Amphotericin B-induced damage of Trypanosoma cruzi epimastigotes

Chem.-BioL Interactions, 71 (1989) 91-- 103 Elsevier Scientific Publishers Ireland Ltd.

91

AMPHOTERICIN B-INDUCED D A M A G E OF T R Y P A N O S O M A CRUZI EPIMASTIGOTES

ROSA MARIA T. HAIDO* and E L I A N A BARRETO-BERGTER**

Departamento de Microbiologia Gera~ Instituto de Microbiologi~ Universidade Federal do Rio de Janeiro, 21944, Rio de Janeiro, R J (Brazil) (Received July 25th, 1988} (Revision received December 14th, 1988) (Accepted December 17th, 1988)

SUMMARY

Amphotericin B (AmB) autoxidation resulted in oxygen consumption, superoxide anion formation and production of thiobarbituric acid (TBA)-reactive material (malondialdehyde). Malondialdehyde formation increased after incubation of the drug with ascorbate-ADP-FeCl 3. Growth of Trypanosoma cruzi epimastigotes in the presence of AmB induced a decrease in the free fatty acid content of the cells (57% in control cells vs. 7% in AmB-treated cells), and in the proportion of unsaturated fatty acids as well as cell killing. No changes were detected on sterol content. No evidence was found for lipid peroxidation as a mechanism of cell injury by this antibiotic. K e y words: Trypanosoma cruzi -- Amphotericin B -- Superoxide anion -F a t t y acids

INTRODUCTION

Amphotericin B (AmB) is a well-known polyene antibiotic highly active in the t r e a t m e n t of systemic fungal infections [1]. Its effectiveness has also been demonstrated in the t r e a t m e n t of mucocutaneous leishmaniasis [2,3]. Horvath and Zierdt [4] reported the effect of AmB in vivo and in culture forms of Trypanosoma cruzi. Cruz et al. [5] demonstrated that AmB is an effective antitrypanosomal agent against the trypomastigote form of T. cruzi and may provide an alternate means of control of blood transfusion-induced Chagas disease. This report was confirmed later by Cover and Gutteridge [6]. *Present address: Universidade do Rio de Janeiro, Rua Frei Caneca 94, 20230, Rio de Janeiro, RJ, Brazil. **To whom correspondence should be addressed. 0009-2797/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

92 AmB has been shown to increase the permeability of the cytoplasmic membrane toward ions and small molecules by specifically interacting with membrane sterols [7,8]. The molecular details of membrane structural changes resulting from interaction with this antibiotic are not well defined [9]. However, recent reports indicate that oxidative damage is involved in the anticellular effect of AmB [10] and other heptaene polyene antibiotics

[11]. Several studies have shown that autoxidation of polyene antibiotics results in the formation of peroxides, epoxides, low-molecular-weight carbonyls and carboxylic acids through free radical-mediated chain reactions [11]. In this regard, some products of polyene antibiotics autoxidation have been chemically characterized [12]. This process is similar to that occurring in polyunsaturated fatty acids with the well-characterized formation of hydro, cyclic and endoperoxides. When these peroxides are subjected to acid heating in the presence of thiobarbituric acid (TBA), they decompose to give a variety of carbonyls, one of which, malondialdehyde, forms a characteristic adduct with TBA. Similar treatment of the oxidatively degraded polyene antibiotics candicidin produces a TBA adduct indistinguishable from that formed by malondialdehyde [11]. Concerning AmB, it has been demonstrated that this antibiotic autoxidizes generating carbon-centered free radicals [13] and malondialdehyde has been detected in incubations of AmB with erythrocytes [10] or human promyelocytic leukemia cells [14]. Changes in biological activity and TBA reactivity of several polyene antibiotics, including AmB, have been described [15]. It has been suggested that Arab might trigger or accelerate a cascade of oxidative reactions linked to its own autoxidation. These reactions might lead to the formation of products that could be responsible for cell killing [16]. In the present work we demonstrate that AmB can autoxidize via a free radical chain reaction generating malondialdehyde and that growth of T. cruzi in the presence of this drug results in a decrease in the amount of polyunsaturated fatty acids of the cells. However, no evidence was found for lipid peroxidation as a mechanism of cell injury exerted by this antibiotic. MATERIALS AND METHODS Culture methods T. cruzi (Y strain) was grown in Warren's modified liquid medium [17]

consisting of brain heart infusion 37 g, hemin (dissolved in triethanolamine) 20 mg and fetal calf serum 20 ml • 1-1. The number of organisms was measured using a Neubauer counting chamber. Fresh media were inoculated with 0.5 × 10~ cells - ml-L For all experiments, AmB was dissolved in sodium deoxycholate (0.08 ~g ml-~). Epimastigote forms used for the experiments were grown in 4 l Erlenmeyers flasks containing 2 l of medium. Five days after inoculation, the cells were collected by centrifugation and washed with 0.154 M NaC1. Sodium deoxycholate did not affect growth of T cruzi.

93

Chemicals All organic solvents were reagent grade from Merck and were distilled before use. AraB was purchased from Squibb Lab. Brazil, or from Sigma Chemical Co. Sodium ascorbate, sodium deoxycholate, cytochrome c, TBA, ADP and superoxide dismutase were obtained from Sigma Chemical Co., St. Louis, MO.

Lipid extraction Cells of T. cruzi were extracted at room temperature with 60 vol. of each of the following solvent mixtures: chloroform/methanol (2 : 1} and chloroform/ methanol {1:2, v/v). Both extracts were combined, evaporated to dryness and weighted.

Characterization of fatty acids and sterol (Fig. 1). Total lipid extract was transesterified for 2 h at room temperature using 70 vol. of 0.025 N methanolic sodium methoxide. Then water was added, the reaction acidified with hydrochloric acid and fatty acid methyl esters and free fatty acids were extracted with chloroform. The total methyl esters and sterols were analyzed by gas chromatography-mass spectrometry (GLC-MS) with a Finnigan instrument, model 4000. The chromatographic separation was carried out using a 30 m × 0.32 mm glass capillary column containing DB-5 fused silica (bonded SE-54). Injections were in the splitless mode at 40°C. The temperature was raised to 80°C and then programmed to 290°C at 4°C • min -1. Helium was used as the carrier gas at a linear velocity of 40 cm • s -1. Electron-impact ionization spectra were recorded by scanning from mass 41 to mass 650 repeatedly every second. The electron energy was 70 eV. For determination of free fatty acids, total fatty acid methyl esters plus sterols were analyzed as their trimethylsilyl (TMS) derivatives, using the same conditions already described. TMS derivatives were made by addition of N10-bis-(trimethylsilyl)-acetamide (BSA) to a solution of fatty acids and sterols in ethyl acetate and the mixture was allowed to stand for 5 min at 50°C. The resulting TMS derivatives were immediately analyzed in order to avoid slow transesterification of methyl esters. F a t t y acids were identified by their fragmentation patterns [18,19] and quantitated by GLC-MS spectra [2Ol.

Malondialdehyde formation Malondialdehyde was measured by reaction with TBA. Aliquots of the incubated suspensions were boiled for 15 min with TBA and trichloroacetic acid, in the presence of 0.01% butylated hydroxytoluene as described by Buege and Aust [9]. After cooling, the tubes were centrifuged (1000 x g for 10 rain) and the malondialdehyde content was determined by the absorbance at 535 nm.

Superoxide anion formation Superoxide anion formation by AmB autoxidation was assessed by measurement of superoxide dismutase inhibitable cytochrome c reduction [21].

94 T.

CRUZI

i (epimastigotes) EXTRACTION (2:1

AND

WITH 1:2,

CHLOROFORM~METHANOL

v/v)

1

FILTRATION

TOTAL

L

LIPID

RESIDUE

EXTRACT

1

TRAHSESTERIFICA

TION

(0.025

IN

2H/ROOM

[

H NaOCH 3

METHANOL)

TEMPEATURE

t STEROL

FATTY METHYL

TMS

ESTERS

FREE

FATTY

ACIDS

DERIVATIZATION

I

J

STEROL (TMS)

ACID

FATTY ETHER

GLC-MS

METHYL

ACID ESTERS

GLC-MS

FATTY

ACID

(TNS)

GLC-HS

Fig. 1. Isolation and characterization of total lipids from T. c r u z i epimastigote.

RESULTS

Acid heating of freshly made solutions of AraB in the presence of T B A resulted in the formation of the characteristic adduct of malondialdehyde

95 with TBA (Fig. 2A) with a peak m a x i m u m at about 535 nm (Fig. 2B). Incubation of AmB for 24 h in b u f f e r increased the formation of the TBAr e a c t i v e material (Table I). T h e d a t a in Table I indicates t h a t ascorbate-ADPFeC13 did not stimulate the autoxidation of AmB. If it had done so, it would have increased the difference b e t w e e n the 0-h and 24-h readings, which it did not do in the case of the Sigma product. The TBA assay used h e r e did not m e a s u r e p e r f o r m e d malondialdehyde, b u t r a t h e r the products of acid-catalized, t h e r m a l decomposition of (oxidized) AmB. The chelated iron f u r t h e r catalyzes this decomposition, and this catalysis is not blocked by adding b u t y l a t e d h y d r o x y t o l u e n e . This i n t e r p r e t a t i o n is s u p p o r t e d by the increased color in the 0-h samples with chelated iron p r e s e n t . It appears t h a t Sigma AraB is more oxidized t h a n is Squibb AmB, although the l a t t e r is known to be partially decomposed as supplied [11]. Incubation of AmB in buffer r e s u l t e d in a slow 02 u p t a k e has b e e n r e p o r t e d previously [13] and in a slow s u p e r o x i d e anion formation as d e t e c t e d by the s u p e r o x i d e dismutase inhibitable c y t o c h r o m e c r e d u c t i o n assay [22] (Table II). Much lower AmB c o n c e n t r a t i o n s w e r e n e c e s s a r y to d e t e c t a significant toxic activity on T. cruzi A m B - t r e a t e d cells. Cholesterol was the main sterol found in both s y s t e m s analyzed. This was identified by its c h r o m a t o g r a p h i c and mass s p e c t r o m e t r i c p r o p e r t i e s (Figs. 3A and 3B; Tables III and IV). Figure 3 shows the GLC-MS c h r o m a t o g r a m s of the f a t t y acid m e t h y l e s t e r s of T. cruzi. 1 8 : 2 and 1 8 : 1 w e r e the m a j o r c o m p o n e n t s of the esterified lipid fraction (Table III). T h e ratio of u n s a t u r a t e d / s a t u r a t e d f a t t y acids found in T.

®

@ 1 0

A 0.4

0.3

if)

~o3

2~

~ 0.2

o,I

<0.1 / o / 0 ~ 0

1

I

0.25 0.50 Amphotericln B (mM)

1.0

1

0

.-0 0

400

I

1

450 500 Wavelength

I

550

Fig. 2. Autoxidation of AmB. AmB was suspended in 0.1 N phosphate buffer (pH 7.2) and 0.154 M NaC1 (final volume: 1 ml). 2 ml of TBA reagent was added and the mixture was heated in a boiling water bath for 15 rain in the presence of 0.01% butylated hydroxytoluene (o) or was not heated (O). (A) TBA-reactive material (malondialdehyde) was measured at 535 ram. (B) Optical absorption spectrum of TBA-reactive material (malondialdehyde) obtained after treating a solution of I mM Arab as described in (A).

(0.1) (0.25) (0.5) (0.1) (0.25) (0,5)

0.15 0.17 0.145 0.11 0.135 0.15

__. 0.01 a +_. 0.01 _ 0.01 _ 0.005 _+ 0.005 __ 0.005

0.155 0.197 0.23 0.13 0.15 0.15

_+ 0.01 +_ 0.01 _.+ 0.01 _+ 0.01 __- 0.01 + 0

24h 0.24 0.26 0.25 0.165 0.195 0.22

0 __ 0,01 __ 0,01 _+ 0.02 _ 0,005 __ 0.01 __ 0.01

T=

T=

T=

0

Ascorbate/ADP/FeCls

Control

aEach value is m e a n _+ S.D, of four d e t e r m i n a t i o n s .

Squibb

Sigma

A m B source (raM)

0.28 0.29 0.32 0.23 0.26 0.28

T=

_ 0.02 _+ 0.01 __ 0.01 __ 0.01 +_ 0.01 _ 0.005

24h

A m B was incubated at room t e m p e r a t u r e , 24°C, in 0.1 M p h o s p h a t e buffer (pH 7.2) and 0.154 M NaC1 (final volume, 1 ml) in t h e a b s e n c e (control) or in t h e p r e s e n c e of 0.1 M s o d i u m a s c o r b a t e , 25 m M A D P and 5 m M FeCl s. A t t h e end of t h e incubation time (0 and 24 h), 2 ml of T B A r e a g e n t w a s a d d e d a n d t h e m i x t u r e w a s h e a t e d in a boiling w a t e r b a t h for 15 min. A f t e r cooling, t h e t u b e s were c e n t r i f u g e d (1000 x g for 10 min) a n d t h e s u p e r n a t a n t w e r e r e a d at 535 nm. 0.01% b u t y l a t e d h y d r o x y t o l u e n e w a s a d d e d to avoid autooxidation catalyzed by m e t a l s d u r i n g h e a t i n g with t h e T B A r e a g e n t [19].

E F F E C T OF A S C O R B A T E P L U S F E R R I C - A D P ON A m B A U T O O X I D A T I O N

TABLE I

¢D

97 TABLE II EFFECT OF AmB ON SUPEROXIDE ANION (O~) PRODUCTION AmB was incubated at room temperature, 24°C, under constant shaking (120 rev./min) in 0.1 M phosphate buffer (pH 7.2) and 0.154 M NaC1 in the presence of 30 mg/ml of cytochrome c. Blanks containing 5 rag. m1-1of superoxide dismutase were substracted. Superoxide anion production was determined by measuring cytochrome c reduction as described in Ref. 20. AmB source (raM) Sigma Squibb

nmol O~/24h (0.5) (1.0) (1.5) (0.5) (1.0) (1.5)

9.4" 18.8 18.8 2.8 5.6 11.3

•Each value is the mean of four determinations.

cruzi lipids was 3.5 in A m B - t r e a t e d e p i m a s t i g o t e s showing a g r e a t e r a m o u n t of s a t u r a t e d f a t t y acids t h a n in control cells (11.0). Palmitic and stearic acids w e r e responsible for t h e increase in total s a t u r a t e d f a t t y acids. Essentially similar r e s u l t s w e r e o b s e r v e d on the free f a t t y acids composition of control (4.7) and A m B - t r e a t e d cells (2.7). 1 8 : 2 , 1 8 : 1 and 1 8 : 0 w e r e d e t e c t e d in h i g h e r c o n c e n t r a t i o n in both s y s t e m s analyzed (Fig. 3 and Table IV). Evenand o d d - m e m b e r e d , b r a n c h e d , s a t u r a t e d and u n s a t u r a t e d f a t t y acids ranging from 12 : 0 to 24 : 0 w e r e d e t e c t e d as c o m p o n e n t s of t h e total lipid fractions of T. cruzi. S a t u r a t e d f a t t y acids with 16 carbon atoms w e r e not p r e s e n t in A m B - t r e a t e d cells. 16 : 0 and iso-16 : 1 w e r e found as components of the free f a t t y acids fraction of control cells. F i g u r e 4 shows t h a t those c o n c e n t r a t i o n s of A r a b t h a t d e c r e a s e d the a m o u n t of p o l y u n s a t u r a t e d f a t t y acids of T. cruzi also inhibited g r o w t h of the p a r a s i t e s in vitro. L i g h t microscopic o b s e r v a t i o n s showed t h a t A r a b induced s e v e r a l alterations in the e p i m a s t i g o t e forms of 7". cruzi. The organisms b e c a m e r o u n d e d and d e v e l o p e d granular, refractile inclusions in t h e i r cytoplasm. Motility was lost in 75% of the organisms. DISCUSSION In a g r e e m e n t with p r e v i o u s r e p o r t s [4--6] the e x p e r i m e n t a l evidence p r e s e n t e d in this s t u d y indicates t h a t AraB is a p o t e n t inhibitor of T. cruzi g r o w t h in vitro. A t a c o n c e n t r a t i o n of 0.04 pg • m1-1 (416 nM), AmB inhibited cell g r o w t h b y 500/o. C o m p l e t e inhibition was achieved with 0.1 ~g • m1-1 (104 nM). AmB induced a d e c r e a s e in the free f a t t y acid c o n t e n t of the cells and in the p r o p o r t i o n of u n s a t u r a t e d f a t t y acids. Although a d e c r e a s e in the polyuns a t u r a t e d f a t t y acid c o n t e n t of cells is commonly found as a consequence of

L

I"

B

A

'I

.......

'

I~

.

Iooo

,::

.

.

.

.

.

.

.

.

.

4

.

+

+,, .

.

.

.

.

9

8

+! + ,, .+

i

b J

j

+0 m

EPI,.

+,ooo

. . . . .

,,

++, :+ ~+,+++~++ ..~.

!iJ

I :i

'! 811

7a

6a~i

+

+P+

3000

lOa

3000

lOa

scAN

SCAN

Fig. 3. Reconstructive GLC-MS ion chromatogram of total fatty acids from T. cruzi. (A) Controls cells; (B) Arab treated cells. Peaks 1 - 9 , fatty acid methyl esters; peaks la--9a, free fatty acids (TMS derivatives}; peak 10, cholesterol; peak 10a, cholesterol TMS ether.

RIG

IO0.O

RIC

100.01

GO

n15 : 0

i16 : 1 n16 : 0

n17 : 1 n17 : 0

n18 : 2 n18 : 1 n18 : 0

cholesterol 18:1 + 18:2

2

3 4

5 6

7 8 9

10

1.64

0.99 1.00 1.02

0.91 0.93

0.83 0.85

0.77

0.68

RRT a

74,75,87,101,115,129,143,157,171,185,199(M÷-43),211 (M~-31),213(M*-29),242(M ÷) 74,75,87,101,115,129,157,171,185,213(M*-43),255(M ÷31),227(M+-29),256(M ÷) 194(M+-74),236(M*-32),237(M ÷-31),268(M ÷) 74,75,87,101,115,129,143,157,171,185,227(M'-43), 239(M+-31),24 l(M÷-29},270(M ÷) 208(M+-74),250(M÷-32),251(M*-31),282(M+) 74,75,87,101,115,129,143,157,185,241(M+-43),253(M +31),255(M'-29),284(M +) 262(M*-32),263(M +-31),294(M+) 222(M+-74),264(M*-32),265(M'-31 ),296(M +~ 74,75,87,101,115,129,143,157,185,255(M+-43),267(M +31),269(M+-29),298(M *) 353(M÷-33),358(M+-18),37 I(M ÷-15),386(M ÷)

Principal f r a g m e n t s (m/e)

~Retention t i m e relative to n18 : 1 (as m e t h y l e s t e r derivatives). bValues are m e a n s of 2 d e t e r m i n a t i o n s . F a t t y acids a r e d e s i g n a t e d by chain l e n g t h and t h e n u m b e r of double bonds. A b b r e v i a t i o n used: (i) iso branched.

18:0

n14 : 0

Fatty acid

1

Peak no.

traces 4 10 2 2 37 26 18 15 3.5

6 6 2

45 32 7 13 11

1

AmB

1

1

Control

% of total fatty acids methyl esters b

F a t t y acid m e t h y l e s t e r s w e r e e s t i m a t e d from GLC-MS (Figure not shown) and v a l u e s w e r e also confirmed by Figs. 3A and 3B.

E F F E C T OF A M P H O T E R I C I N B ON T H E M E T H Y L E S T E R S OF F A T T Y ACIDS A N D S T E R O L F R O M T O T A L LIPIDS OF T. CRUZI

T A B L E III

¢O ¢D

n12 : 0 n14 : 0 n15 : 0 i16 : 1 n16 : 0 n18 : 2 n18 : 1 n18 : 0 n24:30 cholesterol n18 : n18 : 2 n18 : 0

la 2a 3a 4a 5a 6a 7a 8a 9a 10a

0.56 0.79 0.88 0.94 0.96 1.08 1.09 1.11 1.42 1.67

RRT"

73,117,132,145,229(M*43),257(M*-15),272(M÷) 73,117,132,145,257(M*43),285(M..15),300(M ÷) 73,117,132,145,271(M*_43),299(M._15),314(M ÷) 73,117,132,145,283(M÷_43),311(M._15),326(M ÷) 73,117,132,145,285(M÷43),313(M÷ 15),328(M .) 73,117,132,337(M÷_15),352(M .) 73,117,132,145,311(M÷_43),339(M÷.15),354(M+ ) 73,117,132,145,313(M÷_43),341(M+ 15),356(M .) 73,117,132,145,397(M÷_43),425(M÷.15),440(M ÷) 129,329,353,369(M÷_90),443,443,458(M.I

Principal f r a g m e n t s (re~e)

•Relative r e t e n t i o n t i m e to n18 : 1 (as m e t h y l e s t e r derivative). ~n18 : 1 + n18 : 2/n18 : 0 = (% w e i g h t n18 : 1 + n18 : 2/% w e i g h t n18 : 0).

Fatty acid

Peak No.

Values w e r e e s t i m a t e d from GLC-MS (Figs. 3A and 3B).

traces 1 1 5 10 45 22 15 1 13 4.46

Control

30 36 24 2 15 2.75

2 2 4

AraB

% of total f a t t y acid b

E F F E C T OF AraB ON T H E F R E E F A T T Y A C I D A N D S T E R O L F R O M T O T A L LIPIDS OF T. CRUZI A S D E T E R M I N E D BY T R I M E T H Y L SILYL D E R I V A T I Z A T I O N

T A B L E IV

101

7o!

<>

I

6.0 ¸ 5.0

o

'_0 4.0 E

.

~

•

j°

3.0

rn

U

I.O I

I

I

I

48

72 96 Hours Fig. 4. Effect of AmB on T. cruzi growth concentration of AmB (t~g • ml-~): O, 0; A, 0.02; I-3,

0.04; A, 0.06; e, 0.08; l , 0.1; tions.

, sodium deoxycholate(0.08). Values are mean of 4 determina-

lipid peroxidation [23] we see no evidence for lipid peroxidation in our preparations. The relative decrease in 18 : 2 (linolate) would have been associated with a relative increase in 18 : 1 (oleate) if the changes were due to peroxidation, since oleate is much more resistant than linolate to this effect. Oleate decreased in the esterified lipids and increased in the free fatty acids, but the net effect of summing both classes was a relative decrease in total cellular oleate. The data in Tables III and IV are fully consistent with inhibition of fatty acid desaturase activity by AraB. There may also have been stimulation of fatty acid esterification or inhibition of lipolysis to account for the relative decrease in free fatty acids. An interesting aspect of the chemistry of polyene antibiotics is their susceptibility to autoxidation [11,15,24,25]. Arab autoxidation results in the formation of carbon-centered free radicals [13]. Certain carbon-centered free radicals are known to react with oxygen forming peroxyl radicals that decompose to give superoxide anion [26]. A similar reaction could explain the superoxide anion formation detected in our experiments (Table II). However, the production of superoxide anion by incubation of AmB in buffer was very slow and very high concentration of the drug were required to detect this activity. This result in no way guarantees t h a t superoxide anion would be produced within the T. c r u z i cells, at the low concentrations required for cell toxicity. In this regard previous data on AraB-stimulated lysis of erythrocytes [10] did not distinguish between peroxidation-enhanced lysis and lysis-enhanced peroxidation.

102 ACKNOWLEDGEMENTS W e a r e g r a t e f u l t o D r . R. D o c a m p o f o r h e l p f u l d i s c u s s i o n s , t o Mr . L. H o g g e f o r G L C - M S d e t e r m i n a t i o n s a n d M a r i a d e F a t i m a S o a r e s for h e r technical assistance. This w o r k was s u p p o r t e d by G r a n t s from Conselho Nacional de Desenvolvimento C i e n t l f i c o e T e c n b l o g i c o (CNPq), the F i n a n c i a d o r a d e E s t u d o s e P r o j e t o s ( F I N E P ) a n d t h e C o n s e l h o de E n s i n o e P e s q u i s a da U F R J ( C E P G ) . REFERENCES 1 2

W.T. Butler, Pharmacology, toxicity and therapeutic usefulness of Amphotericin B, J. Am. Med. Assoc., 195 (1966) 127-131. S.A.P. Sampaio, J.T. Godoy, L. Paiva, N.L. Dillon and C.S. Lacaz, The treatment of american (mucocutaneous) leishmaniasis with Amphotericin B, Arch. Dermatol., 2 (1960) 195 203. F.A. Neva, D. Wyler and T. Nash, Cutaneous leishmaniasis. A case with persistent organisms after treatment in presence of normal immune response, Am. J. Trop. Med. Hyg., 28(3) (1979) 467--471. A.E. Horvath and C.H. Zierdt, The effect of Amphotericin B on Trypanosoma cruzi in vitro and in vivo, J. Trop. Med. Hyg., 77 (1974) 144--149. F.S. Cruz, J.J. Marr and R.L. Berens, Prevention of transfusion-induced Chagas' disease by Amphotericin B, Am. J. Trop. Med. Hyg., 29(5) (1980) 761--765. B. Cover and W.E. Gutteridge, A primary screen for drugs to prevent transmission of Chagas' disease during blood transfusion, Trans. Roy. Soc. Trop. Med. Hyg., 76(5) (1982) 633635. S.M. Hammond, Biological activity of polyene antibiotics, Prog. Med. Chem., 14~1977) 105179. A.K. Saha, T. Mukherjee and A. Bhaduri, Mechanism of action of Amphotericin B on Leishmania donovani promastigotes, Mol. Biochem. Parasitol., 19(1986) 195-200. Y. Aracava, Y.C.P. Smith and S. Shreier, Effect of Amphotericin B on membranes: A spin probe study, Biochemistry, 20 (1981) 5702--5707. J. Brajburg, S. Elberg, D.R. Schwartz, A. Vertut-Croquin, D. Schlessinger, G.S. Kobayashi and G. Medoff, Involvement of oxidative damage in erythrocyte lysis induced by Amphotericin B, Antimicrob. Agents Chemother., 27(2) (1985) 172-176. M.C. Gutteridge and H. Thomas, Thiobarbituric acid reactivity of auto oxidized candicidin, Biochem. Med., 24 (1980) 194--200. R.W. Rickards, R.M. Smith and B.T. Golding, Macrolide antibiotics studies. XV. The autoxidation of the polyenes of the filipin complex and lagosin, J. Antibiot., 23 (1970) 603--612. M.T. Lamy-Freund, V.F.N. Ferreira and S. Schreier, Mechanism of inactivation of the polyene antibiotic Amphotericin B, evidence for radical formation in the process of autooxidation, J. Antibiot., 37 (1985) 753--757. A. Vertut-Croquin, J. Brajtburg and G. Medoff, Two mechanisms of synergism when Amphotericin B is used in combination with actinomycin D or 1-(2-chloroethyl)-3-cyclohexylLnitrosourea against the human promyelocytic leukemia cell line HL-60, Cancer Res., 46 (1986) 6054-- 6058. J.M. Gutteridge, A.H. Thomas and A. Cuthbert, Free radical damage to polyene antifungal antibiotics: changes in biological activity and thiobarbituric acid reactivity, J. Appl. Biochem., 5 (1983) 53--58. M. Sokol-Anderson, J. Brajtburg and G. Medoff, Amphotericin B-induced oxidative damage and killing of Candida albicans, J. Infect. Dis., 154(1986) 76-83. L. Warren, Metabolism of Schyzotrypanum cruz~ Chagas I, Effect of culture age and substract concentration on respiratory rate, J. Parasitol., 46 (1960) 525--539. - -

3

4 5 6

7 8 9 10

11 12 13

14

15

16 17

103 18 19 20

21 22

23 24 25

26

G. 0dham and E. Stenhagen, Fatty acids, in: G.R. Waller (Ed.), Biochemical Applications of Mass Spectrometry, John Wiley and Sons, New York, 1972, pp. 211--228. J.A. McClaskey, Mass spectrometry of lipids and steroids, Methods Enzymol., 14 (1969) 382 -450. A.B. Vermelho, L. Hogge and E. Barreto-Bergter, Isolation and characterization of a neutral glycosphingolipid from the epimastigote form of Trypanosoma mega, J. Protozool., 33(1986) 208--213, J.A. Buege and S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol., 52 (1978) 302 --310. B.M. Babior and H.J. Cohen, Measurement of neutrophil function: phagocytosis, degranulation, the respiratory burst and bacterial killing, in: M.J. Cline (Ed.), Leukocyte Function, Churchill Livingstone, New York, 1981, pp. 1 - 39. T.F. Slater, Overview of methods used for detecting lipid peroxidation, Methods Enzymol., 105 (1984) 283--293. W.H. Beggs, F.A. Andrews and G.A. Sarosi, Enhancement of Amphotericin B activity with vitamin C and sufhidryl containing compounds, FEMS Microbiol. Lett., 6 (1979) 409--411. L.V. Bronov, T.M. Kroshilova, V.Z. Slonim, G.G. Ivanov, E.V. Komarov and V.G. Krunchak, Study on mechanism of polyene antibiotics inactivation. Changes in physico-chemcial characteristics and destruction of levorin polyenic chromophore on inactivation, Antibiotiks, 33 (1983) 450--501 (In Russian). C. Mottley and R.P. Mason, An electron spin resonance study of free radical intermediates in the oxidation of indol acetic acid by horseradish peroxidase, J. Biol. Chem., 261 (1986) 16860-16864.