that Leishmania parasites use apoptotic signals to increase the infectivity of phagocytes such as macrophages. Thus, the superoxide production by Leishmania detected in our studies is not a surprise and suggests that the cells are responding to factors such as the sapphyrin and heterosapphyrins in their environment via apoptotic mechanisms involving the formation of superoxide by additional extra respiratory chain mechanisms. Benzocarbasapphyrin shows the largest photodynamic enhancement on its in vitro efficacy and cytotoxicity. Interestingly, acenaphthosapphyrin, which has the largest quantum yield of 1O2 in ethanol solution, is quite inefficient as a leishmanicide. This result and the lack of a fluorescence signal from the interior of the parasites after incubation suggest that acenaphthosapphyrin is not able to efficiently enter into the parasite. On the other hand, sapphyrin, oxasapphyrin and thiasapphyrin are the most effective and are therefore promising inhibitors of Leishmania panamensis parasites that cause cutaneous leishmaniasis. Additionally, these compounds have a modest selectivity when compared to their cytotoxicity toward the eukaryotic U937 human promonocyte cell line under our experimental conditions. Thus, these porphyrinoid macrocycles show promise as an alternative treatment for those who suffer from leishmaniasis diseases.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. References
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