Androgen Disruption Research Articles

In silico and in vitro evaluation of potential agonistic and antagonistic estrogenic and androgenic activities of pure cyanotoxins, microcystin-LR and cylindrospermopsin

The potential endocrine disruption activity of cyanotoxins, particularly their effects on estrogen and androgen receptors (ER, AR), remains poorly understood. In the present study, the potential agonistic/antagonistic estrogenic and androgenic activities of MC-LR and CYN have been determined for the first time with validated OECD Test Guidelines No. 455 and 458, respectively. The data show that only MC-LR demonstrated weak estrogenic agonistic effects (LogPC10 value of −9.85 M), while both toxins displayed antagonistic effects on the ER, with LogIC30 values of −4.4 and −6.4 for MC-LR and CYN, respectively. In addition, neither MC-LR nor CYN exhibited agonistic/antagonistic activities in AR. Docking studies revealed potential interactions between both toxins and AR, with CYN showing a higher predicted affinity for this receptor. In vivo studies, particularly those investigating androgen disruption, are warranted to confirm the endocrine disrupting potential of MC-LR and CYN.

A follow-up study on androgen disruption in Vietnamese children from a dioxin exposure area: Decreases in androgen levels with age

A follow-up study on androgen disruption in Vietnamese children from a dioxin exposure area: Decreases in androgen levels with age

Evaluation of the endocrine activity of surface water samples using aquatic eleuthero-embryos - a comparison with in vitro assays.

Over the previous decade numerous new approach methodologies (NAMs) have been developed and validated for the detection of endocrine activity of individual chemicals or environmental samples. These NAMs can be largely separated into three categories, in silico tools, in vitro assays and in vivo assays using organisms or life stages not considered as laboratory animals, each with their own advantages and disadvantages. While in vitro assays provide more mechanistic information, the use of whole organisms, such as fish or amphibian embryos provides a more holistic view of the net effects of an environmental sample on hormonal activity. A panel of bioassays was used to test the endocrine activity of several samples from the Danube River at Novi Sad, Serbia. The results of the in vitro assays have been published previously. Here we present the results of the in vivo assays that were performed at the same time on the same samples. These whole organism assays are based on the use of transgenic fish and amphibian eleuthero-embryos and included the Xenopus Eleuthero-embryo Thyroid Assay (XETA), the Rapid Estrogen ACTivity In Vivo assay (REACTIV) and the Rapid Androgen Disruption Activity Reporter (RADAR) assay. Discrepancies between the different in vitro assays have previously been reported. The results of the in vivo studies also indicate discrepancies between the in vivo and in vitro data with an underestimation of the endocrine activity by the in vitro tests. Therefore, a battery of tests is advised with the initial diagnostic performed with in vivo tests to cover a wider range of modes of action and to allow the appropriate in vitro assay(s) to be selected to confirm the mode of action.

The Endocrine‐Disrupting Chemical Di‐(2‐ethylhexyl) phthalate (DEHP) Increases Corticosteroid Secretion in Human Adrenocortical Cells

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that is widely used as a plasticizer in the production of polyvinyl chloride containing plastics. DEHP is a component of many common household items (toys, packaging, furniture, etc.) and medical devices, resulting in significant and chronic human exposure. Human exposure to DEHP has been linked to androgen disruption and developmental toxicity, including cryptorchidism, decreased anogenital distance, and decreased testosterone levels. Other adrenal corticosteroids are also susceptible to the endocrine-disrupting effects of DEHP. One study has demonstrated that fetal exposure to DEHP results in altered adrenal aldosterone production in adult rats. Due to these potential effects of DEHP on adrenal cortex steroidogenesis, the focus of our studies is to characterize the effect of DEHP on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of DEHP (10 nM-30 μM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. No effects on cell viability were observed with the concentrations studied. Basal aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 75.6±3.5; 10 μM DEHP, 7735.2±151.3; n=8). Ang II-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 4278.9±110.2; 10 μM DEHP, 18651.7±572.2; n=8). ACTH-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 124.0±3.7; 10 μM DEHP, 23455.8±272.7; n=8). Gene expression of most enzymes and cofactors involved in aldosterone synthesis were increased by 10 μM DEHP. Aldosterone synthase (CYP11B2) had the greatest induction at 175.2±2.0 fold. These data support the evidence that DEHP disrupts the regulation of aldosterone secretion and provides further evidence that DEHP is an endocrine disruptor.

Decreased serum testosterone levels associated with 17β-hydroxysteroid dehydrogenase activity in 7-year-old children from a dioxin-exposed area of Vietnam

Since 2008, we have conducted epidemiological cohort studies on the relationship between dioxin exposure and disruption with children in the area sprayed with defoliants during the Vietnam War. In a long-term survey of children through the age of five, we observed androgen disruption due to decreased dehydroepiandrosterone (DHEA) and testosterone levels. In this study of 7-year-old, we separately elucidated androgen disruption for boys and girls, and discussed with respect to hormone disruption with sex differences on the steroid hormone biosynthesis process.This follow-up was conducted with 96 mother-child pairs in Vietnam (hotspot area: 45, non-sprayed area: 51). We took a questionnaire, the physical measurement and assayed 7 steroid hormones in their serum by LC-MS/MS. We examined the relationship between the hormone levels in the serum and dioxin levels in the maternal breast milk. The results showed that the serum DHEA level in the 7-year-old children in the hotspot recovered to levels in the non-sprayed area. The testosterone level of 66.5 pg/mL for boys in the non-sprayed area was 1.5 times the girls level of 44.6 pg/mL, a male-dominant effect. The testosterone level in boys and girls from the hotspot were significantly lower than in the non-sprayed area with no sex difference. The 17β-hydroxysteroid dehydrogenase (17β-HSD) activity was significantly higher in boys than in the girls from the non-sprayed area, but was significantly lower in the hotspot boys than in the non-sprayed area boys. Both the testosterone level and 17β-HSD activity in the boys were inversely correlated with the TEQ total PCDD/Fs in the maternal breast milk.These results indicated that dioxin delayed the expression of the testosterone level and 17β-HSD activity with growth in the 7-year-old boys. The serum DHEA in the 7-year-old children recovered to the levels of the children in the non-sprayed area.

MON-035 Aldosterone Secretion in a Human Adrenal Cell Line Is Enhanced by the Endocrine-Disrupting Chemical Di-(2-Ethylhexyl) Phthalate (DEHP)

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that is widely used as a plasticizer in the production of polyvinyl chloride containing plastics. DEHP is a component of many common household items (toys, packaging, furniture, etc.) and medical devices, resulting in significant and chronic human exposure. Human exposure to DEHP has been linked to androgen disruption and developmental toxicity, including cryptorchidism, decreased anogenital distance, and decreased testosterone levels. Other adrenal corticosteroids are also susceptible to the endocrine-disrupting effects of DEHP. One study has demonstrated that fetal exposure to DEHP results in altered adrenal aldosterone production in adult rats. Due to these potential effects of DEHP on adrenal cortex steroidogenesis, the focus of our studies is to characterize the effect of DEHP on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of DEHP (10 nM-30 μM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. No effects on cell viability were observed with the concentrations studied. Basal aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 75.6±3.5; 10 μM DEHP, 7735.2±151.3; n=8). Ang II-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 4278.9±110.2; 10 μM DEHP, 18651.7±572.2; n=8). ACTH-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 124.0±3.7; 10 μM DEHP, 23455.8±272.7; n=8). Gene expression of most enzymes and cofactors involved in aldosterone synthesis were increased by 10 μM DEHP. Aldosterone synthase (CYP11B2) had the greatest induction at 175.2±2.0 fold. These data support the evidence that DEHP disrupts the regulation of aldosterone secretion and provides further evidence that DEHP is an endocrine disruptor.

Androgen disruption by dioxin exposure in 5-year-old Vietnamese children: Decrease in serum testosterone level

Dioxins have been suspected to be potential substances causing endocrine disruptions in humans. We are conducting the research in one of three dioxin exposure areas (hotspots) in Vietnam. We previously reported that the salivary dehydroepiandrosterone (DHEA) level decreased in 3-year-old Vietnamese children and that it was significantly inversely correlated with polychlorinated dibenzodioxin/dibenzofuran levels in their mother's breast milk. In this study, we investigated the influence of exposure to dioxin on steroid hormone biosynthesis in the same children when they reached 5 years of age, focusing on androgens. Thirty-five and 50 mother–child pairs from dioxin hotspot and non-sprayed areas, respectively, participated in this study. Maternal breast milk was donated at 4 to 16 weeks postpartum in 2008 to measure dioxin levels by gas chromatography/high-resolution mass spectrometry. Serum was collected from 5-year-old children in 2013. Seven steroid hormones were measured by liquid chromatography/mass spectrometry. Most dioxin congeners in breast milk were 2- to 10-fold higher in the hotspot than in the non-sprayed area. DHEA and testosterone (T) were significantly lower in the hotspot and showed negative correlations with most dioxin congeners. Similar results were observed for the activities of cytochrome P450-17, 20 lyase (CYP17 lyase), and 17β-hydroxysteroid dehydrogenase (HSD). Conversely, the elevated androstenedione (A-dione) level and 3β-HSD activity in children from the hotspot were positively correlated with dioxin levels. Moreover, a positive correlation was shown between T and 17β-HSD. It is possible that dioxin inhibits 17β-HSD activity, leading to a decrease in the T level. Multiple regression analysis indicated that dioxin had a strong association with the DHEA, A-dione, and T levels. In conclusion, the present study suggests that dioxin is associated with low levels of DHEA and T and inhibition of the activity of steroidogenic enzymes such as CYP17 lyase and 17β-HSD in 5-year-old children.

Organizational effects of the antiandrogen, vinclozolin, on penis development in the mouse.

Endocrine disrupting chemicals (EDCs) are pollutants found throughout the environment that disrupt normal endocrine processes. In mice, penis development is thought to be most susceptible to EDCs during a critical developmental window occurring on embryonic days (E) 15.5-17.5. However, androgen signaling begins on E13.5 when androgen receptor (AR) protein is found in the genitalia and testosterone is circulating. We hypothesize that disrupting androgen signaling prior to the established critical window sensitizes the penis to future androgen disruption. To test this hypothesis, CD1 dams were exposed to vinclozolin or a corn oil solvent control on E13.5 and E14.5 and AR levels were measured with immunohistochemistry on E14.5. Early antiandrogen exposure reduced AR within nuclei and decreased intensity of AR expression within E14.5 genitalia. To evaluate the influence of antiandrogen exposure before the known critical window of penis development, two groups of pregnant dams (n=3) were exposed to vinclozolin starting at either E13.5 or E14.5 and continued exposure through E16.5. Histology and M.O.U.S.E. scoring were used to quantify penis abnormalities. To account for differences in total doses mice experienced due to differences in length of dosing time, we compared animals that received the same total doses. Exposure to antiandrogens on E13.5 exacerbated malformations when exposure was continued through sexually dimorphic development. Both exposure time and vinclozolin dose are important for severity of vinclozolin-induced penis abnormalities in mice. This work shows that antiandrogen exposure prior to sensitive periods can exacerbate the effects of later antiandrogen exposure on reproductive development.

Identification of endocrine active disinfection by-products (DBPs) that bind to the androgen receptor

The formation of disinfection by-products (DBPs) in drinking water occurs when chemical disinfectants such as chlorine and chloramine react with natural organic matter and anthropogenic pollutants. Some DBPs have been linked to bladder cancer and infertility; however, the underlying mechanism of action is unknown. One possibility is disruption of the endocrine system, with DBPs binding to the androgen receptor and subsequently altering gene expression. Using the androgen receptor-binding assay and in silico molecular docking, the binding affinity of 21 suspected and known DBPs were tested individually at concentrations over the range 0.1 nM–2 mM. 14 DBPs were found to bind at IC50 values ranging from 1.86 mM for 2,3-dichloropropionamide to 13.5 μM for 3,4,5,6-tetrachloro-benzoquinone as compared to the positive control, 4-n-nonylphenol which bound at 31.6 μM. Since DBPs are present in drinking waters as mixtures, the question of how IC50 values for individual DBPs might be affected by the presence of other chemicals is addressed. Seven of the chemicals with the strongest binding affinities and one chemical with no binding affinity were tested in binary mixtures with 4-n-nonylphenol, a known androgenic chemical found in some surface waters. In these binary mixtures, concentration additive binding was observed. While typical levels of individual androgenic DBPs in drinking water are below their measured IC50 values, their combined binding abilities in mixtures could be a source of androgen disruption.

Adipocyte accumulation in corpus cavernosum: First clinical evidence and pathophysiological implications in erectile dysfunction

ObjectivesAnimal models have shown that erectile dysfunction is associated with adipocyte accumulation under tunica albugínea, which could be involved in venous leakage and loss of penile rigidity. In the current study, we compared the histology of the penile sub-albuginean region of drug-refractory erectile dysfunction patients undergoing penile prosthesis implantation with potent patients with Peyronie's disease undergoing curvature correction procedures. Materials and methodsSeventeen refractory erectile dysfunction patients and fourteen potent patients with Peyronie's disease were recruited. Sub-albuginean tissue samples were taken in each surgery. An expert uropathologist analyzed each section. A bivariate analysis was performed. Multivariate logistic regression was used to calculate adjusted odds ratios; p value <0.05 was considered significant. ResultsEleven patients (11/17) in the case group presented cavernous fat cell accumulation, while only one patient (1/14) in the control group presented this finding (p<0.05). Adjusted odds ratio for erectile dysfunction was 40.72; 95% CI 2.28–727.29 (p=0.012). ConclusionsDifferent studies have shown that androgen disruption could be involved in penile structural changes, leading to trabecular smooth muscle apoptosis and trans or de-differentiation into adipocytes. This is the first prospective study in humans to report an association between erectile dysfunction and sub-albuginean adipocyte accumulation. Venous leakage secondary to this phenomenon could be a factor in the pathophysiology of erectile dysfunction, especially in patients that do not respond to medical therapy.

Effects of triazole fungicides on androgenic disruption and CYP3A4 enzyme activity

Triazole fungicides are widely used as broad-spectrum fungicides, non-steroidal antiestrogens and for various industrial applications. Their residues have been frequently detected in multiple environmental and human matrices. The increasingly reported toxicity incidents have led triazole fungicides as emerging contaminants of environmental and public health concern. However, whether triazole fungicides behave as endocrine disruptors by directly mimicking environmental androgens/antiandrogens or exerting potential androgenic disruption indirectly through the inhibition of cytochrome P450 (CYP450) enzyme activity is yet an unresolved question. We herein evaluated five commonly used triazole fungicides including bitertanol, hexaconazole, penconazole, tebuconazole and uniconazole for the androgenic and anti-androgenic activity using two-hybrid recombinant human androgen receptor (AR) yeast bioassay and comparatively evaluated their effects on enzymatic activity of CYP3A4 by P450-Glo™ CYP3A4 bioassay. All five fungicides showed moderate anti-androgenic activity toward human AR with the IC50 ranging from 9.34 μM to 79.85 μM. The anti-androgenic activity remained no significant change after the metabolism mediated by human liver microsomes. These fungicides significantly inhibited the activity of CYP3A4 at the environmental relevant concentrations and the potency ranks as tebuconazole > uniconazole > hexaconazole > penconazole > bitertanol with the corresponding IC50 of 0.81 μM, 0.93 μM, 1.27 μM, 2.22 μM, and 2.74 μM, respectively. We found that their anti-androgenic activity and the inhibition potency toward CYP3A4 inhibition was significantly correlated (R2 between 0.83 and 0.97, p < 0.001). Our results indicated that the risk assessment of triazole pesticides and structurally similar chemicals should fully consider potential androgenic disrupting effects and the influences on the activity of CYP450s.

Acumulación de adipocitos en el cuerpo cavernoso: primera evidencia clínica e implicaciones fisiopatológicas en la disfunción eréctil

ObjectivesAnimal models have shown that erectile dysfunction is associated with adipocyte accumulation under tunica albugínea, which could be involved in venous leakage and loss of penile rigidity. In the current sudy, we compared the histology of the penile sub-albuginean region of drug-refractory erectile dysfunction patients undergoing penile prosthesis implantation with potent patients with Peyronie's disease undergoing curvature correction procedures. Materials and methodsSeventeen refractory erectile dysfunction patients and fourteen potent patients with Peyronie's disease were recruited. Sub-albuginean tissue samples were taken in each surgery. An expert uropathologist analysed each section. A bivariate analysis was performed. Multivariate logistic regression was used to calculate adjusted odds ratios; P value<.05 was considered significant. ResultsEleven patients (11/17) in the case group presented cavernous fat cell accumulation, while only one patient (1/14) in the control group presented this finding (P<.05). Adjusted odds ratio for erectile dysfunction was 40.72; 95% CI 2.28-727.29 (P=.012). ConclusionsDifferent studies have shown that androgen disruption could be involved in penile structural changes, leading to trabecular smooth muscle apoptosis and trans or de-differentiation into adipocytes. This is the first prospective study in humans to report an association between erectile dysfunction and sub-albuginean adipocyte accumulation. Venous leakage secondary to this phenomenon could be a factor in the pathophysiology of erectile dysfunction, especially in patients that do not respond to medical therapy.

Shorter anogenital distance correlates with the severity of hypospadias in pre-pubertal boys.

Do pre-pubertal boys with hypospadias have a shorter anogenital distance (AGD) than boys with normal genitalia? AGD is significantly shorter in boys with hypospadias and decreases with the severity of hypospadias. Animal studies have shown that androgen disruption and exposure to endocrine disrupting chemicals during a critical time period in early gestation, termed the male programming window (MPW), result in hypospadias and reduced AGD; and the severity of hypospadias correlates with the reduction in AGD. However, this correlation has not been established in humans. A prospective descriptive study involving measurement of AGD in pre-pubertal boys (n = 458) presenting to our pediatric urology clinic with hypospadias and normal genitalia was performed over a period of 3 years. AGD was measured in pre-pubertal boys from 5 months to 14 years of age presenting to our clinic with hypospadias (n = 180: four were excluded) and compared with randomly selected boys with normal genitalia (controls, n = 274). Three variants of AGD, from the midpoint of the anus to base of the scrotum (AGD-AS), to the anterior base of penis (AGD-1) and to the posterior base of penis (AGD-2), were measured and assessed for correlation with the severity of hypospadias. Severity of hypospadias was classified as anterior, middle and posterior according to the meatal location. No significant difference in weight (P = 0.123), age (P = 0.162) or height (P = 0.591) between the two groups was observed. Only AGD-AS was significantly shorter in boys with hypospadias compared with controls (mean ± SD: 40.6 ± 9.7 mm versus 45.6 ± 9.4 mm, P < 0.001). This relation persisted after adjusting AGD for weight, height and age (β = 0.016, 95% confidence interval: 0.10-0.21; P < 0.001). The Spearman test showed a significant negative correlation for the severity of hypospadias with all the three AGD measures. Analysis of variance between anterior, middle and posterior subgroups showed a significant reduction in mean AGD-AS (P = 0.003) and AGD-2 (P = 0.008). No data were collected pertaining to in utero exposure to endocrine disrupting chemicals (EDCs) or cigarette smoke, or current diet and environmental exposure to EDCs, which may have influenced the AGD. Family history of genital malformation and use of IVF were not known. There may have been a selection bias as only boys presenting to our clinic were included. The findings suggest that prenatal androgens during early gestation influence development of the male reproductive system and support the existence of a MPW in humans. Of the three AGDs, AGD-AS may be the most reliable biomarker of this in utero androgen action. However, no direct link to any specific exposure leading to shortened AGD in pre-pubertal boys with hypospadias could be determined. Further large scale multi-center studies are needed to understand this association better. Funding was from the Hypospadias Foundation. No conflicts of interest to disclose.

Maternal administration of flutamide during late gestation affects the brain and reproductive organs development in the rat male offspring

We have previously demonstrated that male rats exposed to stress during the last week of gestation present age-specific impairments of brain development. Since the organization of the fetal developing brain is subject to androgen exposure and prenatal stress was reported to disrupt perinatal testosterone surges, the aim of this research was to explore whether abnormal androgen concentrations during late gestation affects the morphology of the prefrontal cortex (PFC), hippocampus (HPC) and ventral tegmental area (VTA), three major areas that were shown to be affected by prenatal stress in our previous studies. We administered 10-mg/kg/day of the androgen receptor antagonist flutamide (4′nitro-3′-trifluoromethylsobutyranilide) or vehicle injections to pregnant rats from days 15–21 of gestation. The antiandrogenic effects of flutamide were confirmed by the analysis of androgen-dependent developmental markers: flutamide-exposed rats showed reduced anogenital distance, delay in the completion of testis descent, hypospadias, cryptorchidism and atrophied seminal vesicles. Brain morphological studies revealed that prenatal flutamide decreased the number of MAP2 (a microtubule-associated protein type 2, present almost exclusively in dendrites) immunoreactive neuronal processes in all evaluated brain areas, both in prepubertal and adult offspring, suggesting that prenatal androgen disruption induces long-term reductions of the dendritic arborization of several brain structures, affecting the normal connectivity between areas. Moreover, the number of tyrosine hydroxylase (TH)-immunopositive neurons in the VTA of prepubertal offspring was reduced in flutamide rats but reach normal values at adulthood. Our results demonstrate that the effects of prenatal flutamide on the offspring brain morphology resemble several prenatal stress effects suggesting that the mechanism of action of prenatal stress might be related to the impairment of the organizational role of androgens on brain development.

Shorter anogenital distance correlates with undescended testis: a detailed genital anthropometric analysis in human newborns

Are the anogenital distance (AGD) and stretched penile length (SPL) shorter in human newborn males with cryptorchidism? AGD is significantly shorter in boys with undescended testis (UDT) and this correlation may indicate that both have a common antecedent early in gestation. Animal studies have reported a critical time period during early gestation termed the male programming window (MPW) where androgen deficiency results in reduced AGD and penile length, as well as cryptorchidism and hypospadias. Two pilot human studies have explored this association but these studies were small and heterogeneous with regard to age, race and had selection bias. A prospective descriptive study involving measurement of AGD and SPL at birth in a racially homogenous sample of 1154 consecutive newborns was performed over a period of 6 months. All measurements were taken by a single trained observer (V.J.). All consecutively born male infants at a community hospital were classified as having descended and or UDT. Testicular position in the undescended group was graded as high scrotal, inguinal or non-palpable. AGD (from the centre of anus to the junction of the smooth and rugated skin of scrotum) and SPL were measured. The AGD index (AGDi) was calculated by dividing AGD by cube root of birthweight. Of the 1154 infants examined, 624 were males and 71 had UDT. AGD was significantly shorter in infants with UDT when compared with infants with descended testis (mean ± SD; 2.21 ± 0.36 versus 2.56 ± 0.31 cm; P < 0.001). AGDi was also significantly shorter in infants with UDT (mean ± SD; 1.68 ± 0.27 versus 1.81 ± 0.20 cm/kg⁻³; P < 0.001). Significance was maintained even when preterm (P < 0.001) and low birthweight boys (LBW) (P < 0.001) were excluded. SPL was also significantly shorter in infants with UDT (Mean ± SD; 3.08 ± 0.52 versus 3.31 ± 0.38 cm; P < 0.001) but the significance was not maintained when preterm (P = 0.119) and LBW boys (P = 0.666) were excluded. Birthweight, gestational age and length adjusted regression models showed significantly shorter AGD in infants with UDT, but SPL was not different. Infants with higher position of testis appeared to have a shorter AGD and SPL but the correlation did not reach statistical significance. No difference in AGD or SPL was noted between boys with unilateral and bilateral UDT. The present study did not include data pertaining to maternal or newborn health status. Also parental drug exposure or occupational exposures to endocrine-disrupting chemicals was not studied. These may possibly affect genital anthropometric measurements. The study strengthens the hypothesis of existence of MPW in humans. Shorter AGD in cryptorchid infants may reflect the effect of androgen disruption or deficiency during MPW. AGD may be a more reliable non-invasive marker of androgen action during MPW than SPL to predict reproductive outcomes in humans.

Acquired Cryptorchidism in a Boy with Disorder of Sex Development

Recently, it has been reported that boys with severe hypospadias are at increased risk for acquired cryptorchidism. The reports suggested that prenatal and postnatal androgen disruption might be correlated with this condition. We experienced a case of ovotesticular disorder of sex development (DSD), which was ultimately diagnosed at surgery for acquired cryptorchidism. Ascent of the scrotal contents of the left side was detected in a 7-yr-old boy with the 46, XX karyotype, who had a history of perineal hypospadias repair. Intraoperative findings revealed the left gonad consisted of 2 segments, and this was histologically diagnosed as ovotestis by biopsy specimen. Resection of the ovarian segment was performed simultaneously. Exploration of the contralateral gonad showed the same findings. This is the first report of acquired cryptorchidism observed in a patient with DSD presenting with ambiguous genitalia.

Are expression and localization of tight and adherens junction proteins in testes of adult boar affected by foetal and neonatal exposure to flutamide?

Several recent studies have indicated that androgen disruption induced by the administration of anti-androgen flutamide during critical developmental stages results in various reproductive abnormalities, mainly in rodents. However, scarce data are available regarding the alterations caused by this toxicant on cell-cell adhesion molecules. Of note, there is no report on the expression and regulation of tight and adherens junction proteins in the boar. Therefore, the purpose of this study was to analyse whether foetal and neonatal exposure to flutamide affects the expression and distribution of ZO-1, occludin, β-catenin, and N-cadherin in testes of adult pigs. Moreover, to evaluate whether androgen signal was altered in the boar, testicular levels of testosterone and oestradiol and the expression of androgen receptor were examined. Flutamide (50 mg/kg bw) was injected into pregnant gilts during gestational days 20-28 and 80-88 (GD20, GD80), and into male piglets on postnatal days 2-10 (PD2). In the testes of all flutamide-exposed boars, expressions of ZO-1, N-cadherin and β-catenin were significantly decreased at mRNA and protein level, whereas expression of occludin was unchanged when compared with the controls. In addition, in severely damaged seminiferous tubules of PD2 pigs, mislocalization of ZO-1, N-cadherin and β-catenin was observed. Changes in junction protein expressions were accompanied by disturbed intratesticular androgen-oestrogen balance, although androgen receptor expression was not altered. Taken together, these results demonstrate that blockade of androgen action by flutamide during both gestational and neonatal periods affects the expression of ZO-1, N-cadherin and β-catenin in adult pig testes. Of concern, neonatal window seems to be most critical for the organization of BTB and consequently for normal spermatogenesis in the boar. It is likely that altered expression of junction proteins is related to insufficient testosterone production and/or excessive oestradiol synthesis, which may result from impaired Leydig cell function.

Assessment of reproductive biomarkers in three‐spined stickleback (Gasterosteus aculeatus) from sewage effluent recipients

The aim of this study was to examine the occurrence of endocrine disruption close to sewage treatment plant effluent discharges along the Finnish Baltic Sea coast using a set of reproductive biomarkers present in adult three-spined stickleback (Gasterosteus aculeatus). Possible variation and sensitivity of the biomarkers during an entire reproductive period were also examined. The analysis of vitellogenin (VTG) for estrogenic activity and spiggin for androgenic activity, together with histopathological analysis indicated that sticklebacks were exposed to estrogenic loads sufficient to cause inappropriate production of VTG and to disrupt normal testicular structure in adult male sticklebacks. No androgenic disruption was observed. The results emphasize the need of a combination of several reproductive biomarkers in fish and repeated sampling for the detection of potential endocrine modulating substances under field condition.

Proximal Hypospadias and Risk of Acquired Cryptorchidism

We hypothesized that boys with proximal hypospadias are at increased risk for acquired cryptorchidism. We retrospectively studied the records of 114 boys who underwent repair for proximal hypospadias and had at least 1 year of followup, and 342 age matched boys receiving well child examinations. We used chi-square analysis to determine if cryptorchidism prevalence differed between the cohorts. Association between predictor (presence and severity of hypospadias, ethnicity/race, age, medical comorbidities) and outcome (acquired cryptorchidism, primary cryptorchidism, retractile testes) variables was modeled using univariate analysis and multivariate logistic regression. A total of 22 subjects (19%) with hypospadias had 26 nonscrotal testes, of which 2 (2%) represented primary cryptorchidism, 16 (14%) acquired cryptorchidism and 8 (7%) retractile testes. A total of 12 controls (3.5%) had 15 nonscrotal testes, of which 6 (1.8%) represented primary cryptorchidism, 1 (0.3%) acquired cryptorchidism and 8 (2.3%) retractile testes. Children with hypospadias had a higher prevalence of acquired cryptorchidism and retractile testes (all p <0.05). Hypospadias (OR 60.67, 95% CI 7.79-472.80) and increasing age (OR 1.02, 95% CI 1-1.03) were associated with development of acquired cryptorchidism. Hypospadias was associated with development of retractile testes (OR 3.11, 95% CI 1.4-8.50), and greater severity of hypospadias correlated with development of acquired cryptorchidism (p = 0.01). Boys with a history of severe hypospadias are at increased risk for acquired cryptorchidism and retractile testes. The risk of acquired cryptorchidism increases directly with hypospadias severity. We suggest that the role of prenatal and postnatal androgen disruption, which may link these conditions, be explored further.

Physical activity in the androgen receptor knockout mouse: Evidence for reversal of androgen deficiency on cancellous bone

Disruption of the androgen receptor (AR) in male mice reduces cortical bone expansion and muscle mass during puberty and results in high bone turnover-related cancellous osteopenia. We hypothesized that voluntary wheel running during growth is able to rescue the effects of AR disruption on bone. To this end, 5-week-old AR knockout (ARKO) mice were randomized to a running group (cage with running wheel) and a sedentary group (cage without wheel) and followed-up until 16 weeks of age. Voluntary wheel running in ARKO mice did not influence body weight, muscle mass or periosteal bone expansion. Interestingly, voluntary running significantly reduced bone turnover in ARKO mice and prevented cancellous bone loss due to a preservation of trabecular number. Thus, voluntary running in ARKO mice was able to reduce cancellous bone resorption, suggesting that sustained exercise may potentially compensate the effects of androgen disruption on cancellous bone.