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Reproductive toxicology of endocrine disruptors: effects of cadmium, phthalates and phytoestrogens on testicular steroidogenesis
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A number of investigations during the last two decades describe adverse trends in male reproductive health, which have been proposed to be caused by environmental factors with endocrine disrupting properties. In contrast to many other toxicants, endocrine disruptors often do not show linear dose-response relationships typical of those found in traditional toxicological studies. For many compounds, low-dose exposure causes effects opposite to the ones seen after high-dose exposure. In addition, the timing of exposure has been found to be critical. Hence, to correctly assess the impact of endocrine disruptors on reproductive health requires in-depth knowledge of their mechanisms of action.

This thesis aimed at identifying the mechanisms underlying the effects of cadmium (Cd), phthalates and phytoestrogens on testicular steroidogenesis. For this purpose, in vitro as well as in vivo models were used. Cd was found to inhibit testosterone synthesis in vivo by down-regulating LH receptor gene expression and reducing the testicular levels of cAMP and StAR protein. In addition, Cd caused a pronounced increase in testicular prostaglandin F (PGF), suggesting that Cd exerts its suppressive effect on steroidogenesis also by inducing the inhibitory PKC pathway. Pre-treatment with zinc (Zn) protected completely against Cd-induced effects on testosterone and PGF. Furthermore, we observed that Cd exposure increased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in the testis. GAPDH is a potent coactivator of androgen receptor-mediated transcription and the up-regulation found in our study is probably a compensatory response to reduced testosterone concentrations. This finding is interesting since GAPDH has been proposed to have an important role in the regulation of apoptosis as well as sperm motility. We discovered that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used phthalate di-(2-ethylhexyl) phthalate (DEHP), stimulates Leydig cell steroidogenesis in vitro, by a cAMP- and StAR-independent mechanism. MEHP exposure caused a similar effect in granulosa cells. Gene expression analysis revealed that MEHP is likely to stimulate steroidogenesis by increasing the amount of cholesterol available for steroid synthesis. In the last investigation, we examined the effects of low-dose phytoestrogen exposure on testosterone synthesis during puberty in male goats. Isoflavones present in clover increased plasma concentrations of testosterone and free as well as total triiodothyronine (T3). T3 has previously been shown to induce testosterone synthesis and it is possible that an elevated T3 secretion underlies the increased plasma testosterone levels.

Reduced fertility and reproductive tract malformations affect both the individual and the society. Hence, a sound knowledge of reproductive toxicants is of crucial importance. The findings presented in this thesis provide new insights into the reproductive toxicology of endocrine disruptors and may be valuable for risk assessment purposes.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet) , 2008. , 90 p.
Keyword [en]
Endocrine disruptors, reproductive toxicology, cadmium, phthalates, DEHP, MEHP, phytoestrogens, steroidogenesis, testosterone, Leydig cell
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1876ISBN: 978-91-7264-631-5 (print)OAI: oai:DiVA.org:umu-1876DiVA: diva2:142275
Public defence
2008-10-31, Major Groove, Building 6L, Department of Molecular Biology, Umeå University S-901 87, Umeå, Sweden, 10:00 (English)
Opponent
Supervisors
Available from: 2008-10-09 Created: 2008-10-09 Last updated: 2010-04-06Bibliographically approved
List of papers
1. Cadmium-induced decrement of the LH receptor expression and cAMP levels in the testis of rats
Open this publication in new window or tab >>Cadmium-induced decrement of the LH receptor expression and cAMP levels in the testis of rats
2003 (English)In: Toxicology, ISSN 0300-483X, Vol. 183, no (1-3), 57-63 p.Article in journal (Refereed) Published
Abstract [en]

Cadmium (Cd) is a widespread environmental pollutant, characterized by its ability to affect various organs. Adverse effect of Cd on the testis including decreased testosterone production are well-known phenomena, but the cellular events explaining these effects have not yet been established. In the present study the initial steps of gonadotropin mediated testosterone biosynthesis were examined in vivo in rats, in relation to Cd dose and time after injection. In the dose–response experiment Male Sprague–Dawley rats received a single subcutaneous (sc) injection of CdCl2 (1, 5 or 10 μmol/kg body weight) and were sacrificed 48 h after injection. A statistically significant decrease in luteinizing hormone (LH) receptor mRNA level in the testicular tissue was demonstrated at the highest dose (10 μmol/kg). In the temporal–response experiment rats were given 10 μmol/kg of CdCl2 sc and sacrificed 0.48, 4.8, 48 or 144 h after injection. LH receptor mRNA levels as well as cyclic adenosine monophosphate (cAMP) levels were found to be significantly lowered at 48 and 144 h. These observations of the mechanisms whereby Cd exerts its effect on the initial steps of testosterone biosynthesis are the first from in vivo experiments.

Keyword
Cadmium; Luteinizing hormone receptor; Cyclic adenosine monophosphate system; Steroidogenesis; Rat; Testis
Identifiers
urn:nbn:se:umu:diva-3524 (URN)10.1016/S0300-483X(02)00440-7 (DOI)
Available from: 2008-10-09 Created: 2008-10-09 Last updated: 2009-06-23Bibliographically approved
2. Pronounced induction of testicular PGF2alpha and suppression of testosterone by cadmium: prevention by zinc
Open this publication in new window or tab >>Pronounced induction of testicular PGF2alpha and suppression of testosterone by cadmium: prevention by zinc
2004 (English)In: Toxicology, ISSN 0300-483X, Vol. 200, no 1, 49-58 p.Article in journal (Refereed) Published
Abstract [en]

In order to investigate the effects of cadmium (Cd) on testicular prostaglandin F(PGF) production, adult male Sprague–Dawley rats were exposed to CdCl2 by subcutaneous injections. Dose–response as well as temporal–response experiments were performed, and PGF levels were determined by radioimmunoassay (RIA). The highest cadmium dose (10 μmol/kg) caused a dramatic elevation of testicular PGF, which was established to occur 48 h after exposure. At this point of time, cadmium-treated animals displayed PGF levels 16.7 times higher than saline-injected controls. No significant differences were found with the lower doses used (1 and 5 μmol/kg). In addition, the influence of pre-treatment with zinc (Zn) was assessed. The very strong stimulatory effect on PGF synthesis (22.3-fold) detected after exposure to 20 μmol/kg cadmium, was completely absent in the group given zinc (1 mmol/kg) prior to cadmium exposure. Plasma testosterone concentrations were determined in the three experiments, and all groups with strongly elevated PGF levels showed drastically lowered concentrations of testosterone. Zinc pre-treatment abolished not only the cadmium-induced rise in PGF but also the testosterone reduction. Additionally, cadmium was found to inhibit the expression of steroidogenic acute regulatory protein (StAR), which is responsible for the rate-limiting step in steroidogenesis. The present findings establish that cadmium can cause a strong induction of testicular PGF production, which might help to explain the well-known antisteroidogenic effect of this heavy metal. Such an inhibitory effect could be due to reduced levels of StAR.

Keyword
Cadmium; Rat; Prostaglandin F2α (PGF2α); Steroidogenic acute regulatory protein (StAR); Testis; Testosterone
Identifiers
urn:nbn:se:umu:diva-3525 (URN)10.1016/j.tox.2004.03.003 (DOI)
Available from: 2008-10-09 Created: 2008-10-09 Last updated: 2009-12-29Bibliographically approved
3. Differential effects of cadmium on the gene expression of seven-transmembrane-spanning receptors and GAPDH in the rat testis.
Open this publication in new window or tab >>Differential effects of cadmium on the gene expression of seven-transmembrane-spanning receptors and GAPDH in the rat testis.
2007 (English)In: Toxicology Letters, ISSN 0378-4274, Vol. 168, no 1, 51-7 p.Article in journal (Refereed) Published
Abstract [en]

Cadmium (Cd) is a widely spread toxicant with endocrine disrupting properties. Under experimental conditions it suppresses sex steroid synthesis in the male as well as the female. Testicular steroidogenesis is primarily regulated by gonadotropins, but is also influenced by catecholamines. We have previously shown that Cd exposure affects rat testosterone synthesis by down-regulating luteinizing hormone (LH) receptor mRNA expression. In this study, rats were given 10 micromol/kg Cd subcutaneously and sacrificed 0.48-144 h later. We investigated the effects of Cd on testicular gene expression of two adrenergic receptors. In addition, mRNA levels of the androgen-regulated house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured. In contrast to the suppressive influence on LH receptor expression Cd lacked effect on the expression of alpha(1A)- and beta(2)-adrenergic receptors. GAPDH gene expression, on the other hand, was up-regulated 1.6-fold after exposure to 10 micromol/kg Cd. These data suggest that the influence of Cd on testicular gene expression involves a specific effect on the LH receptor and not a general effect on seven-transmembrane-spanning receptors. Also, data indicate that the increased expression of GAPDH may be secondary to Cd-induced testosterone deprivation, suggesting future studies of androgen-regulated genes in the toxicity of Cd.

Keyword
Animals, Cadmium/*toxicity, Gene Expression/*drug effects, Glyceraldehyde-3-Phosphate Dehydrogenases/*genetics, Male, RNA; Messenger/metabolism, Rats, Rats; Sprague-Dawley, Receptors; Adrenergic; alpha-1/genetics, Receptors; Adrenergic; beta-2/genetics, Testis/*drug effects/metabolism
Identifiers
urn:nbn:se:umu:diva-16509 (URN)10.1016/j.toxlet.2006.10.015 (DOI)17123754 (PubMedID)
Available from: 2007-10-04 Created: 2007-10-04 Last updated: 2010-01-04Bibliographically approved
4. Mono-(2-ethylhexyl) phthalate stimulates basal steroidogenesis by a cAMP-independent mechanism in mouse gonadal cells of both sexes
Open this publication in new window or tab >>Mono-(2-ethylhexyl) phthalate stimulates basal steroidogenesis by a cAMP-independent mechanism in mouse gonadal cells of both sexes
Show others...
2008 (English)In: Reproduction, ISSN 1470-1626, Vol. 135, no 5, 693-703 p.Article in journal (Refereed) Published
Abstract [en]

Phthalates are widely used as plasticizers in a number of daily-life products. In this study, we investigated the influence of mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used plasticizer di-(2-ethylhexyl) phthalate (DEHP), on gonadal steroidogenesis in vitro. MEHP (25–100 µM) stimulated basal steroid synthesis in a concentration-dependent manner in immortalized mouse Leydig tumor cells (MLTC-1). The stimulatory effect was also detected in KK-1 granulosa tumor cells. MEHP exposure did not influence cAMP or StAR protein levels and induced a gene expression profile of key steroidogenic proteins different from the one induced by human chorionic gonadotropin (hCG). Simultaneous treatment with MEHP and a p450scc inhibitor (aminoglutethimide) indicated that MEHP exerts its main stimulatory effect prior to pregnenolone formation. MEHP (10–100 µM) up-regulated hormone-sensitive lipase and 3-hydroxy-3-methylglutaryl coenzyme A reductase, suggesting that MEHP increases the amount of cholesterol available for steroidogenesis. Our data suggest that MEHP, besides its known inhibitory effect on hCG action, can directly stimulate gonadal steroidogenesis in both sexes through a cAMP- and StAR-independent mechanism. The anti-steroidogenic effect of DEHP has been proposed to cause developmental disorders such as hypospadias and cryptorchidism, whereas a stimulation of steroid synthesis may prematurely initiate the onset of puberty and theoretically affect the hypothalamic–pituitary–gonadal axis.

Identifiers
urn:nbn:se:umu:diva-3527 (URN)10.1530/REP-07-0460 (DOI)
Available from: 2008-10-09 Created: 2008-10-09 Last updated: 2009-06-23Bibliographically approved
5. Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3) levels in male goat kids
Open this publication in new window or tab >>Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3) levels in male goat kids
Show others...
2009 (English)In: Acta Veterinaria Scandinavica, ISSN 1751-0147, E-ISSN 1751-0147, Vol. 51Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids. METHODS: Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3-4 mg/kg/day) for approximately 3 months. Plasma testosterone and total and free triiodothyronine (T3) concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA) was carried out. RESULTS: No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8) phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l). This elevation was preceded by a rise in plasma total T3 that occurred on September 17 (week 6). A slightly higher concentration of free T3 was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10) that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant. CONCLUSION: Our findings suggest that phytoestrogens can stimulate testosterone synthesis during puberty in male goats by increasing the secretion of T3; a hormone known to stimulate Leydig cell steroidogenesis. It is possible that feedback signalling underlies the tendency towards decreased steroid production at the end of the experiment.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-32860 (URN)10.1186/1751-0147-51-51 (DOI)20003293 (PubMedID)
Available from: 2010-03-29 Created: 2010-03-29 Last updated: 2017-12-12Bibliographically approved

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