Activity and mode of action of acridine compounds against Leishmania donovani

In the present work, we have assayed both the in vitro and in vivo action of two acridine compounds against Leishmania donovani. As part of this effort, we have studied the possible action mechanism of these compounds at the ultrastructural and biochemical levels and in relation to the synthesis of macromolecules. The two acridinones inhibit the in vitro growth of the promastigote forms of L. donovani at the highest concentration assayed (100 micrograms/ml). The in vivo results indicate that both compounds reduce the number of amastigotes per gram of spleen, and decrease parasitism, by more than 40%. With respect to the action mechanism, both compounds inhibit the incorporation of [3H]thymidine, inducing alterations at the ultrastructural level in the DNA and mitochondria. Alterations are also caused in the enzymes of the Krebs cycle.

Since 1991, the World Health Organization has considered leishmaniasis one of the most serious diseases worldwide caused by protozoan parasites. The clinical manifestations of this parasitosis vary depending on the etiological agent (21,22,29), the most characteristic symptoms being cutaneous and mucocutaneous leishmaniasis, visceral leishmaniasis, or kalaazar caused by Leishmania donovani and/or Leishmania infantum. Hepato-and splenomegals account for only 1.5 million of the 12 million new cases of leishmaniasis worldwide and produce the most serious illness, which when untreated can cause a 90% mortality rate.
One principal subject of current research in the field of leishmaniasis is the development of new, effective molecules for chemotherapy. At present, treatment is based on the administration of pentavalent antimony and/or pentamidine salts (7,8,33). Recognized antifungal molecules such as amphotericin B prove highly effective against the parasite in patients who show resistance to antimonial treatments, but as in traditional chemotherapy, there are serious disadvantages because of toxic effects and administration difficulties (3,4,12,35).
More recently, investigators have been looking for new molecules active against visceral leishmaniasis. Bauman et al. (1) and Fouce et al. (15) have studied the role of a polyamine analog (alpha-difluoromethylornithine) against L. donovani and L. infantum; Marr (22) and Moorman et al. (26) have demonstrated the therapeutic effectiveness of purine analogs against Chagas' disease and visceral leishmaniasis. Ram et al. (31) synthesized and assayed the leishmanicidal activity of carbazolylpyrimidines, molecules that may not have any toxic effects on patients, as opposed to those currently used (6,36).
Some acridine derivatives have proven effective against such parasites as Leishmania spp. (23,38) and Pneumocystis carinii (30), an opportunistic parasite that causes pneumonitis. The effectiveness of these acridines in other types of parasitosis such as malaria is also known (16).
In the present work, we investigate the activity of two newly synthesized acridinones against L. donovani. In addition, we study the cytotoxic effect of these compounds against a cell line of macrophages, analyzing the mechanism by which these molecules act.

MATERIALS AND METHODS
Parasite, culture and maintenance. The strain of L. donovani used in this study was LCR-L 133 (Leishmania Reference Center, Jerusalem, Israel) isolated in 1967 from a human case of kala-azar in Begemder, Ethiopia, and maintained in our laboratory since 1982 by successive passages in cultures of NNN medium modified with a liquid phase in minimal essential medium (MEM) plus 10% inactivated fetal bovine serum kept in an air atmosphere at 28ЊC. To maintain infectivity, a subculture is made approximately every 2 weeks and at least every 6 months, golden hamsters are inoculated, and parasites are isolated from the spleens 30 to 45 days after inoculation.
For the chemotherapy assays, TC-199 was supplemented with 20% inactivated fetal bovine serum at 56ЊC for 30 min, in Roux flasks (Falcon) (75 cm 2 of surface area). At the exponential growth phase, the liquid medium was centrifuged, and the number of flagellates was counted and distributed in aliquots of 10 6 parasites per ml. The assays were carried out in microtiter trays with 24 wells.
Acridines. The compounds tested were synthesized at the GERCTOP Unité Recherche Associeé, Centre National de la Recherche Scientifique 1411 (J. Barbe, University of Marseille). The structure of these compounds is shown in Fig. 1.
The acridines were dissolved in dimethyl sulfoxide at a concentration of 0.1%, after being determined nontoxic and noninhibitory with respect to growth (25). The concentrations tested were 100, 10, and 1 g/ml dissolved in TC-199 plus 20% inactivated fetal bovine serum. The effects of each compound and the concentrations were evaluated at 24, 48, and 72 h, in a Neubauer hemocytometer chamber for counting and calculating the percentage of growth inhibition, by using the formula %IC ϭ (Tc Ϫ Tp/Tc) ϫ 100, where %IC is the percentage of growth inhibition for each time period and each dosage, Tc is the number of parasites per milliliter in the control wells, and Tp is the average number of parasites per milliliter corresponding to the different products tested and their respective dosages.
Effect of acridines in macromolecule synthesis by parasite forms. To determine the effect of the acridines on the biosynthesis of nucleic acids and proteins, the incorporation of the three radioactively labelled precursors [ (17,18).
DNA, RNA, and protein biosyntheses were determined by studying the incorporation of precipitable material of thymidine, of uridine, and of leucine, re-spectively. Cultures containing 5 ϫ 10 6 parasites per ml were centrifuged, the supernatant was eliminated, and the pellet was resuspended with 1 ml of culture medium to which 100 g or 50 g of the molecules per ml was added. After incubation at 28ЊC for 30 min, the culture medium was eliminated and replaced in each case by culture medium without the acridine compound but with the addition of 5 g of the radioactive analog per ml. At 0, 45, 75, and 135 min, the cultures were centrifuged, and the pellet, after three washes with cold medium, was precipitated with 10% trichloroacetic acid for 2 h at 4ЊC. The precipitates were filtered through Whatman GF/c filters, washed first with 5% trichloroacetic acid and then with 10% ethanol, and dried at 60ЊC under infrared lamps. Once dried, the filters were submerged in scintillation cocktail {2,5-diphenyloxazole [PPO], 4 g; 1,4-bis(5-phenyloxazolyl)benzene [POPOP], 0.1 g; toluene, 1,000 ml}, and the radioactivity incorporated was determined with a beta-spectrometer (Beckman). The radioactivity contained in the pellet was noted as a reflection of the radioactivity present in and incorporated by the precipitable material of the parasites.
Ultrastructural alterations induced in parasites by acridines. For the study of the alterations induced by the compounds, the parasites were treated for 4 h with 100 g of selected acridines per ml as described above. The treatment was followed by centrifugation, washing in fresh medium, and fixation with glutaraldehyde at 2.5% in cacodillate buffer-C1H (0.1 M) in saccharose (0.1 M), adjusted to pH 7.2. After 2 h of fixation, the fixer was eliminated and replaced by fresh fixer, and the culture was maintained for 6 h at 4ЊC. Examination by transmission electron microscopy was done according to the technique of Osuna et al. (28).
Cell line J-774A.1. Cell line J-774A.1 (American Type Culture Collection, tibb 67) was isolated by P. Ralph (Salk Institute, San Diego, Calif.) from macrophages and obtained from a tumor in a female BALB/c mouse in 1968. The macrophages are kept in our laboratory by cryopreservation at 85ЊC and by successive subcultures in RPMI 1640 plus 20% inactivated calf serum at 37ЊC in a moist atmosphere enriched with 5% CO 2 .
Cell culture and cytotoxicity tests. The tests for cytotoxicity against macrophages were designed to determine the dosages to use for in vivo experiments.
Cells from monolayer cell cultures were separated from their support, placed in 25-ml cone-based bottles (Steriling), and centrifuged at 1,500 rpm for 5 min. The culture medium was removed, and Hank's solution was added to a final concentration of 10 6 cells per ml. This cell suspension was distributed in a culture tray (with 24 wells) at a rate of 100 l per well and incubated for 2 h at 37ЊC in a humid atmosphere enriched with 5% CO 2 . The medium was removed, and fresh medium was added together with the product to be studied. The vital stain trypan blue (0.1% P/B in phosphate buffer) was used to be determine cell viability. The number of dead cells was recorded, and the percent viability was calculated in comparison to that of the control culture.
In vivo tests. Wistar rats weighing 50 g were given the compounds in single doses administered for 4 days, (BG-325, 0.318 mg/kg of body weight; BG-374, 0.216 mg/kg) with groups of 10 rats per compound. The effects were assessed by intraperitoneal administration, with olive oil as the solvent. After being starved individually, the rats were allowed food and water ad libitum, with a control group for each compound.
The leishmanicidal activity of the compounds was studied by evaluation of how the spleen amastigotes were affected (32). For this study, the animals were killed 4 days after the end of the treatment, the spleens were removed and measured, and tissue imprints were fixed with methanol and stained with Giemsa. The slices were observed by microcopy, and the percentage of parasitization and the number of parasites per nucleated cells was recorded.
The spleens were weighed, diced with needles into pieces of about 2 mm 2 , washed repeatedly in 0.1 M phosphate-buffered saline (PBS) to remove all blood, homogenized, and centrifuged at 150 ϫ g for 10 min. The pellet was washed several times in PBS (0.1 M) by centrifugation and treated with trypsin at 0.25% in PBS for 5 min with constant agitation at 37ЊC. After the treatment, the pellet was dissolved in 3 volumes of TC-199 enriched to 20% inactivated fetal calf serum (IFCS), to inactive the remaining trypsin.
The amastigotes were purified by centrifugation in discontinuous Percoll gradient previously isotonized (10), rendering amastigotes in the interphase at amounts of 1.07 and 1.05 g/cm 3 . After the amastigotes were collected, the Percoll was eliminated by centrifugation at 150 ϫ g for 10 min before dissolution of the interphase in PBS (0.1 M). The pellet containing the amastigotes was resuspended after their number and viability were evaluated (9), correlating the number of amastigotes obtained to the weight of the spleen. The amastigotes were transferred to TC-199 medium enriched to 10% IFCS supplemented with 20 Ci of [ 3 H]leucine per ml. The amastigotes were incubated for 1 h at 37ЊC in an atmosphere enriched with 5% CO 2 . Afterwards, the radioactive medium was eliminated by centrifugation, and the pellet containing the amastigotes was treated with a solution of NaOH (0.1 N) and sodium dodecyl sulfate (0.1% [wt/vol]). The amount of radioactivity in the sample was determined by liquid scintillation in a Beckman Beta 7500 scintillation counter.
Biochemical tests. The biochemical tests began with 3 ml of TC-199 culture medium (Gibco) containing 10 6 promastigotes of L. donovani in the exponential growth phase. The compounds to be studied were added to the cultures at a concentration of 1 g/ml, except in the control experiments, and incubated for 48 h at 28ЊC. After this time, the culture medium was centrifuged at 150 ϫ g for 10 min, and the supernatant was collected for both qualitative and quantitative analyses of the metabolites produced by the parasite, by proton nuclear magnetic resonance (NMR) spectroscopy and enzymatic methods. The pellet obtained from this centrifugation, composed of the parasites free of medium, was disrupted in a 0.25 M sucrose buffer (pH 7.4) and used for the enzymatic analysis.
(i) 1 H NMR spectroscopy. The proton 1 H NMR spectra were obtained with a Bruker model AM-300 apparatus operated at 300.13 MHz in protons; the temperature of the probe was maintained at 27ЊC. The work was carried out by using the pulse technique and Fourier transformation, with 90ЊC pulses and a spectral frequency of 3,287.5 Hz. The method of presaturation was used to eliminate watermarks, with selective irradiation at the frequency of water for 2.5 s and an interval between pulses of 7.5 s, accumulating 160 FIDs, the total of which was multiplied exponentially with a line broadening of 0.2 before Fourier transformation. The chemical movements are referred to TMS.
(ii) Identification and quantification of metabolism. The resonances of a number of metabolites in L. donovani medium were assigned by the addition of pure compounds, after which the chemical shifts were measured. By recording the spectra with different recycle times, it was established that a recycle time of 8 s caused no significant reduction in intensity, due to partial saturation, for the peaks of interest.
Standard curves were constructed by adding pure compounds, dissolved in D 2 O, to fresh medium; these curves were linear over the range of 0 to 20 mM. All correlation coefficients were greater than 0.98. Quantification of the metabolites was done as described previously (2).

RESULTS
Antiproliferative effects. The incubation of the promastigotes in the culture medium with the acridine compounds BG-325 and BG-374 at concentrations of 1, 10, and 100 g/ml reduced the multiplication of the flagellates; the percentages of reduction, with reference to the control, are presented in Table  1. The control promastigotes were cultured in the presence of dimethyl sulfoxide at the same concentration as that added to the cultures with the acridine compounds. The data show that the maximum concentration of the two compounds completely inhibited growth. Striking variations appeared over time at concentrations of 10 and 1 g/ml for compound BG-374, with the loss of activity attributable either to an instability of the molecule in solution and culture conditions or to a direct  action on the compound by the enzymes of the protozoa, which would at low and medium dosages succeed in inactivating the compound. Promastigote ultrastructure. Control parasites incubated in dimethyl sulfoxide presented an apparently normal ultrastructure (Fig. 2), indicating that many flagellates underwent a normal division process (data not shown). In Fig. 2, a kinetoplast is clearly distinguishable, as are the base of the flagellum, the flagellar pocket, the flagellum itself, and the nucleus. The mitochondrion is visible, with part of the mitochondrial crests discernible.
In the incubation of the parasites with compound BG-325, disorder in the nucleus is patent, while the chromatin begins to condense and become disorderly. Numerous vacuoles are detectable in the interior, some of which show internal curling of the membrane. The mitochondria appear swollen and in disarray, with a loss of the crests. The kinetoplast appears electrodense, in a more pronounced way than in Fig. 3A. In addition, a certain retraction is appreciable in the nuclear membrane, leaving intermembranal lacunae.
Incubation with BG-374 led to the appearance of an electrodense kinetoplast and of mitochondrial generation, as well as numerous vacuoles with compact, electrodense contents (Fig. 4). The nucleus appears bloated and turgid, with condensations revealing chromatic degeneration.
Action mechanisms of compounds. The cytotoxic effects produced by both compounds in the cell line J-774A.1 are reflected by the data in Table 2. Figures 5 and 6 reflect the data for incorporation by the precipitable material of thymidine, uridine, and leucine after incubation of the flagellates with the radioactive analogs for 145 min.
The data for the control cultures in all the cases indicate increments in the number of counts per minute, revealing active synthesis of DNA, RNA, and proteins.
Both compounds reduced the levels of incorporation of the three analogs with respect to the controls. This reduction was more pronounced when the incubation was carried out with compound BG-374, with which synthesis of macromolecules was drastically reduced. The catabolites produced by the treated promastigotes, studied by 1 H NMR and enzymatic methods, are indicated in Fig.  7 and Table 3. Differences are appreciable with respect to the control medium after incubation of the flagellates with the compounds. Incubation with BG-374 lowered the peak corresponding to acetate and to the excretion of alanine and reduced the production of pyruvate and lactate. Meanwhile, incubation with BG-325 considerably increased acetate production and decreased both pyruvate and lactate production, although less markedly than with the other compound.
The data from the quantification of the catabolites, by enzymatic techniques (Table 3) reveal that the excretion of pyruvate and lactate diminished in both cases, whereas the excretion of acetate decreased with BG-374 and increased with BG-325.
The enzymatic activity of the protozoa both treated with the acridines and untreated is presented in Table 3, with malate dehydrogenase registering the most pronounced inhibition.
In vivo studies. The Wistar rats were infected in groups of  The effectiveness of compounds BG-325 and BG-374 are reflected by the data in Table 5. The number of amastigotes per gram of spleen decreased by approximately 45% with BG-325 treatment and by 78% with BG-374 treatment after four treatments. These data were corroborated by the parasite-cell count in the slides of the parasitized spleens, with a reduction of 41% for BG-325 and 63% for BG-374 with respect to the control.
The viability test indicated that the amastigotes isolated  from treated rats incorporated significantly less leucine than did the controls, with inhibition rates of 41 and 50.8% for BG-325 and BG-374, respectively. It is noteworthy that none of the rats treated with the compounds at the doses specified in Material and Methods and after the four administrations died during the course of the experiment.

DISCUSSION
A number of compounds with acridine structure have proved effective against protozoan parasites (5). The assayed compounds have shown antiproliferative activity against the promastigotes of L. donovani, reaching 50% inhibition at 72 h of treatment and a dose of 1 g/ml for the compound BG-325. The growth inhibition rates for L. donovani contrast with those obtained for the epimastigotes of Trypanosoma cruzi by Berny et al. (5), who reported an activity of only 100 g/ml with the BG-374 derivative, whereas in L. donovani both compounds achieved 100% inhibition at 72 h of treatment.
The data referring to the level of synthesis of macromolecules in the flagellates treated show that, of the two compounds, BG-374 had the greater inhibitory effect on DNA and RNA syntheses and on the levels of leucine incorporation in the precipitable material than that reflected by the protein synthesis levels. Acridines are known to inhibit various enzymes vital to living organisms, such as topoisomerase II (37) or the protein kinase C (11,13,19,27). Trypanosomatids in general have a compacted specific DNA in a compartment of the mitochondrion called the kinetoplast. During replication, the minicircles of the kinetoplast separate and by segregation pass into the new cell (14). Topoisomerase II has been located by Melendy et al. (24) in the periphery of the dividing kinetoplast. In this sense, the inhibitors of topoisomerase impede the metacyclogenesis of T. cruzi (18).
The loss of the kinetoplasts due to treatment with acridine derivatives (34) hampered the parasite from passing from one form to the another in the biological cycle (20). Nevertheless, treatment of the trypomastigotes of T. cruzi with the inhibitors of topoisomerase does not block the entry into new cells (18).
The ultrastructural alterations after the treatment of the parasites with the compounds appeared fundamentally in the nucleus and kinetoplast, with electrodense condensations of the chromatin due perhaps to mechanisms of intercalation of these compounds with the DNA, another of the properties traditionally attributed to acridine derivatives. In addition, ultrastructural alterations were noted in the mitochondrin, which appeared swollen and vacuolized, with disorganization of the crypts. This description coincides with the biochemical data for the enzymatic activities and with studies on excreted catabolites, raising the possibility that the primary target of the compounds is the DNA and the secondary one is the mitochondrion.
The relatively low cytotoxicity shown at 6 h of continued acridine treatment, and the absence of mortality among the animals treated, points to the possibility that a greater effect on the parasites is due to the higher rates of division among parasites than among the cells.
The in vivo results show that the two compounds are effective in reducing the number of amastigotes per gram of spleen, 44.23% for BG-325 and 78.08% for BG-374. The number of parasitized cells fell with respect to the control by 41.1% for BG-325 and 63% for BG-374. Similarly, the amastiotes collected from the infected spleens decreased in leucine incorporation by 41.1% (BG-325) and 50.85% (BG-374) after treatment with four daily dosages.
These data illustrate the need for future study on these types of structures, to achieve improved treatments against illnesses caused by protozoa in the genus Leishmania and especially in cases in which the parasite shows resistance to traditional drugs.