Anderson Joel Martino-Andrade

Reproductive toxicity of phthalate esters.

Phthalate esters are ubiquitous environmental contaminants that in general display low-toxicity. Overall, the reproductive effects of these compounds are well characterized in adults animals, with gonadal injury observed after high dose exposure. However, results of recent transgeneration studies indicate that the reproductive system of developing animals is particularly vulnerable to certain phthalates. The phenotypic alterations observed in male offspring rats exposed during the perinatal period have remarkable similarities with common human reproductive disorders, including cryptorchidism, hypospadias and low-sperm counts. Recent results also indicate that high phthalate doses can adversely affect adult and developing female rats. However, the main question involving phthalates is whether the current level of human exposure is sufficient to adversely affect male and/or female reproductive health. Here, we review the reproductive toxicity data of phthalates in adult and developing animals as well as possible modes of action. In addition, we briefly discuss the relevance of animal studies to humans in light of recent epidemiological data and experimental research with low (human relevant) doses. Finally, we point out some critical issues that should be addressed in order to clarify the implications of phthalates for human reproduction.

Coadministration of active phthalates results in disruption of foetal testicular function in rats

The reproductive effects of the coadministration of di-2-(ethylhexyl) phthalate (DEHP) and di-butyl phthalate (DBP) were studied in both foetal and adult male rat offspring exposed in utero. Pregnant Wistar rats were treated by oral gavage from gestation day 13 to 21 with vehicle control, 150 mg DEHP/kg body weight (bw)/day, 100 mg DBP/kg bw/ or a combination of the two compounds (DEHP 150 + DBP 100 mg/kg bw/day). An additional group of dams received 500 mg DBP/kg bw/day. A significant decrease in foetal testicular testosterone levels was observed in animals exposed to 500 mg DBP/kg/day or the phthalate mixture. Similarly, histological analysis of the foetal testis revealed that the coadministration of DEHP and DBP was able to increase the diameter of seminiferous cords and induce gonocyte multinucleation at doses that individually had no significant effects on these variables. However, in the phthalate mixture group, no significant changes were observed in anogenital distance and nipple retention, variables that are used to indicate possible anti-androgenic effects. Also, the adult endpoints investigated, that included reproductive organ weights and the number of spermatids per testis, were unaffected by any treatment regimen. Overall, coadministration of DEHP and DBP in utero significantly reduced testicular testosterone levels and resulted in misshapen seminiferous cords and gonocyte multinucleation in rat foetal testis. Our results also confirm that these foetal endpoints seem to be the most sensitive markers of prenatal phthalate exposure.

In vivo and in vitro estrogenic activity of the antidepressant fluoxetine.

Recent years have seen an increase in the use of antidepressant drugs, especially fluoxetine (FLX), in sensitive populations, such as pregnant and lactating women. Although some evidence suggests a possible endocrine action of FLX, no specific studies have been performed to investigate this hypothesis. In the present study, we investigated the possible (anti)androgenic and (anti)estrogenic actions of FLX using Hershberger, uterotrophic (0.4, 1.7, and 17mg/kg), and reporter gene (7.6-129μM) assays. In the Hershberger assay, no differences were observed in androgen-dependent organ weights. However, the uterotrophic and gene reporter assays indicated a possible estrogenic action of FLX. Uterine weight increased in the 1.7 and 17mg/kg/day groups in the 3-day uterotrophic assay in immature rats. Additionally, noncytotoxic concentrations of FLX induced estrogenic responses and increased the estrogenic response of estradiol in MCF-7 breast cancer cells transfected with luciferase.

Screening for in vivo (anti)estrogenic and (anti)androgenic activities of Tropaeolum majus L. and its effect on uterine contractility.

ETHNOPHARMACOLOGICAL RELEVANCE Tropaeolum majus L. (Tropaeolaceae) is a medicinal herb popularly used in Brazil for treatment of inflammatory and cardiovascular diseases. Despite some published data on its efficacy, there are still few toxicological data describing the safety of this plant. The aim of this study was to evaluate the (anti)estrogenic and (anti)androgenic activity of the hydroethanolic extract obtained from Tropaeolum majus L. (HETM), as well as its possible effects on uterine contractility. MATERIALS AND METHODS Three experimental protocols were performed, (a) uterotrophic assay, (b) Hershberger assay and (c) an ex vivo test to investigate the effects of maternal administration of HETM on uterine contractility at the end of pregnancy. In all protocols three doses of the HETM were administered to Wistar rats: 3, 30 and 300mg/kg. RESULTS In vivo tests for detection of (anti)androgenic and (anti)estrogenic activities did not show any significant alterations. Similarly, no alterations were observed on uterine contractility induced by oxytocin and arachidonic acid. CONCLUSIONS HETM was unable to produce (anti)estrogenic or (anti)androgenic activities in the short-term in vivo screening assays performed. In addition, there was no evidence that HETM can affect uterine contractility following gestational exposure of rats.

Long-term effects of the testicular torsion on the spermatogenesis of the contralateral testis and the preventive value of the twisted testis orchiepididymectomy

PURPOSE To determine whether the testicular torsion causes long-term effects on the spermatogenesis of the contralateral testis, and whether the orchiepididymectomy of the twisted testis could prevent them, using specific spermatogenesis parameters to elucidate the conflicting results in the literature. METHODS Seventy-four pubertal male Wistar rats were randomly selected. The experimental group consisted of 40 rats, divided into four subgroups, submitted to 1.080 degrees counterclockwise left testicular torsion and its scrotal fixation at the beginning of the experiment, and left orchiepididymectomy at one, five, ten and 90 days, respectively. The control group consisted of 24 rats, divided into four sham operation control subgroups. An additional control subgroup consisted of the ten remaining rats, submitted only to the left orchiepididymectomy at the beginning. At 90 days, the contralateral testes of the experimental and control subgroups were collected for the evaluation of their spermatogenesis parameters: testicular weight, seminiferous tubular diameter, Johnsen score and differential counting of the germ cells. RESULTS No statistically significant differences were observed among the experimental and control subgroups for all of the spermatogenesis parameters of the contralateral testes. CONCLUSIONS Testicular torsion does not cause long-term effects on the spermatogenesis of the contralateral testis in pubertal rats, and the orchiepididymectomy of the twisted testis is not necessary for preventive purposes for the contralateral spermatogenesis.

In Utero and Lactational Exposure to Fluoxetine in Wistar Rats: Pregnancy Outcomes and Sexual Development

This study evaluated the reproductive effects of fluoxetine exposure in utero and during lactation on pregnancy outcomes and the sexual development of offspring. Pregnant Wistar rats were treated daily with fluoxetine (0.4, 1.7 and 17 mg/kg/day) or distilled water by gavage from gestation day (GD) 7 to lactation day (LD) 21. A significant reduction in maternal body weight was observed during pregnancy and lactation in dams exposed to 17 mg/kg fluoxetine. Hormone analysis revealed an increase in progestagen and glucocorticoid metabolites on GD 15 and oestrogen and progestagen metabolites on LD 7 in dams treated with 17 mg/kg fluoxetine. Oestrogen metabolites also were increased on LD 7 in dams treated with 0.4 mg/kg fluoxetine. Besides that, an increase in the weight of the adrenal glands and a reduction in uterine weight in dams exposed to highest dose of fluoxetine were observed. Finally, pup birthweight and the viability and weaning indices also were reduced in animals exposed to 17 mg/kg fluoxetine. Overall, maternal hormonal changes were only observed at the highest dose tested, which also induced maternal and foetal toxicity. No significant changes were seen in dams or offspring exposed to therapeutic‐like doses.

Testicular Effects Following In Utero Exposure to the Antivirals Acyclovir and Ganciclovir in Rats

In utero exposure to the antivirals acyclovir and ganciclovir has been reported to induce gross structural defects in rat offspring. The present study investigated the effects of maternal antiviral treatment on gestation day 10 on reproductive and nonreproductive organs in male rat offspring with a particular focus on the testes. Vehicle and two doses of acyclovir and ganciclovir, 75 and 300 mg/kg, were administered to rat dams. The total doses were fractioned into three subcutaneous applications (3 × distilled water, 3 × 25 mg/kg, and 3 × 100 mg/kg) that were administered on gestation day 10 at 8:00 a.m., 1:00 p.m., and 6:00 p.m. The antiviral concentrations were measured in the serum of the dams 1 h after the last administration. Exposure to 300 mg/kg ganciclovir induced germ cell deficiency in both fetal and adult testes, an effect that was not seen in any other treatment group. Adult rats exposed in utero to this high ganciclovir dose exhibited Sertoli cell-only tubules intermingled with seminiferous tubules that displayed a normal size and normal cell counts, alterations that resemble focal Sertoli cell-only syndrome in humans. The serum concentrations of ganciclovir were markedly higher than those of acyclovir, particularly at the high dose tested. However, although 300 mg/kg acyclovir did not induce germ cell deficiency, other specific effects were seen in exposed animals, including incomplete eye opening and reduced thymus weight.

Manipulation of pre and postnatal androgen environments and anogenital distance in rats

We examined the anogenital distance (AGD) plasticity in rats through the manipulation of the androgen environment in utero and during puberty. Dams were treated from gestation days 13-20 with vehicle, flutamide (20mg/kg/day), di-(2-ethylhexyl) phthalate (DEHP, 750mg/kg/day), or testosterone (1.0mg/kg/day). After weaning, male pups were randomly assigned to one of four postnatal groups, which received the same treatments given prenatally. Sixteen treatment groups were established based on the combination of pre- and postnatal exposures. The postnatal treatments were conducted from postnatal days 23-53. In utero flutamide and DEHP exposure significantly shortened male AGD, although this effect was more pronounced in flutamide-exposed rats. Postnatal flutamide, DEHP, and testosterone induced slight but significant reductions in male AGD. Our study indicates that AGD is a stable anatomical landmark that reflects the androgen action in utero, although it can also be slightly responsive to changes in the androgen environment following pubertal exposure.

Supplementation with Pfaffia glomerata (Sprengel) Pedersen does not affect androgenic-anabolic parameters in male rats

ETHNOPHARMACOLOGICAL RELEVANCE Paffia spp (Amaranthacea) has a widespread use of in Brazil as a possible hormonal supplement and a substitute of Panax ginseng, although information on its reproductive effects is missing. AIM OF THE STUDY To evaluated possible anabolic-androgenic or anti-androgenic effects of Pfaffia glomerata (PG) extract using intact eight-months-old male rats and pre-pubertal castrated rats. MATERIALS AND METHODS Three different dose levels of PG (8.5, 30 and 85 mg/kg/day) were administered to eight-months-old rats for 28 days or to castrated males for 7 days (Hershberger assay). In the experiment with intact animals, 24h fecal samples were collected for quantification of fecal metabolites of androgens throughout treatment. At the end of the treatment period, animals were euthanized for evaluation of serum testosterone, reproductive organ weights, number of spermatids per testis, diameter of seminiferous tubules and cross-sectional area of soleus muscle fibers. In the Hershberber assay, androgenic or anti-androgenic effects were evaluated by the weights of androgen-dependent tissues: ventral prostate, seminal vesicle, glans penis and levator ani muscle/bulbocavernosus muscle. RESULTS No effects were observed in the concentrations of fecal metabolites of androgens monitored during the treatment of intact eight-months-old rats. Moreover, at the end of treatment, no changes were seen in any of the investigated parameters. In the Hershberger assay, the PG extract did not induce androgenic or anti-androgenic effects at the dose levels tested. Significant effects were only observed in animals treated with testosterone and testosterone plus flutamide, which were used as positive controls for androgenicity and anti-androgenicity, respectively. CONCLUSIONS At the dose levels tested, PG extract does not induce anabolic-androgenic or anti-androgenic effects in rats.

Fetopathies associated with exposure to angiotensin converting enzyme inhibitor from Tropaeolum majus L.

Abstract The prevalence of the use of herbal medicines is on the rise across the world, especially amongst pregnant women. A fact that draws attention is that many species commonly used by pregnant women, including the Tropaeolum majus L. (Tropaeolaceae), also present inhibitory activity on the angiotensin-converting enzyme (ACE). Herein, we have investigated the effects of T. majus extract (HETM) on fetal development, evaluating its relationship with possible ACE inhibitory activity. Pregnant Wistar rats were treated with different HETM doses (3, 30 and 300 mg/kg/day) from gestational days 8–20. Rats were sacrificed on the day 20 of pregnancy and the following parameters were evaluated: clinical symptoms of maternal toxicity; maternal body weight; feed and water intake; maternal liver, kidney, and ovary weights, maternal ACE activity and aldosterone levels, live fetuses mean; dead fetuses percentage, fetus weight, and fetal malformation. All pregnant rats treated with high HETM doses showed significant reduction in plasma ACE activity accompanied by a decrease in serum aldosterone levels. Moreover, significant changes in fetal development were observed, including growth retardation and renal damage after 20 days of gestation. Thus, data presented demonstrate the significant effects of the use of HETM on fetal development during pregnancy.