R e v is ta d a S o c ie d a d e B r a s il e ir a d e M e d ic in a T r o p ic a l 2 7 ( 4 ) : 2 0 9 - 2 1 5 , o u t- d e z , 1 9 9 4 .
BIOCHEMICAL BEHAVIOR OF
T R Y P A N O S O M A C R U Z ISTRAINS ISOLATED FROM MICE SUBMITTED TO SPECIFIC
CHEMOTHERAPY
Je sila P into M . M a rre tto a n d Sonia G. A n d rad e
To in vestig a te th e influence o fch em o th e ra p y on th e b io ch em ica l b eh a vior o f T rypanosoma cruzi s tr a in s , th r ee g ro u p s o f m ice w ere infected w ith o n e o f th ree s tr a in s o f T. cruzi o f d iffe re n t b io lo g ica l a n d iso en z ym ic p a tte r n s (P eruvian, 2 1 S F a n d C olom bian s tra in s ). E a ch g r o u p w a s s u b d ivid e d in to s u b g ro u p s : 1 - tr ea ted w ith n ifurtim o x; 2 - tr e a te d w ith b e n z n id a z o le a n d 3 - u n trea ted infected con trols. A t the en d o f trea tm en t, th a t la s ted f o r 9 0 d a y s , x e n o d ia g n o s is , s u b ino culatio n o f b lo o d into n ew b o r n m ice a n d h a em o c u ltu r e w er e p e r fo r m e d a s tes ts o f cure. F rom the p o s itiv e tests, 2 2 s a m p les o f T . cruzi w ere is o la te d fr o m
a lls u b g r o u p s . E lec tr op h o r etic a na lys is o f the iso en z ym es PG M , GP1, A L A T a n d A S A T f a i le d to s h o w a n y d iffe r en ce b etw een p a r a s ite s tr a in s iso la ted fr o m tr ea ted a n d u n tre a ted m ic e , w h ich ind ica tes th a t no d etec ta b le c lo n a l s elec tio n o r p a r a s ite g e n e tic m a r k e r s a lter a tio n s c o n c e r n in g the iso en zy m e s a n a lys ed h a ve b een d e te rm in e d by tr e a tm e n t w ith d r u g s o f r e c o g n iz e d a n tip a r a sitic effect, su g g es tin g stab ility o f the p h e n o ty p ic c h a ra c te r is tic s o f th e th r e e b io lo g ic a l ty p e s o f T . cruzi strains.
K ey-w o rd s: Trypanosoma cruzi strains. Isoenzym es. C hem otherapy.
D u rin g e x p e rim e n ta l c h e m o th e ra p y o f T r y p a n o s o m a c r u z i infection in mice, parasite strains show different susceptibility to the drugs now in clinical use, benznidazole and nifurtimox3 5 10. There is good correlation between these responses and the biological behavior o f the T. c r u z i strain6. H owever, a rapid decrease o f parasitemia occurs during treatment, even in the presence o f the most resistant strains, indicating that only a few parasites are really resistant to the drugs. A selection of resistant clones, that could differ in some way from the original strain has been suggested to explain the persistence o f parasites in the vertebrate host after p r o lo n g e d tr e a tm e n t5. A lth o u g h p re v io u s investigations have stressed the stability o f biological and biochemical patterns o f laboratory strains220 21, c lo n a l a n a ly s is r e v e a ls h o m o g e n e o u s an d heterogeneous populations1119. Therefore, selected clones could persist after chemotherapy.
The present investigation was planned to identi fy possible biochemical differences between strains
C e n tro de P e sq u isa s G o n çalo M o n iz , F u n d a ç ã o O sw ald o C ru z , U n iv e rsid a d e F e d e ra l d a B a h ia , S a lv a d o r, B A .
A d d r e s s t o : D r a S o n ia G . A n d ra d e . C e n tro d e P esq u isas G o n ç a lo M o n iz /F IO C R U Z /U F B A . R . V a ld e m a r F alcão 121 B ro tas, 4 0 2 9 5 -0 0 1 S a lv a d o r, B A , B rasil.
R e c e b id o p a ra p u b lic a ç ã o em 2 5 /0 3 /9 4 .
isolated from treated mice, that could be traced to alterations o f the parasite genetic markers. The investigation on the possibility of a selection of different clones by the action o f drugs seems important not only for drug therapy, but also to test the concept of stability o f the biochemical patterns o f T. c r u z i strains.
M ATERIAL AND M ETHODS
M a r r e to J P M , A n d r a d e SG . B io c h e m ic a l b e h a v io r o f Trypanosoma cruzi s tr a in s is o la te d f r o m m ic e s u b m it te d to s p e c ific c h e m o th e r a p y . 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 r o p ip a l 2 7 :2 0 9 -2 1 5 , o u t- d e z , 1 9 9 4 .
Parasitem ia and mortality were evaluated during the experim ent. Table 1 shows general data about the experimental groups, such as the number of animals, inocula, treatment and cure rates for each group. Surviving animals belonging to the treated groups were evaluated three months after the end of treatment by the following parasitological cure te sts: d ire c t p a ra s ite m ia (p e rip h e ra l b lo o d exam ination under cover slides 22x22 in 50 m icroscopic field s, 400X ); haem oculture in W arren’s medium; xenodiagnosis with five 4th - 5th stage R h o d n i u s p r o l ix u s nymphs and subinoculation
o f b lo o d in to n e w b o rn m ice (O .lm g ,
intraperitoneally ).
B io c h e m ic a l c h a r a c t e r i z a t i o n . T h e electro p h o retic p attern s o f isoenzym es w ere determined for several samples o f each strain, individually isolated from treated non-cured mice and from untreated controls either directly from the blood o f positive animals or after haemoculture; from the blood o f mice used for subinoculation or from xenodiagnosis. In either case, the samples were inoculated into mice and then isolated by haemoculture into W arren medium for the obtaining of culture forms and enzymic extracts.
Twenty two samples o f T. c r u z i were isolated
from treated animals as follows: 1st - P eru vian s tra in (seven samples): 1 sample from mouse treated w ith n if u r tim o x ; 1 fro m m o use used fo r
subinoculation o f the blood o f animal treated with benznidazole; 1 from xenodiagnosis o f mouse treated w ith benznidazole; 4 from the peripheral blood of mice treated w ith benznidazole. 2nd - 21 SF s tra in (2 samples):, each one originating from one mouse treated w ith nifurtim ox, after subinoculation into newborn mice. 3rd - C o lom bian s tra in (thirteen samples): 4 samples individually isolated from the blood o f mouse treated w ith nifurtim ox; 1 sample from x e n o d iag n o sis o f m ouse tre a te d w ith nifurtimox; 8 samples from the blood o f mouse treated with benznidazole.
Controls. Three samples o f each strain obtained from untreated mice were also maintained in culture for isoenzymic study.
Cultivation in W arren medium was performed at 2 8 °C. Parasites were washed w ith KRT buffer solution and enzymic extracts w ere obtained according to Godfrey and Kilgour18, and maintained in liquid nitrogen as “pearls” according to M iles et al25.
Isoenzymes tested: alanine aminotransferase (ALAT) -E.C, 2.6.1.2 ; aspartate am inotransferase (A SA T )-E .C .2.6.1.1.; glucosephosphateisomerase (GPI) - E .C .5 .3 .1 .9 .; phosphoglucomutase (PGM) - E .C .2 .7.5 .1.
T hin-layer starch-gel electropho resis was perform ed by application o f 30V/cm , during 90 minutes for ALAT and 60 minutes for AS AT and o f 20V/cm during 150 minutes for GPI and 120 minutes
T a ble 1 - G e n e r a l d a ta on m ic e in fe c te d w ith Trypanosoma cruzi, tr e a te d w ith b e n z n id a z o le a n d n ifu r tim o x . E x p e r im e n ta l g ro u p s.
T. c r u z i strains (Type)
Inoculum (n° trip.)
Experimental groups of treatment
N° mice
Mortality indices (% )
Cure rates (%)
Peruvian 5 x 104 Treat, benz* 40 30 45.4
( I ) Treat, nifurt** 40 15 37.7
Untreated 20 100
-21 SF 1 x 10s Treat, benz 40 30 100.0
( I I ) Treat, nifurt 40 12 75.0
Untreated 20 100
-Colombian 1 x 105 Treat, benz 40 65 30.0
( H I ) Treat, nifurt 40 65 0.0
Untreated 20 100
-* Benz = benznidazole- 4 doses of200m g/kgb.w. followed by daily doses of50mg/kg b.w. during 90 days.
M a r r e t o J P M , A n d r a d e S G . B io c h e m ic a l b e h a v i o r o f T ryp an osom a cru zi s t r a in s i s o l a t e d f r o m m i c e s u b m i t t e d to s p e c i f i c c h e m o t h e r a p y . R e v i s t a d a S o c ie d a d e B r a s i le i r a d e M e d i c in a T r o p i c a l 2 7 : 2 0 9 - 2 1 5 , o u t - d e z , 1 9 9 4 .
for PG M . The enzymes A LA T and AS AT were developed w ith phosphate buffer solution 0.1M and beta N A D , and examined by ultra-violet light; for the enzymes GPI and PG M , TRIS/HC1 buffer solution 0.3M and N A D P w ere used besides the M TT (dimethilthiazole2-yl 2-5 dipheniltetrazolium bromide) 0.36m M , agar gel 0.6% and phenazine metasulfate, 0.03m M .
RESULTS
R esponse to chem otherap y - The cure rates for each strain o f mice treated either w ith nifurtimox or benznidazole are shown in Table 1. It was of 37.7 % for nifurtim ox and 45.4% for benznidazole in mice infected w ith Type I strain; Type II strain
disclosed 75 % and 100 % o f cure rates respectively,
for treatment w ith nifurtim ox and benznidazole; Type III strain showed 0% and 30% o f cure when tr e a te d r e s p e c tiv e ly w ith n if u r tim o x and benznidazole.
B iochem ical analysis -The electrophoretic profiles revealed for the isoenzymes ALAT, AS AT, PGM and GPI w ith the Peruvian strain corresponded to the zymodeme Z2a, according to Tybayrenc et al30. W ith th e2 1 SF strain and the Colombian strain these isoenzymes corresponded to Z2 and Z l, respectively, according w ith M iles et al24. No differences could be detected in the isoenzymic profiles for A LAT, ASAT, PGM and GPI o f the strains, in the several samples isolated from treated mice when compared w ith the control non-treated strains. Examples o f the isoenzymic electrophoretic patterns o f the three strains for the different enzymes are shown in Figures 1 to 5.
DISCUSSION
T he strains o f T. c r u z i are no t necessarily homogeneous populations and different clones can be d isc lo se d th a t d iffe r in th e ir an tig en ic com position11, virulence28, isoenzymic patterns16 19 3i o r the patterns o f kinetoplast DNA26. The clonal structure o f 7. c r u z i strains has been postulated by Tibayrenc et al31, by identification o f 43 genetically different clones from 121 strains isolated from different geographical areas and identified by isoenzymic profiles and dendogram analysis o f
F igu re 1 - G P I is o e n z y m e :a )th e P e r u v ia n s tr a in is o la te d f r o m u n treated c o n tr o b (PE R) o r f r o m an im als tr e a te d w ith b e n z n id a z o le (R O ), d is c lo s e d id e n tic a l e le c tr o p h o r e tic p a tte r n , b e lo n g in g to zy m o d e m e 2 a (Z2a); b ) th e C olom bia n s tr a in i s o l a te d f r o m m ic e t r e a t e d w ith b e n z n id a z o le (R O ) o r n ifu rtim o x (BA Y) s h o w e d th e s a m e e le c tr o p h o r e tic p a tte r n a s th e s tr a in is o la te d f r o m u n tr e a te d c o n tr o l c o r re s p o n d in g to z y m o d e m e 1 (Z l). In ea c h p la te , c o n tr o l s tr a in s a r e in c lu d e d (in a . C o l ( Z l) a n d 21 S F (Z2); in b , P E R (Z 2a) a n d 2 1S F (Z2).
M a r r e t o J P M , A n d r a d e S G . B io c h e m ic a l b e h a v i o r o f T rypanosom a cru zi s t r a i n s i s o l a t e d f r o m m ic e s u b m it te d to s p e c i f i c c h e m o t h e r a p y . R e v i s t a d a S o c ie d a d e B r a s i le i r a d e M e d i c in a T r o p i c a l 2 7 : 2 0 9 - 2 1 5 , q u t - d e z , 1 9 9 4 .
F igu re 2 - P G M - th e is o e n z y m ic p a tte r n s o fzy m o d e m e s Z 1 (C O L ), Z 2 (21 SF) a n d Z 2 a (PE R), a re sh o w n , a ) The C olom bia n str a in is o la te d f r o m m ic e tr e a te d w ith b e n zn id a zo le (RO ) m a in ta in e d th e s a m e p a tte r n d is c lo s e d b y th e u n tr e a te d s tr a in (C O L) c o r re sp o n d in g t o Z l ; b ) th e e le c tr o p h o r e tic p a tte r n s f o r th e P e ru v ia n s tr a in is o la te d f r o m tr e a te d m ic e (R O a n d BA Y) a r e sim ila r to th o se d is c lo s e d b y th e s tr a in fr o m u n tr e a te d c o n tr o l (PER).
the same zymodeme8 9 15 23 29 32. However; the stability o f biological characters o f strains maintained in laboratory, under well controlled conditions, suggests that there is an equilibrium o f the different p o p u la tio n s w ith in o n e s tra in , even a f te r m odifications o f environm ental conditions as passages into axenic cultures, cryopreservation in liquid nitrogen or passages in triatominae20 21. A lthough changes in zymodemes profiles and schizodeme patterns after passages o f strains in C3H m ice has been registered12, the stability o f the isoenzymic characters has also been shown for the Y strain cryopreserved for 6 and 7 years or after being isolated from treated m ice13 14.
T he drugs used in the present study have a know n intracellular action against T. c r u z i. The
F igu re 3 - A S A T a n d A L A T - The 21 S F s tr a in (Z2) m a in ta in ed th e e le c tr o p h o r e tic p r o f ile s f o r b o th is o e n z y m e s w h en is o la te d f r o m m ic e tr e a te d w ith n ifu rtim ox (BA Y) a s c o m p a r e d to c o n tr o l (21 SF). The p r o f ile s o f th e Z 2 a (PE R) a n d Z1 (C O L) a r e a ls o sh ow n .
strains differed in its susceptibility. The Type II strain (21 SF), biochemically characterized as zymodeme 2 according to M iles et al24, showed a high susceptibility, w ith cure rates o f 75 % w ith nifurtimox and 100 % w ith benznidazole. The m ost resistant one w as the C olom bian (T ype III), corresponding to zymodeme 1, showing 0 % o f cure w ith nifurtimox and 30.7% w ith benznidazole. Type I strain (Peruvian), which isoenzymic profile can be compared to Z2a described by Tibayrenc et
al30, showed cure-rates o f 37.7 % w ith nifurtim ox,
M a r r e to J P M , A n d r a d e S G . B io c h e m ic a l b e h a v io r o f Trypanosoma cruzi s tr a in s is o la te d f r o m m ic e s u b m itt e d to s p e c if ic c h e m o th e r a p y . 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 r o p ic a l 2 7 :2 0 9 -2 1 5 , o u t- d e z , 1994.
a a ASAT
mm
—
. . — —w m ■ ■ H l ■ ■
mm mm.
PE R 21 S F COL COL COL COL COL 21 SF COL PER PER PER PER PER PER B A Y RO RO R O RO RO RO RO RO
1 2 1 6 21 8 1 8 2 5 2 6 27 A S A T
b
b ALAT
m m
S “
mm
■ " “s s s s s S
£
___ ___
_ 21 SF COL PER PER PER PER PER PER PER 2 1 S C O L C O L C O L C O L Ç O L RO RO RO RO RO BAY RO RO RO 8 18 25 26 2712 1 6 21 A L A T
f ig u r e 4 - A S A T a n d A L A T - The C olom bian strain is o la te d f r o m m ic e tr e a te d e ith e r w ith n ifu rtim o x (B A Y ) o r b en z n id a z o le (RO) s h o w e d the s am e isoenzym ic p a tte rn ( Z l ) a s th e c o n tr o l s tra in (C O L), iso la ted fr o m u n trea ted m ice. The p ro files o f the Z 2 (21SF) a n d Z 2 a (PER) a r e show n.
Figure 5 - A S A T a n d A L A T - T h e i s o e n z y m i c electrophoretic p r ofiles o f the P eruvian strain (Z 2 a )for both iso en zym es a r e m a in ta in ed fo r the strain isolated fr o m m ice tr ea ted w ith b en zn ida zo le (RO -PER) a s c om p a re d with the c on tro l stra in iso lated fr o m u n trea ted m ice (PER). The p ro file s o f the Z 1 (COL) a n d Z 2 (21 SF) s tra in s a re a b o show n.
s e le c tio n w a s th e n e v id e n t. H o w e v e r, th e investigation o f the post-treatment biochemical behavior o f the strains here studied did not reveal consistent differences that could be correlated with clone selection. Biochemical analysis confirmed that no phenotypic differences, at least for the enzymes used for the present study: GPI, PGM, ALAT and ASAT, have been expressed by the strains subm itted to chem otherapeutic agents. Results o f the present investigation suggest a stability o f biochem ical characteristics o f the strain types studied. Recent observations by McDaniel and Dvorak22 have shown that single cell derived clones o f the Y strain differed in its D N A conter* eventhough the isoenzyme and schizodeme profiles w ere maintained. Investigations on the genotypic characteristics o f the strains here studied by the analysis o f the genomic and kinetoplast D NA could further clarify these aspects.
RESUM O
C om o ob jetivo d e inves tig a r a influ ên cia d a quim ioterapia n o p a d r ã o b ioquím ico d e d ifer en tes cep a s d o Trypanosoma cruzi, tr ês g r u p o s d e ca m u n d o n g o s fo r a m infectados respectivam ente com a s cep a s Peruana, 21 S F e C olom biana, q u e co r re sp o n d e m a d iferen tes p a d r õ es b iológicos e iso en zim á tico s . Cada g ru p o f o i su b d ivid id o em su b g ru p o s: 1 - tra tad o s com nifurtim ox; 2 - tratados com b e n zo n id a zo l; 3 - co n tro les infectados nã o tratados. A o f i n a l d o tratam ento q u e d u r ou 9 0 dias, o s a nim ais fo r a m su b m etid o s a tes tes p a ra s ito ló g ic o s d e cura: xen o d ia g n ó stico , s u b ino cu laçã o d o s a n g u e em ca m undongos recém -n a scid o s e h em o cu ltu ra em m eio W arren. A p a r tir d a p o s itiva çã o d e stes tes tes , fo r a m isoladas 22 a m o stra s d o T. cruzi d o s tr ê s s ub g ru p os. A an á lise eletroforética d o s extratos enzim á tico s ob tidos após c u ltu r a ,p a r a as enzim as P G M , G P I,A L A T e A S A T
M a r r e to J P M , A n d r a d e SG . B io c h e m ic a l b e h a v io r o f Trypanosoma cruzi s tr a in s is o la te d f r o m m ic e s u b m itte d to s p e c ific c h e m o th e r a p y . 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 r o p ic a l 2 7 : 2 0 9 - 2 1 5 , o u t- d e z , 1 9 9 4 .
p a r a s ito s d e u m a m e s m a c e p a is o la d o s d e a n im a is tr a ta d o s o u n ã o tr a ta d o s . C o n clu i-se q u e n ã o h ou v e s e le ç ã o c lo n a l o u a lte r a ç õ e s g e n é tic a s d o s p a r a s ito s , d e te c tá v e is p e la s is o e n z im a s e x a m in a d a s , d e p e n d e n te s d a a ç ã o d e d r o g a s d e r e c o n h e c id a a ç ã o a n tip a ra sitá r ia . Isto s u g e r e u m a e s ta b ilid a d e d o s c a r a c te r e s fe n o típ ic o s d o s tr ê s tip o s b io ló g ic o s d e c e p a s d o T. cruzi.
P a la v r a s -c h a v e s : C e p a s d o Trypanosoma cruzi. Is o e n z im a s . Q u im io te ra pia .
ACKNOW LEDGEM ENT
Thanks are due to C .M .G . Santiago and R. de Castro Silva for technical help in the performance o f isoenzyme electrophoresis.
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