Effects of ethanol or ethylene glycol exposure on PPARγ and aromatase expression in adipose tissue.

The estrogen-synthesizing enzyme aromatase is expressed in adipose tissue where it controls the local concentration of estrogen. It has been suggested that the organic solvents ethanol and ethylene glycol can induce estrogen synthesis by inhibiting PPARγ activity. Since elevated estrogen synthesis in adipose tissue is a risk factor for breast cancer development, it is of interest to further characterize the mechanisms regulating aromatase expression. Here, we explored the mechanisms by which ethanol and ethylene glycol modulate aromatase mRNA expression and the ultimate conversion of androgens into estrogens. NMR spectroscopy revealed that ethanol and ethylene glycol influence the active state of PPARγ. An inhibitory effect on PPARγ was confirmed by adipogenesis assays and PPARγ target gene expression analysis in adipocytes. However, only ethanol increased aromatase mRNA in differentiated human adipocytes. In contrast, ethylene glycol downregulated aromatase in a PPARγ-independent manner. An animal study using female Wistar rats was conducted to assess the acute effects of ethanol and ethylene glycol on aromatase expression in adipose tissue within a physiological context. No changes in aromatase or PPARγ target gene (Adipoq and Fabp4) levels were observed in adipose tissue or ovary in response to the chemical exposures, suggesting an absence of acute PPARγ-mediated effects in these organs. The results suggest that ethanol and ethylene glycol are weak PPARγ antagonists in mouse and human adipocytes as well as in cell-free NMR spectroscopy. Both compounds seem to affect adipocyte aromatase expression in vitro, where ethanol increased aromatase expression PPARγ-dependently and ethylene glycol decreased aromatase expression independently of PPARγ. No acute effects on aromatase expression or PPARγ activity were observed in adipose tissue or ovary in rats in this study design.

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

Health literacy and cognitive function in people with diabetic foot ulcer with focus on knowledge, attitude, and practice in relation to foot self-care.

Introduction: Preventative foot self-care is vital for avoiding diabetic foot ulcer episodes and lowering the risk of amputations. Yet, it demands high levels of health literacy and cognitive function. Objective: To investigate health literacy and cognitive function in persons presenting with a diabetic foot ulcer. Methods: Participants with type 2 diabetes were recruited from the tertiary foot clinic at Steno Diabetes Center North Denmark. The European Health Literacy Survey Questionnaire and Addenbrooke's Cognitive Examination were applied. A semi-structured interview guide was developed to evaluate foot self-care knowledge, attitude, and practice. The qualitative data were analyzed with a deductive approach based on a qualitative thematic analysis model. Subsequently, an integrated analysis of the quantitative and qualitative results was conducted. Results: The participants (n = 12) had a mean age of 62.6 ± 8.4 years, and 11 were males. The mean diabetes duration was 15.9 ± 8.9 years. Eight participants had a recurrent diabetic foot ulcer. The health literacy level was sufficient in nine participants, and cognitive function was normal in five participants. Three different profiles related to foot self-care (proactive, active, or passive, respectively) were constructed by the final integrated analysis: a proactive profile refers to taking preventative action in concordance with knowledge and attitude, an active profile to taking action in response to a situation, but challenged by conflicting levels of knowledge and attitude, and a passive profile to not taking action. Conclusion: The study suggests that people presenting with a diabetic foot ulcer have different foot self-care profiles based on person-specific health literacy, cognitive function, and knowledge, attitude, and practice element characteristics, highlighting the need for individualized education and intervention strategy instead of a one-size-fits-all approach.

Community-based palliative care in two primary care settings - nursing homes and home care: a national survey.

AIMS: Based on selected themes from a national survey, the study aims to describe and analyse similarities and differences in community-based palliative care provided to people living at home in two different care settings - the nursing home setting and the home care setting. METHODS: Responses from four palliative care themes covered by a national survey sent to 717 managers in municipality-based care units were used. The themes were: (a) target groups in palliative care; (b) wishes for end-of-life care; (c) tools/guidelines in palliative care; and (d) palliative care provided to relatives. RESULTS: The response rates were 53% in the nursing home setting and 69% in the home care setting (69%). Both settings had target groups for palliative care, in which significantly more units in the home care settings cared for people with other cultural backgrounds or children. Wishes for end-of-life care were addressed by more than 90% of the units in both settings. There were significantly more nursing home units that addressed questions regarding resuscitation, decision making when you are incapable of making decisions for yourself, and the level of medication. In both settings, around half of the units did not use or did not know if they used tools/guidelines to identify palliative care needs. Half of home care and 65% of nursing home settings did not/were unaware of providing palliative care to relatives. CONCLUSIONS: Both settings serve target populations for palliative care with few differences. Identifying palliative care needs seemed to be a low priority in both settings. A difference was found between the settings regarding end-of-life care questions and palliative care promotion to relatives.

Diagnostic accuracy of BASIC-Q for detection of cognitive impairment in a primary care setting - a cross-validation study.

OBJECTIVES: This study aims to evaluate the diagnostic accuracy and reliability of a new, brief questionnaire, 'Brief Assessment of Impaired Cognition- Questionnaire' (BASIC-Q) for detection of cognitive impairment, primarily developed for use in primary care. BASIC-Q has three components: Self-report, Informant report, and Orientation. Self-report and Orientation are completed by the individual and Informant report is answered by a close relative. METHODS: We included 275 participants ≥ 70 years, without a prior diagnosis of dementia, and with a close relative who agreed to participate as an informant. Participants were included prospectively in 14 general practices in urban and rural Denmark using a convenience sampling method. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), the informant-completed Functional Activities Questionnaire (FAQ) and reported memory concern were used as a reference standard for the classification of the participants' cognitive function. RESULTS: BASIC-Q demonstrated a fair to good diagnostic accuracy to differentiate between people with cognitive impairment and normal cognition with an area under the ROC curve (AUC) of 0.84 (95% CI 0.79-0.89) and a sensitivity and specificity of 0.80 (95% CI 0.72-0.87) and 0.71 (95% CI 0.63-0.78). A prorated BASIC-Q score derived from BASIC-Q without Informant report had significantly lower classification accuracy than the full BASIC-Q. The test-retest reliability of BASIC-Q was good with an intraclass correlation coefficient of 0.84. CONCLUSION: BASIC-Q is a brief, easy-to-use questionnaire for identification of cognitive impairment in older adults. It demonstrated fair to good classification accuracy in a general practice setting and can be a useful case-finding tool when suspecting dementia in primary health care.

Comparison of female rat reproductive effects of pubertal versus adult exposure to known endocrine disruptors.

A prevailing challenge when testing chemicals for their potential to cause female reproductive toxicity is the lack of appropriate toxicological test methods. We hypothesized that starting a 28-day in vivo toxicity study already at weaning, instead of in adulthood, would increase the sensitivity to detect endocrine disruptors due to the possibility of including assessment of pubertal onset. We compared the sensitivity of two rat studies using pubertal or adult exposure. We exposed the rats to two well-known human endocrine disruptors, the estrogen diethylstilbestrol (DES; 0.003, 0.012, 0.048 mg/kg bw/day) and the steroid synthesis inhibitor ketoconazole (KTZ; 3, 12, 48 mg/kg bw/day). Specifically, we addressed the impact on established endocrine-sensitive endpoints including day of vaginal opening (VO), estrous cyclicity, weights of reproductive organs and ovarian histology. After 28 days of exposure, starting either at weaning or at 9 weeks of age, DES exposure altered estrous cyclicity, reduced ovary weight as well as number of antral follicles and corpora lutea. By starting exposure at weaning, we could detect advanced day of VO in DES-exposed animals despite a lower body weight. Some endpoints were affected mainly with adult exposure, as DES increased liver weights in adulthood only. For KTZ, no effects were seen on time of VO, but adrenal and liver weights were increased in both exposure scenarios, and adult KTZ exposure also stimulated ovarian follicle growth. At first glance, this would indicate that a pubertal exposure scenario would be preferrable as timing of VO may serve as sensitive indicator of endocrine disruption by estrogenic mode of action. However, a higher sensitivity for other endocrine targets may be seen starting exposure in adulthood. Overall, starting a 28-day study at weaning with inclusion of VO assessment would mainly be recommended for substances showing estrogenic potential e.g., in vitro, whereas for other substances an adult exposure scenario may be recommended.

Quantitative analysis of ovarian surface photographs as a tool for assessment of chemical effects on folliculogenesis and ovulation in rats.

Female reproductive toxicity assessments rely on histological evaluation of ovaries by hematoxylin & eosin (H&E)-stained cross-sections. This is time-consuming, labor-intensive and costly, thus alternative methods for ovarian toxicity assessment could be valuable. Here, we report on an improved method based on quantification of antral follicles (AF) and corpora lutea (CL) using ovarian surface photographs, called 'surface photo counting' (SPC). To validate a potential utility for the method to detect effects on folliculogenesis in toxicity studies, we investigated ovaries from rats exposed to two well-known endocrine disrupting chemicals (EDCs), diethylstilbestrol (DES) and ketoconazole (KTZ). Animals were exposed to DES (0.003, 0.012, 0.048 mg/kg body weight (bw)/day) or KTZ (3, 12, 48 mg/kg bw/day) during puberty or adulthood. At the end of the exposure, ovaries were photographed under stereomicroscope and subsequently processed for histological assessments to allow for direct comparison between the two methods by quantifying AF and CL. There was a significant correlation between the SPC and histology methods, albeit CL counts correlated better than AF counts, potentially due to their larger size. Effects of DES and KTZ were found by both methods, suggesting applicability of the SPC method to chemical hazard and risk assessment. Based on our study, we propose that SPC can be employed as a fast and cheap tool for assessment of ovarian toxicity in in vivo studies to prioritize chemical exposure groups for further histological assessment.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

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.

AOP key event relationship report: Linking androgen receptor antagonism with nipple retention.

In rat developmental and reproductive toxicity studies, nipple/areola retention (NR) in male offspring is a biomarker for reduced androgen signaling during development. This is because nipples normally regress in male rats in response to androgen signaling during critical stages of development. NR is thus included as a mandatory endpoint in several OECD test guidelines for assessment of chemicals, particularly as a readout for anti-androgenic effects relevant for reproductive toxicity. With the growing interest in developing Adverse Outcome Pathways (AOPs) to aid in chemical risk assessment, a more pragmatic approach has been proposed, whereby essential units of knowledge could be developed independently of complete AOPs, not least emergent key event relationships (KERs). Herein, we have developed a KER linking "androgen receptor antagonism" and "increased areola/nipple retention". The KER is based on a literature review conducted in a transparent semi-systematic manner in peer-reviewed databases with pre-defined inclusion criteria. Twenty-seven papers were included for development of the KER. The results support a qualitative relationship between the two key events (KEs) with a high weight of evidence; i.e., a causal relationship between androgen receptor (AR) antagonism and nipple retention in male rats exists.

Chemical Effects on Breast Development, Function, and Cancer Risk: Existing Knowledge and New Opportunities.

Population studies show worrisome trends towards earlier breast development, difficulty in breastfeeding, and increasing rates of breast cancer in young women. Multiple epidemiological studies have linked these outcomes with chemical exposures, and experimental studies have shown that many of these chemicals generate similar effects in rodents, often by disrupting hormonal regulation. These endocrine-disrupting chemicals (EDCs) can alter the progression of mammary gland (MG) development, impair the ability to nourish offspring via lactation, increase mammary tissue density, and increase the propensity to develop cancer. However, current toxicological approaches to measuring the effects of chemical exposures on the MG are often inadequate to detect these effects, impairing our ability to identify exposures harmful to the breast and limiting opportunities for prevention. This paper describes key adverse outcomes for the MG, including impaired lactation, altered pubertal development, altered morphology (such as increased mammographic density), and cancer. It also summarizes evidence from humans and rodent models for exposures associated with these effects. We also review current toxicological practices for evaluating MG effects, highlight limitations of current methods, summarize debates related to how effects are interpreted in risk assessment, and make recommendations to strengthen assessment approaches. Increasing the rigor of MG assessment would improve our ability to identify chemicals of concern, regulate those chemicals based on their effects, and prevent exposures and associated adverse health effects.

Best practices to quantify the impact of reproductive toxicants on development, function, and diseases of the rodent mammary gland.

Work from numerous fields of study suggests that exposures to hormonally active chemicals during sensitive windows of development can alter mammary gland development, function, and disease risk. Stronger links between many environmental pollutants and disruptions to breast health continue to be documented in human populations, and there remain concerns that the methods utilized to identify, characterize, and prioritize these chemicals for risk assessment and risk management purposes are insufficient. There are also concerns that effects on the mammary gland have been largely ignored by regulatory agencies. Here, we provide technical guidance that is intended to enhance collection and evaluation of the mammary gland in mice and rats. We review several features of studies that should be controlled to properly evaluate the mammary gland, and then describe methods to appropriately collect the mammary gland from rodents. Furthermore, we discuss methods for preparing whole mounted mammary glands and numerous approaches that are available for the analysis of these samples. Finally, we conclude with several examples where analysis of the mammary gland revealed effects of environmental toxicants at low doses. Our work argues that the rodent mammary gland should be considered in chemical safety, hazard and risk assessments. It also suggests that improved measures of mammary gland outcomes, such as those we present in this review, should be included in the standardized methods evaluated by regulatory agencies such as the test guidelines used for identifying reproductive and developmental toxicants.

Using alternative test methods to predict endocrine disruption and reproductive adverse outcomes: do we have enough knowledge?

Endocrine disrupting chemicals (EDCs) are a matter of great concern. They are ubiquitous in the environment, are considered harmful to humans and wildlife, yet remain challenging to identify based on current international test guidelines and regulatory frameworks. For a compound to be identified as an EDC within the EU regulatory system, a plausible link between an endocrine mode-of-action and an adverse effect outcome in an intact organism must be established. This requires in-depth knowledge about molecular pathways regulating normal development and function in animals and humans in order to elucidate causes for disease. Although our knowledge about the role of the endocrine system in animal development and function is substantial, it remains challenging to predict endocrine-related disease outcomes in intact animals based on non-animal test data. A main reason for this is that our knowledge about mechanism-of-action are still lacking for essential causal components, coupled with the sizeable challenge of mimicking the complex multi-organ endocrine system by methodological reductionism. Herein, we highlight this challenge by drawing examples from male reproductive toxicity, which is an area that has been at the forefront of EDC research since its inception. We discuss the importance of increased focus on characterizing mechanism-of-action for EDC-induced adverse health effects. This is so we can design more robust and reliable testing strategies using non-animal test methods for predictive toxicology; both to improve chemical risk assessment in general, but also to allow for considerable reduction and replacement of animal experiments in chemicals testing of the 21st Century.

Genetic investigations of 100 inherited cardiac disease-related genes in deceased individuals with schizophrenia.

Cardiac diseases and sudden cardiac death (SCD) are more prevalent in individuals diagnosed with schizophrenia compared to the general population, with especially coronary artery disease (CAD) as the major cardiovascular cause of death. Antipsychotic medications, genetics, and lifestyle factors may contribute to the increased SCD in individuals with schizophrenia. The role of antipsychotic medications and lifestyle factors have been widely investigated, while the genetic predisposition to inherited cardiac diseases in schizophrenia is poorly understood. In this study, we examined 100 genes associated with inherited cardiomyopathies and cardiac channelopathies in 97 deceased individuals diagnosed with schizophrenia for the prevalence of genetic variants associated with SCD. The deceased individuals had various causes of death and were included in the SURVIVE project, a prospective, autopsy-based study of mentally ill individuals in Denmark. This is the first study of multiple inherited cardiac disease-related genes in deceased individuals with diagnosed schizophrenia to shed light on the genetic predisposition to SCD in individuals with schizophrenia. We found no evidence for an overrepresentation of rare variants with high penetrance in inherited cardiac diseases, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG) consensus guidelines. However, we found that the deceased individuals had a statistically significantly increased polygenic burden caused by variants in the investigated heart genes compared to the general population. This indicates that common variants with smaller effects in heart genes may play a role in schizophrenia.

Long-term physical and psychological outcomes after out-of-hospital cardiac arrest-protocol for a national cross-sectional survey of survivors and their relatives (the DANCAS survey).

INTRODUCTION: The number of out-of-hospital cardiac arrest (OHCA) survivors is increasing. However, there remains limited knowledge on the long-term physical and psychological problems suffered by survivors and their relatives. The aims of the DANCAS (DANish cardiac arrest survivorship) survey are to describe the prevalence of physical and psychological problems, identify predictors associated with suffering them and to determine unmet rehabilitation needs in order to make recommendations on the timing and content of future rehabilitation interventions. METHODS AND ANALYSIS: The DANCAS survey has a cross-sectional design involving a survey of OHCA survivors and their relatives. OHCA survivors will be identified through the Danish Cardiac Arrest Registry as having suffered an OHCA between 1 January 2016 and 31 December 2019. Each survivor will be asked to identify their closest relative to complete the relatives' survey. Contents of survivor survey: EQ-5D-5Level, Hospital Anxiety and Depression Scale, Two Simple Questions, Modified Fatigue Impact Scale, 12-item WHO Disability Assessment Scale 2.0, plus questions on unmet rehabilitation and information needs. Contents of relatives' survey: World Health Organisation-Five Well-Being Index, Hospital Anxiety and Depression Scale, Informant Questionnaire on Cognitive Decline in the Elderly-Cardiac Arrest and the Modified Caregiver Strain Index. Self-report outcome data collected through the surveys will be enriched by data from Danish national registries including demographic characteristics, circumstances of cardiac arrest and comorbidities. The survey will be completed either electronically or by post December 2020-February 2021. ETHICS AND DISSEMINATION: The study will be conducted in accordance with the Declaration of Helsinki. Surveys and registry-based research studies do not normally require ethical approval in Denmark. This has been confirmed for this study by the Region of Southern Denmark ethics committee (20192000-19). Results of the study will be disseminated via several peer-reviewed publications and will be presented at national and international conferences.

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.

Classical toxicity endpoints in female rats are insensitive to the human endocrine disruptors diethylstilbestrol and ketoconazole.

Developmental exposure to endocrine disrupting chemicals can have negative consequences for reproductive health in both men and women. Our knowledge about how chemicals can cause adverse health outcomes in females is, however, poorer than our knowledge in males. This is possibly due to lack of sensitive endpoints to evaluate endocrine disruption potential in toxicity studies. To address this shortcoming we carried out rat studies with two well-known human endocrine disruptors, diethylstilbestrol (DES) and ketoconazole (KTZ), and evaluated the sensitivity of a series of endocrine related endpoints. Sprague-Dawley rats were exposed orally from gestational day 7 until postnatal day 22. In a range-finding study, disruption of pregnancy-related endpoints was seen from 0.014 mg/kg bw/day for DES and 14 mg/kg bw/day for KTZ, so doses were adjusted to 0.003; 0.006; and 0.0012 mg/kg bw/day DES and 3; 6; or 12 mg/kg bw/day KTZ in the main study. We observed endocrine disrupting effects on sensitive endpoints in male offspring: both DES and KTZ shortened anogenital distance and increased nipple retention. In female offspring, 0.0012 mg/kg bw/day DES caused slightly longer anogenital distance. We did not see effects on puberty onset when comparing average day of vaginal opening; however, we saw a subtle delay after exposure to both chemicals using a time-curve analysis. No effects on estrous cycle were registered. Our study shows a need for more sensitive test methods to protect the reproductive health of girls and women from harmful chemicals.

On the Use and Interpretation of Areola/Nipple Retention as a Biomarker for Anti-androgenic Effects in Rat Toxicity Studies.

Areola/nipple retention (NR) is an established biomarker for an anti-androgenic mode of action in rat toxicity studies. It is a mandatory measurement under several OECD test guidelines and is typically assessed in combination with anogenital distance (AGD). Both NR and AGD are considered retrospective biomarkers of insufficient androgen signaling during the masculinization programming window in male fetuses. However, there are still aspects concerning NR as a biomarker for endocrine disruption that remains to be clarified. For instance, can NR be regarded a permanent adverse effect? Is it a redundant measurement if AGD is assessed in the same study? Is NR equally sensitive and specific to anti-androgenic chemical substances as a shortening of male AGD? In this review we discuss these and other aspects concerning the use of NR as a biomarker in toxicity studies. We have collected available literature from rat toxicity studies that have reported on NR and synthesized the data in order to draw a clearer picture about the sensitivity and specificity of NR as an effect biomarker for an anti-androgenic mode of action, including comparisons to AGD measurements. We carefully conclude that NR and AGD in rats for the most part display similar sensitivity and specificity, but that there are clear exceptions which support the continued assessment of both endpoints in relevant reproductive toxicity studies. Available literature also support the view that NR in infant male rats signifies a high risk for permanent nipples in adulthood. Finally, the literature suggests that the mechanisms of action leading from a chemical stressor event to either NR or short AGD in male offspring are overlapping with respect to canonical androgen signaling, yet differ with respect to other mechanisms of action.

Dysregulation of miR-185, miR-193a, and miR-450a in the skin are linked to the depressive phenotype.

BACKGROUND: Dysregulated microRNAs (miRNAs) in dermal fibroblasts of depressive subjects, indicate biomarker potential and can possibly aid clinical diagnostics. To overcome methodological challenges related to human experiments and fibroblast cultures, we here validate 38 miRNAs previously observed to be dysregulated in human fibroblasts from depressed subjects, in the skin of four distinct rat models of depression. METHODS: In the presented study male rats from the adrenocorticotropic hormone (ACTH) model (n = 10/group), the chronic mild stress model (n = 10/group), Wistar Kyoto/Wistar Hannover rats (n = 10/group), and Flinders Resistant/Flinders Sensitive Line rats (n = 8/group) were included. Real-time qPCR was utilized to investigate miRNA alterations in flash-frozen skin-biopsies from the ear and fibroblast cultures. RESULTS: In the ACTH rat model of depression, we identified nine dysregulated miRNAs in the skin and three in the fibroblasts. As the skin presented three times the amount of dysregulated miRNAs compared to the fibroblasts, skin instead of fibroblasts were continuously used for studies with the other rat models. In the skin from the four rat models of depression, 15 out of 38 miRNAs re-exhibited significant dysregulation in at least one of the rat models of depression and 67% were regulated in the same direction as in the human study. miR-450a and miR-193a presented dysregulation across rat models and miR-193a and miR-185 exhibited very strong dysregulation (30-fold and 50-fold, respectively). Lastly, an Ingenuity Pathway Analysis indicated functional overlap between dysregulated miRNAs, and common regulated pathways. CONCLUSION: Flash-frozen skin is a valid alternative to fibroblast cultures as the skin appear to retain more of the miRNA dysregulation present in vivo. A sub-population of 15 miRNAs appear to be specific for the depressive phenotype, as they are dysregulated in both human depressed patients and distinct rat models of depression. We propose miR-450a, miR-185, and miR-193a as biomarker candidates of particular interest.