Cannabidiol as a Promising Adjuvant Therapy for Estrogen Receptor-Positive Breast Tumors: Unveiling Its Benefits with Aromatase Inhibitors.

BACKGROUND: Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype, with aromatase inhibitors (AIs) being one of the therapeutic drug types used in the clinic. However, endocrine resistance may develop after prolonged treatment, and different approaches, such as combining endocrine and targeted therapies, have been applied. Recently, we demonstrated that cannabidiol (CBD) induces anti-tumor actions in ER+ breast cancer cells by targeting aromatase and ERs. Considering this, we studied, in vitro, whether CBD when combined with AIs could improve their effectiveness. METHODS: MCF-7aro cells were used and the effects on cell viability and on the modulation of specific targets were investigated. RESULTS: CBD when combined with anastrozole (Ana) and letrozole (Let) caused no beneficial effect in comparison to the isolated AIs. In contrast, when combined with the AI exemestane (Exe), CBD potentiated its pro-cell death effects, abolished its estrogen-like effect, impaired ERα activation, and prevented its oncogenic role on the androgen receptor (AR). Moreover, this combination inhibited ERK1/2 activation, promoting apoptosis. The study of the hormonal microenvironment suggests that this combination should not be applied in early stages of ER+ breast tumors. CONCLUSIONS: Contrary to Ana and Let, this study highlights the potential benefits of combining CBD with Exe to improve breast cancer treatment and opens up the possibility of new therapeutic approaches comprising the use of cannabinoids.

An Exemestane Derivative, Oxymestane-D1, as a New Multi-Target Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER+) Breast Cancer: Effects on Sensitive and Resistant Cell Lines.

Around 70-85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERß and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic.

Influence on the adult male Leydig cell biomarker insulin-like peptide 3 of maternal exposure to estrogenic and anti-androgenic endocrine disrupting compound mixtures: A retrospective study.

Insulin-like peptide 3 (INSL3) is a peptide biomarker secreted specifically by the mature Leydig cells of the testes. It is constitutive, has low within-individual variance, and effectively measures the functional capacity of Leydig cells to make testosterone. In young adult men there is a large 10-fold range of serum INSL3 concentration, persisting into old age, and implying that later hypogonadal status might be programmed in early life. To determine whether maternal exposure to environmental endocrine disrupting compounds (EDCs) influences adult serum INSL3 concentration, using a retrospective paradigm, INSL3 was measured in young adult male rats (80-90 days) from the F1 generation of females maternally exposed to varied doses of bisphenol A (BPA), butylparaben, epoxiconazole, and fludioxonil as single compounds, as well as estrogenic and anti-androgenic mixtures of BPA and butylparaben, and di(2-ethylhexyl) phthalate and procymidone respectively. A mixture of BPA and butylparaben significantly reduced circulating INSL3 concentration in adult male progeny. The remaining compounds or mixtures tested, though sufficient to induce other effects in the F1 generation were without significant effect. Maternal exposure to low concentrations of some EDCs may be a contributing factor to the variation in the Leydig cell biomarker INSL3 in young adulthood, though caution is warranted translating results from rats to humans.

A Comparative Assessment of Marker Expression Between Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells and the Developing Pig Heart.

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers [platelet-derived growth factor receptor, alpha polypeptide (PDGFR-α), fetal liver kinase 1 (FLK1), ISL1, NK2 homeobox 5 (NKX2.5), cardiac troponin T (CTNT), connexin43 (CX43), and myosin heavy chain 7 (MYHC-B)] in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22, and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and hiPSC, with an earlier onset in the hiPSC compared with reported murine expression. Markers of immature cardiomyocytes, CTNT, and MYHC-B were consistently expressed throughout E16-E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, while this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm and ISL1/NKX2.5 for cardiac progenitors. Furthermore, a combination of two or more of the following, CTNT+/MYHC-B+/ISL1+ could mark immature cardiomyocytes and CTNT+/ISL1- mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo-like cardiac tissue in the future.

Assessment of chemical mixtures using biomarkers of combined biological activity: A screening study in human placentas.

Humans are simultaneously exposed to complex mixtures of chemicals with limited knowledge on potential health effects, therefore improved tools for assessing these mixtures are needed. As part of the Human Biomonitoring for Europe (HBM4EU) Project, we aimed to examine the combined biological activity of chemical mixtures extracted from human placentas using one in vivo and four in vitro bioassays, also known as biomarkers of combined effect. Relevant endocrine activities (proliferative and/or reporter gene assays) and four endpoints were tested: the estrogen receptor (ER), androgen receptor (AR), and aryl hydrocarbon receptor (AhR) activities, as well as thyroid hormone (TH) signaling. Correlations among bioassays and their functional shapes were evaluated. Results showed that all placental extracts agonized or antagonized at least three of the abovementioned endpoints. Most placentas induced ER-mediated transactivation and ER-dependent cell proliferation, together with a strong inhibition of TH signaling and the AR transactivity; while the induction of the AhR was found in only one placental extract. The effects in the two estrogenic bioassays were positively and significantly correlated and the AR-antagonism activity showed a positive borderline-significant correlation with both estrogenic bioassay activities. However, the in vivo anti-thyroid activities of placental extracts were not correlated with any of the tested in vitro assays. Findings highlight the importance of comprehensively mapping the biological effects of "real-world" chemical mixtures present in human samples, through a battery of in vitro and in vivo bioassays. This approach should be a complementary tool for epidemiological studies to further elucidate the combined biological fingerprint triggered by chemical mixtures.

Organophosphate ester flame retardants have antiandrogenic potential and affect other endocrine related endpoints in vitro and in silico.

Organophosphate ester flame retardants (OPFRs) are used to prevent ignition and spreading of fire. They are present in various human matrices suggesting adult, fetal, and neonate exposure. Endocrine related effects have been observed in vivo, but information at the molecular level is lacking for some OPFRs. Also, a better understanding of potential contribution from chemical substructures is needed. The aim of this study was to screen OPFRs for endocrine disruptive potential in vitro and in silico. We selected eleven substances to represent some OPFRs with 1) little information on endocrine activity and others to represent 2) varied chemical substructures. We used in vitro assays for androgen receptor (AR), aryl hydrocarbon receptor (AhR), and Nrf2 activity, effects on steroidogenesis, and transthyretin (TTR) binding, as well as in silico models covering estrogen, thyroid, and CYP3A4 induction related endpoints. Ten OPFRs affected AR and AhR activity, seven affected TTR binding, and five affected 17ß-estradiol levels. Several substances had IC50-values below 10 µM and exhibited efficacious effects. These included TPHP, CDP, TMPP, TIPPP, and EHDPP for AR antagonism, suggesting that the degree of arylation and the size of the substance can play a role for the activity. Chlorinated OPFRs had low/no effect on TTR binding. No clear trend was observed for AhR and steroidogenesis, but all arylated OPFRs were predicted to have alert for estrogen receptor binding in an in silico model with metabolism simulator included. Collectively, our data suggest that OPFRs have endocrine disruptive potential warranting further studies to enable human risk assessment.

Receptor-based in vitro activities to assess human exposure to chemical mixtures and related health impacts.

Humans are exposed to a large number of chemicals from sources such as the environment, food, and consumer products. There is growing concern that human exposure to chemical mixtures, especially during critical periods of development, increases the risk of adverse health effects in newborns or later in life. Historically, the one-chemical-at-a-time approach has been applied both for exposure assessment and hazard characterisation, leading to insufficient knowledge about human health effects caused by exposure to mixtures of chemicals that have the same target. To circumvent this challenge researchers can apply in vitro assays to analyse both exposure to and human health effects of chemical mixtures in biological samples. The advantages of using in vitro assays are: (i) that an integrated effect is measured, taking combined mixture effects into account and (ii) that in vitro assays can reduce complexity in identification of Chemicals of Emerging Concern (CECs) in human tissues. We have reviewed the state-of-the-art on the use of receptor-based in vitro assays to assess human exposure to chemical mixtures and related health impacts. A total of 43 studies were identified, in which endpoints for the arylhydrocarbon receptor (AhR), the estrogen receptor (ER), and the androgen receptor (AR) were used. The majority of studies reported biological activities that could be associated with breast cancer incidence, male reproductive health effects, developmental toxicities, human demographic characteristics or lifestyle factors such as dietary patterns. A few studies used the bioactivities to check the coverage of the chemical analyses of the human samples, whereas in vitro assays have so far not regularly been used for identifying CECs in human samples, but rather in environmental matrices or food packaging materials. A huge field of novel applications using receptor-based in vitro assays for mixture toxicity assessment on human samples and effect-directed analysis (EDA) using high resolution mass spectrometry (HRMS) for identification of toxic compounds waits for exploration. In the future this could lead to a paradigm shift in the way we unravel adverse human health effects caused by chemical mixtures.

Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders.

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (λ-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Distinct Transcriptional Profiles of the Female, Male, and Finasteride-Induced Feminized Male Anogenital Region in Rat Fetuses.

A short anogenital distance (AGD) in males is a marker for incomplete masculinization and a predictor of adverse effects on male reproductive health. For this reason, AGD is used to assess the endocrine disrupting potential of chemicals for risk assessment purposes. The molecular mechanisms underpinning this chemically induced shortening of the AGD, however, remains unclear. Although it is clear that androgen receptor-mediated signaling is essential, evidence also suggest the involvement of other signaling pathways. This study presents the first global transcriptional profile of the anogenital tissue in male rat fetuses with chemically induced short AGD, also including comparison to normal male and female control animals. The antiandrogenic drug finasteride (10 mg/kg bw/day) was used to induce short AGD by exposing time-mated Sprague Dawley rats at gestation days 7-21. The AGD was 37% shorter in exposed male fetuses compared with control males at gestation day 21. Transcriptomics analysis on anogenital tissues revealed a sexually dimorphic transcriptional profile. More than 350 genes were found to be differentially expressed between the 3 groups. The expression pattern of 4 genes of particular interest (Esr1, Padi2, Wnt2, and Sfrp4) was also tested by RT-qPCR analyses, indicating that estrogen and Wnt2 signaling play a role in the sexually dimorphic development of the anogenital region. Our transcriptomics profiles provide a stepping-stone for future studies aimed at characterizing the molecular events governing development of the anogenital tissues, as well as describing the detailed Adverse Outcome Pathways for short AGD; an accepted biomarker of endocrine effects for chemical risk assessment.

Effects on metabolic parameters in young rats born with low birth weight after exposure to a mixture of pesticides.

Pesticide exposure during fetal life can lead to low birth weight and is commonly observed in reproductive toxicology studies. Associations have also been found in low birth weight babies born from pesticide-exposed gardeners. Since low birth weight is also linked to metabolic disorders, it can be speculated that early life exposure to pesticides could increase the risk of becoming obese or developing diabetes later in life. We have analyzed potential long-term effects of gestational and lactational exposure to a low dose mixture of six pesticides that individually can cause low birth weight: Cyromazine, MCPB, Pirimicarb, Quinoclamine, Thiram, and Ziram. Exposed male offspring, who were smaller than controls, displayed some degree of catch-up growth. Insulin and glucagon regulation was not significantly affected, and analyses of liver and pancreas did not reveal obvious histopathological effects. Efforts towards identifying potential biomarkers of metabolic disease-risk did not result in any strong candidates, albeit leptin levels were altered in exposed animals. In fat tissues, the key genes Lep, Nmb and Nmbr were altered in high dosed offspring, and were differentially expressed between sexes. Our results suggest that early-life exposure to pesticides may contribute to the development of metabolic disorders later in life.

Enniatin B and beauvericin are common in Danish cereals and show high hepatotoxicity on a high-content imaging platform.

Mycotoxins are fungi-born metabolites that can contaminate foods through mould-infected crops. They are a significant food/feed-safety issue across the globe and represent a substantial financial burden for the world economy. Moreover, with a changing climate and fungal biota, there is now much discussion about emerging mycotoxins that are measurable at significant levels in crops world-wide. Unfortunately, we still know very little about the bioavailability and toxic potentials of many of these less characterized mycotoxins, including the large family of enniatins. In this study, we present new occurrence data for enniatin A, A1, B, B1 and beauvericin in four Danish crops: oat, wheat, and barley from the 2010 harvest, and rye from 2011 harvest. The occurrence of the four enniatins were B > B1 > A1 > A. Enniatin B was detected in 100% of tested samples regardless of crop type. In addition to occurrence data, we report a proof-of-concept study using a human-relevant high-content hepatotoxicity, or "quadroprobe," assay to screen mycotoxins for their cytotoxic potential. The assay was sensitive for most cytotoxic compounds in the 0.009-100 µM range. Among eight tested mycotoxins (enniatin B, beauvericin, altenariol, deoxynivalenol, aflatoxin B1, andrastin A, citrinin, and penicillic acid), enniatin B and beauvericin showed significant cytotoxicity at a concentration lower than that for aflatoxin B1, which is the archetypal acute hepatotoxic and liver-carcinogenic mycotoxin. Hence, the quadroprobe hepatotoxicity assay may become a valuable assessment tool for toxicity assessment of mycotoxins in the future. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1658-1664, 2017.

Perinatal exposure to mixtures of anti-androgenic chemicals causes proliferative lesions in rat prostate.

BACKGROUND: Elevated levels of endogenous or exogenous estrogens during fetal life can induce permanent disturbances in prostate growth and predispose to precancerous lesions. Recent studies have indicated that also early anti-androgen exposure may affect prostate cancer risk. METHODS: We examined the influence of perinatal exposure to mixtures of anti-androgenic and estrogenic chemicals on prostate development. Wistar rats were exposed from gestation day 7 to postnatal day 22 to a mixture of 8 anti-androgenic compounds (AAMix), a mixture of four estrogenic compounds (EMix), or paracetamol or a mixture of all 13 compounds (TotalMix) in mixture ratios reflecting human exposure levels. RESULTS: Ventral prostate weights were reduced by the TotalMix and AAMix in pre-pubertal rats. Histological changes in prostate appeared with increasing age and indicated a shift from the normal age-dependent epithelial atrophy towards hyperplasia. These lesions showed similarities to pre-cancerous lesions in humans. Increased proliferation was observed already in pre-puberty and it was hypothesized that this could be associated with reduced ERß signaling, but no clear conclusions could be made from gene expression studies on ERß-related pathways. The influences of the estrogenic chemicals and paracetamol on prostate morphology were minor, but in young adulthood the estrogen mixture reduced ventral prostate mRNA levels of Igf1 and paracetamol reduced the mRNA level ofPbpc3. CONCLUSIONS: Mixtures of endocrine disrupters relevant for human exposure was found to elicit persistent effects on the rat prostate following perinatal exposure, suggesting that human perinatal exposure to environmental chemicals may increase the risk of prostate cancer later in life.

Mixtures of environmentally relevant endocrine disrupting chemicals affect mammary gland development in female and male rats.

Estrogenic chemicals are able to alter mammary gland development in female rodents, but little is known on the effects of anti-androgens and mixtures of endocrine disrupting chemicals (EDCs) with dissimilar modes of action. Pregnant rat dams were exposed during gestation and lactation to mixtures of environmentally relevant EDCs with estrogenic, anti-androgenic or dissimilar modes of action (TotalMix) of 100-, 200- or 450-fold high end human intake estimates. Mammary glands of prepubertal and adult female and male offspring were examined. Oestrogens increased mammary outgrowth in prepubertal females and the mRNA level of matrix metalloproteinase-3, which may be a potential biomarker for increased outgrowth. Mixtures of EDCs gave rise to ductal hyperplasia in adult males. Adult female mammary glands of the TotalMix group showed morphological changes possibly reflecting increased prolactin levels. In conclusion both estrogenic and anti-androgenic chemicals given during foetal life and lactation affected mammary glands in the offspring.

Are structural analogues to bisphenol a safe alternatives?

BACKGROUND: Bisphenol A (BPA) is a chemical with widespread human exposure suspected of causing low-dose effects. Thus, a need for developing alternatives to BPA exists. Structural analogues of BPA have already been detected in foods and humans. Due to the structural analogy of the alternatives, there is a risk of effects similar to BPA. OBJECTIVES: The aim was to elucidate and compare the hazards of bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF), bisphenol S (BPS) and 4-cumylphenol (HPP) to BPA. METHODS: In vitro studies on steroidogenesis, receptor activity, and biomarkers of effect, as well as Quantitative Structure-Activity Relationship (QSAR) modeling. RESULTS: All test compounds caused the same qualitative effects on estrogen receptor and androgen receptor activities, and most of the alternatives exhibited potencies within the same range as BPA. Hormone profiles for the compounds indicated a specific mechanism of action on steroidogenesis which generally lead to decreased androgen, and increased estrogen and progestagen levels. Differential effects on corticosteroid synthesis were observed suggesting a compound-specific mechanism. Overall, BPS was less estrogenic and antiandrogenic than BPA, but BPS showed the largest efficacy on 17α-hydroxyprogesterone (17α-OH progesterone). Finally, there were indications of DNA damage, carcinogenicity, oxidative stress, effects on metabolism, and skin sensitization of one or more of the test compounds. CONCLUSIONS: Interference with the endocrine system was the predominant effect of the test compounds. A substitution of BPA with these structural analogues should be carried out with caution.

A computational approach to mechanistic and predictive toxicology of pesticides.

Emerging challenges of managing and interpreting large amounts of complex biological data have given rise to the growing field of computational biology. We investigated the applicability of an integrated systems toxicology approach on five selected pesticides to get an overview of their modes of action in humans, to group them according to their modes of action, and to hypothesize on their potential effects on human health. We extracted human proteins associated to prochloraz, tebuconazole, epoxiconazole, procymidone, and mancozeb and enriched each protein set by using a high confidence human protein interactome. Then, we explored modes of action of the chemicals, by integrating protein-disease information to the resulting protein networks. The dominating human adverse effects affected were reproductive disorders followed by adrenal diseases. Our results indicated that prochloraz, tebuconazole, and procymidone exerted their effects mainly via interference with steroidogenesis and nuclear receptors. Prochloraz was associated to a large number of human diseases, and together with tebuconazole showed several significant associations to Testicular Dysgenesis Syndrome. Mancozeb showed a differential mode of action, involving inflammatory processes. This method provides an efficient way of overviewing data and grouping chemicals according to their mode of action and potential human adverse effects. Such information is valuable when dealing with predictions of mixture effects of chemicals and may contribute to the development of adverse outcome pathways.

Late-life effects on rat reproductive system after developmental exposure to mixtures of endocrine disrupters.

This study examined late-life effects of perinatal exposure of rats to a mixture of endocrine-disrupting contaminants. Four groups of 14 time-mated Wistar rats were exposed by gavage from gestation day 7 to pup day 22 to a mixture of 13 anti-androgenic and estrogenic chemicals including phthalates, pesticides, u.v.-filters, bisphenol A, parabens, and the drug paracetamol. The groups received vehicle (control), a mixture of all 13 chemicals at 150-times (TotalMix150) or 450-times (TotalMix450) high-end human exposure, or 450-times a mixture of nine predominantly anti-androgenic chemicals (AAMix450). Onset of puberty and estrous cyclicity at 9 and 12 months of age were assessed. Few female offspring showed significantly regular estrus cyclicity at 12 months of age in the TotalMix450 and AAMix450 groups compared with controls. In 19-month-old male offspring, epididymal sperm counts were lower than controls, and in ventral prostate an overrepresentation of findings related to hyperplasia was observed in exposed groups compared with controls, particularly in the group dosed with anti-androgens. A higher incidence of pituitary adenoma at 19 months of age was found in males and females in the AAMix450 group. Developmental exposure of rats to the highest dose of a human-relevant mixture of endocrine disrupters induced adverse effects late in life, manifested as earlier female reproductive senescence, reduced sperm counts, higher score for prostate atypical hyperplasia, and higher incidence of pituitary tumors. These delayed effects highlight the need for further studies on the role of endocrine disrupters in hormone-related disorders in aging humans.

Effects of perinatal ethinyl estradiol exposure in male and female Wistar rats.

Perinatal exposure to endocrine disrupting chemicals with estrogenic activity can adversely affect reproductive development, but few studies evaluating estrogen-sensitive endpoints have been performed in Wistar rats. Therefore, time-mated Wistar rats (n=10) were gavaged during gestation and lactation with 0, 5, 15 or 50µg/kg bw/day of ethinyl estradiol. This potent estrogen was found to induce an increased number of nipples and reduced ovary weight in female offspring. Malformations of female genitalia were found in young as well as adult offspring, as an increased AGD was seen at birth and a deeper urethral slit length was seen in adulthood. In prepubertal male offspring, estrogen-regulated gene expression in ventral prostate was increased dose-dependently and a decreased ventral prostate weight was seen at 15µg/kg. Female external sexual characteristics and prostate development were found to be targets for exposure to estrogenic compounds and may be of interest in studies on estrogenic environmental compounds.

Concentration addition, independent action and generalized concentration addition models for mixture effect prediction of sex hormone synthesis in vitro.

Humans are concomitantly exposed to numerous chemicals. An infinite number of combinations and doses thereof can be imagined. For toxicological risk assessment the mathematical prediction of mixture effects, using knowledge on single chemicals, is therefore desirable. We investigated pros and cons of the concentration addition (CA), independent action (IA) and generalized concentration addition (GCA) models. First we measured effects of single chemicals and mixtures thereof on steroid synthesis in H295R cells. Then single chemical data were applied to the models; predictions of mixture effects were calculated and compared to the experimental mixture data. Mixture 1 contained environmental chemicals adjusted in ratio according to human exposure levels. Mixture 2 was a potency adjusted mixture containing five pesticides. Prediction of testosterone effects coincided with the experimental Mixture 1 data. In contrast, antagonism was observed for effects of Mixture 2 on this hormone. The mixtures contained chemicals exerting only limited maximal effects. This hampered prediction by the CA and IA models, whereas the GCA model could be used to predict a full dose response curve. Regarding effects on progesterone and estradiol, some chemicals were having stimulatory effects whereas others had inhibitory effects. The three models were not applicable in this situation and no predictions could be performed. Finally, the expected contributions of single chemicals to the mixture effects were calculated. Prochloraz was the predominant but not sole driver of the mixtures, suggesting that one chemical alone was not responsible for the mixture effects. In conclusion, the GCA model seemed to be superior to the CA and IA models for the prediction of testosterone effects. A situation with chemicals exerting opposing effects, for which the models could not be applied, was identified. In addition, the data indicate that in non-potency adjusted mixtures the effects cannot always be accounted for by single chemicals.

In vitro-in vivo correlations for endocrine activity of a mixture of currently used pesticides.

Two pesticide mixtures were investigated for potential endocrine activity. Mix 3 consisted of bitertanol, propiconazole, and cypermethrin, and Mix 5 included malathion and terbuthylazine in addition to the three pesticides in Mix 3. All five single pesticides and the two mixtures were investigated for their ability to affect steroidogenesis in vitro in H295R cells. The pesticides alone and both mixtures affected steroidogenesis with both mixtures causing increase in progesterone and decrease in testosterone. For Mix 5 an increase in estradiol was seen as well, indicating increased aromatase activity. The two mixtures were also investigated in pregnant rats dosed from gestational day 7 to 21, followed by examination of dams and fetuses. Decreased estradiol and reduced placental testosterone were seen in dams exposed to Mix 5. Also a significant increase in aromatase mRNA-levels in female adrenal glands was found for Mix5. However, either of the two mixtures showed any effects on fetal hormone levels in plasma or testis, or on anogenital distance. Overall, potential aromatase induction was found for Mix 5 both in vitro and in vivo, but not for Mix 3, an effect likely owed to terbuthylazine in Mix 5. However, the hormonal responses in vitro were only partly reflected in vivo, probably due to some toxicokinetic issues, as the pesticide levels in the amniotic fluid also were found to be negatively affected by the number of compounds present in the mixtures. Nonetheless, the H295R assay gives hints on conceivable interference with steroidogenesis, thus generating hypotheses on in vivo effects.

Levels of pesticides and their metabolites in Wistar rat amniotic fluids and maternal urine upon gestational exposure.

Concentrations of pesticides and selected metabolites in rat urine and amniotic fluid were determined as biomarker upon oral administration of Wistar rats to two pesticide mixtures consisting of three to five pesticides (bitertanol, propiconazole, cypermethrin, malathion, and terbuthylazine). The pesticides and their metabolites were found in rat amniotic fluid and urine, generally in dose-response concentrations in relation to dosage. The measurement of the substances in the amniotic fluid indicated that the fetus was exposed to the pesticides as well as their metabolites. Moreover, the pesticides detected in urine demonstrated the exposure as well as the ability of the rat to excrete these compounds.