Majken Dalgaard

Synergistic disruption of external male sex organ development by a mixture of four antiandrogens

Background By disrupting the action of androgens during gestation, certain chemicals present in food, consumer products, and the environment can induce irreversible demasculinization and malformations of sex organs among male offspring. However, the consequences of simultaneous exposure to such chemicals are not well described, especially when they exert their actions by differing molecular mechanisms. Objectives To fill this gap, we investigated the effects of mixtures of a widely used plasticizer, di(2-ethylhexyl) phthalate (DEHP); two fungicides present in food, vinclozolin and prochloraz; and a pharmaceutical, finasteride, on landmarks of male sexual development in the rat, including changes in anogenital distance (AGD), retained nipples, sex organ weights, and malformations of genitalia. These chemicals were chosen because they disrupt androgen action with differing mechanisms of action. Results Strikingly, the effect of combined exposure to the selected chemicals on malformations of external sex organs was synergistic, and the observed responses were greater than would be predicted from the toxicities of the individual chemicals. In relation to other hallmarks of disrupted male sexual development, including changes in AGD, retained nipples, and sex organ weights, the combined effects were dose additive. When the four chemicals were combined at doses equal to no observed adverse effect levels estimated for nipple retention, significant reductions in AGD were observed in male offspring. Conclusions Because unhindered androgen action is essential for human male development in fetal life, these findings are highly relevant to human risk assessment. Evaluations that ignore the possibility of combination effects may lead to considerable underestimations of risks associated with exposures to chemicals that disrupt male sexual differentiation.

Combined Exposure to Anti-Androgens Exacerbates Disruption of Sexual Differentiation in the Rat

Objective The aim of this study was to assess whether the joint effects of three androgen receptor antagonists (vinclozolin, flutamide, procymidone) on male sexual differentiation after in utero and postnatal exposures can be predicted based on dose–response data of the individual chemicals. Methods Test chemicals and mixtures were administered by gavage to time-mated nulliparous, young adult Wistar rats from gestational day 7 to the day before expected birth, and from postnatal days 1–16. Changes in anogenital distance (AGD) and nipple retention (NR) in male offspring rats were chosen as end points for extensive dose–response studies. Vinclozolin, flutamide, and procymidone were combined at a mixture ratio proportional to their individual potencies for causing retention of six nipples in male offspring. Results With AGD as the end point, the joint effects of the three anti-androgens were essentially dose additive. The observed responses for NR were slightly higher than those expected on the basis of dose addition. A combination of doses of each chemical, which on its own did not produce statistically significant AGD alterations, induced half-maximal mixture effects. At individual doses associated with only modest effects on NR, the mixture induced NR approaching female values in the males. Conclusions Effects of a mixture of similarly acting anti-androgens can be predicted fairly accurately on the basis of the potency of the individual mixture components by using the dose addition concept. Exposure to anti-androgens, which individually appears to exert only small effects, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.

The acute effects of mono(2-ethylhexyl)phthalate (MEHP) on testes of prepubertal Wistar rats

A single oral dose of 400 mg/kg body weight of mono(2-ethylhexyl)phthalate (MEHP), the testis toxic metabolite of di(2-ethylhexyl)phthalate, was given to 28-day-old male Wistar rats and the testis toxic effects were investigated 3,6, and 12 h after exposure. Detachment and sloughing of germ cells were observed, and in the Sertoli cells the cytoplasmatic intermediate filament vimentin collapsed. In the immunohistochemical investigation the androgen receptor distribution was unchanged between the control group and treated groups. The expression of the testosterone-repressed-prostatic-message-2 gene in rat testis increased after 3 h, but returned to control levels after 6 and 12 h. Caspase-3 activity increased 3 and 12 h after MEHP exposure. This increase could not be correlated to an increase in DNA fragmentation or increase in apoptotic numbers of germ cells. In conclusion, the effect of MEHP in testis is apparently not involving the androgen receptor. Vimentin localisation in the Sertoli cells, and increased levels of caspase-3 activity appear to be sensitive and early markers of MEHP testis toxicity.

Impact of diisobutyl phthalate and other PPAR agonists on steroidogenesis and plasma insulin and leptin levels in fetal rats

Endocrine disrupting chemicals can induce malformations and impairment of reproductive function in experimental animals and may have similar effects in humans. Recently, the environmental obesogen hypothesis was proposed, suggesting that environmental chemicals contribute to the development of obesity and insulin resistance. These effects could be related to chemical interaction with nuclear receptors such as the peroxisome proliferator activated receptors (PPARs). As several testosterone-reducing drugs are PPAR activators, we aimed to examine whether four PPAR agonists were able to affect fetal testosterone production and masculinization of rats. Additionally, we wished to examine whether these chemicals affected fetal plasma levels of insulin and leptin, which play important roles in the developmental programming of the metabolic system. Pregnant Wistar rats were exposed from gestation day (GD) 7-21 to diisobutyl phthalate (DiBP), butylparaben, perfluorooctanoate, or rosiglitazone (600, 100, 20, or 1 mg/kg bw/day, respectively). Endocrine endpoints were studied in offspring at GD 19 or 21. DiBP, butylparaben and rosiglitazone reduced plasma leptin levels in male and female offspring. DiBP and rosiglitazone additionally reduced fetal plasma insulin levels. In males, DiBP reduced anogenital distance, testosterone production and testicular expression of Insl-3 and genes related to steroidogenesis. PPARalpha mRNA levels were reduced by DiBP at GD 19 in testis and liver. In females, DiBP increased anogenital distance and increased ovarian aromatase mRNA levels. This study reveals new targets for phthalates and parabens in fetal male and female rats and contributes to the increasing concern about adverse effects of human exposure to these compounds.

Low-dose perinatal exposure to di(2-ethylhexyl) phthalate induces anti-androgenic effects in male rats.

Perinatal di(2-ethylhexyl) phthalate (DEHP) exposure was examined in time-mated Wistar rats gavaged from gestation day 7 to postnatal day 16 with doses from 3 to 900 mg/kg-d. These doses covered the whole dose-response curve for the demasculinizing effects of DEHP including low-dose effects. At a relatively low dose of 10 mg/kg-d, DEHP caused adverse anti-androgenic effects on male rat development as male anogenital distance was decreased, the incidence of nipple retention was increased, weight of levator ani/bulbocavernosus muscles and prostate was reduced and mild external genitalia dysgenesis was observed. Higher doses of DEHP induced histopathological effects on the testes, reduced testis weight, and expression of androgen-regulated genes in the prostate. The results provide new evidence of low-dose effects of DEHP and are consistent with the EU NOAEL of 5 mg/kg for DEHP. Our results also indicate a reason for concern about human exposure to DEHP.

Reproductive and behavioral effects of diisononyl phthalate (DINP) in perinatally exposed rats

Diisononyl phthalate (DINP) is a plasticizer abundantly used in consumer products as a substitute for other plasticizers prohibited in certain products due to reproductive toxicity. As anti-androgenic effects of DINP are suspected, DINP effects on reproduction and sexually dimorphic behavior were studied. Pregnant Wistar rats were gavaged from gestation day 7 to postnatal day (PND) 17 with vehicle, 300, 600, 750 or 900 mg DINP/kg bw/day. In fetal testes histopathological effects typical of phthalates were observed. In male offspring, DINP caused increased nipple retention, reduced anogenital distance, reduced sperm motility and increased sperm count. DINP affected spatial learning as female offspring performed better than controls and similarly to control males in the Morris Water Maze, indicating masculinization of behavior in DINP exposed females. These results show that DINP causes anti-androgenic effects on reproductive development, though less potent than DEHP, DBP and BBP, and further safety evaluation of DINP appears warranted.

In utero reproductive study in rats exposed to nonylphenol.

Alkylphenol ethoxylates are widely used non-ionic surfactants. Nonylphenol ethoxylate constitutes 82% of the production of all alkylphenol ethoxylates and the breakdown product of nonylphenol ethoxylate, nonylphenol (NP) has been shown to be estrogenic in both in vitro and in vivo screening assays. To determine the potential reproductive toxicity of NP, a one-generation in utero study was conducted. Rats were dosed from gestation day 11 through 18 with NP at 3, 15, or 75 mg/kg/day or diethylstilbestrol (DES) at 30 microg/kg/day. DES was used as a positive control. Both substances were given orally by gavage. Male offspring were sacrificed at postnatal day (PND) 11, 21, or 110 and reproductive parameters were evaluated. Pup birth weight and body weight and percent motile sperm at age of 110 day were significantly reduced by DES. The absolute weight of the right epididymis was significantly reduced in the DES group. The absolute weight of the right epididymis were also significantly decreased in the animals exposed to 75 or 15 mg/kg/day NP, effects which disappeared when organ weight was related to body weight. This study showed a dose-dependent effect of nonylphenol on male reproductive development at doses of 75 and 15 mg/kg bw/day based on absolute epididymal weight.

Di(2-ethylhexyl) adipate (DEHA) induced developmental toxicity but not antiandrogenic effects in pre- and postnatally exposed Wistar rats

Di(2-ethylhexyl) adipate (DEHA) has replaced the phthalates in thin plasticized polyvinyl chloride films used for food packaging, mainly because some phthalates induce testis toxicity and antiandrogenic effects. A dose-range finding study followed by a dose-response/effect study in Wistar rats investigated whether pre- and postnatal DEHA doses of 0, 800, or 1200mg/kg/day body weight and doses of 0, 200, 400, or 800mg/kg/day (main study) elicited developmental toxicity including antiandrogenic effects. In the main study, DEHA induced a prolonged gestation period (800mg/kg/day) and a dose-related increase in postnatal death (400 and 800mg/kg/day). DEHA also induced a permanent decrease in offspring body weight (800mg/kg/day). No antiandrogenic endpoints were affected. We conclude that DEHA induced developmental toxicity and the NOAEL is 200mg/kg. DEHA did not induce antiandrogenic effects similar to those of di(2-ethylhexyl) phthalate even though the chemical structures have similarities and the two chemicals have a common metabolite.

Toxicity study of di(2-ethylhexyl)phthalate (DEHP) in combination with acetone in rats

In two separate studies with exposure duration 9 weeks or 4 weeks, male Wistar rats were dosed with di(2-ethylhexyl)phthalate (DEHP) by gavage and exposed to drinking water with or without acetone (0.5% wt/v in the 9-week study, 1.0% wt/v in the 4-week study). In the 9-week study the doses of DEHP were 0, 125, 250, 500 or 1000 mg/kg b.wt. In the 4-week study the doses of DEHP were increased to 1000, 5000 and 10,000 mg/kg b.wt. In the 9-week study, the relative liver weight was increased in the rats exposed to 500 and 1000 mg/kg b.wt. No interaction of DEHP and acetone was observed in any of the measured parameters. In the 4-week study DEHP, at the highest dose level, resulted in severe general toxicity. The group exposed to DEHP in combination with acetone was more affected. Male fertility was decreased. Body weight was decreased, and the relative weight of the liver, kidney, heart, brain and adrenals increased. The relative weight of the testes decreased in the 5000 and 10,000 mg/kg b.wt. groups. The weight of seminal vesicles and epididymals decreased at 10,000 mg/kg b.wt. In animals exposed to 5000 and 10,000 mg DEHP/kg b.wt. a severe atrophy of the seminiferous tubules and a slight diffuse Leydigs cell hyperplasia was observed. The cellular debris and conglomerates of desquamated cells found in the lumen of the seminiferous tubules were immunostained positive for vimentin. This indicates that Sertoli cell cytoplasm is included in the conglomerates an interesting finding not previously described. No specific interaction of DEHP and acetone was observed in any of the measured parameters.

Quantification of antiandrogen effect determined by Lightcycler technology

During the last decade, the possible effects of xenobiotics on male reproductive health have resulted in great concern. More recently, evidence of antiandrogen effect in vivo by certain chemicals has been reported. The classical Hershberger in vivo assay determining organ weight changes can be improved by measuring hormone levels as well as determining changes in gene expression of androgen-responsive genes. A real-time RT-PCR method using LightCycler technology (Roche) suitable for quantitative determination of gene expression is described. The technique combines rapid thermocycling with online fluorescence detection of PCR product formation. In this study, investigation of expression of prostate specific binding protein polypeptide C3 (PBP C3) and testosterone-repressed prostatic message 2 (TRPM-2) in the ventral prostate was performed in 60-days-old castrated Wistar rats treated daily with testosterone with or without addition of flutamide or vinclozolin for 7 days in total. We show that we can quantify the level of gene expression by use of LightCycler technology, supported by changes in reproductive organ weights as well as in hormone levels, and that analysis of gene expression levels is an even more sensitive endpoint.