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Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro
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R.A. Valdez a, P. Jiménez a, A.M. Fernández Presas b, L. Aguilar b, K. Willms a, M.C. Romano a,⇑ a b
Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV del I.P.N., Apdo. Postal 14-745, 07360 México, D.F., Mexico Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM, Ciudad Universitaria, México 04510, D.F., Mexico
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Keywords: Taeniids Taenia solium tapeworms Corticosteroid synthesis Sex steroid synthesis Helmynths Parasites
a b s t r a c t Cysticercosis is a disease caused by the larval stage of Taenia solium cestodes that belongs to the family Taeniidae that affects a number of hosts including humans. Taeniids tapeworms are hermaphroditic organisms that have reproductive units called proglottids that gradually mature to develop testis and ovaries. Cysticerci, the larval stage of these parasites synthesize steroids. To our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursors to steroid hormones. Therefore, the aim of this paper was to investigate if T. solium tapeworms were able to transform steroid precursors to corticosteroids and sex steroids. T. solium tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were cultured in the presence of tritiated progesterone or androstenedione. At the end of the experiments the culture media were analyzed by thin layer chromatography. The experiments described here showed that small amounts of testosterone were synthesized from 3H-progesterone by complete or segmented tapeworms whereas the incubation of segmented tapeworms with 3H-androstenedione, instead of 3 H-progesterone, improved their capacity to synthesize testosterone. In addition, the incubation of the parasites with 3H-progesterone yielded corticosteroids, mainly deoxicorticosterone (DOC) and 11-deoxicortisol. In summary, the results described here, demonstrate that T. solium tapeworms synthesize corticosteroid and sex steroid like metabolites. The capacity of T. solium tapeworms to synthesize steroid hormones may contribute to the physiological functions of the parasite and also to their interaction with the host. Ó 2014 Published by Elsevier Inc.
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1. Introduction
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Taenia solium (T. solium) tapeworms belong to the family Taenidae and develop and live for long periods in the gut of the human. T. solium cysticerci are the larval stage of tapeworms and live in the pork muscles and in the human brain causing a disease called neurocysticercosis. Throughout the course of cysticercosis infections, multiple host–parasite interactions occur that affect the human host immune response and cause severe endocrine and immune disturbances (White, 2000; Terrazas, 2008; Cárdenas et al., 2012). Once the scolex of the T. solium worm attaches to the duodenum of the definitive human host, growth proceeds from the germinative tissue in the neck by continuous duplication of undifferentiated cells, which migrate from the medullary region to the tegument (Merchant et al., 1997). Adult Taenias are hermaphroditic organisms with one of the highest reproduc⇑ Corresponding author. E-mail address: mromano@fisio.cinvestav.mx (M.C. Romano).
tive capacities among invertebrate species, worms can grow several meters in length and produce millions of infective eggs in a lifetime, which are released in the feces. The tapeworm develops reproductive units called proglottids, these units gradually mature to develop testis and later ovaries. Different experimental models had been used to study T. solium tapeworm and cysticerci infections in the laboratory (Zubarian and Q6 Willms, 2008). Experimental infections of mice with Taenia crassiceps ORF or WFU cysticerci had been extensively used to study immune aspects of the parasite–host interactions (Terrazas, 2008). In our laboratory we have been studying the capacity of cysticerci to synthesize steroid hormones (Romano et al., 2003, 2008). We have investigated the capacity of T. crassiceps ORF cysticerci to synthesize sex steroids in vitro and found that these parasites transform tritiated androstenedione to testosterone (Gómez et al., 2000). We also found that T. solium cysticerci from infected pork meat, produce androgens and estrogens in vitro (Valdéz et al., 2006). In addition, T. solium and T. crassiceps ORF cysticerci transform steroid precursors such as progesterone,
http://dx.doi.org/10.1016/j.ygcen.2014.04.014 0016-6480/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Valdez, R.A., et al. Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.04.014
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dehydroepiandrosterone, and androstenedione (Jiménez et al., 2006). To this regard, we had recently shown that that the larval stage of T. solium tapeworms express a 17b-hydroxysteroid dehydrogenase that catalyzes the transformation of androstenedione to testosterone and belongs to the short chain dehydrogenases/ Q7 reductase family (Aceves-Ramos et al., 2014). It is well known that 3b-hydroxysteroid-dehydrogenase (3b-HSD) is a key enzyme in the biosynthesis of gonad and adrenal steroid hormones (Labrie et al., 1994; Conley and Bird, 1997; Penning, 1997; Payne and Hale, 2004; Simard et al., 2005). To our knowledge there is not much information available related to Taenia tapeworms steroidogenic capacity. We have demonstrated the presence of active 3b-HSD in T. crassiceps WFU cysticerci and in tapeworm tissues, where the enzyme was found in the subtegumentary areas of the neck and immature proglottids (Fernández Presas et al., 2008). We have shown that experimental T. crassiceps WFU tapeworms obtained from the intestine of infected hamsters, synthesize androstenediol and 17b-estradiol when incubated in the presence of tritiated dehydroepiandrosterone (Fernández Presas et al., 2008). These results, strongly suggest the presence and activity of D5 steroid pathway enzymes in T. crassiceps WFU tapeworms, like the 3b-hydroxysteroid-dehydrogenase/D5–4 isomerase-like enzyme (3b-HSD), which converts androstenediol to testosterone, and dehydroepiandrosterone (DHEA) to androstenedione (Fernández Presas et al., 2008). However, to our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursor to sex steroid hormones. On the other hand, we have recently shown that T. crassiceps WFU cysticerci obtained from mice peritoneal cavity synthesize corticosteroids in vitro, and that metyrapone and ketoconazole blocks their synthesis (Valdéz et al., 2012; Aceves-Ramos et al., 2013). The cysticerci and tapeworms steroidogenic capacity might play a role in the permanence of the parasites in muscle and brain tissues and in the host intestine. It is well known that cortisol, corticosterone and aldosterone are essential hormones in most tetrapods. Their importance in stress response, immunity, metabolism and differentiation is well known (Sapolsky et al., 2000). Glucocorticoids (GCs) are synthesized by the adrenal cortex (Stocco and Clark, 1996) or the interrenal tissues of amphibians, reptiles and fishes. Interestingly, local CS synthesis has been reported in several tissues like the brain and cardiovascular tissues (for a review see Davies and MacKenzie, 2003). Furthermore, corticosteroid synthesis and the corresponding steroidogenic enzymes and their genes were found in thymic epithelial cells (Vacchio et al., 1994; Pazirandeh et al., 1999) in the developing lung cells of mammals (Provost and Tremblay, 2005) and in human skin keratinocytes (Hannen et al., 2011). Corticosteroids are also important for many developmental events. Data reported in the literature strongly suggest that corticosteroids participate in germ cell development and on selected ovarian steroidogenic enzymes (Michael and Cooke, 1994). Wada (2008) published a complete vision of GCs as mediators for vertebrate ontogenic 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 fish through Na+-K+ ATPase activity and ion transport among other events critical for survival. Besides their multiple physiological effects, it is well known that corticosteroids have a critical role in inflammation, modulating the response of immune cells to infections. Therefore, we considered important to investigate if tapeworms have the capacity to synthesize corticosteroids and release them to the culture media. In the present paper we have investigated the steroidogenic capacity of T. solium tapeworms. We show here that these parasites
synthesize corticosteroids and androgens from tritiated steroid precursors.
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2. Materials and methods
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2.1. Animals and specimen preparation
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The growth of tapeworm in the hamster intestine. Adult T. solium tapeworms were recovered from the intestine of golden hamsters (Mesocricetus auratus) inoculated 25–45 days before by oral administration of 8–10 cysticerci obtained either from naturally infected pork. Hamsters were pretreated for 5 days with 40 mg/kg of albendazole (Zentel, oral suspension, Smith, Kline, Beecham, Mexico) and injected with 2 mg of methyl-prednisolone acetate (Depomedrol, Upjohn, Mexico) on the day of infection and every 2 weeks for the duration of the experiments. Animals were kept on commercial food pellets and water under controlled conditions and killed by injection of sodium barbital (Anestesal, Cat. Q-0001-065 for Veterinary use, Pfizer, Mexico) and the small intestine opened longitudinally to recover the tapeworms from the duodenal cavity (Willms et al., 2003; Fernández Presas et al., 2008).
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2.2. Steroid synthesis by tapeworms
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T. solium tapeworms were thoroughly rinsed in PBS, and then washed twice with Dulbecco’s Modified Eagle Medium plus antibiotics (DMEM+A), 1 ml/100 ml of media (penicillin–streptomycin– amphotericin B, Gibco, In Vitro, USA).The parasites or the sections were transferred to tissue culture dishes containing 2 ml of DMEM+A and 0.1% bovine serum albumin. The tapeworms were pre-cultured for 48 h at 37 °C in a 95% air-5% CO2 atmosphere. In some experiments, the parasites were divided in sections from the head (Section I) to the tail (Sections II, III and IV). After this period the parasites were transferred to new dishes and 3H-progesterone (1,2,6,7-3H progesterone, 93.0 Ci/mmol, New Products, MS, Dupont, Boston, MA; 3H-P4) or 3H-androstenedione (1,2,6,73 H-(N)-androst-5-ene-3b-ol-17-one, 96 Ci/mmol, Perkin Elmer, Boston, MA, USA; 3H-A4) as steroid precursors were added to each parasite and further incubated for different periods in the conditions described above. Blanks containing the culture media plus 3 H-P4 were simultaneously incubated. The parasites were removed and media transferred to vials where steroids were extracted with 2 volumes of cold anhydrous ether. The organic phase was transferred to new vials and evaporated in a nitrogen atmosphere at 37 °C. The samples were reconstituted in 100 ll of absolute ethanol. Metabolites synthesized from the precursors were identified by thin layer chromatography.
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2.3. Thin layer chromatography (TLC)
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Tritiated progesterone (3H-P4) or androstenedione (3H-A4) were used as steroid precursors. Progesterone, corticosteroids (Cortisol, corticosterone, 11-deoxicorticosterone (DOC) and 11-deoxycortisol) and hydroxiprogesterone (OH-P4), androstenedione (A4), testosterone (T) estrogens (17b-estradiol and estrone), purchased from Steraloids (Steraloids, Wilton, NH, USA) were used as authentic standards. TLC was carried out using silica gel plates (20 20 Kieselgel 60 F254, Merck, Darmstadt, Germany). Aliquots of 40 ll of the ethanol samples were supplemented with the standard steroids and further separated in different TLC systems. Plates were developed with bencene:acetone 50:50 v/v or toluene:acetone:methanol (78:20:2 v/v) to separate corticosteroids and with benzene–methanol (9:1 v/v) to separate sex steroid hormones. The standard steroids were detected in the plates by ultraviolet light and also exposed to 10% H2SO4 followed by heating at
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Please cite this article in press as: Valdez, R.A., et al. Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.04.014
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120 °C. Regions corresponding to the authentic standards were cut and placed in vials containing scintillation liquid and radioactivity was counted in a liquid scintillation counter. The recovery of radioactivity was estimated by comparing the difference between initial and final cpm. Results are expressed as the percentage of substrate transformation for each identified metabolite after incubation with the corresponding tritiated precursor.
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2.4. Statistics
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Statistical analysis were performed using Prism version 4 2003 (GraphPad Software Inc.). Data represent as means ± SE. Probability values of p < 0.05 were considered significant. Kolmogorov Smirnov normality test was performed to analyse the data distribution followed by Kruskal Wallis nonparametric test.
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3.1. Synthesis of corticosteroids and sex steroids from tritiated progesterone
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Fig. 1 shows the sex steroid metabolites found after individual incubation of intact T. solium tapeworms (n = 8) in the presence of 3H-P4 if plates were developed in a solvent system that detected sex steroids. A modest synthesis of tritiated T4 was found in the culture media, whereas 17b-estradiol and estrone were under the detection limit of the technique. In some experiments the tapeworms were divided in four sections before incubation with 3 H-P4 for 3 h and the metabolite synthesis was similar to that found in the uncut tapeworms (Fig. 2).
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METABOLITE Fig. 1. Sex steroids synthesis from tritiated progesterone by T. solium tapeworms. Each complete tapeworm was individually incubated for 3 h in the presence of 3Hprogesterone and the tritiated metabolites were separated by TCL. Data indicate mean ± SEM (n = 8 tapeworms).
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4. Discussion
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The results of the present study showed that T. solium tapeworms have the ability to synthesize in vitro sex steroid like metabolites using tritiated progesterone or androstenedione as precursors. The transformation of the labeled steroid precursors 3 H-P4 or 3H-A4 to tritiated sex steroids such as OH-P4, A4 and T4 (Figs. 1–3) strongly suggests that these taeniid worms have at least part of a functional D4 steroidogenic enzymatic pathway. It follows that the worms should have steroidogenic enzymes such as 17, 20-lyase that converts progesterone to 17-OH progesterone, 17a-hydroxilase that transforms 17-OH progesterone to androstenedione, and 17b-HSD that converts androstenedione to testosterone. In fact, we have recently shown that the larval stage of T. solium tapeworms expresses a functional 17b-hydroxysteroid dehydrogenase that catalyzes the transformation of androstenedione to testosterone, and belongs to the short chain dehydrogenases/reductase family (Aceves-Ramos et al., 2014). Therefore, if the enzyme is expressed in the larvae, we propose that it should be also present in the tapeworm and could catalyze the synthesis of testosterone from androstenedione observed in the present study. These enzymes are part of the D4 steroidogenic pathway in most mammalian and avian species. Mammalian gonads have both D5 and D4 steroidogenic pathways, but which one predominates depends on the organ, in example the former is predominant in the ovary and the latter in the testis (Payne, 2004). Previous work from our group strongly suggests that T. solium and T. crassiceps
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However, if T. solium segments were incubated with 3H-A4 instead of 3H-P4, testosterone synthesis was higher compared with that of the experiments where tritiated progesterone was the precursor (Fig. 3). Using this experimental approach testosterone transformation of 3H-A4 to testosterone was ten to 20 times higher. Interestingly, there was a trend toward higher levels of testosterone synthesis in the distal segments II and III compared to the proximal segments (Fig. 3). The incubation of each of 12 T. solium tapeworms in the presence of 3H-P4 for 48 h yielded 17OH-progesterone, 11-deoxycorticosterone and 11-deoxycortisol as main metabolites judged by the percent of transformation. However, small amounts of corticosterone and cortisol were also found in both solvent systems used to separate the corticosteroid metabolites. Fig. 4A shows the results found when TLC plates were developed in bencene:acetone (50:50 v/v) and Fig. 4B when developed in toluene:acetone:methanol (78:20:2 v/v). Both solvent systems showed similar amounts of tritiated 17OH-progesterone, DOC and 11-deoxycortisol, corticosterone and cortisol. In both systems DOC was the most abundant metabolite.
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METABOLITE Fig. 2. Sex steroid synthesis by segmented T. solium tapeworms. The worms were divided in four segments from the head to the tail and individually incubated for 3 h with 3 H-progesterone and the metabolites were separated by TLC. I II III IV . Data indicate mean ± SEM (n = 3 tapeworms).
Please cite this article in press as: Valdez, R.A., et al. Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.04.014
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3H-Testosterone Fig. 3. Segmented Taenia solium tapeworms synthesize testosterone from tritiate androstenedione. The worms were divided in four segments from the head to the tail, and the segments were individually incubated in the presence of 3H-androstendione for 3 h. TLC analysis of media showed mainly testosterone. Data indicate mean ± SEM (n = 8 tapeworms).
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Fig. 4. Corticosteroid synthesis by Taenia solium tapeworms. The complete worms were individually incubated for 48 h with H-progesterone and the metabolites were separated by TCL using bencene:acetone (50:50 v/v) (A) or toluene:acetone:methanol (78:20:2) v/v. Data indicate mean ± SEM (n = 12 tapeworms).
cysticerci use D5 and D4 steroidogenic pathways to synthesize sex steroids (Valdéz et al., 2006; Jiménez et al., 2006; Aceves-Ramos et al., 2014). Interestingly, when the tapeworms were segmented and incubated with 3H-A4 instead of 3H-P4, testosterone synthesis by tapeworms was higher which could be explained by the shorter pathway to testosterone synthesis used when androstenedione was the steroid precursor and also probably by the more efficient penetration of the precursor to the segmented tapeworm. The results shown in Fig. 3, where a trend toward higher levels of testosterone synthesis was observed in the distal segments II and III compared to the proximal segments may be related to the abundance of testis that increased from the immature to the more
mature proglottids to finally disappear in the last segments of a complete mature tapeworm (Willms et al., 2003). We have shown recently that T. crassiceps WFU cysticerci transformed tritiated progesterone to corticosteroids, being DOC the most abundant metabolite (Valdéz et al., 2012). The results in Fig. 4A and B showed that T. solium tapeworms developed in the hamster gut have the capacity to use a steroid precursor like P4 to synthesize corticosteroids, and strongly suggest that the tapeworms have some of the enzymes involved in the steroidogenic pathways that conduct to their synthesis, in example CYP21A2 (21-hydroxyilase) or CYP2C19. As in the case of cysticerci, DOC and 11-deoxycortisol were the most abundant metabolites found in the culture media what suggest a poor activity of the enzymes
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involved in corticosterone and cortisol synthesis. An ‘‘incomplete’’ corticosteroid synthesis occurs in some mammals as the rat, mouse, and also in birds that produce corticosterone as the main corticosteroid. In these animals, corticosterone covers both gluco and mineralocorticoid functions. Many species that synthesize corticosteroids lack complete steroidogenic pathways; for example teleosts fishes lacks aldosterone, and therefore cortisol may have both, GC and MC actions (for a review see Vinson, 2011). Close et al. (2010) provided evidence that 11-deoxycortisol is the main corticosteroid hormone in the lamprey, an agnathan member that belongs to the oldest vertebrate lineage. Data in the literature had demonstrated that DOC is a potent mineralocorticoid and also a glucocorticoid in mammals and in other species (Vinson, 2011; Brookes et al., 2012), and that DOC serum concentration increases in the rainbow trout during spermiation (Campbell et al., 1980; Milla et al., 2008) and is an agonist of the MC in this fish (Sturm et al., 2005). Interesting phylogenetic studies on corticosteroid receptors performed by several authors suggested that a MC receptor complex evolved first (Thornton, 2001; Kumar and Hedges, 1998; Bridghamm et al., 2006; Stolte et al., 2006). Close et al. (2010) had demonstrated that 11-deoxycortisol participates in lamprey modulation of gill Na+-K+-ATPase activity. Furthermore, studies in the rainbow trout have shown that DOC concentrations raise at the end of the reproductive cycle and this finding was related to milt fluidity (Milla et al., 2008). Several authors had shown the effect of corticosteroids in Na+-K+-ATPase pump and ion movements in smoltification, a process that prepares young fishes for the transition from fresh water to salt water is controlled by cortisol among another hormones (as reviewed by Mommsen et al., 1999; Mc Cormick, 2001; Varsamos et al., 2005). Deoxycorticosterone also participates in osmoregulation, and gill Na-K ATPase mrRNA levels in salmon (Mc Cormick, 2001) and in Mozambique tilapia and striped bass (Kiilerich et al., 2011). Interestingly, a Na+-K+-ATPase had been cloned and detected in cysticerci by Western Blot and immunocytochemistry (Willms et al., 2004) suggesting that these parasites maintain their internal media using mechanisms similar to those described for mammals and reptiles. Results from the literature shown above allow to hypothesize that DOC and deoxycortisol may have a role in cysticerci osmoregulation and Na+-K+ equilibrium by the regulation of the tapeworm enzyme expression and function. In summary, the results described here add new information on the reproductive physiology of T. solium tapeworms. In addition, this study showed that T. solium tapeworms have the ability to transform 3H-P4, to corticosteroid like metabolites being 11-DOC the most abundant. It is well known that corticosteroids are critical hormones for the growth, glucose metabolism, immune function and electrolyte balance in many species. Thereafter, corticosteroids may be critical hormones for the tapeworm development and survival. In addition, it is well known that parasites module the host immune response and a recent paper reviewed the amelioration effect of parasitosis in the unwanted inflammatory responses that occurs in autoimmune or allergic diseases (Reyes-Hernandez et al., 2013). Therefore, together with many other molecules parasite’s corticosteroids may be contributing to modulate the host immune response.
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5. Conclusions
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The results of this study indicate that the incubation of the tapeworms with 3H-progesterone rendered small amounts of sex steroid like metabolites. Additional experiments showed that incu-
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bation of the worms with 3H-androstenedione instead of 3H-progesterone improved their capacity to synthesize testosterone. Furthermore, our results indicate that T. solium tapeworms maintained in culture have the ability to transform 3H-P4 to tritiated corticosteroid like metabolites, mainly DOC and 11-deoxycortisol. Tritiated DOC and 11-deoxycortisol were the main corticosteroids synthesized by T. solium tapeworms cultured in the presence of 3H-P4.
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Acknowledgments
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The authors wish to acknowledge MVZ José Agustin Jiménez for the development of tapeworms in hamsters. This project was partially supported by CONACYT project #79686.
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References
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Penning, T.M., 1997. Molecular endocrinology of hydroxysteroid dehydrogenases. Endocr. Rev. 18, 281–305. Payne, A., 2004. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Rev. 25, 947–970. Pazirandeh, Xue, Y., Rafter, I., Sjoväl, J., Jondal, M., Okret, S., 1999. Paracrine glucocorticoid activity produced by thymic epithelial cells. FASEB J. 13, 893– 901. Provost, P.R., Tremblay, Y., 2005. Genes involved in the adrenal pathway of glucocorticoid synthesis are transiently expressed in the developing lung. Endocrinology 146, 2239–2245. Romano, M.C., Valdéz, R.A., Cartas, A.L., Gómez, Y., Larralde, C., 2003. Steroid hormone production by parasites: the case of Taenia crassiceps and Taenia solium cysticerci. J. Steroid Biochem. Mol. Biol. 85, 221–225. Romano, M.C., Valdéz, R.A., Hinojosa, L., Gómez, Y., Jiménez, P., 2008. Are hormones relevant for the search and design of anti-parasitic drugs? Curr. Top. Med. Chem. 8, 408–414. Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory and preparative actions. Endocr. Rev. 21, 55–89. Simard, J., Rickkets, M.L., Gingras, S., Soucy, P., Feltus, F.A., Melner, F.H., 2005. Molecular biology of the 3beta-hydroxysteroid dehydrogenase delta 5/delta 4 isomerase gene family. Endocr. Rev. 26, 525–582. Stocco, D.M., Clark, B.J., 1996. Regulation of the acute production of steroids in steroidogenic cells. Endocr. Rev. 17, 221–244. Stolte, E.H., Kemenade, B.M., Savelkoul, H.F.J., Flik, G., 2006. Evolution of glucocorticoid receptors with different glucocorticoid sensitivity. J. Endocrinol. 190, 17–28. Sturm, B.J., Bury, N., Dengreville, L., Fagart, J., Flouriot, G., Rafestin-Oblin, M.E., Prunet, P., 2005. 11-Deoxycorticosterone is a potent agonist of the rainbow
trout (Oncorhynchus mykiss) mineralocorticoid receptor. Endocrinology 146, 47–55. Terrazas, L.I., 2008. Susceptibility and resistance to cysticercosis. Curr. Top. Med. Chem. 8, 408–414. Thornton, J.W., 2001. Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proc. Natl. Acad. Sci. U.S.A. 98, 5671–5676. Vacchio, M.S., Papadopoulos, V., Ashwell, J.D., 1994. Steroid production in the thymus: implications for thymocyte selection. J. Exp. Med. 179, 1835–1846. Valdéz, R.A., Jiménez, P., Cartas, A.L., Gómez, Y., Romano, M.C., 2006. Taenia solium cysticerci synthesize androgens and estrogens in vitro. Parasitol. Res. 98, 472– 476. Valdéz, R.A., Hinojosa, L., Gómez, Y., Willms, K., Romano, M.C., 2012. Taenia crassiceps WFU cysticerci synthesize corticosteroids in vitro: metyrapone regulates the production. Gen. Comp. Endocrinol. 176, 409–414. Varsamos, S., Nebel, C., Charmantier, G., 2005. Ontogeny of osmoregulation in postembryonic fish: a review. Comp. Biochem. Physiol. A 141, 401–429. Vinson, G.P., 2011. The mislabelling of deoxycorticosterone: making sense of corticosteroid structure and function. Wada, H., 2008. Glucocorticoids: mediators of vertebrate ontogenic transitions. Gen. Comp. Endocrinol. 156, 441–453. Willms, K., Shoemaker, Ch.B., Skelly, P.J., Landa, A., 2004. Cloning and expression of a Na+, K+-ATPase-subunit from Taenia solium (TNaK1). Mol. Biochem. Parasitol. 138, 79–82. White, A.C., 2000. Neurocysticercosis: updates on epidemiology, pathogenesis, diagnosis and management. Annu. Rev. Med. 51, 187–206. Willms, K., Caro, J.A., Robert, L., 2003. Ultrastructure of spermatogonia and spermatocyte lobules in Taenia solium strobilae (Cestoda, Cyclophyllidea, Taeniidae) from golden hamsters. Parasitol. Res. 90, 479–488.
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Contents lists available at ScienceDirect
General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen 5 6
Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro
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R.A. Valdez a, P. Jiménez a, A.M. Fernández Presas b, L. Aguilar b, K. Willms a, M.C. Romano a,⇑ a b
Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV del I.P.N., Apdo. Postal 14-745, 07360 México, D.F., Mexico Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM, Ciudad Universitaria, México 04510, D.F., Mexico
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Keywords: Taeniids Taenia solium tapeworms Corticosteroid synthesis Sex steroid synthesis Helmynths Parasites
a b s t r a c t Cysticercosis is a disease caused by the larval stage of Taenia solium cestodes that belongs to the family Taeniidae that affects a number of hosts including humans. Taeniids tapeworms are hermaphroditic organisms that have reproductive units called proglottids that gradually mature to develop testis and ovaries. Cysticerci, the larval stage of these parasites synthesize steroids. To our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursors to steroid hormones. Therefore, the aim of this paper was to investigate if T. solium tapeworms were able to transform steroid precursors to corticosteroids and sex steroids. T. solium tapeworms were recovered from the intestine of golden hamsters that had been orally infected with cysticerci. The worms were cultured in the presence of tritiated progesterone or androstenedione. At the end of the experiments the culture media were analyzed by thin layer chromatography. The experiments described here showed that small amounts of testosterone were synthesized from 3H-progesterone by complete or segmented tapeworms whereas the incubation of segmented tapeworms with 3H-androstenedione, instead of 3 H-progesterone, improved their capacity to synthesize testosterone. In addition, the incubation of the parasites with 3H-progesterone yielded corticosteroids, mainly deoxicorticosterone (DOC) and 11-deoxicortisol. In summary, the results described here, demonstrate that T. solium tapeworms synthesize corticosteroid and sex steroid like metabolites. The capacity of T. solium tapeworms to synthesize steroid hormones may contribute to the physiological functions of the parasite and also to their interaction with the host. Ó 2014 Published by Elsevier Inc.
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1. Introduction
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Taenia solium (T. solium) tapeworms belong to the family Taenidae and develop and live for long periods in the gut of the human. T. solium cysticerci are the larval stage of tapeworms and live in the pork muscles and in the human brain causing a disease called neurocysticercosis. Throughout the course of cysticercosis infections, multiple host–parasite interactions occur that affect the human host immune response and cause severe endocrine and immune disturbances (White, 2000; Terrazas, 2008; Cárdenas et al., 2012). Once the scolex of the T. solium worm attaches to the duodenum of the definitive human host, growth proceeds from the germinative tissue in the neck by continuous duplication of undifferentiated cells, which migrate from the medullary region to the tegument (Merchant et al., 1997). Adult Taenias are hermaphroditic organisms with one of the highest reproduc⇑ Corresponding author. E-mail address: mromano@fisio.cinvestav.mx (M.C. Romano).
tive capacities among invertebrate species, worms can grow several meters in length and produce millions of infective eggs in a lifetime, which are released in the feces. The tapeworm develops reproductive units called proglottids, these units gradually mature to develop testis and later ovaries. Different experimental models had been used to study T. solium tapeworm and cysticerci infections in the laboratory (Zubarian and Q6 Willms, 2008). Experimental infections of mice with Taenia crassiceps ORF or WFU cysticerci had been extensively used to study immune aspects of the parasite–host interactions (Terrazas, 2008). In our laboratory we have been studying the capacity of cysticerci to synthesize steroid hormones (Romano et al., 2003, 2008). We have investigated the capacity of T. crassiceps ORF cysticerci to synthesize sex steroids in vitro and found that these parasites transform tritiated androstenedione to testosterone (Gómez et al., 2000). We also found that T. solium cysticerci from infected pork meat, produce androgens and estrogens in vitro (Valdéz et al., 2006). In addition, T. solium and T. crassiceps ORF cysticerci transform steroid precursors such as progesterone,
http://dx.doi.org/10.1016/j.ygcen.2014.04.014 0016-6480/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Valdez, R.A., et al. Taenia solium tapeworms synthesize corticosteroids and sex steroids in vitro. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.04.014
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dehydroepiandrosterone, and androstenedione (Jiménez et al., 2006). To this regard, we had recently shown that that the larval stage of T. solium tapeworms express a 17b-hydroxysteroid dehydrogenase that catalyzes the transformation of androstenedione to testosterone and belongs to the short chain dehydrogenases/ Q7 reductase family (Aceves-Ramos et al., 2014). It is well known that 3b-hydroxysteroid-dehydrogenase (3b-HSD) is a key enzyme in the biosynthesis of gonad and adrenal steroid hormones (Labrie et al., 1994; Conley and Bird, 1997; Penning, 1997; Payne and Hale, 2004; Simard et al., 2005). To our knowledge there is not much information available related to Taenia tapeworms steroidogenic capacity. We have demonstrated the presence of active 3b-HSD in T. crassiceps WFU cysticerci and in tapeworm tissues, where the enzyme was found in the subtegumentary areas of the neck and immature proglottids (Fernández Presas et al., 2008). We have shown that experimental T. crassiceps WFU tapeworms obtained from the intestine of infected hamsters, synthesize androstenediol and 17b-estradiol when incubated in the presence of tritiated dehydroepiandrosterone (Fernández Presas et al., 2008). These results, strongly suggest the presence and activity of D5 steroid pathway enzymes in T. crassiceps WFU tapeworms, like the 3b-hydroxysteroid-dehydrogenase/D5–4 isomerase-like enzyme (3b-HSD), which converts androstenediol to testosterone, and dehydroepiandrosterone (DHEA) to androstenedione (Fernández Presas et al., 2008). However, to our knowledge there is no information about the capacity of T. solium tapeworms to metabolize progesterone or other precursor to sex steroid hormones. On the other hand, we have recently shown that T. crassiceps WFU cysticerci obtained from mice peritoneal cavity synthesize corticosteroids in vitro, and that metyrapone and ketoconazole blocks their synthesis (Valdéz et al., 2012; Aceves-Ramos et al., 2013). The cysticerci and tapeworms steroidogenic capacity might play a role in the permanence of the parasites in muscle and brain tissues and in the host intestine. It is well known that cortisol, corticosterone and aldosterone are essential hormones in most tetrapods. Their importance in stress response, immunity, metabolism and differentiation is well known (Sapolsky et al., 2000). Glucocorticoids (GCs) are synthesized by the adrenal cortex (Stocco and Clark, 1996) or the interrenal tissues of amphibians, reptiles and fishes. Interestingly, local CS synthesis has been reported in several tissues like the brain and cardiovascular tissues (for a review see Davies and MacKenzie, 2003). Furthermore, corticosteroid synthesis and the corresponding steroidogenic enzymes and their genes were found in thymic epithelial cells (Vacchio et al., 1994; Pazirandeh et al., 1999) in the developing lung cells of mammals (Provost and Tremblay, 2005) and in human skin keratinocytes (Hannen et al., 2011). Corticosteroids are also important for many developmental events. Data reported in the literature strongly suggest that corticosteroids participate in germ cell development and on selected ovarian steroidogenic enzymes (Michael and Cooke, 1994). Wada (2008) published a complete vision of GCs as mediators for vertebrate ontogenic 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 fish through Na+-K+ ATPase activity and ion transport among other events critical for survival. Besides their multiple physiological effects, it is well known that corticosteroids have a critical role in inflammation, modulating the response of immune cells to infections. Therefore, we considered important to investigate if tapeworms have the capacity to synthesize corticosteroids and release them to the culture media. In the present paper we have investigated the steroidogenic capacity of T. solium tapeworms. We show here that these parasites
synthesize corticosteroids and androgens from tritiated steroid precursors.
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2. Materials and methods
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2.1. Animals and specimen preparation
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The growth of tapeworm in the hamster intestine. Adult T. solium tapeworms were recovered from the intestine of golden hamsters (Mesocricetus auratus) inoculated 25–45 days before by oral administration of 8–10 cysticerci obtained either from naturally infected pork. Hamsters were pretreated for 5 days with 40 mg/kg of albendazole (Zentel, oral suspension, Smith, Kline, Beecham, Mexico) and injected with 2 mg of methyl-prednisolone acetate (Depomedrol, Upjohn, Mexico) on the day of infection and every 2 weeks for the duration of the experiments. Animals were kept on commercial food pellets and water under controlled conditions and killed by injection of sodium barbital (Anestesal, Cat. Q-0001-065 for Veterinary use, Pfizer, Mexico) and the small intestine opened longitudinally to recover the tapeworms from the duodenal cavity (Willms et al., 2003; Fernández Presas et al., 2008).
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2.2. Steroid synthesis by tapeworms
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T. solium tapeworms were thoroughly rinsed in PBS, and then washed twice with Dulbecco’s Modified Eagle Medium plus antibiotics (DMEM+A), 1 ml/100 ml of media (penicillin–streptomycin– amphotericin B, Gibco, In Vitro, USA).The parasites or the sections were transferred to tissue culture dishes containing 2 ml of DMEM+A and 0.1% bovine serum albumin. The tapeworms were pre-cultured for 48 h at 37 °C in a 95% air-5% CO2 atmosphere. In some experiments, the parasites were divided in sections from the head (Section I) to the tail (Sections II, III and IV). After this period the parasites were transferred to new dishes and 3H-progesterone (1,2,6,7-3H progesterone, 93.0 Ci/mmol, New Products, MS, Dupont, Boston, MA; 3H-P4) or 3H-androstenedione (1,2,6,73 H-(N)-androst-5-ene-3b-ol-17-one, 96 Ci/mmol, Perkin Elmer, Boston, MA, USA; 3H-A4) as steroid precursors were added to each parasite and further incubated for different periods in the conditions described above. Blanks containing the culture media plus 3 H-P4 were simultaneously incubated. The parasites were removed and media transferred to vials where steroids were extracted with 2 volumes of cold anhydrous ether. The organic phase was transferred to new vials and evaporated in a nitrogen atmosphere at 37 °C. The samples were reconstituted in 100 ll of absolute ethanol. Metabolites synthesized from the precursors were identified by thin layer chromatography.
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2.3. Thin layer chromatography (TLC)
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Tritiated progesterone (3H-P4) or androstenedione (3H-A4) were used as steroid precursors. Progesterone, corticosteroids (Cortisol, corticosterone, 11-deoxicorticosterone (DOC) and 11-deoxycortisol) and hydroxiprogesterone (OH-P4), androstenedione (A4), testosterone (T) estrogens (17b-estradiol and estrone), purchased from Steraloids (Steraloids, Wilton, NH, USA) were used as authentic standards. TLC was carried out using silica gel plates (20 20 Kieselgel 60 F254, Merck, Darmstadt, Germany). Aliquots of 40 ll of the ethanol samples were supplemented with the standard steroids and further separated in different TLC systems. Plates were developed with bencene:acetone 50:50 v/v or toluene:acetone:methanol (78:20:2 v/v) to separate corticosteroids and with benzene–methanol (9:1 v/v) to separate sex steroid hormones. The standard steroids were detected in the plates by ultraviolet light and also exposed to 10% H2SO4 followed by heating at
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120 °C. Regions corresponding to the authentic standards were cut and placed in vials containing scintillation liquid and radioactivity was counted in a liquid scintillation counter. The recovery of radioactivity was estimated by comparing the difference between initial and final cpm. Results are expressed as the percentage of substrate transformation for each identified metabolite after incubation with the corresponding tritiated precursor.
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Statistical analysis were performed using Prism version 4 2003 (GraphPad Software Inc.). Data represent as means ± SE. Probability values of p < 0.05 were considered significant. Kolmogorov Smirnov normality test was performed to analyse the data distribution followed by Kruskal Wallis nonparametric test.
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3.1. Synthesis of corticosteroids and sex steroids from tritiated progesterone
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Fig. 1 shows the sex steroid metabolites found after individual incubation of intact T. solium tapeworms (n = 8) in the presence of 3H-P4 if plates were developed in a solvent system that detected sex steroids. A modest synthesis of tritiated T4 was found in the culture media, whereas 17b-estradiol and estrone were under the detection limit of the technique. In some experiments the tapeworms were divided in four sections before incubation with 3 H-P4 for 3 h and the metabolite synthesis was similar to that found in the uncut tapeworms (Fig. 2).
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METABOLITE Fig. 1. Sex steroids synthesis from tritiated progesterone by T. solium tapeworms. Each complete tapeworm was individually incubated for 3 h in the presence of 3Hprogesterone and the tritiated metabolites were separated by TCL. Data indicate mean ± SEM (n = 8 tapeworms).
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4. Discussion
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The results of the present study showed that T. solium tapeworms have the ability to synthesize in vitro sex steroid like metabolites using tritiated progesterone or androstenedione as precursors. The transformation of the labeled steroid precursors 3 H-P4 or 3H-A4 to tritiated sex steroids such as OH-P4, A4 and T4 (Figs. 1–3) strongly suggests that these taeniid worms have at least part of a functional D4 steroidogenic enzymatic pathway. It follows that the worms should have steroidogenic enzymes such as 17, 20-lyase that converts progesterone to 17-OH progesterone, 17a-hydroxilase that transforms 17-OH progesterone to androstenedione, and 17b-HSD that converts androstenedione to testosterone. In fact, we have recently shown that the larval stage of T. solium tapeworms expresses a functional 17b-hydroxysteroid dehydrogenase that catalyzes the transformation of androstenedione to testosterone, and belongs to the short chain dehydrogenases/reductase family (Aceves-Ramos et al., 2014). Therefore, if the enzyme is expressed in the larvae, we propose that it should be also present in the tapeworm and could catalyze the synthesis of testosterone from androstenedione observed in the present study. These enzymes are part of the D4 steroidogenic pathway in most mammalian and avian species. Mammalian gonads have both D5 and D4 steroidogenic pathways, but which one predominates depends on the organ, in example the former is predominant in the ovary and the latter in the testis (Payne, 2004). Previous work from our group strongly suggests that T. solium and T. crassiceps
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However, if T. solium segments were incubated with 3H-A4 instead of 3H-P4, testosterone synthesis was higher compared with that of the experiments where tritiated progesterone was the precursor (Fig. 3). Using this experimental approach testosterone transformation of 3H-A4 to testosterone was ten to 20 times higher. Interestingly, there was a trend toward higher levels of testosterone synthesis in the distal segments II and III compared to the proximal segments (Fig. 3). The incubation of each of 12 T. solium tapeworms in the presence of 3H-P4 for 48 h yielded 17OH-progesterone, 11-deoxycorticosterone and 11-deoxycortisol as main metabolites judged by the percent of transformation. However, small amounts of corticosterone and cortisol were also found in both solvent systems used to separate the corticosteroid metabolites. Fig. 4A shows the results found when TLC plates were developed in bencene:acetone (50:50 v/v) and Fig. 4B when developed in toluene:acetone:methanol (78:20:2 v/v). Both solvent systems showed similar amounts of tritiated 17OH-progesterone, DOC and 11-deoxycortisol, corticosterone and cortisol. In both systems DOC was the most abundant metabolite.
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METABOLITE Fig. 2. Sex steroid synthesis by segmented T. solium tapeworms. The worms were divided in four segments from the head to the tail and individually incubated for 3 h with 3 H-progesterone and the metabolites were separated by TLC. I II III IV . Data indicate mean ± SEM (n = 3 tapeworms).
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3H-Testosterone Fig. 3. Segmented Taenia solium tapeworms synthesize testosterone from tritiate androstenedione. The worms were divided in four segments from the head to the tail, and the segments were individually incubated in the presence of 3H-androstendione for 3 h. TLC analysis of media showed mainly testosterone. Data indicate mean ± SEM (n = 8 tapeworms).
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Fig. 4. Corticosteroid synthesis by Taenia solium tapeworms. The complete worms were individually incubated for 48 h with H-progesterone and the metabolites were separated by TCL using bencene:acetone (50:50 v/v) (A) or toluene:acetone:methanol (78:20:2) v/v. Data indicate mean ± SEM (n = 12 tapeworms).
cysticerci use D5 and D4 steroidogenic pathways to synthesize sex steroids (Valdéz et al., 2006; Jiménez et al., 2006; Aceves-Ramos et al., 2014). Interestingly, when the tapeworms were segmented and incubated with 3H-A4 instead of 3H-P4, testosterone synthesis by tapeworms was higher which could be explained by the shorter pathway to testosterone synthesis used when androstenedione was the steroid precursor and also probably by the more efficient penetration of the precursor to the segmented tapeworm. The results shown in Fig. 3, where a trend toward higher levels of testosterone synthesis was observed in the distal segments II and III compared to the proximal segments may be related to the abundance of testis that increased from the immature to the more
mature proglottids to finally disappear in the last segments of a complete mature tapeworm (Willms et al., 2003). We have shown recently that T. crassiceps WFU cysticerci transformed tritiated progesterone to corticosteroids, being DOC the most abundant metabolite (Valdéz et al., 2012). The results in Fig. 4A and B showed that T. solium tapeworms developed in the hamster gut have the capacity to use a steroid precursor like P4 to synthesize corticosteroids, and strongly suggest that the tapeworms have some of the enzymes involved in the steroidogenic pathways that conduct to their synthesis, in example CYP21A2 (21-hydroxyilase) or CYP2C19. As in the case of cysticerci, DOC and 11-deoxycortisol were the most abundant metabolites found in the culture media what suggest a poor activity of the enzymes
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involved in corticosterone and cortisol synthesis. An ‘‘incomplete’’ corticosteroid synthesis occurs in some mammals as the rat, mouse, and also in birds that produce corticosterone as the main corticosteroid. In these animals, corticosterone covers both gluco and mineralocorticoid functions. Many species that synthesize corticosteroids lack complete steroidogenic pathways; for example teleosts fishes lacks aldosterone, and therefore cortisol may have both, GC and MC actions (for a review see Vinson, 2011). Close et al. (2010) provided evidence that 11-deoxycortisol is the main corticosteroid hormone in the lamprey, an agnathan member that belongs to the oldest vertebrate lineage. Data in the literature had demonstrated that DOC is a potent mineralocorticoid and also a glucocorticoid in mammals and in other species (Vinson, 2011; Brookes et al., 2012), and that DOC serum concentration increases in the rainbow trout during spermiation (Campbell et al., 1980; Milla et al., 2008) and is an agonist of the MC in this fish (Sturm et al., 2005). Interesting phylogenetic studies on corticosteroid receptors performed by several authors suggested that a MC receptor complex evolved first (Thornton, 2001; Kumar and Hedges, 1998; Bridghamm et al., 2006; Stolte et al., 2006). Close et al. (2010) had demonstrated that 11-deoxycortisol participates in lamprey modulation of gill Na+-K+-ATPase activity. Furthermore, studies in the rainbow trout have shown that DOC concentrations raise at the end of the reproductive cycle and this finding was related to milt fluidity (Milla et al., 2008). Several authors had shown the effect of corticosteroids in Na+-K+-ATPase pump and ion movements in smoltification, a process that prepares young fishes for the transition from fresh water to salt water is controlled by cortisol among another hormones (as reviewed by Mommsen et al., 1999; Mc Cormick, 2001; Varsamos et al., 2005). Deoxycorticosterone also participates in osmoregulation, and gill Na-K ATPase mrRNA levels in salmon (Mc Cormick, 2001) and in Mozambique tilapia and striped bass (Kiilerich et al., 2011). Interestingly, a Na+-K+-ATPase had been cloned and detected in cysticerci by Western Blot and immunocytochemistry (Willms et al., 2004) suggesting that these parasites maintain their internal media using mechanisms similar to those described for mammals and reptiles. Results from the literature shown above allow to hypothesize that DOC and deoxycortisol may have a role in cysticerci osmoregulation and Na+-K+ equilibrium by the regulation of the tapeworm enzyme expression and function. In summary, the results described here add new information on the reproductive physiology of T. solium tapeworms. In addition, this study showed that T. solium tapeworms have the ability to transform 3H-P4, to corticosteroid like metabolites being 11-DOC the most abundant. It is well known that corticosteroids are critical hormones for the growth, glucose metabolism, immune function and electrolyte balance in many species. Thereafter, corticosteroids may be critical hormones for the tapeworm development and survival. In addition, it is well known that parasites module the host immune response and a recent paper reviewed the amelioration effect of parasitosis in the unwanted inflammatory responses that occurs in autoimmune or allergic diseases (Reyes-Hernandez et al., 2013). Therefore, together with many other molecules parasite’s corticosteroids may be contributing to modulate the host immune response.
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5. Conclusions
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The results of this study indicate that the incubation of the tapeworms with 3H-progesterone rendered small amounts of sex steroid like metabolites. Additional experiments showed that incu-
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bation of the worms with 3H-androstenedione instead of 3H-progesterone improved their capacity to synthesize testosterone. Furthermore, our results indicate that T. solium tapeworms maintained in culture have the ability to transform 3H-P4 to tritiated corticosteroid like metabolites, mainly DOC and 11-deoxycortisol. Tritiated DOC and 11-deoxycortisol were the main corticosteroids synthesized by T. solium tapeworms cultured in the presence of 3H-P4.
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Acknowledgments
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The authors wish to acknowledge MVZ José Agustin Jiménez for the development of tapeworms in hamsters. This project was partially supported by CONACYT project #79686.
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