General and Comparative Endocrinology 176 (2012) 409414

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General and Comparative Endocrinology

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Taenia crassiceps WFU cysticerci synthesize corticosteroids in vitro: Metyrapone

regulates the production
R.A. Valdez a, L. Hinojosa a, Y. Gmez c, K. Willms b, M.C. Romano a,
a
Dpto. de Fisiologa, Biofsica y Neurociencias, Cinvestav, Apdo. Postal 14-740, 07000 Mexico D.F., Mexico
b
Dpto. de Microbiologa y Parasitologa, Facultad de Medicina, UNAM, 04510 Mxico D.F., Mexico
c
Dpto. Bioprocesos, Unidada Interdisciplinaria de Biotecnologa. I.P.N., 07340 Mexico D.F., Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Taenia solium and Taenia crassiceps WFU cysticerci and tapeworms have the ability to synthesize sex ste-
Available online 2 February 2012 roid hormones and have a functional 3b-hydroxisteroid dehydrogenase. Corticosteroids (CS) like cortico-
sterone and dexamethasone have been shown to stimulate in vitro estrogen production by Taenia
Keywords: crassiceps WFU cysticerci. The aim of this work was to study the ability of T. crassiceps WFU cysticerci
Corticosteroids to synthesize corticosteroids, and the effect of the inhibitor metyrapone on the CS synthesis. For this pur-
Glucocorticoids pose T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, thoroughly washed
Mineralocorticoids
and pre-incubated in multiwells for 24 h in DMEM plus antibiotics/antimycotics. The tritiated CS precur-
Parasites
Taenia crassiceps cysticerci
sor progesterone (3H-P4) was added to the culture media and parasites cultured for different periods.
Blanks containing the culture media plus the 3H-P4 were simultaneously incubated. Blanks and parasite
culture media were ether extracted and analyzed by thin layer chromatography (TLC) in two different
solvent systems. Corticosterone production was measured in the culture media by RIA. In some experi-
ments metyrapone (0.10.5 mM) was added for 24, 48 or 72 h. Results showed that cysticerci mainly syn-
thesized tritiated 11-deoxy corticosterone (DOC) and small amounts of corticosterone that was also
detected by RIA. Small amounts of 3H-11-deoxy cortisol were also found. Corticosteroid synthesis was
time dependent. The addition of metyrapone signicantly inhibited tritiated DOC, deoxycortisol and cor-
ticosterone synthesis. These results show for the rst time that parasites have the capacity to synthesize
CS that is modulated by metyrapone. Data suggest that DOC is the main corticosteroid in the parasites.
2012 Elsevier Inc. All rights reserved.

1. Introduction the transcription of several proteins; GC receptors are widely dis-

Cortisol, corticosterone and aldosterone are essential hormones
found in most tetrapods. Their importance in stress response,
immunity, metabolism and differentiation is well known [22].
Glucocorticoids (GCs) are steroid hormones synthesized by the glo-
merular zone of the adrenal cortex [24] or the interrenal tissues of
amphibians, reptiles and shes. However local CS synthesis has
been found in several tissues like the brain and cardiovascular tis-
sues (for a review see Davies and MacKenzie [7]). Furthermore cor-
ticosteroid synthesis and corresponding steroidogenic enzymes
and their genes were found in thymic epithelial cells [27,19] in
the developing lung cells of mammals [20] and in human skin
keratinocytes [10].
Corticosteroids exert their effect by binding to specic recep-
tors that share all the characteristics of a large family of mole-
cules that nally recognizes a site called glucocorticoid receptor
element in DNA. The binding of the receptor to this site initiates
Corresponding author. Address: Dpto. de Fisiologa, Biofsica y Neurociencias,
Cinvestav del IPN, Apdo. Postal 14-740, 07360 Mxico D.F., Mexico.
E-mail address: mromano@sio.cinvestav.mx (M.C. Romano).
tributed in most cells of vertebrates and are classied as GC
receptors (Type II) and mineralocorticoid receptors (Type I), that
have great afnity for glucocorticoids and mineralocorticoids,
respectively; however Type I receptors act like mineralocorticoid
receptors in some tissues but have a high afnity for GC receptors
in others; non-genomic actions of GCs have also been reported.
Cortisol is the most abundant glucocorticoid in most verte-
brates and corticosterone is the main corticosteroid in rodents,
birds and amphibia and has also important mineralocorticoid
activities. A wide variety in the type of corticosteroids and their
function can be found, for example teleosts lacks aldosterone and
in these shes cortisol may have GC and MC actions. Close et al.
[6] provided evidence that 11-deoxycortisol is the main corticoste-
roid hormone in the lamprey, an agnathan member that belongs to
the oldest vertebrate lineage.
Phylogenetic studies on corticosteroid receptors performed by
several authors [26,13,3,23] suggested that a MC receptors
complex evolved rst. Therefore other corticosteroids should be
considered as active GCs or MCs and an ancestral hormone, 11-
deoxycorticosterone may be important in this complex panorama.
In this regard it has been shown that DOC serum concentration

0016-6480/$ - see front matter 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2012.01.015
410 R.A. Valdez et al. / General and Comparative Endocrinology 176 (2012) 409414
increases in the rainbow trout during spermiation [4,16] and that
DOC is an agonist of the MC in this sh [25].
Effects of corticosterone and dexamethasone on androgen syn-
thesis by mouse Leydig cell has been described by Payne and Sha
[18]. Interestingly, corticosteroid action on female and male mam-
mal reproduction aspects seems to be different in chronic and
acute stress [1]. Data reported in the literature strongly suggest
that these hormones participate in germ cell development as well
as on selected ovarian steroidogenic enzymes [15]. Wada [30] pub-
lished a complete vision of GCs as mediators for vertebrate onto-
genic transitions, for example they promote maturation of organs
before birth in mammals and hatching in birds, participates in
metamorphosis and facilitate acquisition of osmoregulatory ability
in sh through Na+K+ ATPase activity and ion transport among
other events critical for survival.
We have shown that Taenia crassiceps WFU and Taenia solium
cysticerci and tapeworms, in vitro synthesize sex steroid hormones
[12,28,21] and have demonstrated the presence and activity of
3b-hydroxysteroid dehydrogenase in their tissues [9].
On the other hand, we have recently demonstrated that the
addition of corticosterone or dexamethasone to the culture media
of T. crassiceps WFU cysticerci increased the 17b-estradiol synthe-
sis in a time and dose dependent-manner [11].
Corticosteroid presence and function is not well understood in
the case of most invertebrates and to our knowledge is unknown
in parasites. To this end we have investigated the ability of T.
crassiceps WFU cysticerci, to synthesize corticosteroids in vitro. In
addition we have investigated the effect of metyrapone, an inhibi-
tor of CS synthesis on corticosteroid synthesis.
2. Materials and methods
2.1. Parasites
Cysticerci from the WFU strain of T. crassiceps were collected
from the peritoneal cavity of female Balb/c mice after 57 months
postinfection and rinsed ve times in phosphate-buffered-saline,
pH 7.2 (PBS). Cysticerci were pre-incubated for 24 h at 37 C in
an atmosphere of 5% CO2 and air, in Dulbeccos Modied Eagles
Medium (DMEM; GIBCO BRL, Grand Island, NY, USA), plus 0.1% bo-
vine serum albumin (BSA, SigmaAldrich Chemical Co., St. Louis,
MO, USA), 25 mM of N-[2-hidroxyethyl]piperazine-N0 -[2-ethane-
sulfonic acid] (HEPES; Sigma) and 1% antibioticantimycotic solu-
tion (penicillin at 10,000 U/ml, plus streptomycin at 10,000 lg/ml,
amphotericin 25 lg/ml; GIBCO).
Following the pre-incubation period, the parasites were washed
with DMEM and transferred to new wells (250 lL cysticerci/well)
containing fresh culture medium. Some cultures were treated for
24, 48 or 72 h with different concentrations of metyrapone (0.1
0.5 mM) solved in ethanol, this solvent was added to control
groups at a nal concentration of 0.6%. Finally, fresh medium con-
taining 0.1 lCi of 3H-progesterone (3H-progesterone, 1, 2, 6, 7-3H
progesterone 93.0 Ci/mmol, Amersham Pharmacia Biotech) and
the cysticerci further incubated for different periods. Blanks con-
taining the culture media plus the 3H-P4 were simultaneously
incubated. At the end of the experiments, culture media were
recovered and ether extracted. The samples were reconstituted in
100 microliter of absolute ethanol [12].
2.2. Thin layer chromatography
17OH-progesterone, 11-deoxycorticosterone, corticosterone,
11-deoxycortisol, and cortisol were used as internal standards.
Thin layer chromatography (TLC) was carried out using Silica gel
60 F254 pre-coated sheet plates (Merck, Darmstadt, Germany) as
described previously for T. crassiceps cysticerci [12]. Aliquots of
20 ll of the ethanolic samples were supplemented with standard
steroids and further fractionated in a TLC system. The plates were
developed in two different solvent systems (benzene:acetone
50:50 v/v or toluene:acetone:methanol 78:20:2 v/v; Merck,
Darmstadt, Germany). The standard steroids (Steraloids, Wilton,
NH) were detected in the plates by ultraviolet light and exposed
to 10% H2SO4 followed by heating at 120 C. Regions corresponding
to authentic standards were cut and placed in vials containing
scintillation liquid and radioactivity was counted in a liquid scintil-
lation spectrometer. The recovered radioactivity was estimated by
comparing the difference between initial and nal cpm.
Results were expressed as the percentage of substrate transfor-
mation for each metabolite, after incubation in the presence of the
precursor. Since the recovery of the radioactivity of each precursor
was higher than 85% no corrections were made. Results were sub-
mitted to analysis of variance and Students t-tests.
2.3. Radioimmunoanalysis (RIA)
In another experiments cysticerci were precultured as de-
scribed above and further incubated for 48 h in DMEM with
1 mM cold pregnenolone as the precursor to determine corticoste-
rone in the cysticerci culture media. The pool of culture media was
ether extracted to obtain enough samples to measure corticoste-
rone by RIA. For costicosterone RIA, tritiated corticosterone (3H-
corticosterone (1, 2, 6, 7-3H(N)) New England Nuclear USA
250 lCi 70 Ci/mmol) was used as the tracer and the anti-cortico-
sterone antibody was purchased from Sigma (Product # 8784,
Saint Louis Missouri, USA). The percent of the antibody cross-reac-
tivity was: corticosterone 100%, 11-deoxycorticosterone 20%, corti-
sol 4.5%, progesterone 15.7%, 20a-hydroxyprogesterone 8.8%, 20b-
hydroxyprogesterone 5.2%, testosterone 7.9%, androstenedione
2.6%, aldosterone 4.4%. The experiments were repeated three
times.
2.4. Statistics
Statistical analysis were performed using Prism version 4 2003
(GraphPad Software Inc.). Data are presented as means SD. Prob-
ability values of p < 0.05 were considered to be signicant. One-
way analysis of variance Anova followed by Dunnets test was used
to compare all metyrapone dose columns vs. control column.
3. Results
Fig. 1 shows the corticosteroid metabolites found in cysticerci
culture media after 24, 48 or 72 h in the presence of 3H-progester-
one. The plates were developed in benzene:acetone 50:50 v/v
(Fig. 2A) or in toluene:acetone:methanol 78:20:2 v/v (Fig. 2B). Tri-
tiated progesterone was mainly metabolized to 3H-11 DOC and 3H-
11-deoxicortisol, small quantities of corticosterone and cortisol
were also found. Data in Fig. 1 shows that the corticosteroid syn-
thesis by T. crassiceps WFU was time dependent.
The corticosteroid synthesis was similar using the two solvent
systems which conrms the nature of metabolites found (Fig. 2).
The effects of metyrapone on corticosteroid synthesis by cysti-
cerci is shown in Figs. 3 and 4. The addition of the inhibitor
signicantly decreased the synthesis of 3H-11 DOC, but the effect
was not evident after 72 h, whereas 3H-11-deoxicortisol was still
signicantly depressed after 48 and 72 of exposition to metyra-
pone (Figs. 3 and 4). The highest dose of metyrapone decreased
3
H-corticosterone synthesis at 24 h, no signicant effect of the
drug was observed after this incubation time (Fig. 5).
 R.A. Valdez et al. / General and Comparative Endocrinology 176 (2012) 409414 411

a b
80 25
% Of Transformation * **

% of Transformation
20
60
15 *
40
10
20
5

0 0
24 48 72 24 48 72
HOURS HOURS

c d
% of Transformation

% of Transformation
4

3
3

2 2

1 1

0 0
24 48 72 24 48 72
HOURS HOURS

Fig. 1. Corticosteroids synthesis by Taenia crassiceps WFU cysticerci. The parasites were cultured in the presence of 3H-progesterone and synthesized tritiated corticosteroids:
(a) DOC, (b) deoxycortisol, (c) corticosterone and (d) cortisol. The synthesis of DOC and deoxycortisol signicantly increased with time in culture. Representative experiment
of three independent repetitions. n = 6 p < 0.05.

a 25
% of Transformation

20

15

10

0
cortisol corticosterone deoxycortisol DOC

b 40
& of Transformation

30

20

10

0
cortisol corticosterone deoxycortisol DOC

Fig. 2. Taenia crassiceps WFU cysticerci corticosteroid synthesis evaluated in two different solvent systems. The parasites were incubated for 24 h (a) or 48 h (b) with 3H-
progesterone and the plates developed in benzene:acetone 50:50 v/v (white bars), or toluene:acetone:methanol 78:20:2 v/v (black bars).

Corticosterone concentration was measured by RIA in the 48 h. Corticosterone production was 8.07 1.17 pg/ml per 100
pooled culture media of T. crassiceps WFU cysticerci incubated for cysticerci (mean SD of three independent experiments).
412 R.A. Valdez et al. / General and Comparative Endocrinology 176 (2012) 409414

a 40
b 40
% of Transformation

% of Transformation
30 * 30

* *
20 20

10 10

0 0
CONTROL MET 0.1 mM MET 0.2 mM MET 0.5 mM CONTROL MET 0.1 mM MET 0.2 mM MET 0.5 mM

c
% of Transformation 80

60

40

20

0
CONTROL MET 0.1 mM MET 0.2 mM MET 0.5 mM

Fig. 3. The effect of metyrapone on 3H-DOC synthesis by T. crassiceps WFU cysticerci. The parasites were incubated for (a) 24, (b) 48 or (c) 72 h in the presence of different
doses of metyrapone. The plates were developed in benzene:acetone 50:50 v/v. Representative experiment repeated twice, n = 6 p < 0.05.

a b 16
6
% of Transformation

% of Transformation

12
4 * ** **
8

2
4

0 0
CONTROL MET 0.1 mM MET 0.2 mM MET 0.5 mM CONTROL MET 0.1 mM MET 0.2 mM MET 0.5 mM

c 25
% of Transformation

20

15
** **
10 **
5

0
CONTROL MET 0.1 mM MET 0.2 mM MET 0.5 mM

Fig. 4. The effect of metyrapone on 3H-deoxycortisol synthesis by T. crassiceps WFU cysticerci. The parasites were incubated for (a) 24, (b) 48 or (c) 72 h in the presence of
different doses of metyrapone. The plates were developed in benzene:acetone 50:50 v/v. Representative experiment repeated twice, n = 6 p < 0.05, p < 0.01.

4. Discussion the end of the reproductive cycle and this nding was related to
milt uidity [16].
This paper shows for the rst time that T. crassiceps WFU cysti- The effect of corticosteroids in Na+K+-ATPase pump and ion
cerci have the capacity to synthesize corticosteroids such as DOC, movements in smoltication, a process that prepares young shes
11-deoxycortisol and corticosterone being DOC the most abundant for the transition from fresh water to salt water is controlled by
corticosteroid. As in mammals, this function has been cortisol among another hormones (as reviewed by Mommsen
demonstrated in avian, sh, reptiles and amphibians and even in et al. [17]; Mc Cormick [14], Varsamos et al. [29]). A role for DOC
an ancient vertebrate ancestor, the lamprey, that synthesizes 11- in osmoregulation, and gill NaK ATPase mrRNA levels in salmon
deoxycortisol. This steroid is known to participate in lamprey have been demonstrated by Mc Cormick et al. [14]. 11-Deoxycorti-
modulation of gill Na+K+-ATPase activity [6]. Furthermore, studies costerone, the main metabolite found in cysticerci culture media is
in the rainbow trout have shown that DOC concentrations rise at an important mineralocorticoid that modulates the Na+K+-ATPase
 R.A. Valdez et al. / General and Comparative Endocrinology 176 (2012) 409414
3 References
% of Transformation
2,5
2 J. 5 (1991) 26912698.
1,5
1
0,5
0 and oocyte maturation, J. Endocrinol. 85 (1980) 371378.
CONTROL MET 0.1 mM MET 0.2 mM
Fig. 5. 3H-corticosterone synthesis after the parasites were exposed to metyrapone
for 24 h. The plates were developed in benzene:acetone 50:50 v/v. Representative
experiment repeated twice, n = 6 p < 0.05.
pump. Interestingly, the presence of a Na+K+-ATPase pump have
been shown by Willms et al. [31] suggesting that cysticerci main-
tain their internal media using mechanisms similar to those de-
scribed for mammals and reptiles. Results from the present study
and literature data shown above suggest that DOC may have a role
in cysticerci osmoregulation and Na+K+ equilibrium.
Furthermore, the corticosteroid synthesis by parasites together
with other hormones like insulin, growth and thyroid hormones
may be useful for the physiological changes the cysticerci needs
to move from one developmental stage to the next one. Of no less
importance could be the role of GCs in glucose metabolism, since
these parasites are extremely dependent on glucose supply.
Another important function of GC synthesis by the parasites
could be the control of the host immune cells since the deletereous
effect of GCs of these hormones on lymphocytes and macrophages
have been extensively demonstrated in several species.
Radioimmunoassay results also showed that DOC and cortico-
sterone were detectable in the culture media of cysticerci. The
TLC ndings illustrated that DOC amounts were much higher than
corticosterone, which suggest that 11b-hydroxylase activity is poor.
Metyrapone is an inhibitor of cortisol biosynthesis used to treat
hypercortisolism. It is well established that acts on P450c11-
hydroxylase inhibiting the conversion of DOC to corticosterone
and of 11-deoxycortisol to cortisol. There is evidence that besides
this effect, metyrapone interferes in a dose-related manner with
another enzymes involved in corticosteroid synthesis as the cleav-
age of cholesterol into pregnenolone in rat adrenal mitochondria
[8] and also inhibits P450-scc in a human adrenocortical cancer cell
line [8]. In addition, Brown and Fishman [2] showed that metyra-
pone decreased 11-deoxycortisol, an effect probably explained by
the rechanneling of steroid synthesis intermediates into the andro-
gen pathway. These effects of metyrapone could explain the inhi-
bition of 3H-DOC and 3H-11-deoxycortisol synthesis found in the
present study. Although the corticosterone synthesis by cysticerci
was modest, an effect of the highest dose of metyrapone was also
found suggesting an effect of the inhibitor on a parasites P450c11-
hydroxylase like enzyme.
In conclusion present results show that T. crassiceps WFU cysti-
cerci metabolize steroid precursors to corticosteroids in vitro and
that 3H-11-DOC was the most abundant metabolite found in the
culture media. The corticosteroid synthesis was modied by the
presence of metyrapone, a corticosteroid metabolism inhibitor. Cor-
ticosteroid synthesis may be important for the parasite physiology
and may also contribute to defend the parasites from the host im-
mune cells and therefore facilitate their survival in the host for long
periods.
Acknowledgments
Partially supported by Conacyt Grant # 69347. The authors
acknowledge IBT Viridiana Salvador, IBT Bulmara Gaona and MVZ
Jos A. Jimnez for technical assistance.
413
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