R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T ro p ic a l 17: 8 9 -9 3 , A b r -J u n , 1 9 8 4
ULTRASTRUCTURAL ALTERATIONS OF INTRACELLULAR STAGES
AND EFFECTS ON BLOOD FORMS OF
T
r y p a n o s o m aC R U Z I
INDUCED
IN VIVO
BY
2-AMINO-5-(l-METHYI^5-NITRO-2-IMIDAZOLYL)-l,3,4-THIADIAZOLE 1
Thaisa de Alm eida M aria2, Leny de Sousa Filardi
3
and Zigm an Brener
4
A n e le c tr o n m ic r o s c o p y s tu d y s h o w s th a t th e a d m in is tr a tio n o f a sin g le d o se ( 5 0 0 m g /k g , p .o .) o f 2 - a m in o - 5 - ( l- m e th y l- 5 - n itr o - 2 - im id a z o ly l) - l, 3, 4 -th ia d ia z o le in-d u c e s in m ic e in fe c te in-d w ith T r y p a n o s o m a c r u z i r e su lts in d e g e n e ra tiv e le s io n s o f th e in tr a c e llu la r sta g es. U ltr a s tr u c tu r a l a lte r a tio n s a re d e te c te d a s e a r ly a s 6 h o u r s a fte r th e d r u g a d m in is tr a tio n a n d d e s tr u c tio n o f th e p a r a s ite s o c c u rs w ith in 18 - 3 6 hours. T r y p o m a s tig o te s a re c le a r e d f r o m th e b lo o d s tr e a m 4 to 6 h o u r s a fte r tr e a tm e n t. T h e c o m b in e d e ffe c t o n b o th d e v e lo p m e n ta l s ta g e s is a p p a r e n tly r e sp o n sib le f o r th e in v iv o
e jfe c ts o f th is d r u g w h ic h is th e m o s t a c tiv e d r u g e v e r te s te d in o u r la b o r a to r y in e x p e r im e n ta l C h a g a s ’ d ise a se .
K e y w o r d s : T r y p a n o s o m a c ru zi. N i t r o im id a z o - th ia d ia z o le . E l e c t r o n m ic r o s c o p y .
R e c e n t in v e s tig a tio n h a s d e m o n s t r a t e d a re - m a r k a b le c u r a tiv e e f f e c t o f 2 - a m in o -5 -1 ( l - m e t h y l - 5 -
n i t r o - 2 - i m i d a z o l y l ) - l , 3 , 4 - t h ia d ia z o le in m ic e e x p e -
r im e n ta lly i n o c u la te d w it h T r y p a n o s o m a c r u z fi. T h e d r u g h a s b e e n f u r th e r in v e s tig a te d in viv o a n d w e n o w r e p o r t th e fin e s tr u c tu r e a l t e r a tio n s o f T. c r u z i in tr a c e l l u l a r s ta g e s in t r e a t e d a n im a is a s w e ll a s a n
in te n s e s u p p r e s s iv e a c tio n o n c ir c u la tin g b lo o d fo rm s in d u c e d b y th e c o m p o u n d . T h e e a r l y a n d in te n s e
a c tiv ity o f th is d r u g o n b o th p a r a s i t e s ta g e s e x p la in s its u n u s u a l h ig h c u r a tiv e e f f e c t in e x p e r im e n ta l C h a g a s ’
d is e a s e .
M A T E R I A L S A N D M E T H O D S
U ltr a s tr u c tu r a l stu d ie s . M a le a lb in o m ic e w e ig h in g 18 - 2 0 g w e re i n tr a p e r ito n e a l ly i n o c u la te d w ith 5 x 1 0 4 b lo o d tr y p o m a s tig o te s o f th e C o lo m b ia n a
s t r a i n 1. 2 - a m in o - 5 - ( 1 - m e th y l- 5 - n itr o - 2 - im id a z o ly l) -
1, 3 , 4 - t h ia d ia z o le ( C L 6 4 ’8 5 5 , “ M e g a z o l” ) w a s g i v e n b y o r a l r o u te in a s in g le d o s e o f 5 0 0 m g /k g , 1 2 d a y s
a f te r in o c u la tio n . U n tr e a te d m ic e in o c u la te d w ith th e s a m e in o c u lu m w e r e u s e d a s C o n tro ls. B o th tr e a te d a n d
c o n tro l a n im a is w e re s a c rific ed 6 , 1 2 , 1 8 , 2 4 a n d 3 6 h o u rs
1. S u p p o r t e d b y C N P q - F I N E P .
2 . D e p a r t a m e n t o d e M o r f o lo g ia , I C B / U F M G . 3 . D e p a r t a m e n t o d e Z o o lo g i a , I C B / U F M G .
4 . C e n t r o d e P e s q u i s a s R e n é R a c h o u , F I O C R U Z , A v . A u g u s to d e L im a , 1 7 1 5 - C .P . 1 7 4 3 , 3 0 .0 0 0 - B e lo H o r iz o n t e , B ra s il ( A d d r e s s fo r C o r r e s p o n d e n c e ) .
R e c e b id o p a r a p u b l i c a ç ã o e m 1 5 / 1 2 / 8 3 .
a f te r t r e a tm e n t. T h e h e a r t w a s r e m o v e d a n d fix e d in 2 .5 % g lu ta r a ld e h y d e in 0 . 1 M p h o s p h a te b u ffe r. T h e
o r g a n w a s s e c tio n e d a t t h e le v e i o f th e a tr io - v e n tr ic u la r s e p tu m ; th e a t r i a w e r e t h e n k e p t fo r 1 2 h o u r s a t 4 ° C in th e g lu ta r a ld e h y d e s o lu ti o n , p o s t- f ix e d w ith 1 % o s m iu n te tr o x id e in 0 . 1 M p h o s p h a te b u ff e r fo r 3 h o u rs ,
d e h y d r a t e d in a lc o h o l s e ri e s a n d , f in a lly , e m b e d d e d in E p o n . U l t r a t h i n s e c ti o n s w e r e o b ta in e d in a M T
2
P o r t e r B lu m u ltr a - m ic r o to m e , s ta in e d in u r a n il a c e ta te a n d R e y n o l d ’s s ta in , a n d e x a m in e d in a Z e i s s E M - 1 0
e le c tr o n m ic r o s c o p e .
E ffe c t on b lo o d s tr e a m f o r m s . T h e te c h n iq u e d e s c r i b e d b y F i l a r d i & B r e n e r ? w a s u s e d , a s fo llo w s: m a le a lb in o , w e ig h in g 18 - 2 0 g , w e r e i n o c u la te d w ith 1 x 1 0 5 b lo o d s t r e a m f o r m s o f s tr a in s C o lo m b ia n a1, V I ^ I O 1 1 a n d C L3. A t th e p e a k o f p a r a s i t e m i a (w h ic h w a s a t d a y s 9 - 1 1 w it h C L s t r a i n a n d 15 - 2 0 w ith
C o lo m b ia n a a n d V L - 1 0 s tr a in s ) th e a n im a is re c e iv e d a s in g le d o s e o f 5 0 0 m g /k g o f th e d r u g b y o r a l r o u te . A
g ro u p o f in o c u l a t e d b u t u n t r e a t e d a n im a is w a s u s e d a s
c o n tr o l. T h e p a r a s i t e m i a w a s d e te r m in e d a c c o r d in g to
B r e n e r 2 b e f o r e a n d 6 h o u r s a f te r d r u g a d m in is tr a ti o n .
T h e r e d u c tio n in th e n u m b e r o f p a r a s ite s w a s e s- ti m a te d b y c o m p a r in g th e n u m b e r o f p a r a s i t e s a f te r
t r e a t m e n t w it h t h a t d e t e c t e d b e f o r e d r u g a d m in is tr a tio n .
R E S U L T S
A r e m a r k a b le f in d in g o f th e u ltr a s tr u c tu r a l
s tu d y w a s th e o c c u r r e n c e o f e x tr e m e ly e a r ly a lte r a
Ma r ia T A, F i l a r d iL S , B r e n e r Z . U ltr a s tr u c tu r a la lte r a tio n s o f in tr a c e llu la r s ta g e s a n d e ffe c ts on b lo o d f o r m s o f T r y p a n o s o m a c r u z i in d u c e d in v iv o b y 2 - a m in o - 5 - ( l- m e th y l- 5 - n itr o - 2 - im id a z o ly l) - l, 3, 4 -th ia d ia z o le . R e v is ta d a S o c ie d a d e B r a s ile ir a de M e d ic in a T r o p ic a l 17: 8 9 -9 3 , A b r-J u n , 1 9 8 4
( F ig . 1) a n u m b e r o f u l t r a s t r u c t u r a l a l t e r a tio n s w e r e
o b s e r v e d a s s o o n a s 6 h o u r s a f t e r d r u g a d m in is tr a tio n in th e tr e a te d a n im a is . T h e in itia l le s io n s w e r e v a c u o -
li z a ti o n o f th e c y to p la s m f o llo w e d b y s w e llin g o f th e n u c le a r e n v e lo p e , ro u g h e n d o p la s m ic r e tic u lu m a n d G o lg i c is te m s ( F ig . 2 ); a s im ila r s w e llin g w a s d e te c te d
in th e a r e a a r o u n d th e fib rils n e tw o r k o f th e k in e to p la s t. T h e s e u l tr a s tr u c tu r a l c h a n g e s a r e p r o g r e s s iv e ly m o r e
s e v e re in th e a n im a is s a c r if ic e d a t th e f u r th e r p e r io d s o f
e x a m in a tio n a f te r t r e a tm e n t, s h o w in g a c l e a r r e d u c tio n o f th e n u m b e r o f r ib o s o m e s a n d o t h e r c y to p la s m ic
c o m p o n e n ts a f te r 18 h o u r s ( F ig . 3 ). E v e n in th o s e d e g e n e r a te d p a r a s i t e s s o m e s tr u c tu r e s r e m a in a p p a -
r e n tl y i n t a c t s u c h a s th e s u b - p e llic u la r m ic r o tu b u le s , p la s m a tic m e m b r a n e a n d th e k i n e t o p l a s t fib ril n e tw o rk . A t 3 6 h o u r s d e g e n e r a te d fo rm s d is p la y in g
p a r t i a l o r to ta l ly s is a r e h i g h ly p r e d o m i n a n t ( F i g . 4 ) . I n th e u n tr e a te d c o n tr o l g ro u p th e g r e a t m a jo r it y o f th e
in tr a c e llu la r s ta g e s r e m a in e d m o r p h o lo g ic a l n o r m a l.
t .
.
; \ *■ * ' ■ ■ ;r ' í ■ - N ■ ,„r .
F ig. 2 - T r e a t e d , 6 h o u r s . T. c r u z i in t r a c e l lu l a r a m a s ti g o te s s h o w in g e a r l y v a c u o li z a ti o n o f th e c i s t e m s o f th e e n d o p la s m ic r e tic u lu m , n u c le a r e n v e lo p e a n d k in e to p la s t. X 5 ,7 0 0 .
Ma r ia T A , F i l a r d i L S , B r e n e r Z . U ltr a s tr u c tu r a l a lte r a tio n s o fin tr a c e llu la r sta g e s a n d effects on b lo o d fo r m s o / T r y p a n o s o m a c ru z i in d u c e d in v iv o b y 2 - a m in o - 5 - ( l- m e th y l- 5 - n itr o - 2 - im id a z o ly l) - l, 3, 4 -th ia d ia z o le . R e v is ta d a S o c ie d a d e B ra sile ir a d e M e d ic in a T r o p ic a l 17: 8 9 -9 3 , A b r-J u n , 1 9 8 4
F ig 3 - T r e a t e d , 18 h o u r s . T. c r u z i in t r a c e l l u l a r a m a s ti g o te s w ith P ro g r e s s i v e le s io n s o f th e m e m b r a n o u s s t r u c tu r e s o f th e e n d o p la s m ic re t ic u l u m a n d n u c l e a r e n v e lo p e ; a n d r a r e f a c ti o n o f th e c y to p l a s m ic c o m p o n e n t s . X 1 3 ,0 0 0 .
F ig . 5 s h o w s th e e f f e c ts o f C L 6 4 ’8 5 5 o n th e
p a r a s i t e m i a in m ic e in o c u la te d w it h T. c r u z i s tr a in s
V L -1 0 , C o lo m b ia n a a n d C L . A m a r k e d a n d ra p id
d e c lin e in th e n u m b e r o f c ir c u la ti n g tr y p o m a s ti g o te s w a s o b s e r v e d a s s o o n a s 6 h o u r s a f te r d r u g a d m in is -
tr a tio n , s h o w in g t h a t th e e a r ly e f f e c ts o n b lo o d fo rm s p a r a lle l t h a t d e s c r i b e d fo r th e in tr a c e llu la r s ta g e s .
* " •
t:
V ■'
*
. v
. ^
■--*>
^
m
- - i
F ig 4 - T r e a t e d , 3 6 h o u rs . T. c r u z i d e g e n e r a t e d a m a s tig o te s . L y s is o f m e m b r a n e s a n d c y to p l a s m ic c o m p o n e n ts . X 1 0 .7 0 0 .
H o u r s a f t e r t r e a t m e n t
Ma r ia T A , F i l a r d i L S , B r e n e r Z . U ltr a str u c tu ra l a lte r a tio n s o f in tr a c e llu la r sta g e s a n d ejfe c ts on b lo o d f o r m s o f T r y p a n o s o m a c r u z i in d u c e d in v iv o by 2 - a m in o - 5 - ( l- m e th y l- 5 - n itr o - 2 - im id a z o ly l) - l, 3, 4 -th ia d ia zo le . R e v is ta d a S o c ie d a d e B r a s ile ir a de M e d ic in a T r o p ic a l 17: 8 9 -9 3 , A b r-J u n , 1 9 8 4
D I S C U S S I O N
T h e c o m p o u n d 2 - a m i n o - 5 - ( l - m e t h y l - 5 - n i t r o - 2 - i m i d a z o l y l ) - l , 3 , 4 - t h ia d ia z o le is a p p a r e n tly th e m o s t a c tiv e d r u g s o f a r te s te d a g a in s t T. c r u z i e x p e rim e n ta l in fec tio n s® . A c c o r d in g to o u r d a t a it c u r e s 8 5 - 1 0 0 % o f th e i n o c u la te d a n im a is t r e a t e d f o r 2 0
d a y s w ith d o s e s o f 5 0 - 1 0 0 m g /k g ; s in g le d o s e s o f 5 0 0 m g /k g c u r e 8 8 .8 % o f m ic e i n o c u la te d w ith th e
T. c r u z i Ys tr a in . M o r e o v e r , i t a l s o c u r e s 8 5 - 1 0 0 % o f th e a n im a is i n o c u la te d w ith s tr a in s C o lo m b ia n a a n d V L - 1 0 w h ic h a r e h ig h ly r e s i s t a n t to n it r o f u r a n a n d
2 - n it r o im id a z o l d e r i v a t i v e s 1 11. I n th is p a p e r w e d e s c r ib e th e e ff e c ts o f th is d r u g o n b o th i n tr a c e llu la r
a n d c ir c u la ti n g T. c r u z i s ta g e s . T h e e a r ly a n d m a r k e d e ffe c ts o f s in g le d o s e s a p p a r e n t l y p r o v id e a n e x -
p la n a ti o n fo r h ig h c u r a ti v e r a te s d e te c te d a f te r s in g le d r u g a d m in is tr a tio n a n d s tro n g ly in d ic a te a n ir re v e r- s ib le c u r a tiv e a c tio n o f th is c o m p o u n d .
T h e p a t t e r n o f th e in tr a c e l l u l a r p a r a s i t e d e s - tr u c tio n w ith c o m p o u n d C L 6 4 ’8 5 5 s e e m s to b e
s im ila r to t h a t fo u n d in a n im a is o r in tis s u e c u lt u r e tr e a te d w ith n itr o f u r a z o n e 4 a n d n i f u r t i m o x 1 2 , B a s i-
c a lly , th e y c o n s is t o f a n in c r e a s e in th e n u m b e r o f c y to p la s m ic v a c u o le s , r e d u c tio n o f th e ri b o s o m e s a n d
s w e llin g o f th e o r g a n e ll e s . T h e p r o c e s s e n d s in th e ly s i s o f th e p a r a s i t e s w ith p e r s is te n c e o f th e p la s m a m e m b r a n e a n d th e k in e to p la s t. T h e m o le c u la r m e -
c h a n is m o f th e le s io n s in d u c e d b y C L 6 4 ’8 5 5 is n o t y e t k n o w n . D o c a m p o e t a l^ d e m o n s t r a t e d t h a t in c u b a tio n
o f T. c r u z i h o m o g e n a te s w ith a n itr o f u r a n d e r iv a ti v e ( “ n if u rt im o x ” ) f a v o r s th e a p p e a r a n c e o f a n it r o a - r o m a tic a n io n r a d ic a l w h ic h r e a c t s w ith o x y g e n a n d
in d u c e s a n in c r e a s e d r a te p r o d u c tio n o f s u p e r o x id e a n io n a n d h y d r o g e n p e r o x id e , w h ic h a re in v o lv e d in
th e t r y p a n o c id a l e ffe c t. B e n z n id a z o le ( a n it r o im id a - z o le d e r iv a tiv e ) , h o w e v e r , is n o t r e d u c e d to th e n itr o
a n io n r a d ic a l a n d d o e s n o t in d u c e g e n e r a tio n o f s u p e ro x id e a n d h y d r o g e n p e ro x id e . T h is s u g g e s ts a
d iff e re n t ( b u t n o t y e t d e f in e d ) m e c h a n is m o f to x ic it y
fo r n it r o im id a z o le d e r iv a tiv e s 1®. S in c e C L 6 4 ’8 5 5 is a n itr o im id a z o le w ith a th ia d ia z o le c o m p o n e n t, th e
m e c h a n is m o f th is n o v e l c o m p o u n d s h o u ld b e s p e - c ific a lly in v e s tig a te d in c o m p a r is o n w ith th e n itr o f u r a n
a n d n it r o im id a z o le a c tiv e c o m p o u n d s .
T h e e f fe c t o f d ru g s o n b lo o d fo r m s h a s b e e n le s s
in v e s tig a te d . H a b e r k o m & G õ n n e rt® u s i n g a s p e c ia l re fle x m ic r o s c o p e o b s e r v e d in liv in g b lo o d t r y p o m a s
tig o te s fr o m m ic e t r e a t e d w ith n if u r ti m o x le s io n s s u c h as c y to p la s m v a c u o liz a tio n a n d m e m b r a n e sp littin g . I n
o u r e x p e r im e n ts th e d e c lin e in th e n u m b e r o f c ir
c u la tin g tr y p o m a s tig o te s o c c u r s v e r y s o o n a f te r d r u g
a d m in is tr a tio n a n d p a r a lle ls th e e a r ly a c tio n a g a in s t in tr a c e l l u l a r s ta g e s . D a t a fr o m o u r l a b o r a t o r y s h o w
t h a t m a c r o p h a g e s p la y a n im p o r t a n t r o le in th e c l e a r a n c e o f b lo o d p a r a s i t e s b y re m o v in g th e c ir
c u la ti n g t r y p o m a s tig o te s in a p r o c e s s w h ic h b e g in s v e r y s o o n a f te r th e d ru g a d m in is tr a tio n ^ . T h e e f f e c t o n
b o t h i n tr a c e llu la r a n d c ir c u la tin g s ta g e s is r e s p o n s ib le f o r t h e r a p id a n d e x tr e m e ly in te n s e a c tiv ity o f th is
c o m p o u n d .
R E S U M O
U m e stu d o c o m m ic ro sc o p ia e le tr ô n ic a m o s tr o u q u e a a d m in is tr a ç ã o d e d o se ú n ic a ( 5 0 0 m g /k g , p .o .) d o c o m p o s to 2 - a m in o - 5 - ( l- m e til- 5 - n itr o - 2 -im i-d a z o lil) - l, 3, 4 - tia i-d ia z o le in i-d u z, em c a m u n i-d o n g o s in o c u la d o s c o m T . c ru z i, le sõ e s d e g e n e r a tiv a s d a s f o r m a s in tr a c e lu la r e s d o p a r a s ita . A lte r a ç õ e s u ltra e s tr u tu r a is s ã o o b s e r v a d a s 6 h o r a s a p ó s a a d m in is tr a ç ã o d a d ro g a e d e s tr u iç ã o o corre em 1 8 - 3 6 horas. T r ip o m a s tig o ta s ta m b é m d e sa p a re c e m d o sa n g u e c ir c u la n te d o s a n im a is 4 - 6 h o r a s a p ó s o tr a ta m e n to . O e fe ito e m a m b o s o s e s tá g io s e v o lu tiv o s d o T . c r u z i é a p a r e n te m e n te r e s p o n s á v e l p e lo s efe ito s in v iv o d e sse d e r iv a d o q u e é o m a is a tiv o c o m p o s to j á te sta d o em n o sso la b o ra tó rio n a d o e n ç a d e C ha ga s.
P a l a v r a s c h a v e s : T r y p a n o s o m a c r u z i. N i- tr o im id a z o l - T ia d ia z o le . M ic r o s c o p i a e l e tr ô n ic a .
R E F E R E N C E S
1. A n d r a d e S G , F ig u e i r a R M . E s t u d o e x p e r i m e n t a l s o b r e a a ç ã o te r a p ê u t i c a d a d ro g a R o 7 - 1 0 5 1 n a in f e c ç ã o p o r d if e r e n te s c e p a s d o T r y p a n o s o m a c r u z i. R e v is t a d o I n s tit u to d e M e d i c in a T r o p ic a l d e S ã o P a u lo 19: 3 3 5 - 3 4 1 , 1 9 7 7 .
2 . B r e n e r Z . T h e r a p e u t i c a c ti v it y a n d c r i te r io n o f c u re o n m ic e e x p e r i m e n t a ll y in f e c te d w ith T r y p a n o s o m a c ru z i. R e v is t a d o I n s tit u to d e M e d i c in a T r o p i c a l d e S ã o P a u lo 4: 3 8 9 - 3 9 6 , 1 9 6 2 .
3 . B r e n e r Z , C h ia r i E . V a r ia ç õ e s m o r fo ló g ic a s o b s e r v a d a s e m d if e r e n te s a m o s tr a s d o T r y p a n o s o m a c r u z i. R e v is t a d o I n s tit u to d e M e d i c in a T r o p i c a l d e S ã o P a u l o 5: 2 2 0 - 2 2 4 , 1 9 6 3 .
4 . B r e n e r Z , T a f u r i W L , M a r i a T A . A n e le c tr o n m i c ro s c o p e s tu d y o f T r y p a n o s o m a c r u z i i n t r a c e l lu l a r fo rm s in m ic e tr e a t e d w ith a n a c ti v e n it r o f u r a n c o m p o u n d . R e v is t a d o I n s tit u to d e M e d i c in a T r o p ic a l d e S ã o P a u lo 11: 2 4 5 - 2 4 9 , 1 9 6 9 .
ni-Ma r ia T A, F ila r d i L S , B r e n e r Z . U ltr a s tr u c tu r a l a lte r a tio n s o f in tr a c e llu la r sta g e s a n d effec ts on b lo o d f o r m s o f T r y p a n o s o m a cru z i in d u c e d in v iv o b y 2 - a m in o - 5 -(l-m e th y l- 5 -n itr o ~ 2 -im id a z o ly l)-l, 3, 4 -th ia d ia z o le . R e v is ta d a S o c ie d a d e B ra sile ira d e M e d ic in a T r o p ic a l 17: 8 9 -9 3 , A b r-J u n , 1 9 8 4
fu r tim o x to x i c it y in d if f e r e n t fo r m s o f T r y p a n o s o m a c ru z i. B io c h e m is t r y P h a r m a c o lo g y 3 0 : 1 9 4 7 - 1 9 5 1 ,
1 9 8 1 .
6 . F il a r d i L S . B r e n e r Z . A n it r o i m i d a z o le - t h ia d i a z o le d e riv a tiv e w ith c u r a tiv e a c ti o n in e x p e r i m e n t a l T ry p a n o s o m a c r u z i in f e c ti o n s . A n n a ls o f T r o p ic a l M e d i c in e a n d P a r a s ito l o g y 7 6 : 2 9 3 - 2 9 7 , 1 9 8 2 .
7 . F i l a r d i L S , B r e n e r Z . A r a p id m e th o d fo r te s ti n g in vivo th e s u s c e p tib i li ty o f d if f e r e n t s tr a in s o f T r y p a n o s o m a c r u z i to a c ti v e c h e m o t h e r a p e u t ic a g e n ts . M e m ó r ia s d o I n s tit u to O s w a l d o C r u z ( in p re s s ) .
8 . H a b e r k o m A , G õ n n e r t R . A n im a l e x p e r i m e n t a l in v e s ti- g a ti o n s in th e a c ti v it y o f n i f u rt im o x a g a in s t T ry p a n o s o m a c r u z i. A rz n e im it te l F o r s c h u n g 2 2 : 1 5 7 0 - 1 5 8 1 ,
1 9 7 2 .
9 . L a g e s - S ilv a E , F il a r d i L S , G i l b e r t M E , G ilb e r tB , B re n e r Z . P a r t ic i p a ti o n o f m a c r o p h a g e s in th e c le a r a n c e o f
T r y p a n o s o m a c r u z i fro m th e b lo o d o f m ice s u b m itte d to s p e c i fic tr e a tm e n t. X R e u n iã o A n u a l s o b re P e s q u is a B á s ic a e m D o e n ç a s d e C h a g a s , C a x a m b ú , n o v e m b ro 1 9 8 3 .
10. M o r e n o S N J , D o c a m p o R , M a s o n R P , L e o n W , S to - p a n n i A O M . D if f e r e n t b e h a v io u r o f b e n z n id a z o le a s free r a d i c a l g e n e r a t o r w ith m a m m a l ia n a n d T ry p a n o s o m a c r u z i m ic r o s o m a l p r e p a r a ti o n s . A rc h iv e s o f B io c h e m is tr y a n d B io p h y s ic s 2 1 8 : 5 8 5 - 5 9 1 , 1 9 8 3 .
11 . S c h le m p e r J r B R . C a r a c t e r i z a ç ã o d e c e p a s d o T ry p a n o s o m a c r u z i is o la d a s d e p a c ie n te s c o m d if e re n te s fo r m a s c li n ic a s d a d o e n ç a d e C h a g a s . T e s e d e D o u to ra d o , U n iv e r s id a d e F e d e r a l d o R io d e J a n e i r o , R io d e J a n e i r o , 1 9 8 2 .
