Reu. Inst. Med. tlop. Sd.o Pø.ulo 29í5): 289 -294. s etefl¿br o-outubto, 1981
RADIOMETRIC DETECTION OF METABOLIC ACTIVITY OF PARACOCCIDIOIDES BRASILIENSIS
AND ITS SUSCEPTIBILITY TO AMPHOTERICIN B AND DIETHYLSTILBESTROL
Edìraldo E. CAMARGO, Mariâ K. SATO, Gilila M. B. DDL NEGRO & Cârlos da Silvâ LACAZ.
Pâracoccidioidomycosis (South American blastomycosis) is a systemic disease,
strikingly more frequent in males, caused by the dimorphic tungus ParÀcoccidioides brasiliensis. A radiometric assay system has been applied to study the metabolic activity and the effect of drugs on ttlis fungus "in vitro". The Y form of the yeast,
glown in liquid Sâbouraud medium was inoculated into sterile reaction vials contai ning the 68 aerobic medium along with 2.0 poi of rac-substrates. Control vials,
prepared in the same way, contained autoclaved tungi. To study the effects ofampho-tericin B (AB) (0.1 and 10 pglml) and diethylstilbestrol (DSB) (1.0, 5.0 and 10 r¿glml) extra controls with live fungi and no drug s¡ere used. AII vials were incubated at
35"C and metabolism measured daily with a Bactec instrument. laCO, production by P. brasiliensis was slo$¡ and could be followed for as long as 50 days. AB at l0f¿g/ml and DSB ât 5 pglml inhibited the metâbolism and hâd a cidal effect on
this tungus. The results with DSB might explain the low incidence of the disease
in females. This technique shows promise fo¡ studyi¡g metabolic pathways, investi-gating more convenient lac-substrates to expedite radiometric detection and for monitoring the effects of other drugs and fâcto¡s on the metabolism ofP. brasiliensis "in vitro".
KEY woRDS: Paracoccidioides btasiliensis; R¿diometric assay; r{C-Substrates; Ämphotericin B; Diethylstilbestrol
SUMMARY
Paracoccidioidomycosis is a systemic
disea-se, strikingly more fiequent in males than fema-les, caused by the dimorphi¿ fungus
Paracocci-diolales brasiliensis. Despite the various studies on the biochemical composition of both its Y
and M forms (12) and several attempts at impro-ving culture media (15), P. brasiliensis grows
ve-ry slowly in all media so fâr used for its cultiva-tion "in vitro".
Centro de Medicina Nuctear, Låbomtórlo de Micologia Médica do Instituto de Medicina Tropical de Sáo Paulo e Lâborâtódo
de Investigâçáo Médica (LIM-53) da Faculdade de Medicina da Unive$idade de Sáo Paulo
AddÌess for coÈespondence: Dr. Edwåldo E. Câ¡nargo. centro de Medicina Nuclear da Universidade de sáo Paulo. caixa Posta.l
INTRODUCTION
Since the early 1970's, aradiometric methöd, which measures laco, evolved from a conve-nient reâction system, has been used for rapid dbtection of bacterial growth in clinical micro-biology (?). Today the method is widely used
ai
a clinical tool in many hospitals for fast detec-tion and drug susceptibility testingofmost com-mon clinical pathogens (8). The method wasge-a.
CAMARGO. E E.i SATO. M. K.: DEL NEGRO. G. M. B. E L¡.CAZ,. C. da S. - Râdiometdc det€ction of metâbolic ãctiviüy ot Pârâcoccidioid€s brasitieDsis and its sìrsceptibihty to amphotedcin B ând diethylshlbestrol. Rev. lnsr- Med. troÞ. Sáo Pâulo. 29:289-294. 198?.
nus Mycobacterium with very promising results
(4). This led, in turn, to clinical application with mycobâcteriâ, which are now becoming routine in several centers (18). However, the ¡adiometric method has not been used with P. brasiliensis, Because of
its
high sensitivity,this
method might be a useful tool to betterunderstand meta-bolic requirements of this organism, asit
did with mycobacteria (3, 6).The purÞose of this investigation was to
de-terminethefeâsibilityofdetectingthemetabolic activity of P. brasiliensis with the radiometric method using a commerciâlly âvailable radioac-tive medium, and the effect of factors that might interfere with is metabolism "in vitro".
M.A,TER,IALS AND METHODS
Preparation ofFungi:
Drugs:
The effects of amphotericin B (Squibb) a¡d diethylstilbestrol (Sigma) on the metabolism of P. brasiliensis were studied. Amphotericin B
was diluted with sterile saline so that the desired concentrations of 0.1 pglml and
l0
pglml weredelivered in 0.6 ml. Similarly, diethylstilbestrol n'as diluted with methânol so that concentra-tions of 1.0, 5.0 and 10 pglml were delivered in
75 pl. Control vials for diethylstilbestrol
contai-ned the same volume of methanol.
At the end of the incubation period of the experiments, the fungi from vials containing 10 pglml amflhotericin B and from vials containing
5.0 pglml diethylstilbestrol were recovered after centrifugation at 1,200 xg, washed 3 times with sterile saline, inoculated sepârately into new 68 medium vials and incubâted at 35.C for f,wo weeks.
The yeâst fo¡m (Y) of P, brasiliensis, strain
18, was obtâined on solid Sabouraud medium (Difco) from Laboratório de Micologiâ Médica
do lnstituto de Medicina Tropical de Sáo Pâulo. The fungi were transferred to liquid Sâbou¡aud medium (Difco), and incubated at 35"C for tv¡o
weeks. The fungâl suspension was centrifuged at 1,200 xg and the supernatant discarded.
the
pelletwas ressuspended with 10 ml ofsterile sali-ne and the centrifugation procedure repeated3 times. The microorgânisms vlrere counted di-rectly using a Neubâuer châmber. The final
sus-pension was diluted with sterile sâline to yield
2
x
10? microorganisms Þer ml.Reaction System:
Radiometric Measur€ment:
The reaction system for rac02 detection
con-sisted of 30 ml of the 68 aerobic medium in a 50 ml multidose sterile vial along with 2-0
uci
of r4C-substrates(JohnstonLaboratories)ând 1.0 ml offungal suspension. All vials were prefrared
atleast in triplicate. Control vials were prepared in the same {'ay, but with autoclaved fungi
ad-ded. When studying the effect of drugs on the tungâl metabolism, extrâ controls with live fungi and no drug nrere always prepâred for comÞa
nson.
All
vials were incubated at 35.C ând theracO, produced by fungal metâbolism was
mea-suredradiometrically. The measurementdevice,
a Bactec 301 B (Johnston Laboratories), consis ted of ân ionization châmber, a vacuum pump,
a set of sterile sampling needles anà a
logari¿h-mic scate up to 1,000 index units (100 index units = 25 nanocudes of rac activity). The needles pe-. netrated the ¡ubberstopper ofthe multidose ste-rile vial and the raCO, was aspirated into the ioni¿ation chamber under vacuum. The atmos-phere
in
the viâIs $'as replaced with room ai¡filtered through a 0.22
\m
pore size filtermem-brane. Radioactivity was measured by the ioni-zation chamber as index units. More details of the meâsurement procedure have been
pubti-shed eìsevt'here (5). The viâls were sampled daily for as long as 50 days. Results were expressed
either as index units for the metabolic activity
experiments or as percent activity of control
vials for the drug experiments. Background
rea-dings of the Bactec ranged from zero to
I
index units.All
readings above 12 units wereconsi-dered as frositive for growth. Pilot Exper¡ments:
Pilot experiments with the M form of this fungus were performed as describe above. The 290
CAMAROO, E. E.i SATO, M. K.i DEL NEGRO, O. M. B. & LACAZ, C. da S- - Radiomei c detection of mètabolic activitv of parscocctatioi¿t€s bråsili¿nsis anat its susceptiÞility to amphote¡lcin B and diethvlstilbestlol &€v. Insl. Med. aroD Sáo
Paulo, 29:289-294, 1987.
colonies were scrapped from solid Sabouraud medium, suspended in sterile saline, dilùted to approximately 2 x 10? organisms per ml, inocu
lated into úhe 6B aerobic medium andincubated at room temperâture. The vials were sampled daily for ? days.
RESULTS
Metabolic activity of P. brasiliensis ìn 6E} aerobic medium was slow. After 2 days incubâ-tion, 1aco, produgtion was fairly constant at about 40 index units per dav up
tb
20 days. Then,it
fell off to reach backgound levels by50 days. Figure 1 represents the differential weeklyr{Co, production, that is, the amount of lrCO, produced within a given week. As shown
in the ñgure, differential metabolism peaked at 2'r'eeks. In every week, r'co2 sampliná was
per-formed daily for 5 days, stârting over again at the beginntng of the next week Figure 2 repre-sents the cumulative.,rtco, production, that is'
ute addiüon ofthe difrerential raco, production within a given time intervâl.
o-o E o-= Fig. 2
- Cumuìative s¡eekÌy rrCO, prodr¡ction þy the Y fo¡m ofP. brasilie¡sis in 68 aercbic medìr¡m. Index u ts diffeÉnce
ftom one experimental viål to another waB negligible, Each expeliment wâs repeated at least twlce.
Amphotericin B at 0.1 pglml r¿i'as unable to block the metabolic activity of P. brasiliensis. After the flrstweek, vials with the drug displayed
àbolut 6OEo of the uCO, production of control vials, increasing to aboutS0Eø at tv/o weeks and 957¿ at three weeks. At 10 pglml, however. meta bolism was inhibited and dropped to 359o oÍ X}Je
control vials by the end of the first week and
to less than 10% at two weeks (Figure 3).
Diethylstilbestrcl
at
1.0 pglml vùas unableto inhibit the metabolism ofP. brasiliensis; the-re was no difrerence between control vials and hormone vials.
At
5.0 pglml, the metabolism dropped to 357o of control vials after one week, and to about ïEo ãt lwo weeks. There was no metabolism at all when hormonal concentrations/as l0 pglml (Figure 4).
Fungirecovered from vials containing 10 pgl
ml amphotericin B and from vials containing 5.0 r¿glml diethylstilbestrol showed no metabolic
Flg. I - DifieÉútlal w€eklv ÍCo, production bv the Y form of P. bústlleNi! ln 68 ¡e¡obic ñedlum. Thêle ìvas a peâk activl¿y st two w€eks, wltjr Ê progesslve decli¡e to beckg¡ound
I€v€ls. Index units difierence Êom olre expcrimental vial to anothelwssnegll8lble. Each expe¡lrDentwasEpeated aileâst
9 U/e€*s
CAMARGO' E E i SATO, M K i DEL NEGRo, G. M. B. & LAcAz, c. ala S. - Râdiometric rteüeciion of metabotic acüviüy
ol Paracoccidioid€s brâsiliensis and its susceptibility to amphotericih B anct diethylstiltrestrol. Rev- Inst. M€d. aroD. são Paulo, 29:289 294. 198?. d E E
il
N
N
-t
il
il
N
F¡e 3 - EfrecL of amphotencin B on rhe ,¡CO2 producLion of bhe Y folm ofP. brasilie¡sia in 68 âe¡obic medium. A¿ 0.1
pglml, there wês no inhibitio¡ ofmetaÞolic acbivity of the
tun-gus. At l0 ,¡gl¡nl ühree was mârked illhtbiùion ofmetabotism, speclally alÌer 2 ând 3 w€eks incub âtion. Index un¡tsdiffe¡ence
ftom one expelimentat vial to anothet wss negtigible, Eâch
expefimentwas reÞeâted at least t\¡rice
il
I
il
LI
dium, which contins r4c-substrâtes, ¡adiometric detection of 14CO, production by this fungus is
feasible within â few days. In contrast, the
con-ventionâl culture technique in Sâbouraud-agar
takes 20 to 30 days for g¡owth detection.
There-fore, the radiometric method has the potential
for fast, effective screening of new r4o-substrates, more adequate growth mediâ and in the investi-gation ofother facto$ that might interfere with the metabolic âctivity of this fungus ,,in vitro".
However, when compared to other fâstidious
mi-croorganisms such as Canalida albicans (ll), My-cobacterium tuberculosis (6) and Mycobacte-¡ium leptaemurium (2-4), metabolic activity of P- brasiliensis as measured by raCO, evolved ftom 68 aerobic medium was considerably slo-wer.
It
is conceivable that the 68 medium, al-though adequate to support growth of a broad spectrum of the most common clinicalpatho-gens, is not entirely suited for this fungus. Also, the unlabeled subshâtes in the medium might compete u¡ith the I'C-substrates for oxidation. Or, the ¡4c-substrates provided by the manufac-turer and which most Ukely inctude (U-14C)
glu-cose, r4c-formate and one of the simpler (U-raC)
L-amino acids, may not be adequate for the
me-tabolic requirements of p, brasiliensis.
The search for the best r4c-substrate to mo-nitor the metabolism of a given microorganism may be â difncult process. Ideâlly, the simplest
medium able to support its growth, and free of
each of the unlabeled substrates ¿o be tested
in radioactive form, shouìd be used. Differenú rlc-substrates would then
be added separately
to the medium alohg with the mic¡oorga¡xism a¡d a simple yes-or-no answer obtained after in-cqbation. This v¡as the procedure used to deter-mine that (U-r4C) glycerol, (U-laC) acetate, (1-tac)
fatty acids and trc-formate were more effective than (U-taC) glucose, (U-t4C) pFuvate and (U-raC)
glycine with M. tuberculosis and M.
lepraemu-rium (except r.C-formate) (2-4, 6). Biochemical studies have shown that p. b¡asiliensis utilizes fatty acids such as oleic, palmitic, ând linoleic
(12) and that growth can bestimulated by amino acids such as asparagine and glycine (9). One can then speculate thatperhaps by usingsepara-tely these substrates labeled with carbon-I4, hi-gher 1rCO2 outputs
and fâster results would be
obtained. Nl o¡rh'r Dc'd
¡rm'iñ'"rs
ri
-ä'
ËïN
tN
tN
I
lt
å1tN
tN
lN
|
ll
:1iN
lN
lN
lñ
ll
=1LN
LN
N
tN
II
Ii]llir
l||l
lt
IlNllll
K
NLKtt
E cù'ú' Fig. 4- Effect of diethylsiitÞestrol on ì,CO, p¡oduction þy the Y form of P. bmsili€Dsis ih 68 ae¡obic medium. At 1.0
pglml there q¡as no effect. A ¿ 5.0 and 10 ,¡slml therc ìFas marked
iDlibitlon of metabollc activity, more pronounced after 2 and
3 weeks lncubation. Index units diffe¡ence from one expe¡i mentâI vial to a¡othet was negligible. Each expeúment was repeated at least tr¡ice.
There was no raCO, production from vials containing the M form of P. brasiliensis.
DISCUSSION
Radiometric studies on metabolic activity of P. brasiliensis haye not been reÞorted. The
present i¡vestigation has demonstrated that by
using the commercially available 6B aerobic me-292
CAMARGO. E. E.: SATO, M_ K.; DEL NEGRO, G. M. B. & LACAZ, C- dâ S. - Râdiometric detection of metabolic ac¿ivrty
of parâcoccidioid€s brãsiliensis ând its suscepbibility to âmphotericin B and diethyìstûbestrol Rcv. lnsl. M¿d. t¡op. São
Paulo. 29:289'294, r98?.
The effect ofsteroid hormones on the growth
of P. brasiliensis was reported by MUCHMORE
et al. (16) $'ho found that at 25"C and 37"C estra-diol and diethyl$tilbestroì in concentrations of 10 pglml completely inhibited the growth of this fungus. Horrever, testosterone and choleste¡ol
iÍ
the same concentration showed no effect on any ofthe isolâtes tested. The rationale for their investigation Ìvas the striking preponderance ofmale patients with the disease: in ourinstitution
(13) and in other series (1, 1?), ratios male/female of l3:1 have been found. Such â phenomenon cannot be explainded by different exposure
râ-tes to the fungus, since the incidence of delayed
hypersensitivity to intradermal paracoccidioi-din Ís the same in both sexes. MUCHMORE et al. (16) did not mean to imply that the simple inhibitory effects of natural estrogen were res
ponsible for the low incidence of the disease in
femâles, since the concentrations used in their investigation were much higher than those that occur in the human body. The present study has
confirmed thei.r findings, even at a lower
concen-tration of 5 pglml of diethylstilbestrol. Because of the cidal effect of5 r¿glml of diethylstilbeshol,
it
is conceivable that, not taking into account other defense mechanisms, this hormone alonewould provide a basis for explanation of a lo$¡
incidence of ihe disease in females.
It
is worthyof mention the study of STADALNIK et al. (19)
on a very similar disease, coccidioidomycosis. After the 197? dust storm in the Sacramento Va Uey, which randomly exposed an entire popuìa-tion to spores ofCoccidio¡des immitis, an epide
mic ofthe disease occurred and 12 patients Ì'ere referred to their hospital. The striking fact was that 11 of these patients r¡ere males and only
one female. In an randomized exposure such as
this, a l:1 ratio would be expect€d, and a 11:1 ratio could only be understood if the female
po-pulation wås protected, possibly by estrogens.
may be hindered by pa¡tial inactivation of some
sort (10).
This investigation has demonstrated that the metabolic activity of P. brasiliensis and the effects of factors that might interfere with its metabolism can be studied with a radiometric system. Howeve¡, the slow rqCO, production
using the commercially available 68 aerobic
me-dium strongly suggests that the reâction system here utilized is not optimal. Investigation of other lao-subsirates, such âs fatty acids and ami-no âcids, that might be more adequate for the metabolic requirements of this organism, seems
to be warrânted in future experiments.
R,ESUMO
Detecçáo radiométrica da at¡vidaale metabólica alo Paracoccidioides brasiliensis e
da sua sensibilialaale à Anfotericina B e ao
Dietilestilbestrol
A paracoccidiodomicose (blastomicose sul-americana) é uma doençâ sistêmica muito mais freqüente no sexo masculino, causada pelo fun
go dimórfico PÀ¡acoccirlioides brasiliensis, Um sistema radiométrico foi utilizado para estudar a atividade metabólica e o efeito de'drogas sobre este fungo "in vitro". A forma Y do fungo, culti-vadâ em Sâbourâud líquido, foi inoculâdâ em frascos estéreis contendo o meio aeróbio 6I},
jun-tamente com 2,0
uci
de substâ¡ciâs marcadas com carbono-14. Frascos-controle, preparadosda mesma forma, foram inoculados com fungos âutoclâvados. Para estudar os efeitos da
anfote-ricina B (AB) (0,1 e 10 pglml) e do dietilestil-bestrol (DEB) (1, 5 e 10 ¡,g/ml), controles
âdicio-nais foram preparados, contendo fungos viáveis
mas náo a droga. Todos os frâscos foram
incuba-dos a 35'C e o metabolismo medido diariâmente
com uma máqìrina Bactec. A produçáo de laco, pelo P. brasiliensis foi lenta e pôde ser acompa-nhada por 50 diâs. Concentraçoes de 10 pglml de AB e 5 r¿glml de DEB inibiram o metabolismo e liveram efeito fungicida. Os resultados com DEB poderiam explicar a baixâ incìdência da doença em mulheres. Esta técnica é promissora para estudar as vias metabólicas, investiga¡
substâncias marcadas mais adequadas para tor-nar mais rápida a detecçâo ¡âdiométrica do
fun-go e para acompanhar os efeitos de outras drogas Effective levels of amphotericin B range
from 2.0 to 4.0 pglml of blood (10, l4). The cidal
effect of 10 r¿glml described in the present inves tigation represents less than the clinically
effec-tive dose; when one considers the heavy i¡ocu-lum of2 x 10?fungi/ml, a concentration not found
r¡nder clinicâ-l conditions. This suggests that ei-ther the 68 medium, notbeing completely suited
for P. brÂs¡l¡ensis, makes the organism more
CAMARGO E E; SATO M- K.: DEL NEGRO, c. M. B. & LACAZ, C. ala S. - R.adiomelrlc detection of metabo¡c âctivity ot Paracoccidioides brâsiliensis ând i¿s susceptìbilitv Lo âmphote¡icin B anal clieühytstilbestlol- Rev. Inst. M¿d. trop. são
Pâulo, 29:289 294. 198?.
a:.
e fatores sobte o metabolismo do p. trrasiliensis "in vitro".
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WAGNER JR., H. N.
- Râdiometric measure¡nenü ofme taþolic activit} of Mycob¡cteriuD l€Þrâemu¡ium. Appr.
Micrcbiol., 2E: 452-45õ, 19?4.
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