Please cite this article as: Tempone, A.G., Pimenta, D.C., Lebrun, I., Sartorelli, P., Taniwaki, N.N., de Andrade, H.F., Antoniazzi, M.M., Jared, C. Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad rhinella jimi parotid macrogland secretion, Toxicon (2008), doi: 10.1016/j.toxicon.2008.05.008

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Accepted Manuscript

Title: Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad rhinella jimi parotid macrogland secretion

Authors: André Gustavo Tempone, Daniel Carvalho Pimenta, Ivo Lebrun, Patrícia Sartorelli, Noemi N. Taniwaki, Heitor Franco de Andrade Jr.Marta Maria Antoniazzi, Carlos Jared

PII: S0041-0101(08)00369-3

DOI: 10.1016/j.toxicon.2008.05.008

Reference: TOXCON 3233

To appear in: Toxicon

Received Date: 24 March 2008

Revised Date: 19 May 2008

Accepted Date: 20 May 2008

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Antileishmanial and Antitrypanosomal Activity of Bufadienolides Isolated from the

Toad Rhinella jimi Parotid Macrogland Secretion

André Gustavo Tempone1,2,3∗, Daniel Carvalho Pimenta4, Ivo Lebrun4, Patrícia

Sartorelli5, Noemi N. Taniwaki6, Heitor Franco de Andrade Jr.7, Marta Maria Antoniazzi3

& Carlos Jared3

1- Laboratório de Toxinologia Aplicada, Departamento de Parasitologia, Instituto Adolfo Lutz Av. Dr. Arnaldo 355, 8º andar CEP 01246-000 São Paulo Brazil. atempone@ial.sp.gov.br 2- Departamento de Morfologia e Genética. Universidade Federal de São Paulo. R. Botucatu, 740 - CEP 04023-900 3- Laboratório de Biologia Celular, Instituto Butantan, Av Vital Brasil, 1500, São Paulo – SP – 05503-900 – Brazil 4- Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av Vital Brasil, 1500, São Paulo – SP – 05503-900 – Brazil 5- Departamento de Ciências Exatas e da Terra – Escola Paulista de Química - Universidade Federal de São Paulo Campus Diadema – Rua Prof. Artur Ridel 275, Jd. Eldorado, CEP 09972-270, Diadema, SP, Brazil. 6- Seção de Microscopia Eletrônica, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355, CEP 01246-000, São Paulo, Brazil. 7- - Lab. Protozoologia, Instituto de Medicina Tropical – Universidade de Sao Paulo - Av. Dr. Enéas de Carvalho Aguiar, 470 CEP 05304-000 - São Paulo Brazil. ∗Corresponding author: 1- Andre G. Tempone (atempone@ial.sp.gov.br/

atempone@usp.br). Lab. Toxinologia Aplicada, Departamento de Parasitologia, Instituto

Adolfo Lutz Av. Dr. Arnaldo 355, 8º andar CEP 01246-000 São Paulo Brazil.

Phone:55+11+3068-2888 Fax. 55+11+3068-2890

Keywords: Rhinella jimi, bufadienolides, telocinobufagin, hellebrigenin, Leishmania,

Trypanosoma cruzi

Ethical statement: The author certifies that this paper consists of original, unpublished

work, which is not under consideration for publication elsewhere. The animal procedures

are approved by Institute Adolfo Lutz. The authors consent to the transfer of the

copyright to the publisher.

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Abstract

Amphibian skin secretions are considered a rich source of biologically active compounds

and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are

cardioactive steroids from animals and plants that have also been reported to possess

antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic

diseases found in tropical and subtropical regions. The efforts toward the discovery of

new treatments for these diseases have been largely neglected, despite the fact that the

only available treatments are highly toxic drugs. In this work we have isolated, through

bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotid

macrogland secretion. Mass spectrometry and 1H and 13C-NMR spectroscopic analyses

were able to demonstrate that the active molecules are telocinobufagin and

hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi

promastigotes, but only hellebrigenin was active against Trypanosoma cruzi

trypomastigotes. These steroids were active against the intracellular amastigotes of

Leishmania, with no activation of nitric oxide production by macrophages. Neither

cytotoxicity against mouse macrophages nor hemolytic activities were observed. The

ultrastructural studies with promastigotes revealed the induction of mitochondrial

damage and plasma membrane disturbances by telocinobufagin, resulting in cellular

death. This novel biological effect of Rhinella jimi steroids could be used as a template

for the design of new therapeutics against Leishmaniasis and American

Trypanosomiasis.

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1. Introduction.

Amphibian cutaneous secretions have been demonstrated as a potential source

of peptides and organic compounds active against bacteria (Batista et al., 1999), human

pathogenic yeasts, and protozoan parasites (Ghosh et al., 1997, Brand et al., 2002,

Tempone et al., 2007). Bufadienolides are cardioactive C-24 steroids, found in only few

plant and animal families. The Egyptians are thought to be the first civilization to use

squill, a bufadienolide-containing plant, in the treatment of heart diseases, but some

Asian cultures (where the Chinese were the first) still use different toad venoms for the

preparation of the remedy Ch’an Su (Ye et al., 2006, Krenn & Kopp, 1998).

Bufadienolides have been described in about 10 species of the genus Rhinella (formerly

Bufo) (Krenn & Kopp, 1998), one snake (Rhabdophis tigrinus) (Hutchinson et al., 2007)

and an arthropod (Photinus) (Eisner et al., 1978); with anticancer (Hashimoto et al.,

1997) and antimicrobial (Cunha Filho et al., 2005) activities having been described.

Leishmaniasis is a widespread but neglected disease found in 16 developed and 72

developing countries, affecting 12 million people (Croft et al., 2006, Trouiller et al.,

2001). The disease is caused by protozoan parasite, Leishmania spp., and is transmitted

by sandfly of the Phlebotominae family (Dobson et al., 2003). The visceral form of

Leishmaniasis (VL) is a serious disease, causing a severe and, if untreated, fatal

hepatosplenomegaly. The therapeutic arsenal against the disease is limited, and

consists of highly toxic drugs. The antimonial therapy has been unchanged for over a

hundred years, and has severe cardiotoxicity and nephrotoxicity as other side effects.

Pentamidine and amphotericin B are very active drugs in clinical use, but considered

second line therapies due to high toxicity (Balaña-Fouce et al., 1998). Miltefosine, an old

anticancer drug, has been used in India against VL (clinical phase IV), but teratogenicity

and the emergence of in vitro resistance have been major concerns (Vanlerberghe et al.,

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2007). Furthermore, clinical failures against other species of Leishmania from New

World have recently been demonstrated (Soto and Berman, 2006).

Chagas disease, or American Trypanosomiasis, is a parasitic infection typically

spread by triatomine bugs, and affects 16-18 million people throughout Latin America.

Current chemotherapies based on the nitroaromatic compounds benznidazole and

nifurtimox have provided unsatisfactory results and suffer from considerable side effects

and low efficacy (Duschak and Couto, 2007). Consequently, there is an urgent need for

new drugs to treat this neglected disease. In this work, we isolated for the first time two

novel antiparasitic bufadienolides from the secretions of the parotid macroglands

(Toledo and Jared, 1995) of Rhinella jimi (Chaparro et al., 2007), an anuran commonly

found in north-eastern Brazil. Through biological driven assays, we have isolated two

antileishmanial compounds and were able to purify and characterize the active

molecules as telocinobufagin and hellebrigenin. Telocinobufagin induced strong

ultrastructural damage in the parasites with no cytotoxicity against mammalian cells.

This is the first report of the presence of these Bufadienolides in Rhinella jimi.

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2. Material and Methods.

2.1 Materials

Sodium dodecyl sulphate (SDS), acetic acid and trifluoroacetic acid (TFA) were

purchased from Merck (Germany). Lipopolysaccharide (LPS), 3-[4,5-Dimethylthiazol-2-

yl]-2,5-diphenyltetrazolium bromide (Thiazol blue; MTT), M-199 and RPMI-PR− 1640

medium (without phenol red) were purchased from Sigma (St Louis, MO USA).

Pentavalent antimony (Aventis-Pharma-Brazil) and pentamidine (Sideron-Brazil) were

used as the standard drugs. Acetonitrile was purchased from Carlo Erba (Italy). Other

analytical reagents were purchased from Sigma (St Louis, MO, USA) unless specifically

stated otherwise.

2.2. Animals

2.2.1 Animals and parotid macrogland secretion

BALB/c mice and Golden hamsters were supplied by the Animal Breeding Facility

at the Instituto Adolfo Lutz of São Paulo. They were maintained (under conventional

conditions) in sterilized cages under a controlled environment, receiving water and food

ad libitum. Animal procedures were performed under the approval of the Research

Ethics Commission, and according to the Guide for the Care and Use of Laboratory

Animals from the National Academy of Sciences (http://www.nas.edu).

2.1.2 Parotid secretion

Rhinella jimi secretion. The parotid secretions of 6 female specimens of Rhinella

jimi were obtained directly in the wild, during field work, in Angicos (Rio Grande do

Norte State, Brazil), a semi-arid region, where the temperature and the relative air

humidity are practically constant, around 30 ºC and 30%, respectively. Both parotid

macroglands of each specimen were manually compressed causing the release of the

whitish and pasty secretion in the form of jets, which were collected on the surface of a

plastic sheet. The secretion, still on the sheet, was dried in the open air in a shaded

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location for 24 hours. The dried secretion was then transferred to a vial and stored at –

20 ºC. The collection of the animals was made after obtaining the appropriate permits,

IBAMA license (0110/2004-CGFAU/LIC-02027-023238/2003).

2.2

2.3 HPLC analysis: Steroids Isolation

Dried Rhinella jimi parotid secretion was weighed, and 300 mg were extracted with

1M acetic acid. The crude extract was centrifuged at 12,900 g for 30 minutes at 4°C and

the supernatant collected for further steps. The extract was applied to a C18 Strata X

cartridge (Phenomenex-USA) and eluted with a step gradient of acetonitrile in water,

containing 0.1% TFA. The fraction eluted at 6:4 (acetonitrile:water - v/v) presented

antileishmanial activity and was further lyophilized. HPLC analyses were performed in a

binary Shimadzu Proeminence LC 20 system, coupled to a Photodiode array (PDA)

detector SPD-M20A, employing an ACE C18 column (4.6 x 250 mm 5µm particle size).

The mobile phase consisted of solvent A (H2O:TFA; 1000:1; 0.1% TFA) and solvent B

(Acetonitrile:H2O:TFA; 900:100:1) with a linear gradient of B over A 5 to 80% in 20 min,

after a 5 min isocratic elution under constant flow rate (1 mL/min). PDA detection was

performed in the range from 190-400 nm. Peaks were manually collected and dried for

bioguided assays.

2.4 Mass spectrometry

Previously lyophilized samples were dissolved into 0.1% formic acid and deposited

into the 384 well plate of the autosampler for MS analyses in a MSQ Surveyor mass

spectrometer system (Thermo Finnigan). Typically, 20 µL sample aliquots were injected

under a constant flow of 60 μL/min. Data were acquired under positive mode, after ESI

ionization. Instrument control, data acquisition and processing were performed by the

XCalibur suite (Thermo Finnigan).

2.5 NMR Spectroscopy

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1H NMR spectra were recorded on Bruker DRX-500 and Bruker DPX-300

operating at 500 and 300 MHz, respectively. 13C NMR spectra were recorded on a

Bruker DPX-300 operating at 75 MHz. All spectra were performed in CD3OD with

tetramethylsilane (TMS) as an internal standard. The chemical shifts (δ) are given in

ppm and coupling constants (J) are given in Hz.

2.6 Parasites

Leishmania (L.) chagasi (MHOM/BR/1972/LD) was maintained in golden hamsters.

Approximately 60-70 days post-infection, amastigotes were obtained from the hamster

spleens by differential centrifugation and the parasite burden was determined using the

Stauber method (1958). Promastigotes were maintained in M-199 medium

supplemented with 10% (v/v) calf serum and 0.25% (v/v) hemin at 24 °C.

2.7 Determination of Leishmania Growth Inhibition: 50% Inhibitory Concentration

The growth inhibition of Leishmania (L.) chagasi promastigotes was determined as

described elsewhere (Tempone et al, 2004), using pentamidine as the standard drug.

Promastigotes were counted in a Neubauer haemocytometer and seeded at 2 x 105

cells/well in 96-well microplates. The isolated steroids were incubated with

promastigotes in concentrations ranging from 0.488 to 200 μg/mL (based on dry weight)

for 5 h, and parasites were centrifuged at 3,000 g for 10 minutes. The pellet was

dissolved in 150 µL of RPMI-PR- medium and further incubated in a 96-well microplate

for 72 h at 24 °C. Parasite growth inhibition was determined using the MTT assay at 570

nm (Tada et al., 1986). The antileishmanial activity against intracellular amastigotes was

determined with infected macrophages, using pentavalent antimony as the standard

drug. The parasite burden was defined as the mean number of infected macrophages

per 1000 cells, as determined by light microscopy of Giemsa-stained laminas (Tempone

et al, 2004).

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2.8 Antitrypanosomal Activity

Trypomastigotes obtained from LLC-MK2 (ATCC CCL 7) cultures were counted in

a Neubauer haemocytometer and seeded at 1 x 106 cells/well in 96 well microplates.

The steroids were incubated at the highest concentration of 150 μg/mL for 24 h at 37 °C

in a 5% CO2 humidified incubator with benznidazole as the standard drug. The

trypomastigote viability was based on the cellular conversion of the soluble tetrazolium

salt MTT into the insoluble formazan by mitochondrial enzymes (Lane et al., 1996). The

formazan extraction was carried out with 10% (v/v) SDS for 18 h (100 μL/well) at 24 °C

(Tada et al., 1986).

2.9 Cytotoxicity Assays

Macrophages were obtained from the peritoneal cavity of BALB/c mice in RPMI-

PR- 1640 medium (without phenol red) and dispensed at 4 x 105 cells per well in 96 well

microplates at 37 °C in a 5% CO2 incubator for 2 h to allow for attachment. Cells were

washed twice with medium at 37 °C and further incubated with the isolated steroids in a

range of 3.9 µg/mL to 200 µg/mL for 48 h at 37 °C. The viability of the macrophages was

determined using the MTT assay (Tada et al., 1986). Briefly, 3-[4,5-Dimethylthiazol-2-yl]-

2,5-diphenyltetrazolium bromide (MTT) was dissolved in phosphate-buffered saline

(PBS) at 5 mg/mL and incubated with cells (20 µL/well) for 4 h at the same conditions.

The extraction of the mitochondrial formazan was done with 80 µL of 10% SDS with a

further 24 h incubation. The Optical Density was read at 570 nm (Multiskan) using

control wells without drugs (100% viability) and without cells (blank). Pentamidine was

used as the standard drug.

2.10 Macrophage Nitric Oxide Production

The nitric oxide (NO) production of macrophages was measured in the presence of

the isolated steroids by the Griess reaction (Panaro et al., 1999). Peritoneal

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macrophages were incubated for 24 h at 37 °C with the steroids (40 μg/mL). LPS (50

μg/mL) was used to induce NO up-regulation. The absorbance was determined at 540

nm using a spectrophotometer Multiskan MS (Uniscience) microplate reader.

2.11 Electron Microscopy Studies

L. (L.) chagasi promastigotes were incubated with telocinobufagin for different

periods (0, 1, 3, 5, h) at 24 °C in 24 well plates. Subsequently, promastigotes were fixed

in 2.5% glutaraldehyde in 0,1 M sodium cacodylate/0.2 M sucrose buffer, pH 7.2. One

percent osmium tetroxide post fixation was followed by 1% uranyl acetate treatment. The

samples were then dehydrated through ascending series of acetone and embedded in

Epon. Thin sections were stained with uranyl acetate and lead citrate. The material was

examined in a JEOL 1011 transmission electron microscope.

2.12 Hemolytic Activity

The hemolytic activity of the isolated steroids was evaluated in BALB/c

erythrocytes (Moreira et al., 2007). A 3% suspension of mice erythrocytes was incubated

for 2 h with the isolated compounds in 96 well U-shape microplates at 25 °C and the

supernatant was read at 550 nm in a spectrophotometer Multiskan MS (Uniscience)

microplate reader.

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3.0 Results

3.1 Purification of Antileishmanial Compounds

The fractionation of the acetic acid extract of the Rhinella jimi parotid macrogland

secretion, after SPE step gradient of acetonitrile in C18 Strata X cartridge, resulted in an

antileishmanial fraction eluting at 60% ACN. The HPLC purification of the active fraction

yielded 19 major peaks, with the antileishmanial compounds eluting at 18.44 and 22.63

min (Fig. 1). According to the biological monitoring of all fractions PDA analyses showed

an intense UV absorption at 300 nm, which corresponds to the λmax for the conjugated

lactone ring of such compounds.

3.2 Spectroscopic identification

The spectroscopic analysis of the substances found in the Rhinella jimi parotid

macrogland secretion led to the identification of two bufadienolides that are referred to

as telocinobufagin and hellebrigenin (Fig. 2). The structures were determined by

analysis of 1H and 13C-NMR spectra. Among the characteristic features that indicated a

steroid skeleton with an α-pyrone ring are δ [7.43 (d, J = 2.5 Hz, H-21), δ 7.99 (dd, J =

2.5; 9.7 Hz, H-22) and δ 6.28 (d, J = 9.7 Hz, H-23)] for telocinobufagin and [δ 7.42 (d, J =

2.5 Hz, H-21), δ 7.97 (dd, J = 3.0; 12.0 Hz, H-22) and δ 6.27 (d, J = 12.0 Hz, H-23)] for

hellebrigenin. This last substance also showed a signal at δ 10.05, corresponding to a

aldehyde group at C-19, while telocinobifagin showed the signal of a methyl group at δ

0.93 s (CH3-19). Also observed in the 1H- NMR spectra of the bufadienolides were broad

singlets at δ 4.18 and 4.20, corresponding to H-3, in addition to another methyl signal at

δ 0.71 s or 0.67 s (CH3-18) for telocinobufagin and hellebrigenin, respectively. In the 13C-

NMR spectrum of telocinobufagin, 24 signals were observed referring to the 24 carbons

of the steroid, including a carbonyl group (δC 164.9) corresponding to C-24, four

unsaturated carbons (δC 125.1, 150.6, 149.5, 115.6) corresponding to C-20, C-21, C-22

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and C-23, respectively, and three carbinol groups (δC 69.2, 76.3 and 86.2) corresponding

to C-3, C-4 and C-14, respectively. Similar signals were observed for hellebrigenin,

except for the signal at δ 210.3, corresponding to the aldehyde group at C-19. A full set

of 13C NMR spectroscopic assignments are given in table 2. The molecular formula of

telocinobufagin was determined as C24H34O5, since the mass spectrum showed a mass

component at m/z 403.51 corresponding to the protonated [M+H]+, while the mass

spectrum of hellebrigenin (C24H32O6) showed a peak at m/z 417.39 corresponding to the

protonated [M+H+].

3.3 The antiprotozoan activity and mammalian toxicity

The bioguided purification resulted in the characterization of telocinobufagin and

hellebrigenin, which showed growth inhibition with IC50 values of 61.21 µg/mL and 126.2

µg/mL, respectively, against Leishmania (L.) chagasi promastigotes (Table 1). The

treatment of amastigote-infected macrophages with steroids for 96 h resulted in a

reduction in the parasite burden of 37.0% (SD ± 1.2) for telocinobufagin and 21.5% (SD

± 4.3) for hellebrigenin at the highest concentration of 150 µg/mL (Tab. 1).

Telocinobufagin was inactive against trypomastigotes of T. cruzi, but hellebrigenin

presented an IC50 value of 91.75 µg/mL. The mammalian cytotoxicity studies

demonstrated no hemolytic activity for the steroids at the highest concentration of 200

µg/mL. In addition, no cytotoxicity was found for the steroids when incubated with

BALB/c macrophages for over 48 h at the highest concentration of 200 µg/mL. The MTT

assay resulted in a 100% viability of cells when compared to control wells, with an

unaffected morphology observed by light microscopy (data not shown).

3.4 Nitric Oxide Production

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Telocinobufagin and hellebrigenin did not stimulate the production of nitric oxide in

macrophages, nor did they interfere with the activation of macrophages after LPS

incubation (Fig. 3).

3.5 Ultrastructural Studies

Ultrastructural studies of promastigotes incubated for 1 h with the most active

steroid, telocinobufagin, revealed a loss of nucleolar structures, as a low density

nucleoplasm was observed (Fig. 4). An intense chromatin condensation of the

kinetoplast characterized as an electron dense structure was observed. The overall

shape of the parasite was completely modified, as an amastigote-like aspect was clearly

observed with swelling of the cell body (Fig. 4B). The parasitic plasma membrane was

preserved as a general rule, except for the presence of small pores in the internal

plasma membrane (Fig 4C, arrow). After a 3 h incubation (Fig. 4D), no huge

modifications were observed, but increased damage to the plasma membrane and an

intensification of cytoplasmic vacuoles were apparent. After a 5 h incubation (Fig. 4E),

an intense cell edema, with a total loss of cellular structures and an intense swelling of

mitochondria was observed. Normal ultrastructural organelles in Leishmania are

demonstrated in Figure 4A as a control (untreated group).

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4.0 Discussion

Amphibian cutaneous secretions have proven to be an astonishing source of

biologically active compounds, with noxious or toxic secretions containing biogenic

amines, peptides, bufadienolides, tetrodotoxins and alkaloids (Daly et al., 1995). In this

work, we have described, for the first time, the antileishmanial and antitrypanosomal

activities of Rhinella jimi parotid macrogland secretion. Rhinella jimi is an endemic toad

of the Brazilian caatinga, i.e., the north-eastern semi-arid region. Through bioguided

isolation, we were able to purify two active molecules, and by analyses of the mass

spectra and 1H and 13C-NMR spectroscopic data we have identified two steroids:

telocinobufagin (Ye et al., 2004; Cunha Filho et al., 2005), and hellebrigenin (Meng et

al., 2001). This is the first report of such bufadienolides and their antiprotozoan activity in

R. jimi parotid secretions.

Telocinobufagin was previously isolated from another species of Rhinella, including

Rhinella rubescens (Cunha Filho et al., 2005), while hellebrigenin has been isolated from

plant sources, including Urginea altisiima (Shimada et al., 1979). Hellebrigenin has been

found in a glycoside form (hellebrin) and some variations, such as hellebortin A, B and

C, have been isolated from seeds of Helleborus torquatus, a species belonging to a

genus known for its important biological activities (Meng et al. 2001).

Our results showed growth inhibition of Leishmania (L.) chagasi promastigotes

with a leishmanicidal effect, and with the inhibition of the parasitic mitochondrial

dehydrogenases as determined by the tetrazolium assay (MTT) (Tada et al., 1986).

Trypanosoma cruzi trypomastigotes were also susceptible to the isolated steroid

hellebrigenin, but not to telocinofufagin. The MTT assay revealed a trypanocidal activity

after 24 h, with a strong altered morphology by light microscopy. Telocinobufagin and

marinobufagin have been isolated from skin secretions of Rhinella rubescens (former

Bufo rubescens). Both steroids presented antimicrobial activities with MIC values

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ranging from 64 to 128 µg/mL (Cunha-Filho et al., 2005). Despite the differences in the

biological models, our IC50 values were consistent with those observed against bacteria.

Hellebrigenin has been found in plants of Helleborus and Kalanchoe genus

(Steyn and Heerden, 1998), and in the skin secretions of the toad Rhinella viridis (Gella

et al., 1995). Hellebrigenin and telocinobufagin have demonstrated potent anticancer

activity against liver carcinoma cells at 1.6 x 10-4 µg/mL and 3.5 10-4 µg/mL, respectively

(Kamano et al., 1998). Bufalin, a cardiotonic steroid found in toads of Bufanidae family,

has induced apoptosis in human leukemia cells, and growth inhibitory and differentiation-

inducing activities in leukemia cells (Yamada et al., 1997), malignant melanoma cells

(Zhang et al., 1992) and human squamous skin carcinoma cells (Akiyama et al., 1999).

In spite of several studies reporting different biological activities for the bufadienolides,

this is the first report of the presence of bufadienolides and of the antiparasitic effects of

telocinobufagin and hellebrigenin from R. jimi parotid macrogland secretion.

The ultrastructural study of promastigotes was carried out with different periods

of incubation with telocinobufagin. The initial cellular damage of promastigotes

demonstrated the loss of nucleolar structures with amastigotes-like forms, suggesting

increased cell permeation. Species of the trypanosomatid parasite genera Trypanosoma

and Leishmania exhibit a particular range of cell shapes that are defined by their internal

cytoskeletons. The cytoskeleton is characterized by a subpellicular corset of

microtubules that are cross-linked to each other and to the plasma membrane, with an

extremely precise organization of microtubules (Gull, 1999). Based in this initial altered

morphology of parasites, and the interference with the mitochondrial oxidation of

tetrazolium bromide (MTT), we suggest that the activity of telocinobufagin in Leishmania

might involve the mitochondrial metabolism and the disturbance of the parasite plasma

membrane, resulting in a cellular edema and death. However, further studies are

necessary to elucidate the mechanism of action of telocinobufagin in Leishmania.

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Hellebrigenin and telocinobufagin demonstrated an antiparasitic effect against

intracellular amastigotes, but to a lesser efficacy when compared to axenic

promastigotes. This effect could be due to the lack of a transporter as a receptor in the

macrophage membrane for hellebrigenin and telocinobufagin, resulting in the steroids’

weaker activity. The metabolism of amastigotes has different characteristics from the

extracellular promastigotes, as the hard intracellular milieu of macrophages press on to

a superior defensive mechanism. These facts could have also contributed to the higher

resistance observed against the intracellular form of Leishmania. Superior efficacy of

drugs entrapped in phosphatidylserine-liposomes has been observed, as a result of a

targeted delivery to intracellular amastigotes via macrophage scavenger receptors

(Tempone et al., 2004). This approach could be an alternative tool to deliver higher

amounts of these bufadienolides into amastigotes.

The effect of a diminished parasite burden in macrophages after incubation with

the isolated steroids was investigated by measuring nitric oxide (NO) levels. Nitric oxide

is a known macrophage defensive metabolite largely produced against intracellular

aggressors, and is a basic marker for macrophage activation (Balestieri et al., 2002).

Neither telocinobufagin nor hellebrigenin showed significant activation of peritoneal

macrophages, as confirmed by the lack of NO production after incubation. Bacterial LPS

was used as an internal control for NO up-regulation by macrophages (Balestieri et al.,

2002), but both steroids showed no interference with the NO levels of stimulated

macrophages. These results suggest that both steroids result in a killing effect on the

parasite, but mechanisms other than macrophage activation might be involved in the

intracellular antiparasitic activitiy.

Bufadienolides have been described as very cytotoxic compounds, especially

against cancer cells, at nanomolar concentrations (Kamano et al., 1998). In our data

neither telocinobufagin nor hellebrigenin demonstrated cytotoxicity against mice

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peritoneal macrophages, the host cells for Leishmania spp. (Murray and Etienne, 2000).

Likewise, no hemolytic activity of mice erythrocytes was found at concentrations higher

than the IC50 (against the parasites) determined for both steroids. This could be

explained by the fact that bufadienolides have been found in mammalians as an

endogenous steroid. Recently, telocinobufagin has been found in mammalians and

exhibits elevated plasma levels in patients with terminal renal failure (Komiyama et al.,

2005). It has been suggested that cardiotonic steroids (CS) such as bufadienolides

participate in many physiological process like sodium excretion, blood pressure

regulation and signal transduction (Dmitrieva and Doris, 2002). Furthermore, studies

with breast cancer cells from patients receiving CS such as digitalis demonstrated a

more benign characteristic and a 9.6-fold reduction in the recurrence of breast cancer

after mastectomy (Stenkvist et al., 1982). Although telocinobufagin is an endogenous

substance of mammalians, chemical modifications could contribute to a higher efficacy.

Structure activity relationships (SAR) through chemical biotransformation of

bufadienolides have been described using in vitro anticancer models (Ye et al., 2004).

The hydroxylation of different sites than the usual ones described in the literature

resulted in the most effective bufadienolides. These data suggests that structural

modifications of telocinobufagin or hellebrigenin could also enhance its antileishmanial

effect.

Our data contributes to the novel biological activities of amphibian steroids such

as bufadienolides. Telocinobufagin and hellebrigenin presented an antileishmanial

activity and were not cytotoxic against mammalian macrophages. If adequately studied,

these novel antileishmanial compounds could contribute significantly as tools for drug

design and the development of new therapeutics against neglected diseases like

Leishmaniasis.

Acknowledgments.

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This work was supported by grants from Fundação de Amparo a Pesquisa do

Estado de São Paulo 05/00974-9 (AGT) and CNPq grants 303516/2005-4 and

473614/2006-5 (DCP). The authors wish to thank Ms. Anete A. Gomes for manuscript

review.

Figure Legends

Fig. 1. Chromatogram profile of the Rhinella jimi fraction (60% ACN – SPE) employing

an ACE C18 column. PDA detection was performed in the range from 190-400 nm. A.

The chromatogram was obtained at 214 nm. Black arrow- hellebrigenin; gray arrow-

telocinobufagin.

Fig. 2. Structures of the bufadienolides telocinobufagin(1) and hellebrigenin (2).

Fig. 3. Nitric oxide (NO) production of peritoneal macrophages incubated with isolated

steroids by the Griess reaction. The absorbance was determined at 540 nm using a

spectrophotometer microplate reader.

Fig. 4. Ultrastructural studies of L. chagasi promastigotes incubated with telocinobufagin

for different periods. A. control (untreated promastigotes); B, C. 1 h incubation (arrow-

membrane pores); D. 3 h incubation; E. 5 h incubation. k- kinetoplast, m- mitochondria,

n- nucleus, f- flagellum, fp- flagellar pocket, L- lipid inclusion, v- vacuoles, pm- plasma

membrane, mt- microtubules.

Table Legends

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Table 1: Antiparasitic, hemolytic and cytotoxic effects of telocinobufagin and

hellebrigenin.

Table 2. 13C NMR spectroscopic data for the bufadienolides telocinobufagin and

hellebrigenin (δ in CD3OD, 75 MHz).

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Steroids IC50 (µg/mL) L. chagasi

Promastigote (95%CI)

% Treatment of Leishmania-Infected Macrophages at 150

µg/mL

IC50 (µg/mL) T. cruzi

Trypomastigotes (95%CI)

Macrophage Toxicity

IC50 (µg/mL)

Hemolytic Activity (µg/mL)

telocinobufagin 61.2 (42.9-87.2) 37.0 % (SD ± 1.2) >200 >200 >200

hellebrigenin 126.2 (121.9-130.7) 21.5 (SD ± 4.3) 91.75 (64.5-130.5) >200 >200

IC50- 50% Inhibitory Concentration; 95%CI - 95% Confidence Interval. Pentamidine IC50 0.096 µg/mL against promastigotes; IC50 8.72 µg/mL against macrophages. Glucantime IC50 21.0 µg/mL against amastigotes-infected macrophages. Benznidazole IC50 36.33 µg/mL against tripomastigotes. Cells (promastigotes/trypomastigotes and macrophages) were incubated with steroids and the viability was determined by MTT assay. Infected-macrophages were treated with steroids and the parasite burden determined by light microscopy. Hemolytic activity was determined using BALB/c erythrocytes incubated with steroids. 50% Inhibitory Concentration data was determined using the Graph Pad Prism Software 3.0.

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δ in CD3OD, 75 MHz C telocinobufagin hellebrigenin 1 26.3 18.6 2 29.9 27.7 3 69.2 68.1 4 36.1 37.6 5 76.3 76.0 6 33.3 38.7 7 24.9 25.3 8 42.1 43.1 9 37.9 40.7

10 42.2 56.4 11 23.1 23.7 12 40.4 41.7 13 48.9 48.3 14 86.2 85.8 15 30.9 32.4 16 28.6 29.8 17 52.2 52.7 18 17.4 17.2 19 17.4 210.3 20 125.1 125.0 21 150.6 150.7 22 149.5 149.4 23 115.6 115.6 24 164.9 163.6

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0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min

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