Changes in bone mass associated with obesity and weight loss in humans: Applicability of animal models.

The implications of obesity and weight loss for human bone health are not well understood. Although the bone changes associated with weight loss are similar in humans and rodents, that is not the case for obesity. In humans, obesity is generally associated with increased bone mass, an outcome which is exacerbated by advanced age and menopause. In rodents, by contrast, bone mass decreases in proportion to severity and duration of obesity, and is influenced by sex, age and mechanical load. Despite these discrepancies, rodents are frequently used to model the situation in humans. In this review, we summarise the existing knowledge of the effects of obesity and weight loss on bone mass in humans and rodents, focusing on the translatability of findings from animal models. We then describe how animal models should be used to broaden the understanding of the relationship between obesity, weight loss, and skeletal health in humans. Specifically, we highlight the aspects of study design that should be considered to optimise translatability of the rodent models of obesity and weight loss. Notably, the sex, age, and nutritional status of the animals should ideally match those of interest in humans. With these caveats in mind, and depending on the research question asked, our review underscores that animal models can provide valuable information for obesity and weight-management research.

Kisspeptin-52 partially rescues the activity of the hypothalamus-pituitary-gonadal axis in underweight male rats dosed with an anti-obesity compound.

Energy metabolism and reproduction are closely linked and reciprocally regulated. The detrimental effect of underweight on reproduction complicates the safety evaluation of anti-obesity drugs, making it challenging to distinguish pathological changes mediated through the intended drug-induced weight loss from direct drug effects on reproductive organs. Four-weeks dosing of normal weight Sprague Dawley rats with a glucagon-like peptide 1 (GLP-1)/glucagon receptor co-agonist induced a robust weight loss, accompanied by histological findings in prostate, seminal vesicles, mammary glands, uterus/cervix and vagina. Characterization of the hypothalamus-pituitary-gonadal (HPG) axis in male rats revealed reduced hypothalamic Kiss1 mRNA levels and decreased serum luteinizing hormone (LH) and testosterone concentrations following co-agonist dosing. These alterations resemble hypogonadotropic hypogonadism typically seen in adverse energy deprived conditions, like chronic food restriction. Concomitant daily administration of kisspeptin-52 from day 21 to the end of the four-week co-agonist dosing period evoked LH and testosterone responses without normalizing histological findings. This incomplete rescue by kisspeptin-52 may be due to the rather short kisspeptin-52 treatment period combined with a desensitization observed on testosterone responses. Concomitant leptin treatment from day 21 did not reverse co-agonist induced changes in HPG axis activity. Furthermore, a single co-agonist injection in male rats slightly elevated LH levels but left testosterone unperturbed, thereby excluding a direct acute inhibitory effect on the HPG axis. Our data suggest that the reproductive phenotype after repeated co-agonist administration was driven by the intended weight loss, however, we cannot exclude a direct organ related effect in chronically treated rats.

The Effect of Diet-induced Obesity on Toxicological Parameters in the Polygenic Sprague-Dawley Rat Model.

The obese rodent serves as an indispensable tool for proof-of-concept efficacy and mode-of-action pharmacology studies. Yet the utility of this disease model as an adjunct to the conventional healthy animal in the nonclinical safety evaluation of anti-obesity pharmacotherapies has not been elucidated. Regulatory authorities have recommended employing disease models in toxicology studies when necessary. Our study investigated standard and exploratory toxicology parameters in the high-fat diet (HFD)-induced obese, polygenic Sprague-Dawley rat model in comparison to chow diet (CD)-fed controls. We sought to establish feasibility of the model for safety testing and relevance to human obesity pathophysiology. We report that both sexes fed a 45% kcal HFD for 29 weeks developed obesity and metabolic derangements that mimics to a certain extent, common human obesity. Minor clinical pathologies were observed in both sexes and considered related to CD versus HFD differences. Histopathologically, both sexes exhibited mild obesity-associated findings in brown and subcutaneous white fat, bone, kidneys, liver, lung, pancreas, salivary parotid glands, and skeletal muscle. We conclude that chronic HFD feeding in both sexes led to the development of an obese but otherwise healthy rat. Therefore, the diet-induced obese Sprague-Dawley rat may serve as a suitable model for evaluating toxicity findings encountered with anti-obesity compounds.

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.

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.

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.

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.

Endocrine-disrupting activities in vivo of the fungicides tebuconazole and epoxiconazole.

The triazole fungicides tebuconazole and epoxiconazole were investigated for reproductive toxic effects after exposure during gestation and lactation. Rats were dosed with epoxiconazole (15 or 50 mg/kg bw/day) or tebuconazole (50 or 100 mg/kg bw/day) during pregnancy from gestational day (GD) 7 and continued during lactation until postnatal day (PND) 16. Some dams were randomly chosen for cesarean section at GD 21 to evaluate effects on sexual differentiation in the fetuses. Other dams delivered normally, and the pups were examined (e.g., anogenital distance [AGD] and hormone levels) at birth, at PND 13 or PND 16, and semen quality was assessed in adults. Both tebuconazole and epoxiconazole affected reproductive development in the offspring after exposure in utero. Both compounds virilized the female offspring as shown by an increased AGD PND 0. Furthermore, tebuconazole had a feminizing effect on male offspring as shown by increased nipple retention. This effect was likely caused by the reduced testosterone levels seen in male fetuses. Tebuconazole increased the testicular concentrations of progesterone and 17alpha-hydroxyprogesterone in male fetuses, indicating a direct impact on the steroid synthesis pathway in the Leydig cells. The high dose of epoxiconazole had marked fetotoxic effects, while the lower dose caused increased birth weights. The increased birth weights may be explained by a marked increase in testosterone levels in dams during gestation. Common features for azole fungicides are that they increase gestational length, virilize female pups, and affect steroid hormone levels in fetuses and/or dams. These effects strongly indicate that one major underlying mechanism for the endocrine-disrupting effects of azole fungicides is disturbance of key enzymes like CYP17 involved in the synthesis of steroid hormones.

Dysgenesis and histological changes of genitals and perturbations of gene expression in male rats after in utero exposure to antiandrogen mixtures.

We investigated the ability of a mixture of three androgen receptor antagonists to induce disruption of male sexual differentiation after perinatal exposure. The aim was to assess whether the joint effects of vinclozolin, flutamide, and procymidone can be predicted based on dose-response data of the individual chemicals. Chemicals were administered orally to pregnant Wistar rats from gestational day 7 to postnatal day 16. Changes in reproductive organ weights and of androgen-regulated gene expression in prostates from male rat pups were chosen as end points for extensive dose-response studies. With all end points, the joint effects of the three antiandrogens were dose additive. Histological evaluations showed that dysgenesis and hypoplasia of prostates, seminal vesicles, and epididymis were seen with the highest mixture doses. No changes were observed in any single-compound low-dose group for these lesions, nor were there histopathological changes in the testes. Pronounced dysgenesis of external genitals was observed with all doses of the mixture, and severe dysgenesis was seen with a mixture for which the individual compounds caused no effects. A combination of doses of each chemical that on its own did not produce significant reductions in the weights of seminal vesicles and PBP C3 expression induced a marked mixture effect. Thus, antiandrogens cause additive effects on end points of various molecular complexities such as alterations at the morphological and the molecular level. Exposure to antiandrogens, which appears to exert only small effects when judged on a chemical-by-chemical basis, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.

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.

Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis.

Diethylhexyl phthalate (DEHP) is widely used as a plasticizer in consumer products and is known to disturb the development of the male reproductive system in rats. The mechanisms by which DEHP exerts these effects are not yet fully elucidated, though some of the effects are related to reduced fetal testosterone production. The present study investigated the effects of four different doses of DEHP on fetal testicular histopathology, testosterone production and expression of proteins and genes involved in steroid synthesis in fetal testes. Pregnant Wistar rats were gavaged from GD 7 to 21 with vehicle, 10, 30, 100 or 300 mg/kg bw/day of DEHP. In male fetuses examined at GD 21, testicular testosterone production ex vivo and testicular testosterone levels were reduced significantly at the highest dose. Histopathological effects on gonocytes were observed at 100 and 300 mg/kg bw/day, whereas Leydig cell effects were mainly seen at 300 mg/kg bw/day. Quantitative RT-PCR revealed reduced testicular mRNA expression of the steroidogenesis related factors SR-B1, StAR, PBR and P450scc. Additionally, we observed reduced mRNA expression of the nuclear receptor SF-1, which regulates certain steps in steroid synthesis, and reduced expression of the cryptorchidism-associated Insl-3. Immunohistochemistry showed clear reductions of StAR, PBR, P450scc and PPARgamma protein levels in fetal Leydig cells, indicating that DEHP affects regulation of certain steps in cholesterol transport and steroid synthesis. The suppression of testosterone levels observed in phthalate-exposed fetal rats was likely caused by the low expression of these receptors and enzymes involved in steroidogenesis. It is conceivable that the observed effects of DEHP on the expression of nuclear receptors SF-1 and PPARgamma are involved in the downregulation of steroidogenic factors and testosterone levels and thereby underlie the disturbed development of the male reproductive system.

Prochloraz: an imidazole fungicide with multiple mechanisms of action.

Prochloraz is an imidazole fungicide that is widely used in Europe, Australia, Asia and South America within gardening and agriculture. Screening studies have shown that prochloraz elicits multiple mechanisms of action in vitro, as it antagonizes the androgen and the oestrogen receptor, agonizes the Ah receptor and inhibits aromatase activity. In vivo prochloraz acts as an antiandrogen in the Hershberger assay by reducing weights of reproductive organs, affecting androgen-regulated gene expressions in the prostate and increasing luteinizing hormone levels. In order to investigate the developmental effects of prochloraz, pregnant Wistar dams were dosed perinatally with 30 mg/kg prochloraz. Results showed that prochloraz significantly reduced plasma and testicular testosterone levels in gestational day 21 male foetuses, whereas testicular progesterone was increased. Gestational length was increased by prochloraz. In male pups a significant increase in nipple retention was found, and the weight of the bulbourethral glands was decreased. Behavioural studies showed that the activity level and sweet preference of adult males were significantly increased, indicating that exposure during gestation and lactation causes permanent effects in adulthood. Overall, these results indicate that prochloraz feminizes the male offspring after perinatal exposure, and that these effects are due, at least in part, to diminished fetal steroidogenesis. Thus, a novel endocrine disruptor has been identified that is mechanistically interesting as it elicits dual mechanisms of action and acts as an antiandrogen both by blocking the androgen receptor and by inhibiting fetal steroidogenesis. That a fungicide with such effects is so widely used is a cause for concern, and its use should be reduced, thereby minimizing the risk of human exposure.

Diisobutyl phthalate has comparable anti-androgenic effects to di-n-butyl phthalate in fetal rat testis.

UNLABELLED: Phthalates are widely used as plasticizers in various consumer products and building materials. Some of the phthalates are known to interfere with male reproductive development in rats, and di-n-butyl phthalate (DBP), diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) were recently banned for use in toys in the EU mainly due to their reproductive toxicity. Diisobutyl phthalate (DiBP) has similar structural and application properties as DBP, and is being used as a substitute for DBP. However, knowledge on male reproductive effects of DiBP in experimental animals is lacking. METHODS: In the current study, four groups of pregnant Wistar rats were exposed to either 0mg/kg bw/day or 600 mg/kg bw/day of DiBP from gestation day (GD) 7 to either GD 19 or GD 20/21. Male offspring was examined at GD 19 or GD 20/21 for effects on testicular testosterone production and testicular histopathology. Changes in anogenital distance (AGD) were evaluated as an indication of feminisation of males. RESULTS: Anogenital distance was statistically significantly reduced at GD 20/21 together with reductions in testicular testosterone production and testicular testosterone content. Histopathological effects (Leydig cell hyperplasia, Sertoli cell vacuolisation, central location of gonocytes and presence of multinuclear gonocytes) known for DBP and DEHP were observed in testes of DiBP-exposed animals at GD 20/21. Additionally, immunohistochemical expression of P450scc and StAR proteins in Leydig cells was reduced by DiBP. At GD 19, these effects on anogenital distance, testosterone levels and histopathology were less prominent. CONCLUSION: In this study, GD 20/21 rather than GD 19 appears to be the optimal time for investigating changes in anogenital distance, testosterone levels, and testicular histopathology. DiBP has similar testicular and developmental effects as DBP and DEHP, and although more developmental and especially postnatal studies are needed to clearly identify the reproductive effects of DiBP, this study indicates a reason for concern about the use of DiBP as a substitute for DBP.

Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz.

The fungicide prochloraz has got multiple mechanisms of action that may influence the demasculinizing and reproductive toxic effects of the compound. In the present study, Wistar rats were dosed perinatally with prochloraz (50 and 150 mg/kg/day) from gestational day (GD) 7 to postnatal day (PND) 16. Caesarian sections were performed on selected dams at GD 21, while others were allowed to give birth to pups that were followed until PND 16. Prochloraz caused mild dysgenesis of the male external genitalia as well as reduced anogenital distance and retention of nipples in male pups. An increased anogenital distance indicated virilization of female pups. Effects on steroidogenesis in male fetuses became evident as decreased testicular and plasma levels of testosterone and increased levels of progesterone. Ex vivo synthesis of both steroid hormones was qualitatively similarly affected by prochloraz. Immunohistochemistry of fetal testes showed increased expression of 17alpha-hydroxylase/17,20-lyase (P450c17) and a reduction in 17beta-hydroxysteroid dehydrogenase (type 10) expression, whereas no changes in expression of genes involved in testicular steroidogenesis were observed. Increased expression of P450c17 mRNA was observed in fetal male adrenals, and the androgen-regulated genes ornithine decarboxylase, prostatic binding protein C3 as well as insulin-like growth factor I mRNA were reduced in ventral prostates PND 16. These results indicate that reduced activity of P450c17 may be a primary cause of the disrupted fetal steroidogenesis and that an altered androgen metabolism may play a role as well. In vitro studies on human adrenocortical carcinoma cells supported the findings in vivo as reduced testosterone and increased progesterone levels were observed. Overall, these results together indicate that prochloraz acts directly on the fetal testis to inhibit steroidogenesis and that this effect is exhibited at protein, and not at genomic, level.

Gene expression changes in rat prostate after activation or blocking of the androgen and estrogen receptor.

Several endpoints of different molecular complexity were studied in the Hershberger assay in order to evaluate the specificity and suitability of this test as a broad screening model. Androgen and estrogen receptors were activated or blocked, and expression of typical estrogen- or androgen responsive genes (complement C3, ERalpha, ERbeta, AR, TRPM-2, PBP C3, ODC, and IGF-1 mRNA) was analyzed in rat ventral prostate by real time RT-PCR. Administration of estradiol benzoate (EB) to castrated testosterone-treated rats had no effect on reproductive organ weights or gene expression levels and the anti-estrogen, ICI 182780, only affected ODC expression. Therefore, estrogenic or anti-estrogenic compounds would not be expected to seriously affect the outcome of a Hershberger test. However, EB given alone to castrated rats resulted in various effects. EB increased seminal vesicle weight, an effect reversed by ICI 182780, and affected TRPM-2, PBP C3, ODC, IGF-1, AR, and ERalpha mRNA levels. AR expression in the prostate seemed to be under regulation of both estrogens and androgens, as ICI 182780 inhibited the testosterone-induced AR expression, and flutamide inhibited the EB-induced AR expression. These data indicate that estrogens have various effects in castrated male rats and that expression of several genes is under multi-hormonal control in the ventral prostate. However, interactions between estrogens and androgens do not play a major role in the Hershberger assay, as simultaneous TP administration abolished the effects of EB. First choice of gene expression profiles in the Hershberger assay to study androgenic or anti-androgenic effects would be the traditional, TRPM-2 and PBP C3, supplemented with the new complement C3.

Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring.

Prochloraz is a commonly used fungicide that has shown multiple mechanisms of action in vitro. It antagonizes the androgen and the estrogen receptors, agonizes the Ah receptor, and inhibits aromatase activity. In vivo prochloraz acts antiandrogenically in the Hershberger assay by reducing weights of reproductive organs, affecting androgen-regulated gene expressions, and increasing luteinizing hormone (LH) levels. The purpose of this study was to investigate reproductive toxic effects after exposure during gestation and lactation to prochloraz alone and a mixture of five pesticides (deltamethrin, methiocarb, prochloraz, simazine, and tribenuron-methyl). Prochloraz (30 mg/kg/day) or the mixture (20 mg/kg/day) was dosed to pregnant Wistar dams from gestational day (GD) 7 until postnatal day (PND) 16. Some dams were taken for cesarean section at GD 21, and others were allowed to give birth. Results showed that prochloraz and the mixture significantly reduced plasma and testicular testosterone levels in GD 21 male fetuses, whereas testicular progesterone was increased. Gestational length was increased by prochloraz. Chemical analysis of the rat breast milk showed that prochloraz was transferred to the milk. In males a significant increase of nipple retention was found, and the bulbourethral gland weight was decreased, whereas other reproductive organs were unaffected. In addition cytochrome P450 (CYP)1A activities in livers were induced by prochloraz, possibly as a result of Ah receptor activation. Behavioral studies showed that the activity level and sweet preference of adult males were significantly increased. Overall these results strongly indicate that prochloraz feminizes the male offspring after perinatal exposure, and that these effects are due, at least in part, to diminished fetal steroidogenesis.

Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate.

Di(2-ethylhexyl) phthalate (DEHP) is a well-known testicular toxicant inducing adverse effects in androgen responsive tissues. Therefore, di(2-ethylhexyl) adipate (DEHA) is currently being evaluated as a potential substitute for DEHP. Similarities in structure and metabolism of DEHP and DEHA have led to the hypothesis that DEHA can modulate the effects of DEHP. Wistar rats were gavaged with either vehicle, DEHP (300 or 750mg/kg bw/day) or DEHP (750mg/kg bw/day) in combination with DEHA (400mg/kg bw/day) from gestation day (GD) 7 to postnatal day (PND) 17. Decreased anogenital distance (AGD) and retention of nipples in male offspring were found in all three exposed groups. Dosed males exhibited decreased weights of ventral prostate and m. levator ani/bulbocavernosus. Histopathological investigations revealed alterations in testis morphology in both juvenile and adult animals. The litter size was decreased and postnatal mortality was increased in the combination group only, which is likely a combined effect of DEHP and DEHA. However, no combination effect was seen with respect to antiandrogenic effects, as males receiving DEHP in combination with DEHA did not exhibit more pronounced effects in the reproductive system than males receiving DEHP alone.

Early testicular effects in rats perinatally exposed to DEHP in combination with DEHA--apoptosis assessment and immunohistochemical studies.

This study aimed to characterize the effects of di(2-ethylhexyl) phthalate (DEHP) on the fetal rat testes and relate them to the effects seen in adults. Histopathological effects in fetal testes were examined with immunohistochemistry for anti-Mullerian hormone (AMH), 3beta-hydroxysteroid dehydrogenase, smooth muscle actin (SMA), proliferating cell nuclear antigen (PCNA), histone H3 and vimentin. Additionally, testicular apoptosis levels were assessed in fetal, prepubertal and adult rats. As the plasticizer di(2-ethylhexyl) adipate (DEHA) has similarities with DEHP in chemical structure and metabolism, we investigated if the testicular effects of DEHP were modulated by co-administration with DEHA. Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg/day), or DEHP (750 mg/kg/day) in combination with DEHA (400mg/kg/day), and male offspring were examined at gestation day (GD) 21, postnatal day (PND) 22, 26 and 190. In fetal testes, Leydig cells were found in large clusters containing AMH positive Sertoli cells. At GD 21, seminiferous chords appeared enlarged with an apparently increased number of gonocytes. However, proliferation of gonocytes did not appear increased. A few animals had a high number of TUNEL positive apoptotic cells in degenerating seminiferous tubules at PND 22 and 190, whereas most exposed animals had low levels of germ cell apoptosis at GD 21, PND 22 or PND 26, as evaluated by DNA laddering, TUNEL staining, Caspase-3 immunohistochemistry and Caspase-3 activity measurement. No differences between DEHP and DEHP+DEHA exposed groups were observed.

The combined antiandrogenic effects of five commonly used pesticides.

In this study, mixture effects of five dissimilarly acting pesticides were analyzed for antiandrogenic effects in vitro and in vivo. Deltamethrin, methiocarb, prochloraz, simazine, and tribenuron-methyl are all commonly used for agricultural and horticultural purposes. Concentration-response curves for the inhibition of R1881-induced transcriptional activity of the androgen receptor (AR) in vitro of each pesticide alone and in an equimolar mixture were obtained. The IC25 values for deltamethrin, methiocarb, prochloraz, and the mixture were 5.8, 5.8, 3.5, and 7.5 microM, respectively. Simazine and tribenuron-methyl were ineffective. Applying the isobole method resulted in an isobole coefficient of 0.94 at IC25 for the effect of the mixture, indicating additive effects of the compounds. Comparison of observed effects and effects calculated by assuming additivity also strongly indicated additive effects of the pesticides in vitro. In vivo, each of the five pesticides and a mixture of the pesticides were tested for antiandrogenic effects in castrated testosterone-treated Wistar rats. The mixture induced a significant change of weights of the levator ani/bulbocavernosus muscle and adrenal glands. Changes in gene expression in ventral prostates were observed as distinct effects on levels of ornithin decarboxylase (ODC) mRNA and effects on levels of prostate binding protein subunit C3 (PBP C3) mRNA. No pesticide-induced effect on the level of testosterone-repressed prostatic message 2 (TRPM-2) mRNA was observed, whereas flutamide increased TRPM-2 levels. In conclusion, the pesticides were found to act additively in vitro. In vivo, the organ weight changes indicated that the pesticides had an accumulating effect that was not observed for the individual pesticides. Several pesticide-induced gene expression changes were observed, indicating that these are either very sensitive antiandrogenic end-points or that these changes are induced by a pathway not related to AR.