Exposure to topiramate and acetazolamide causes endocrine disrupting effects in female rats during estrus.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is a disease characterized by elevated intracranial pressure (ICP) and is a disease of young females. The first line pharmacological treatments include acetazolamide and topiramate and given the nature of IIH patients and the dosing regimen of these drugs, their effect on the endocrine system is important to evaluate. We aimed to assess the effects of acetazolamide and topiramate on steroid profiles in relevant endocrine tissues. METHODS: Female Sprague Dawley rats received chronic clinically equivalent doses of acetazolamide or topiramate by oral gavage and were sacrificed in estrus. Tissue specific steroid profiles of lateral ventricle CP, 4th ventricle CP, CSF, serum, uterine horn and fundus, ovaries, adrenal glands and pituitary glands were assessed by quantitative targeted LC-MS/MS. We determined luteinizing hormone (LH) and follicle stimulating hormones (FSH) levels in paired serum by ELISA. RESULTS: Topiramate increased the concentration of estradiol and decreased the concentration of DHEA in lateral choroid plexus. Moreover, it decreased the concentration of androstenediol in the pituitary gland. Topiramate increased serum LH. Acetazolamide decreased progesterone levels in serum and uterine fundus and increased corticosteroid levels in the adrenal glands. CONCLUSION: These results demonstrate that both acetazolamide and topiramate have endocrine disrupting effects in rats. Topiramate primarily targeted the choroid plexus and the pituitary gland while acetazolamide had broader systemic effects. Furthermore, topiramate predominantly targeted sex hormones, whereas acetazolamide widely affected all classes of hormones. A similar effect in humans has not yet been documented but these concerning findings warrants further investigations.

Ancient forest plants possess cytotoxic properties causing liver cancer HepG2 cell apoptosis.

Here, we collected 154 plant species in China ancient forests looking for novel efficient bioactive compounds for cancer treatments. We found 600 bioactive phyto-chemicals that induce apoptosis of liver cancer cell in vitro. First, we screen the plant extract's in vitro cytotoxicity inhibition of cancer cell growth using in vitro HepG2 cell lines and MTT cytotoxicity. The results from these initial MTT in vitro cytotoxicity tests show that the most efficient plants towards hepatoma cytoxicity is Cephalotaxus sinensis, mint bush (Elsholtzia stauntonii) and winged spindle tree (Euonymus alatus). We then used in cell-counting kit-8 (CCK-8) to further understand in vivo tumor growth using nude mice and GC-MS and LC-QTOF-MS to analyze the composition of compounds in the extracts. Extracted chemically active molecules analyzed by network pharmacology showed inhibition on the growth of liver cancer cells by acting on multiple gene targets, which is different from the currently used traditional drugs acting on only one target of liver cancer cells. Extracts from Cephalotaxus sinensis, mint bush (Elsholtzia stauntonii) and winged spindle tree (Euonymus alatus) induce apoptosis in hepatoma cancer cell line HepG2 with a killing rate of more than 83% and a tumor size decrease by 62-67% and a killing rate of only 6% of normal hepatocyte LO2. This study highlight efficient candidate species for cancer treatment providing a basis for future development of novel plant-based drugs to help meeting several of the UN SDGs and planetary health.

Cycle-dependent sex differences in expression of membrane proteins involved in cerebrospinal fluid secretion at rat choroid plexus.

BACKGROUND: Female sex is a known risk factor of brain disorders with raised intracranial pressure (ICP) and sex hormones have been suggested to alter cerebrospinal fluid (CSF) dynamics, thus impairing ICP regulation in CSF disorders such as idiopathic intracranial hypertension (IIH). The choroid plexus (CP) is the tissue producing CSF and it has been hypothesized that altered hormonal composition could affect the activity of transporters involved in CSF secretion, thus affecting ICP. Therefore, we aimed to investigate if expression of various transporters involved in CSF secretion at CP were different between males and females and between females in different estrous cycle states. Steroid levels in serum was also investigated. METHODS: Female and male rats were used to determine sex-differences in the genes encoding for the transporters Aqp1 and 4, NKCC1, NBCe2, NCBE; carbonic anhydrase enzymes II and III (CA), subunits of the Na+/K+-ATPase including Atp1a1, Atp1b1 and Fxyd1 at CP. The estrous cycle stage metestrus (MET) and estrous (ES) were determined before euthanasia. Serum and CP were collected and subjected to RT-qPCR analysis and western blots. Serum was used to measure steroid levels using liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Significant differences in gene expression and steroid levels between males and ES females were found, while no differences were found between male and MET females. During ES, expression of Aqp1 was lower (p < 0.01) and NKCC1 was higher in females compared to males. CAII was lower while CAIII was higher in ES females (p < 0.0001). Gene expression of Atp1a1 was lower in ES compared to male (p = 0.0008). Several of these choroidal genes were also significantly different in MET compared to females in ES. Differences in gene expression during the estrus cycle were correlated to serum level of steroid hormones. Protein expression of AQP1 (p = 0.008) and CAII (p = 0.035) was reduced in ES females compared to males. CONCLUSIONS: This study demonstrates for the first time that expression at CP is sex-dependent and markedly affected by the estrous cycle in female rats. Further, expression was related to hormone levels in serum. This opens a completely new avenue for steroid regulation of the expression of CSF transporters and the close link to the understanding of CSF disorders such as IIH.

Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based chip.

The development of cell-based microfluidic assays offers exciting new opportunities in toxicity testing, allowing for integration of new functionalities, automation, and high throughput in comparison to traditional well-plate assays. As endocrine disruption caused by environmental chemicals and pharmaceuticals represents a growing global health burden, the purpose of the current study was to contribute towards the miniaturization of the H295R steroidogenesis assay, from the well-plate to the microfluidic format. Microfluidic chip fabrication with the established well-plate material polystyrene (PS) is expensive and complicated; PDMS and thiol-ene were therefore tested as potential chip materials for microfluidic H295R cell culture, and evaluated in terms of cell attachment, cell viability, and steroid synthesis in the absence and presence of collagen surface modification. Additionally, spike-recovery experiments were performed, to investigate potential steroid adsorption to chip materials. Cell aggregation with poor steroid recoveries was observed for PDMS, while cells formed monolayer cultures on the thiol-ene chip material, with cell viability and steroid synthesis comparable to cells grown on a PS surface. As thiol-ene overall displayed more favorable properties for H295R cell culture, a microfluidic chip design and corresponding cell seeding procedure were successfully developed, achieving repeatable and uniform cell distribution in microfluidic channels. Finally, H295R perfusion culture on thiol-ene chips was investigated at different flow rates (20, 10, and 2.5 µL/min), and 13 steroids were detected in eluting cell medium over 48 h at the lowest flow rate. The presented work and results pave the way for a time-resolved microfluidic H295R steroidogenesis assay.

Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models.

BACKGROUND: Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. METHODS: In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. RESULTS: Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. CONCLUSION: Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

Ibuprofen alters human testicular physiology to produce a state of compensated hypogonadism.

Concern has been raised over increased male reproductive disorders in the Western world, and the disruption of male endocrinology has been suggested to play a central role. Several studies have shown that mild analgesics exposure during fetal life is associated with antiandrogenic effects and congenital malformations, but the effects on the adult man remain largely unknown. Through a clinical trial with young men exposed to ibuprofen, we show that the analgesic resulted in the clinical condition named "compensated hypogonadism," a condition prevalent among elderly men and associated with reproductive and physical disorders. In the men, luteinizing hormone (LH) and ibuprofen plasma levels were positively correlated, and the testosterone/LH ratio decreased. Using adult testis explants exposed or not exposed to ibuprofen, we demonstrate that the endocrine capabilities from testicular Leydig and Sertoli cells, including testosterone production, were suppressed through transcriptional repression. This effect was also observed in a human steroidogenic cell line. Our data demonstrate that ibuprofen alters the endocrine system via selective transcriptional repression in the human testes, thereby inducing compensated hypogonadism.

Exposure of consumers to substandard antibiotics from selected authorised and unauthorised medicine sales outlets in Ghana.

OBJECTIVE: To assess the quality of antibiotics sampled from authorised sales outlets (ATs) (i.e. hospitals/health centres, pharmacies and licensed chemical shops) and unauthorised sales outlets (UATs) (mainly street vendors) in Ghana and to explore the health-seeking behaviour of medicine consumers. METHODS: The contents of 14 active pharmaceutical ingredients (APIs) in 348 sampled products were determined using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Data on health-seeking practices were collected through entry and exit interviews and field observations from ATs and UATs. RESULTS: It was observed that 66.38% of all sampled antibiotic products were substandard; they either contained less (<90%) or more API (>110%) than the label claim. Medicines from UATs recorded substantially less API contents than those from ATs (F(2,419)  = 43.01, P < 0.0001). For example, 90.54% of street vendor samples contained < 90% of the APIs. 75.93% of consumers often sought self-treatment with drugs without a prescription from UATs, as they perceived UATs as easily accessible, trustworthy and knowledgeable, and their medicines as inexpensive. These consumers rather thought of the formal healthcare providers as alternative sources. CONCLUSIONS: Consumers who purchase from UATs are at high risk of receiving substandard medicines. The quality of medicines in the national healthcare system, in the supply chain and in the distribution system needs to be monitored regularly to reduce the incidence of substandard medicines and their impact on antimicrobial resistance. The fight against substandard medicines needs to incorporate a full understanding of socioeconomic factors that drive consumer decisions regarding their health and choice of healthcare providers.

OBJECTIF: Evaluer la qualité des antibiotiques prélevés auprès des vendeurs autorisés (VA) (c'est-à-dire les hôpitaux/centres de santé, les pharmacies et les magasins de produits chimiques agréés) et des vendeurs non autorisés (VNA) (principalement les vendeurs de rue) au Ghana et étudier le comportement des utilisateurs de médicaments en quête de santé. MÉTHODES: Le contenu de 14 principes actifs (PA) pharmaceutiques dans 348 produits échantillonnés a été déterminé à l'aide d'une méthode validée de chromatographie liquide et de spectrométrie de masse en tandem (LC-MS/MS). Les données sur les pratiques de recherche de santé ont été collectées par le biais d'entretiens d'entrée et de sortie, et d'observations sur le terrain des VA et des VNA. RÉSULTATS: Il a été observé que 66,38% de tous les produits antibiotiques échantillonnés étaient inférieurs aux normes; ils contenaient soit moins (<90%), soit plus de PA (>110%) que ce qui était indiqué sur la notice. Les médicaments provenant des VNA ont enregistré une quantité de PA sensiblement inférieure à celle des VA (F(2,419)  = 43.01, P < 0,0001). Par exemple, 90,54% des échantillons de vendeurs de rue contenaient <90% de PA. 75,93% des utilisateurs ont souvent cherché à se soigner eux-mêmes avec des médicaments sans ordonnance des VNA, car ils ont perçu les VNA comme étant facilement accessibles, fiables et bien informés, et leurs médicaments comme étant peu coûteux. Ces utilisateurs considéraient également les prestataires de soins de santé officiels comme des sources alternatives. CONCLUSIONS: Les utilisateurs qui s'approvisionnent auprès des VNA courent un risque élevé de recevoir des médicaments de qualité inférieure. La qualité des médicaments dans le système national de santé, dans la chaîne d'approvisionnement et dans le système de distribution doit être contrôlée régulièrement pour réduire l'incidence des médicaments de qualité inférieure et leur impact sur la résistance aux antimicrobiens. La lutte contre les médicaments de qualité inférieure doit intégrer une compréhension complète des facteurs socioéconomiques qui déterminent les décisions des utilisateurs concernant leur santé et le choix des prestataires de soins de santé.

The anti-epileptic drug lamotrigine inhibits the CYP17A1 lyase reaction in vitro.

The potential endocrine disrupting effects of the commonly prescribed anti-epileptic drug lamotrigine (LAM) were investigated using the H295R steroidogenic in vitro assay and computational chemistry methods. The H295R cells were exposed to different concentrations of LAM, and a multi-steroid LC-MS/MS method was applied to quantify the amount of secreted steroid hormones. LAM affected several steroid hormones in the steroidogenesis at therapeutic concentrations. All progestagens as well as 11-deoxycorticosterone and corticosterone increased 100-200% with increasing concentrations of LAM suggesting a selective inhibitory effect of LAM on CYP17A1, in particular on the lyase reaction. Recombinant CYP17A1 assay confirmed the competitive inhibition of LAM toward the enzyme with IC50 values of 619 and 764 µM for the lyase and the hydroxylase reaction, respectively. Levels of androstenedione and testosterone decreased at LAM concentrations above the therapeutic concentration range. The ability of LAM to bind to CYP17A1, CYP19A1, and CYP21A2 was investigated using docking and molecular dynamics simulations. This in silico study showed that LAM was able to bind directly to the heme iron in the active site of CYP17A1, but not CYP21A2, thus supporting the results of the in vitro studies. The molecular dynamics simulations also suggested binding of LAM to the heme iron in the CYP19A1 active site. No inhibition of the aromatase enzyme was, however, observed in the H295R assay. This could be due to a sequential effect within the steroidogenesis caused by the inhibition of CYP17A1, which reduced the amounts of androgens available for CYP19A1.

Malaria causes long-term effects on markers of iron status in children: a critical assessment of existing clinical and epidemiological tools.

BACKGROUND: Most epidemiological studies on the interplay between iron deficiency and malaria risk classify individuals as iron-deficient or iron-replete based on inflammation-dependent iron markers and adjustment for inflammation by using C-reactive protein (CRP) or α-1-acid glycoprotein (AGP). The validity of this approach and the usefulness of fibroblast growth factor 23 (FGF23) as a proposed inflammation-independent iron marker were tested. METHODS: Conventional iron markers and FGF23 were measured in children with acute falciparum malaria and after 1, 2, 4, and 6 weeks. Children, who were transfused or received iron supplementation in the follow-up period, were excluded, and iron stores were considered to be stable throughout. Ferritin levels 6 weeks after admission were used as a reference for admission iron status and compared with iron markers at different time points. RESULTS: There were long-term perturbations in iron markers during convalescence from acute malaria. None of the tested iron parameters, including FGF23, were independent of inflammation. CRP and AGP normalized faster than ferritin after malaria episodes. CONCLUSION: Malaria may bias epidemiological studies based on inflammation-dependent iron markers. Better markers of iron status during and after inflammation are needed in order to test strategies for iron supplementation in populations at risk of malaria.

Atorvastatin decreases steroid production in H295R cells and in major endocrine tissues of male rats.

Obesity is increasing worldwide, and since obesity is associated with dyslipidemia, the consumption of cholesterol-lowering pharmaceuticals has increased. The aim of this study was therefore to study potential endocrine disrupting effects of one of the world's most frequently prescribed drugs, the cholesterol-lowering drug, atorvastatin (ATO) in vitro using the H295R steroidogenesis assay and in vivo using male Sprague-Dawley rats. We analyzed all major steroids in the mammalian steroidogenesis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vitro, ATO significantly decreased all steroids in the H295R steroidogenesis at concentrations close to human plasma Cmax values, with an IC50 value for testosterone of 0.093 ± 0.033 µM. Additionally, we determined steroid hormone levels in testis, adrenals, brain and plasma from rats after 14 days of exposure to three therapeutically relevant doses of ATO and observed pronounced decreasing steroid levels in particular in testis and adrenals but also in brain and plasma. In testis, all major steroidogenic enzymes were up-regulated, indicating autocrine and/or paracrine compensation for the decrease in steroid production by this tissue. In adrenals, StAR and CYP11A1 gene expression were decreased, whereas little effects were observed in the brain. Furthermore, we analyzed plasma LH and ACTH levels to investigate feedback via the PT and HPA axes. No effects were observed on LH levels, indicating little compensation via the PT axis. In contrast, ACTH levels increased during ATO exposure, indicating that the HPA axis to some extend compensated for the decrease in adrenal steroid production. Overall, ATO exerted pronounced effects on steroid production both in vitro and in vivo at therapeutically relevant doses. This clearly demonstrates the high potency of ATO to affect steroid homeostasis during therapeutic treatment. Further clinical and epidemiological studies should be conducted to investigate the relevance of these observations in patients treated with cholesterol-lowering pharmaceuticals.

Steroid hormones and persistent organic pollutants in plasma from North-eastern Atlantic pilot whales.

Persistent organic pollutants (POPs) are known to have endocrine disruptive effects, interfering with endogenous steroid hormones. The present study examined nine steroid hormones and their relationships with the concentrations of selected POPs in pilot whales (Globicephala melas) from the Faroe Islands, NE Atlantic. The different steroids were detected in 15 to all of the 26 individuals. High concentrations of progesterone (83.3-211.7pmol/g) and pregnenolone (PRE; 4.68-5.69pmol/g) were found in three adult females indicating that they were pregnant or ovulating. High androgen concentrations in two of the males reflected that one was adult and that one (possibly) had reached puberty. In males a significant positive and strong correlation between body length and testosterone (TS) levels was identified. Furthermore, positive and significant correlations were found between 4-OH-CB107/4'-OH-CB108 and 17ß-estradiol in males. In adult females significant positive correlations were identified between PRE and CB149 and t-nonachlor, between estrone and CB138, -149, -187 and p,p'-DDE, between androstenedione and CB187, and between TS and CB-99 and -153. Although relationships between the POPs and the steroid hormones reported herein are not evidence of cause-effect relationships, the positive correlations between steroids and POPs, particularly in females, suggest that POPs may have some endocrine disrupting effects on the steroid homeostasis in this species.

Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays.

Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS(2) method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55-530 pg/mL (LLOQ).

Hydroxylated polychlorinated biphenyls decrease circulating steroids in female polar bears (Ursus maritimus).

As a top predator in the Arctic food chain, polar bears (Ursus maritimus) are exposed to high levels of persistent organic pollutants (POPs). Because several of these compounds have been reported to alter endocrine pathways, such as the steroidogenesis, potential disruption of the sex steroid synthesis by POPs may cause implications for reproduction by interfering with ovulation, implantation and fertility. Blood samples were collected from 15 female polar bears in Svalbard (Norway) in April 2008. The concentrations of nine circulating steroid hormones; dehydroepiandrosterone (DHEA), androstenedione (AN), testosterone (TS), dihydrotestosterone (DHT), estrone (E1), 17α-estradiol (αE2), 17ß-estradiol (ßE2), pregnenolone (PRE) and progesterone (PRO) were determined. The aim of the study was to investigate associations among circulating levels of specific POP compounds and POP-metabolites (hydroxylated PCBs [OH-PCBs] and hydroxylated PBDEs [OH-PBDEs]), steroid hormones, biological and capture variables in female polar bears. Inverse correlations were found between circulating levels of PRE and AN, and circulating levels of OH-PCBs. There were no significant relationships between the steroid concentrations and other analyzed POPs or the variables capture date and capture location (latitude and longitude), lipid content, condition and body mass. Although statistical associations do not necessarily represent direct cause-effect relationships, the present study indicate that OH-PCBs may affect the circulating levels of AN and PRE in female polar bears and that OH-PCBs thus may interfere with the steroid homeostasis. Increase in PRO and a decrease in AN concentrations suggest that the enzyme CYP17 may be a potential target for OH-PCBs. In combination with natural stressors, ongoing climate change and contaminant exposure, it is possible that OH-PCBs may disturb the reproductive potential of polar bears.

Brain region-specific perfluoroalkylated sulfonate (PFSA) and carboxylic acid (PFCA) accumulation and neurochemical biomarker responses in east Greenland polar bears (Ursus maritimus).

Perfluoroalkyl substances (PFASs) is a growing class of contaminants in the Arctic environment, and include the established perfluorinated sulfonates (PFSAs; especially perfluorooctane sulfonate (PFOS)) and carboxylic acids (PFCAs). PFSAs and PFCAs of varying chain length have been reported to bioaccumulate in lipid rich tissues of the brain among other tissues such as liver, and can reach high concentrations in top predators including the polar bear. PFCA and PFSA bioaccummulation in the brain has the potential to pose neurotoxic effects and therefore we conducted a study to investigate if variations in neurochemical transmitter systems i.e. the cholinergic, glutaminergic, dopaminergic and GABAergic, could be related to brain-specific bioaccumulation of PFASs in East Greenland polar bears. Nine brain regions from nine polar bears were analyzed for enzyme activity (monoamine oxidase (MAO), acetylcholinesterase (AChE) and glutamine synthetase (GS)) and receptor density (dopamine-2 (D2), muscarinic cholinergic (mAChR) and gamma-butyric acid type A (GABA-A)) along with PFSA and PFCA concentrations. Average brain ∑PFSA concentration was 25ng/g ww where PFOS accounted for 91%. Average ∑PFCA concentration was 88ng/g ww where PFUnDA, PFDoDA and PFTrDA combined accounted for 79%. The highest concentrations of PFASs were measured in brain stem, cerebellum and hippocampus. Correlative analyses were performed both across and within brain regions. Significant positive correlations were found between PFASs and MAO activity in occipital lobe (e.g. ∑PFCA; rp=0.83, p=0.041, n=6) and across brain regions (e.g. ∑PFCA; rp=0.47, p=0.001, ∑PFSA; rp=0.44, p>0.001; n=50). GABA-A receptor density was positively correlated with two PFASs across brain regions (PFOS; rp=0.33, p=0.02 and PFDoDA; rp=0.34, p=0.014; n=52). Significant negative correlations were found between mAChR density and PFASs in cerebellum (e.g. ∑PFCA; rp=-0.95, p=0.013, n=5) and across brain regions (e.g. ∑PFCA; rp=-0.40, p=0.003, ∑PFSA; rp=-0.37, p=0.007; n=52). AChE activity and D2 density were negatively correlated with single PFCAs in several brain regions, whereas GS activity was positively correlated with PFASs primarily in occipital lobe. Results from the present study support the hypothesis that PFAS concentrations in polar bears from East Greenland have exceeded the threshold limits for neurochemical alterations. It is not known whether the observed alterations in neurochemical signaling are currently having negative effects on neurochemistry in East Greenland polar bears. However given the importance of these systems in cognitive processes and motor function, the present results indicate an urgent need for a better understanding of neurochemical effects of PFAS exposure to wildlife.

Mixture Effects of 3 Mechanistically Different Steroidogenic Disruptors (Prochloraz, Genistein, and Ketoconazole) in the H295R Cell Assay.

Mixture effects of 3 model endocrine disruptors, prochloraz, ketoconazole, and genistein, on steroidogenesis were tested in the adrenocortical H295R cell line. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and 17ß-estradiol) were analyzed using gas chromatography and tandem mass spectrometry (GC-MS/MS) to investigate the effects throughout the steroidogenic pathway. Current modeling approaches often rely on models assuming compounds acting independently and that the individual effects in some way can be summarized to predict a mixture effect. In H295R cells with an intact steroidogenic pathway, such assumptions may not be feasible. The purpose of this study was therefore to evaluate whether effects of a mixture with differing modes of action followed or deviated from additivity (concentration addition) and whether the H295R cell line was suitable for evaluating mixture toxicity of endocrine disruptors with different modes of action. The compounds were chosen because they interfere with steroidogenesis in different ways. They all individually decrease the concentrations of the main sex steroids downstream but exert different effects upstream in the steroidogenic pathway. Throughout the study, we observed lowest observed effect concentrations of mixtures at levels 2 to 10 times higher than the predicted EC(50), strongly indicating antagonistic effects. The results demonstrate that chemical analysis combined with the H295R cell assay is a useful tool also for studying how mixtures of endocrine disruptors with differing modes of action interfere with the steroidogenic pathway and that existing models like concentration addition are insufficient in such cases. Furthermore, for end points where compounds exert opposite effects, no relevant models are available.

CSF hyperdynamics in rats mimicking the obesity and androgen excess characteristic of patients with idiopathic intracranial hypertension.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is a syndrome exhibiting elevated intracranial pressure (ICP), visual disturbances, and severe headache. IIH primarily affects young obese women, though it can occur in individuals of any age, BMI, and sex. IIH is characterized by systemic metabolic dysregulation with a profile of increased androgen hormones. However, the contribution of obesity/hormonal perturbations to cerebrospinal fluid (CSF) dynamics remains unresolved. METHODS: We employed obese female Zucker rats and adjuvant testosterone to reveal IIH causal drivers. ICP and CSF dynamics were determined with in vivo experimentation and magnetic resonance imaging, testosterone levels assessed with mass spectrometry, and choroid plexus function revealed with transcriptomics. RESULTS: Obese rats had undisturbed CSF testosterone levels and no changes in ICP or CSF dynamics. Adjuvant testosterone treatment of obese rats elevated the CSF secretion rate, although with no effect on the ICP, due to elevated CSF drainage capacity of these rats. CONCLUSIONS: Obesity in itself therefore does not suffice to recapitulate the IIH symptoms in rats, but modulation of CSF dynamics appears with adjuvant testosterone treatment, which mimics the androgen excess observed in female IIH patients. Obesity-induced androgen dysregulation may thus contribute to the disease mechanism of IIH and could potentially serve as a future therapeutic target.

Acetazolamide and topiramate lower intracranial pressure through differential mechanisms: The effect of acute and chronic administration.

BACKGROUND AND PURPOSE: Diseases of raised intracranial pressure (ICP) cause severe morbidity and mortality. Multiple drugs are utilised to lower ICP including acetazolamide and topiramate. However, the evidence for their use is unclear. We aimed to assess the ICP modulatory effects and molecular effects at the choroid plexus (CP) of acetazolamide and topiramate. EXPERIMENTAL APPROACH: Female rats were implanted with telemetric ICP probes for physiological, freely moving 24/7 ICP recordings. Randomised cross-over studies were performed, where rats received acute (24 h) high doses of acetazolamide and topiramate, and chronic (10 days) clinically equivalent doses of acetazolamide and topiramate, all via oral gavage. Cerebrospinal fluid (CSF) secretion assays, and RT-qPCR and western blots on in vitro and in vivo CP, were used to investigate drug actions. KEY RESULTS: We demonstrate that acetazolamide and topiramate achieved maximal ICP reduction within 120 min of administration, and in combination doubled the ICP reduction over a 24-h period. Chronic administration of acetazolamide or topiramate lowered ICP by 25%. Topiramate decreased CSF secretion by 40%. Chronic topiramate increased the gene expression of Slc12a2 and Slc4a10 and protein expression of the sodium-dependent chloride/bicarbonate exchanger (NCBE), whereas chronic acetazolamide did not affect the expression of assessed genes. CONCLUSIONS AND IMPLICATIONS: Acetazolamide and topiramate are effective at lowering ICP at therapeutic levels. We provide the first evidence that topiramate lowers CSF secretion and that acetazolamide and topiramate may lower ICP via distinct molecular mechanisms. Thus, the combination of acetazolamide and topiramate may have utility for treating raised ICP.

Corticosteroid production in H295R cells during exposure to 3 endocrine disrupters analyzed with LC-MS/MS.

The adrenocortical human cell line H295R is a valuable tool for screening endocrine disrupting compounds. In general, previous research focus has been on the production of the 2 sex steroids, 17ß-estradiol and testosterone, and less attention has been paid to other important steroid end points in the steroidogenesis with a wide range of physiological functions, such as the glucocorticoids (corticosterone and cortisol). A newly developed and validated solid phase extraction (SPE) liquid chromatography-mass spectroscopy (LC-MS/MS) method was used to measure the production of cortisol and corticosterone in the H295R cell line. The method was applied by studying the effects of 2 model endocrine disrupters, ketoconazole and prochloraz, the pharmaceutical budesonide, and the inducer forskolin on the steroid production in this cell line. Dose-response curves were obtained for the correlation between hormone concentrations and the concentration of the individual disruptors. Exposing cells to ketoconazole resulted in a decrease in cortisol and corticosterone concentrations in a dose-dependent manner with EC50 values of 0.24 and 0.40 µmol/L, respectively. The same applied for cells exposed to prochloraz with EC50 values of 0.06 and 0.09 µmol/L for cortisol and corticosterone, respectively. Budesonide also inhibited glucocorticoid secretion. The EC50 value for cortisol was 19.50 µmol/L, whereas the EC50 value for corticosterone was 71.42 µmol/L. Forskolin induced the secretion of both cortisol (EC50 = 4.09 µmol/L) and corticosterone (EC50 = 0.28 µmol/L). The results obtained demonstrated the validity of the method. Based on these findings, quality criteria for the production of these steroids in this cell line were suggested.

Optimization of HPLC-MS/MS method for determination of antimalarial adulterants in herbal products.

The use of herbal products is booming all over the world because of being believed as safer than conventional drugs and free of side effects. However, there are untrustworthy manufacturers who adulterate herbal products by adding conventional drugs which might eventually lead to microbial resistance and herb-to-drug interactions. There is a need to develop methods for detecting adulterants in herbal products. A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous identification and determination of conventional antimalarials (chloroquine, quinine, sulfadoxine, pyrimethamine, mefloquine, lumefantrine, amodiaquine, artemisinin, dihydroartemisinin, artesunate and artemether) in herbal products was developed. Stable isotopically labelled compounds (artemether-d3, quindine-d3, and sulfadoxine-d3) were used as internal standards (ISs) for quantitative analysis. Extraction of analytes was performed using methanol: water: formic acid (90:10:0.1, v/v) and chromatographic separation was done in a gradient mode using mobile phase A: Ultrapure water containing 0.1% formic acid and 1 mM ammonium formate and mobile phase B: Acetonitrile/methanol (50:50) containing 0.1% formic acid and 1 mM ammonium formate. The calibration curves were linear (r2 ≥ 0.991) over the range of 0.001-0.3 µg mL-1 for all compounds. The limit of detection (LOD) ranged from 0.002 to 0.02 µg mL-1 while the limit of quantification (LOQ) ranged from 0.006 to 0.08 µg mL-1. Accuracy, expressed as recovery of spiked herbal products ranged from 52 to 128%. The precision, expressed as percent relative standard deviation (%RSD) at two concentration levels, ranged from 1.0 to 13.8%. The matrix effect expressed as the matrix factor (MF) ranged from 0.77 to 0.97. The developed method was used to identify and quantify conventional antimalarials in herbal product samples from Tanzania. Ten out of 50 herbal products were found to contain amodiaquine, sulfadoxine, pyrimethamine, mefloquine, dihydroartemisinin, artemether and lumefantrine. The developed method is considered a valuable tool for getting a better understanding of the adulteration of conventional antimalarials in herbal products.