Human-relevant concentrations of the antifungal drug clotrimazole disrupt maternal and fetal steroid hormone profiles in rats.

Clotrimazole is a non-prescription and broad-spectrum antifungal drug sold under brand names such as Canesten® and Lotrimin®. It is used to treat different types of fungal infections, from oral thrush to athlete's foot and vaginal mycosis. The level of exposure to clotrimazole is uncertain, as the exact usage amongst self-medicating patients is unclear. Recent studies have raised potential concern about the unsupervised use of clotrimazole during pregnancy, especially since it is a potent inhibitor of CYP enzymes of the steroidogenesis pathway. To address some of these concerns, we have assessed the effects of intrauterine exposure to clotrimazole on developing rat fetuses. By exposing pregnant rats to clotrimazole 25 or 75 mg/kg bw/day during gestation days 7-21, we obtained internal fetal concentrations close to those observed in humans. These in vivo data are in strong agreement with our physiologically-based pharmacokinetic (PBK)-modelled levels. At these doses, we observed no obvious morphological changes to the reproductive system, nor shorter male anogenital distance; a well-established morphometric marker for anti-androgenic effects in male offspring. However, steroid hormone profiles were significantly affected in both maternal and fetal plasma, in particular pronounced suppression of estrogens was seen. In fetal testes, marked up-concentration of hydroxyprogesterone was observed, which indicates a specific action on steroidogenesis. Since systemic clotrimazole is rapidly metabolized in humans, relevant exposure levels may not in itself cause adverse changes to the reproductive systems. Its capacity to significantly alter steroid hormone concentrations, however, suggests that clotrimazole should be used with caution during pregnancy.

Perfluorononanoic acid in combination with 14 chemicals exerts low-dose mixture effects in rats.

Humans are simultaneously exposed to several chemicals that act jointly to induce mixture effects. At doses close to or higher than no-observed adverse effect levels, chemicals usually act additively in experimental studies. However, we are lacking knowledge on the importance of exposure to complex real-world mixtures at more relevant human exposure levels. We hypothesised that adverse mixture effects occur at doses approaching high-end human exposure levels. A mixture (Mix) of 14 chemicals at a combined dose of 2.5 mg/kg bw/day was tested in combination with perfluorononanoic acid (PFNA) at doses of 0.0125 (Low PFNA), 0.25 (Mid PFNA) and 5 (High PFNA) mg/kg bw/day by oral administration for 14 days in juvenile male rats. Indication of a toxicokinetic interaction was found, as simultaneous exposure to PFNA and the Mix caused a 2.8-fold increase in plasma PFNA concentrations at Low PFNA. An increase in testosterone and dihydrotestosterone plasma concentrations was observed for Low PFNA + Mix. This effect was considered non-monotonic, as higher doses did not cause this effect. Reduced LH plasma concentrations together with increased androgen concentrations indicate a disturbed pituitary-testis axis caused by the 15-chemical mixture. Low PFNA by itself increased the corticosterone plasma concentration, an effect which was normalised after simultaneous exposure to Mix. This combined with affected ACTH plasma concentrations and down-regulation of 11ß HSD mRNA in livers indicates a disturbed pituitary-adrenal axis. In conclusion, our data suggest that mixtures of environmental chemicals at doses approaching high-end human exposure levels can cause a hormonal imbalance and disturb steroid hormones and their regulation. These effects may be non-monotonic and were observed at low doses. Whether this reflects a more general phenomenon that should be taken into consideration when predicting human mixture effects or represents a rarer phenomenon remains to be shown.

In vitro antiandrogenic effects of the herbicide linuron and its metabolites.

Although the herbicide linuron is banned for use in the EU due to its reproductive and developmental toxicity, it can still be found in randomly sampled foods grown in and outside the EU. It is not clear if metabolites of linuron can contribute to the endocrine disrupting effects following exposure to the parent compound. To address this gap, we analysed linuron and the metabolites 1-(3,4-dichlorophenyl) urea (DCU), 3,4-dichloroaniline (DCA) and 1-(3,4-dichlorophenyl)-3-methoxyurea (DCXU) for androgen receptor (AR) activities and effects on steroidogenesis. Generally, linuron and the metabolites showed qualitatively similar antiandrogenic profiles, but potencies varied. All compounds were AR antagonists, with linuron showing highest potency (IC50 of 2.8 µM). The overall picture of effects on steroidogenesis showed that linuron and metabolites increased the levels of estrogens and corticosteroids, whereas the synthesis of androgens was inhibited. The metabolite DCU was by far the most potent inhibitor of testosterone synthesis (IC50 of 6.7 µM compared to IC50 of 51.1 µM for linuron). We suggest that it is likely that the metabolites contribute to the antiandrogenic effects of linuron in vivo, especially by inhibiting testosterone synthesis.

Dietary fibre directs microbial tryptophan metabolism via metabolic interactions in the gut microbiota.

Tryptophan is catabolized by gut microorganisms resulting in a wide range of metabolites implicated in both beneficial and adverse host effects. How gut microbial tryptophan metabolism is directed towards indole, associated with chronic kidney disease, or towards protective indolelactic acid (ILA) and indolepropionic acid (IPA) is unclear. Here we used in vitro culturing and animal experiments to assess gut microbial competition for tryptophan and the resulting metabolites in a controlled three-species defined community and in complex undefined human faecal communities. The generation of specific tryptophan-derived metabolites was not predominantly determined by the abundance of tryptophan-metabolizing bacteria, but rather by substrate-dependent regulation of specific metabolic pathways. Indole-producing Escherichia coli and ILA- and IPA-producing Clostridium sporogenes competed for tryptophan within the three-species community in vitro and in vivo. Importantly, fibre-degrading Bacteroides thetaiotaomicron affected this competition by cross-feeding monosaccharides to E. coli. This inhibited indole production through catabolite repression, thus making more tryptophan available to C. sporogenes, resulting in increased ILA and IPA production. The fibre-dependent reduction in indole was confirmed using human faecal cultures and faecal-microbiota-transplanted gnotobiotic mice. Our findings explain why consumption of fermentable fibres suppresses indole production but promotes the generation of other tryptophan metabolites associated with health benefits.

Exposure to the phthalate metabolite MEHP impacts survival and growth of human ovarian follicles in vitro.

Phthalates are found in everyday items like plastics and personal care products. There is an increasing concern that continuous exposure can adversely affect female fertility. However, experimental data are lacking to establish causal links between exposure and disease in humans. To address this gap, we tested the effects of a common phthalate metabolite, mono-(2-ethylhexyl) phthalate (MEHP), on adult human ovaries in vitro using an epidemiologically determined human-relevant concentration range (2.05 nM - 20.51 mM). Histomorphological assessments, steroid and cytokine measurements were performed on human ovarian tissue exposed to MEHP for 7 days in vitro. Cell viability and gene expression profile were investigated following 7 days of MEHP exposure using the human granulosa cancer cell lines KGN, and COV434, the germline tumor cell line PA-1, and human ovarian primary cells. Selected differentially expressed genes (DEGs) were validated by RT-qPCR and immunofluorescence in human ovarian tissue. MEHP exposure reduced follicular growth (20.51 nM) and increased follicular degeneration (20.51 mM) in ovarian tissue, while not affecting steroid and cytokine production. Out of the 691 unique DEGs identified across all the cell types and concentrations, CSRP2 involved in cytoskeleton organization and YWHAE as well as CTNNB1 involved in the Hippo pathway, were chosen for further validation. CSRP2 was upregulated and CTNNB1 downregulated in both ovarian tissue and cells, whereas YWHAE was downregulated in cells only. In summary, one-week MEHP exposure of human ovarian tissue can perturb the development and survival of human follicles through mechanisms likely involving dysregulation of cytoskeleton organization and Hippo pathway.

PPARγ antagonists induce aromatase transcription in adipose tissue cultures.

Aromatase is the rate-limiting enzyme in the biosynthesis of estrogens and a key risk factor for hormone receptor-positive breast cancer. In postmenopausal women, estrogens synthesized in adipose tissue promotes the growth of estrogen receptor positive breast cancers. Activation of peroxisome proliferator-activated receptor gamma (PPARγ) in adipose stromal cells (ASCs) leads to decreased expression of aromatase and differentiation of ASCs into adipocytes. Environmental chemicals can act as antagonists of PPARγ and disrupt its function. This study aimed to test the hypothesis that PPARγ antagonists can promote breast cancer by stimulating aromatase expression in human adipose tissue. Primary cells and explants from human adipose tissue as well as A41hWAT, C3H10T1/2, and H295R cell lines were used to investigate PPARγ antagonist-stimulated effects on adipogenesis, aromatase expression, and estrogen biosynthesis. Selected antagonists inhibited adipocyte differentiation, preventing the adipogenesis-associated downregulation of aromatase. NMR spectroscopy confirmed direct interaction between the potent antagonist DEHPA and PPARγ, inhibiting agonist binding. Short-term exposure of ASCs to PPARγ antagonists upregulated aromatase only in differentiated cells, and a similar effect could be observed in human breast adipose tissue explants. Overexpression of PPARG with or without agonist treatment reduced aromatase expression in ASCs. The data suggest that environmental PPARγ antagonists regulate aromatase expression in adipose tissue through two mechanisms. The first is indirect and involves inhibition of adipogenesis, while the second occurs more acutely.

Key bacterial taxa determine longitudinal dynamics of aromatic amino acid catabolism in infants' gut.

The development of the gut microbiota in early life is linked to metabolic, neuronal, and immunological development. Recent studies have shown that bacterial production of short-chain fatty acids (SCFAs) and aromatic amino acid (AAA) catabolites in the gut can mediate host-microbe interactions. However, the dynamics of these microbiota-derived metabolites and the key bacterial taxa producing AAA catabolites during infancy are largely unknown. Here, we investigated the longitudinal dynamics of the microbiota and microbiota-derived SCFAs and AAA catabolites in more than 200 fecal samples from 25 healthy breast- or mixed-fed Danish infants during the first 6 months of life. We found that the gut microbiota composition and metabolism were highly individual but showed significant development over time. SCFAs and specific groups of AAA catabolites showed distinct temporal abundance patterns. Furthermore, we identified bacterial taxa responsible for the generation of AAA catabolites by associating the dynamics of gut microbial taxa and AAA catabolites and subsequently validating these associations in vitro by cultivation of strains representing the associated taxa. In addition to specific Bifidobacterium species being the main producers of aromatic lactic acids, we identified Peptostreptococcus anaerobius as the main producer of aromatic propionic acids, Ruminococcus gnavus as a main producer of tryptamine, and Enterococcus species as main tyramine producers in infants' gut. Thus, our results showcase the temporal dynamics of key gut microbial metabolites in early life and demonstrate that the appearance and abundance of specific AAA catabolites result from the appearance and abundance of specific key bacterial taxa in infants' gut.

Simultaneous determination of heterocyclic aromatic amines and N-nitrosamines in fried bacon cubes and slices using LC-(ESI/APCI)-MS/MS.

Bacon manufacturing involves several processing steps including nitrite curing, followed by cooking processes, typically frying. During these processes, harmful processing contaminants such as N-nitrosamines (NAs) and heterocyclic aromatic amines (HAAs) can be formed. Consequently, we developed and validated a multi-class method for quantification of the most frequently reported HAAs and NAs in fried bacon. Satisfactory repeatability and reproducibility with limits of quantification between 0.1 and 0.5 ng g-1 for most of the compounds were achieved. Quantification in pan-fried bacon cubes and slices revealed generally low levels of individual HAAs (≤1.5 ng g-1), except in ready-to-eat bacon (0.9-2.9 ng g-1). Differences in amounts of individual HAAs were observed in cubes and slices, most likely due to meat thickness. Among volatile NAs (VNAs), only N-nitrosopiperidine (NPIP), N-nitrosopyrolidine (NPYR), and N-nitrosodibutylamine (NDBA) were found at generally low concentrations (≤5 ng g-1). In contrast, non-volatile NAs (NVNAs) were present in all tested samples at considerably higher amounts, for example, N-nitroso-thiazolidine-4-carboxylic acid (NTCA) at 12-77 ng g-1. N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA) were not detected in any samples. Statistical evaluation and principal component analysis revealed some differences among tested samples. Dietary exposure estimation of the Danish population to HAAs and NAs showed the highest exposure in the teenage group (10-17 years).

Exposure to the pesticides linuron, dimethomorph and imazalil alters steroid hormone profiles and gene expression in developing rat ovaries.

Inhibition of androgen signaling during critical stages of ovary development can disrupt folliculogenesis with potential consequences for reproductive function later in life. Many environmental chemicals can inhibit the androgen signaling pathway, which raises the question if developmental exposure to anti-androgenic chemicals can negatively impact female fertility. Here, we report on altered reproductive hormone profiles in prepubertal female rats following developmental exposure to three pesticides with anti-androgenic potential: linuron (25 and 50 mg/kg bw/d), dimethomorph (60 and 180 mg/kg bw/d) and imazalil (8 and 24 mg/kg bw/d). Dams were orally exposed from gestational day 7 (dimethomorph and imazalil) or 13 (linuron) until birth, then until end of dosing at early postnatal life. Linuron and dimethomorph induced dose-related reductions to plasma corticosterone levels, whereas imazalil mainly suppressed gonadotropin levels. In the ovaries, expression levels of target genes were affected by linuron and dimethomorph, suggesting impaired follicle growth. Based on our results, we propose that anti-androgenic chemicals can negatively impact female reproductive development. This highlights a need to integrate data from all levels of the hypothalamic-pituitary-gonadal axis, as well as the hypothalamic-pituitary-adrenal axis, when investigating the potential impact of endocrine disruptors on female reproductive development and function.

An engineered Escherichia coli Nissle 1917 increase the production of indole lactic acid in the gut.

The expanding knowledge of the health impacts of the metabolic activities of the gut microbiota reinforces the current interest in engineered probiotics. Tryptophan metabolites, in particular indole lactic acid (ILA), are attractive candidates as potential therapeutic agents. ILA is a promising compound with multiple beneficial effects, including amelioration colitis in rodent models of necrotizing enterocolitis, as well as improved infant immune system maturation. In this work, we engineered and characterized in vitro and in vivo an Escherichia coli Nissle 1917 strain that produces ILA. The 2-step metabolic pathway comprises aminotransferases native of E. coli and a dehydrogenase introduced from Bifidobacterium longum subspecies infantis. Our results show a robust engineered probiotic that produces 73.4 ± 47.2 nmol and 149 ± 123.6 nmol of ILA per gram of fecal and cecal matter, respectively, three days after colonization in a mouse model. In addition, hereby is reported an engineered-probiotic-related increase of ILA in the systemic circulation of the treated mice. This strain serves as proof of concept for the transfer of capacity to produce ILA in vivo and as ILA emerges as a potent microbial metabolite against gastrointestinal inflammation, further development of this strain offers efficient options for ILA-focused therapeutic interventions in situ.

Combination of IVF and IVM in naturally cycling women.

This study investigated the combination of an unstimulated IVF cycle with in-vitro maturation (IVM) of additional immature cumulus­oocyte­complexes (COC) from the same cycle collected at the same time as the spontaneous preovulatory follicle. This could potentially improve rates of embryo transfer and pregnancy/live births compared with conventional unstimulated IVF treatment and at the same time eliminate the risk of ovarian hyperstimulation syndrome. This prospective trial included 77 women with regular menstrual cycles. Age at inclusion was between 20 and 37 years. Results showed a retrieval rate of mature oocytes of 50/80 (62.5%) per cycle started and immature COC were collected in 74/80 (92.5%) cycles. The embryo transfer rate was 28/80 (35.0%) with mature oocytes and increased in total to 43/80 (53.8%) with IVM oocytes. Corresponding birth rates per transfer were 3/28 (10.7%) and 4/43 (9.3%). Birth rates per aspiration were 3/76 (3.9%) and 4/76 (5.3%). It is concluded that the protocol described here shows proof of concept, but the impact of the IVM procedure only reached a significant level regarding embryo transfer, not with live births. The reason for this is yet unclear, but asynchrony between endometrial factors and IVM oocytes together with unknown competence of IVM embryos is suspected.

Atypical TGF-ß signaling controls neuronal guidance in Caenorhabditis elegans.

Coordinated expression of cell adhesion and signaling molecules is crucial for brain development. Here, we report that the Caenorhabditis elegans transforming growth factor ß (TGF-ß) type I receptor SMA-6 (small-6) acts independently of its cognate TGF-ß type II receptor DAF-4 (dauer formation-defective-4) to control neuronal guidance. SMA-6 directs neuronal development from the hypodermis through interactions with three, orphan, TGF-ß ligands. Intracellular signaling downstream of SMA-6 limits expression of NLR-1, an essential Neurexin-like cell adhesion receptor, to enable neuronal guidance. Together, our data identify an atypical TGF-ß-mediated regulatory mechanism to ensure correct neuronal development.

Antimicrobial effect of nisin in processed cheese - Quantification of residual nisin by LC-MS/MS and development of new growth and growth boundary model for Listeria monocytogenes.

This study tested the hypothesis that growth of Listeria monocytogenes in processed cheese with added nisin can be predicted from residual nisin A concentrations in the final product after processing. A LC-MS/MS method and a bioassay were studied to quantify residual nisin A concentrations and a growth and growth boundary model was developed to predict the antilisterial effect in processed cheese. 278 growth rates were determined in broth for 11 L. monocytogenes isolates and used to determine 13 minimum inhibitory concentration (MIC) values for nisin between pH 5.5 and 6.5. To supplement these data, 67 MIC-values at different pH-values were collected from the scientific literature. A MIC-term was developed to describe the effect of pH on nisin MIC-values. An available growth and growth boundary model (doi: https://doi.org/10.1016/j.fm.2019.103255) was expanded with the new MIC-term for nisin to predict growth in processed cheese. To generate data for model evaluation and further model development, challenge tests with a total of 45 growth curves, were performed using processed cheese. Cheeses were formulated with 11.2 or 12.0 ppm of nisin A and heat treated to obtain residual nisin A concentrations ranging from 0.56 to 5.28 ppm. Below 15 °C, nisin resulted in extended lag times. A global regression approach was used to fit all growth curves determined in challenge tests. This was obtained by combining the secondary growth and growth boundary model including the new term for the inhibiting effect of nisin on µmax with the primary logistic growth model with delay. This model appropriately described the growth inhibiting effect of residual nisin A and showed that relative lag times depended on storage temperatures. With residual nisin A concentrations, other product characteristics and storage temperature as input the new model correctly predicted all observed growth and no-growth responses for L. monocytogenes. This model can support development of nisin A containing recipes for processed cheese that prevent growth of L. monocytogenes. Residual nisin A concentrations in processed cheese were accurately quantified by the developed LC-MS/MS method with recoveries of 83 to 110% and limits of detection and quantification being 0.04 and 0.13 ppm, respectively. The tested bioassay was less precise and nisin A recoveries varied for 53% to 94%.

Perinatal exposure to known endocrine disrupters alters ovarian development and systemic steroid hormone profile in rats.

Disrupted ovarian development induced by chemical exposure may impair fertility later in life. Since androgens are essential for early ovarian development, we speculated that perinatal exposure to a binary mixture of the known anti-androgens DEHP and procymidone could alter steroid synthesis, disrupt ovarian follicle recruitment and ultimately maturation in female rat offspring. Wistar rat dams were exposed by oral gavage from gestation day 7 to postnatatal day 22 to two mixture doses known to alter reproductive development in male offspring (low: 10 mg/kg bw/day of procymidone and 30 mg/kg bw/day of DEHP; high: 20 mg/kg bw/day of procymidone and 60 mg/kg bw/day of DEHP). The Effects on plasma steroid hormones, ovarian follicle distribution and expression of markers related to steroid synthesis were examined in female offspring. In prepubertal offspring, we observed an increased number of newly recruited (primary) follicles in exposed animals compared to controls, and the plasma steroid hormone profile was altered by exposure: levels of progesterone, corticosterone and estrone were dose dependently elevated, whereas androgen levels were unaffected. In adulthood, a trend towards a smaller number of early-stage follicles may point to accelerated loss of follicle reserves, which is disconcerting. The changes in follicle distribution in exposed ovaries may reflect the combined influence of androgen receptor antagonism and altered ovarian steroid synthesis. This study adds to a growing body of evidence showing altered ovarian development following exposure to human relevant chemicals with possible severe consequences for female fertility.

Anticonvulsant effects of Searsia dentata (Anacardiaceae) leaf extract in rats.

Searsia species are used in South Africa to treat epilepsy. Previous studies have demonstrated an in vitro N-methyl-D-aspartic acid (NMDA) receptor antagonistic effect of the ethanolic leaf extract. The aim of this study was to evaluate the potential anticonvulsant properties of the ethanolic extract of S. dentata in various animal models of epilepsy. The extract was submitted to a screening in anticonvulsant assays including NMDA-, kainic acid (KA)-, pentylenetetrazol (PTZ)- and bicuculline (BIC)-induced seizures in rats. The extract protected 47% of the PN 18 Wistar pups (postnatal day 18, date of birth PN 0) (p < 0.05, n > 10) against NMDA-induced seizures and significantly delayed the onset of PTZ-induced seizures (p < 0.05, n > 8) at a dose of 250 mg/kg. A dose optimum was detected at 500 mg/kg for protection against KA-(63% protection, p < 0.05, n > 8) and BIC-induced seizures (50% protection, p < 0.05, n > 8) in young adult and PN 18 rats, respectively. The ethanolic extract of S. dentata showed anticonvulsive properties in several models of epilepsy. These results are compatible with previous findings of NMDA receptor antagonism. Due to the complex composition of the extract, the effect might be caused by more than one compound.

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.

Amides from Piper capense with CNS activity - a preliminary SAR analysis.

Piper capense L.f. (Piperaceae) is used traditionally in South Africa as a sleep inducing remedy. Bioassay-guided fractionation of the roots of P. capense led to the isolation of piperine (1) and 4,5-dihydropiperine (2), which showed moderate affinity for the benzodiazepine site on the GABA(A) receptor (IC(50) values of 1.2 mM and 1.0 mM, respectively). The present study suggests that strict structural properties of the amides are essential for affinity. Taken together, these observations suggest that the carbon chain must contain not less than four carbons, and that a conjugated double bond, adjacent to the amide group, is necessary for binding to the receptor and that the amine part should be bulky.

In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole.

Azoles are effective antifungal agents used in both medicine and agriculture. They typically work by inhibiting cytochrome P450 enzymes, primarily CYP51 of the ergosterol biosynthesis pathway, thus damaging the fungal cell membrane. However, apart from their desired antifungal properties, several azoles also exhibit endocrine disrupting properties in mammals, both in vitro and in vivo. Here, we have tested two currently used agricultural azole fungicides, triticonazole and flusilazole, for their in vitro anti-androgenic activity and potential effects on reproductive parameters. Both fungicides showed strong androgen receptor (AR) antagonism and disruption of steroid biosynthesis in vitro. Following gestational exposure to flusilazole (15 or 45 mg/kg bw/day) or triticonazole (150 or 450 mg/kg bw/day) in time-mated Sprague Dawley rats, triticonazole induced shorter male anogenital distance (AGD). Flusilazole exposure did not affect the AGD, but altered fetal male blood hormone profile, with increased androstenedione and decreased estrone levels. Flusilazole and triticonazole have dissimilar effects on reproductive parameters in vivo, but both show endocrine disrupting activities.

Review on plants with CNS-effects used in traditional South African medicine against mental diseases.

The majority of the population in South Africa use traditional health care to treat various mental conditions. In this review, we present ethnobotanical information on plants used by the traditional healers in South Africa to treat mental illnesses, specifically epilepsy, depression, age-related dementia and debilitative mental disorders. Details of the recent scientific studies conducted on some of these plants are reviewed. Extracts of Searsia chirindensis, Cotelydon orbiculata and Leonotis leonurus have shown in vivo anticonvulsant activity. Extracts from Searsia dentata and Searsia pyroides showed spontaneous epileptiform discharge in mouse cortical slices, and acted as NMDA-receptor antagonists. Apigenin, amentoflavone and agathisflavone with affinity to the benzodiazepine site on the GABA(A)-receptor were isolated from Searsia pyroides. Naringenin with affinity to the GABA(A)-benzodiazepine receptor was isolated from Mentha aquatica. Agapanthus campanulatus, Boophone disticha, Mondia whitei and Xysmalobium undulatum exhibited antidepressant-like activity in three in vivo models for depression. Amaryllidaceae alkaloids with activity to the serotonin transporter were isolated from Boophone disticha. The alkaloid mesembrine, which act as a serotonin reuptake inhibitor, was isolated from Sceletium tortuosum. Investigations of plants used to treat age-related dementia and debilitative mental disorders lead to the isolation of a number of Amaryllidaceae alkaloids with acetylcholinesterase inhibitory activity from Boophone disticha and Crinum species. Extracts of Mentha aquatica, Gasteria croucheri, Ruta graveolens and Scotia brachypetala inhibited MAO-B. Naringenin was isolated from Mentha aquatica as a MAO inhibitor. Only a small number of the more than 300 southern African plant species reported to treat or affect the CNS have been scientifically evaluated. Very few of the active compounds have been isolated and identified.

The effect of extracts of Searsia species on epileptiform activity in slices of the mouse cerebral cortex.

ETHNOPHARMACOLOGICAL RELEVANCE: Searsia dentata and Searsia pyroides are used in traditional South African medicine to treat convulsions and epilepsy. Previous studies have demonstrated that extracts of these plants comprise compounds that bind to the flumazenil-sensitive site on the GABA(A) receptor. However, their use as anticonvulsant medicinal plants cannot be adequately explained by these findings. AIMS: The aim of this study was to examine the possible involvement of the glutamatergic system of extracts from the plants. MATERIALS AND METHODS: The mouse cortical wedge preparation was used for functional characterization of the extracts. The affinity towards the NMDA and the AMPA receptor was investigated using classical [(3)H]-GP39653 and [(3)H]-AMPA binding assays, respectively. RESULTS: The extracts of Searsia dentata and Searsia pyroides inhibited the spontaneous epileptiform discharges in mouse cerebral cortical slices with ED(50) of 0.62 and 1.67mg dry extract/mL, respectively. Both extracts displaced [(3)H]-GP39653 binding and significantly inhibited the NMDA-induced response during co-administration in cortical slices. CONCLUSION: In this study, the NMDA receptor antagonistic effect of the crude ethanolic extracts of these two South African medicinal plants was demonstrated.