United States

Background: Within a spectrum of cancers implicated by VHL germline pathogenic variants, one rare cancer which may present in patients are pheochromocytomas (PHEOs). These tumors occur in the adrenal glands and predispose patients to a variety of morbid clinical symptoms due to the excess secretion of catecholamines. As a part of these patients&#39; management, imaging is important to localize the tumors of which there are both morphologic (CT and MRI) and molecular (PET/CT with ¹⁸F-FDG, 68Ga-DOTATATE, ¹⁸F-DOPA, and ¹⁸F-DA) imaging modalities.
Purpose: To determine which imaging modalities provided the optimal detection of VHL-related PHEO.
Methods: The study compared detection of 19 PHEOs (18 histologically proven and 1 is a contralateral tumor awaiting resection) in 12 patients. The CT and MRI were evaluated by a body radiologist and the PET/CT of¹⁸F-FDG, 68Ga-DOTATATE, ¹⁸F-DOPA, and ¹⁸F-DA were evaluated by a nuclear medicine physician in a blinded fashion. Both experts graded using a five-point Likert scale (1 – not PHEO and 5 – PHEO). For analysis, the Likert scale was dichotomized with 1-3 designating negative for PHEO and 4-5 positive for PHEO. The findings were reported as detection rates of positive PHEOs found on imaging to the gold standard of pathologically (aside from one contralateral PHEO awaiting surgery) proven PHEOs. The McNemar test was performed to compare the statistical relevance of detection rates between histology and the imaging modalities. Two-sided p values &lt;0.05 were considered significant.
Results: The detection rates were CT 17/19 [89.5%, 95% CI = 66.9%-98.7%], MRI 17/17 [100%, 95% CI = 80.5%-100%], ¹⁸F-FDG PET/CT 16/17 [94.1%, 95% CI = 80.5%-100%], 68Ga-DOTATATE 5/9 [55.6%, 95% CI = 21.2%-86.3%], ¹⁸F-DOPA 12/14 [85.7%, 95% CI = 57.2%-98.2%], and ¹⁸F-DA 4/6 [66.7%, 95% CI = 22.3%–95.7%]. All McNemar tests showed p-values greater than 0.05, which were not statistically significant.
Conclusion: The study was performed in a small patient set with VHL-related PHEO showed MRI, with the highest detection percent followed by ¹⁸F-FDG PET/CT. More trials with a larger patient set are needed to support the statistical relevance as it was not seen in this study. Furthermore, performing a study on a patient cohort with additional VHL associated cancers could prove clinically beneficial for patients.

ENDO 2022

Morphologic or Molecular Imaging - Diagnostic Capabilities in von Hippel-Lindau related pheochromocytoma

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Background: Within a spectrum of cancers implicated by VHL germline pathogenic variants, one rare cancer which may present in patients are pheochromocytomas (PHEOs). These tumors occur in the adrenal glands and predispose patients to a variety of morbid clinical symptoms due to the excess secretion of catecholamines. As a part of these patients' management, imaging is important to localize the tumors of which there are both morphologic (CT and MRI) and molecular (PET/CT with ¹⁸F-FDG, 68Ga-DOTATATE, ¹⁸F-DOPA, and ¹⁸F-DA) imaging modalities.
Purpose: To determine which imaging modalities provided the optimal detection of VHL-related PHEO.
Methods: The study compared detection of 19 PHEOs (18 histologically proven and 1 is a contralateral tumor awaiting resection) in 12 patients. The CT and MRI were evaluated by a body radiologist and the PET/CT of¹⁸F-FDG, 68Ga-DOTATATE, ¹⁸F-DOPA, and ¹⁸F-DA were evaluated by a nuclear medicine physician in a blinded fashion. Both experts graded using a five-point Likert scale (1 – not PHEO and 5 – PHEO). For analysis, the Likert scale was dichotomized with 1-3 designating negative for PHEO and 4-5 positive for PHEO. The findings were reported as detection rates of positive PHEOs found on imaging to the gold standard of pathologically (aside from one contralateral PHEO awaiting surgery) proven PHEOs. The McNemar test was performed to compare the statistical relevance of detection rates between histology and the imaging modalities. Two-sided p values <0.05 were considered significant.
Results: The detection rates were CT 17/19 [89.5%, 95% CI = 66.9%-98.7%], MRI 17/17 [100%, 95% CI = 80.5%-100%], ¹⁸F-FDG PET/CT 16/17 [94.1%, 95% CI = 80.5%-100%], 68Ga-DOTATATE 5/9 [55.6%, 95% CI = 21.2%-86.3%], ¹⁸F-DOPA 12/14 [85.7%, 95% CI = 57.2%-98.2%], and ¹⁸F-DA 4/6 [66.7%, 95% CI = 22.3%–95.7%]. All McNemar tests showed p-values greater than 0.05, which were not statistically significant.
Conclusion: The study was performed in a small patient set with VHL-related PHEO showed MRI, with the highest detection percent followed by ¹⁸F-FDG PET/CT. More trials with a larger patient set are needed to support the statistical relevance as it was not seen in this study. Furthermore, performing a study on a patient cohort with additional VHL associated cancers could prove clinically beneficial for patients.

technical paper

Adrenocortical carcinoma (ACC) is a rare, aggressive, and poorly understood cancer of the adrenal cortex. Abnormal epigenetic patterning is a major predictor of dismal disease outcomes. Patients with genome-wide CpG island hypermethylation (CIMP-high) experience rapidly recurrent and routinely fatal disease. A deeper understanding of how epigenetic programs coordinate tumorigenesis is essential to develop improved medical therapies. We identify that DNA hypermethylation is directed to targets of a histone modifying complex, PRC2, suggesting cross-talk between DNA and histone methylation in CIMP-high ACC. In contrast to our expectations, we discovered that DNA methylation and PRC2-directed histone methylation are mutually exclusive. Additionally, we identified that EZH2 (a canonical member of the PRC2) forms a separate complex with tissue-specific proteins to coordinate sustained proliferation and steroidogenic differentiation in vitro and in vivo. These complexes persist through advanced stages of human malignancy and are conserved in mouse models of adrenal carcinogenesis. Taken together, our studies illustrate how CpG island hypermethylation exposes a pharmacologically targetable tissue-specific therapeutic vulnerability, and stabilizes a differentiation state required for sustained proliferation. Ultimately, we hope this work illuminates novel strategies for tissue-specific disruption of the aberrant epigenetic wiring supporting this devastating disease.

Epigenetic Programs Stabilize a Differentiated Cell State Required for Sustained Proliferation in Adrenocortical Carcinoma

Bisphenol A (BPA) is known to produce adverse effects on fertility, cardiovascular, and metabolic health. The constraints placed on BPA production led companies to switch to BPA's structural analogs, Bisphenol S (BPS) and Bisphenol F (BPF). These chemicals are used in various equipment, food and drink cans, and paper, permitting widespread exposure. We recently found that prenatal exposure to low levels of these chemicals can increase blood pressure in the offspring.  However, the underlying neurobiological mechanism is unclear. We hypothesized that these chemicals could alter the levels of monoamines, specifically in brain regions that regulate cardiovascular function such as the paraventricular nucleus (PVN) of the hypothalamus. Pregnant Sprague Dawley rats were exposed to saline, 5 µg/kg BW of BPA or BPS, or 1 µg/kg BW of BPF from gestation day 6 to 21. Their adult Male and Female offspring were implanted with a radiotelemetry device to measure blood pressure and heart rate levels. Following telemetry, animals were sacrificed, and the brains were collected, frozen and sectioned. The PVN was microdissected, and analyzed for monoamine levels using HPLC-EC. Males exposed to BPA had significant increases in the levels of serotonin (5HT) in the PVN compared to the control group, while a marked increase in 5HT levels were only observed in females exposed to BPF and not BPA (p = 0.0002). No significant changes were observed in Dopamine (DA) or norepinephrine (NE) levels. There were significant sex differences (p<0.0001) and treatment effects (p=0.0003) in the DOPAC/DA turnover ratio with males exposed to BPA, BPS, and BPF having markedly less turnover rates compared to control. No changes in DOPAC/DA turnover was apparent in females. There were significant sex (p<0.0001) and treatment effects (p<0.0001) in the 5HIAA/5HT turnover ratio, with males in the BPA and BPS groups having significantly less turnover compared to controls. In contrast, Females exposed to BPS and BPF had significantly lower 5HIAA/5HT turnover ratios but higher turnover ratios in the groups exposed to BPA. These results indicate that 5-HT production increases markedly in males exposed to BPA and in females exposed to BPF.  This is associated with reduced metabolism of 5-HT.  This suggests that the significant increases in 5-HT activity in the PVN could possibly contribute to the development of hypertension in these animals.

Sex-specific Effects of Bisphenol-A and its Analogs on Monoamine Levels in the Paraventricular Nucleus

Exposure to endocrine disruptors (EDs), including the well-known plasticizers bisphenol A (BPA) and di(2-Ethylhexyl) phthalate (DEHP), during the highly sensitive gestational period, can produce long-lasting behavioral changes in offspring. In this study, we hypothesized that gestational exposure to BPA and/or DEHP at various doses would alter stress-related behaviors and monoaminergic activity in a region-specific manner in exposed offspring. To test this, female Sprague-Dawley dams were orally administered saline (control group - 10 µL/kg BW), BPA (5 µg/kg body weight (BW)), DEHP (5 µg/kg BW or 7.5 mg/kg BW), a combination of BPA and low-dose (LD) DEHP (5 µg BPA + 5 µg DEHP/kg BW), or a combination of BPA and high-dose (HD) DEHP (5 µg BPA + 7.5 mg DEHP/kg BW) during days 6 through 21 of pregnancy. The male and female offspring of these dams underwent a battery of behavioral tests in adulthood. Brains collected from the offspring were microdissected to obtain the medial prefrontal cortex (mPFC), paraventricular hypothalamic nucleus (PVN), and hippocampus (HC), which were analyzed for monoamine and metabolite levels using High-Performance Liquid Chromatography (HPLC). While BPA and B+D (LD) female offspring demonstrated anxiolytic effects in the Open Field Test, DEHP (HD) male offspring showed feminized anxiety-like behavior in the Elevated Plus Maze. The offspring also displayed impaired fear responses in the Shock Probe Defensive Burying (SPDB) test. Males in most ED groups buried less than their control counterparts. Furthermore, low-dose ED treatments led to a robust preference for passive coping strategies in both male and female offspring. Within the brain, male offspring in the BPA, LD and HD DEHP, and B+D (HD) groups had diminished dopamine (DA) levels in the PVN, which corresponded with their reduced SPDB burying levels. Moreover, DEHP (LD) male offspring had elevated DA turnover in the mPFC, whereas B+D (LD) female offspring had enhanced norepinephrine levels in the HC, which may underlie their behavioral outcomes in the SDPB. The findings of this study have implications for sex differences in mood and anxiety disorders following prenatal exposures to BPA, DEHP, and their mixtures.

Sex- and Dose-Dependent Effects of In Utero Endocrine Disruptor Exposure on Offspring Behavior and Brain Monoamines

In the United States, Black women are 39% more likely to die from breast cancer compared to White women (AACR Cancer Disparities Progress Report 2020). Furthermore, Black women are at a higher risk of developing breast cancer under the age of 40 than any other racial or ethnic group. While the underlying cause of these disparities is multifactorial, exposure to endocrine disrupting chemicals (EDCs) in hair and personal care products has been associated with increased risk of breast cancer. Parabens are known EDCs that are commonly used as preservatives in hair and other personal care products. The Environmental Working Group scores methylparaben (MP), propylparaben (PP), and butylparaben (BP) moderate (MP) to high (BP and PP) on a hazardous scale of chemicals found in personal care products. Furthermore, these parabens are restricted in personal care products with set maximum concentration standards in Japan, the European Union, and Southeast Asia. Studies have shown that parabens impact breast cancer cell proliferation, death, migration/invasion, and metabolism, as well as gene expression in vitro. However, these studies were conducted using cell lines of European ancestry; to date, no studies have utilized breast cancer cell lines of West African ancestry to examine effects of parabens. We hypothesize that, like what has been shown for breast cancer cell lines with European ancestry, parabens will promote pro-tumorigenic effects in breast cancer cell lines with West African ancestry. To test this hypothesis, we treated MCF-7 (European ancestry) and HCC1500 (West African ancestry) luminal breast cancer cell lines with biologically relevant doses of either MP, PP, or BP. We also co-treated with the estrogen receptor (ER) antagonist ICI 182,780 to determine whether observed effects are mediated by ER. We found BP increases HCC1500 cell viability, but not MCF-7 cell viability. This effect was not blocked by co-treatment with ICI 182,780. We also observed that BP and PP, but not MP, increased expression of estrogen-regulated genes in both MCF-7 and HCC1500. The increase in gene expression was reduced with co-treatment of ICI 182,780 in both cell lines, suggesting that these effects are ER-mediated. Preliminary studies suggest that parabens promote migration of both cell lines, with a potentially greater effect on HCC1500 cells. Taken together, these data demonstrate that parabens, particularly BP and PP, promote pro-tumorigenic effects in diverse luminal breast cancer cell lines. These findings have future translational relevance as we are part of a community-led initiative called Bench to Community. Bench to Community brings together basic researchers in endocrinology and social-behavioral scientists, community stakeholders including breast cancer survivors to develop interventions to reduce adverse exposures to EDCs in hair and other personal care products in Black women.

Parabens Promote Pro-Tumorigenic Effects in Luminal Breast Cancer Cell Lines With Diverse Genetic Ancestry

Background: Perfluorooctanoic acid (PFOA), is a persistent fluorinated compound with oil and water repelling properties found in cookware, food packaging and municipal water systems. Adult animals exposed to PFOA develop hepatomegaly, fatty liver, peroxisome proliferation, and immunotoxicity. Rodents exposed to PFOA in utero have altered hepatic lipid metabolism, increased hepatic de novo lipogenesis and susceptibility to non-alcoholic fatty liver disease (NAFLD), but underlying molecular mechanisms remain unknown. With increasing rates of obesity, diabetes, and NAFLD it is critical to examine the mechanisms by which in utero exposure to PFOA contributes to the development of metabolic syndrome in offspring.
Objectives: To characterize how PFOA exposure during hepatocyte differentiation leads to the development of NAFLD through alterations in DNA methylation profiles and changes in the availability of transcription factor binding sites.
Methods: HepaRG cells (human-derived hepatocyte progenitor cells) were treated with 0.5uM PFOA or vehicle for 48 hours followed by differentiation. Undifferentiated and differentiated hepatocytes exposed to PFOA were assessed relative to controls (n=4). RNASeq was completed; DESeq2 identified differentially expressed genes via false discovery rate (FDR) of <0.05 after Bonferroni correction. Genome-wide DNA methylation analysis via MethylSeq was completed to identify differentially methylated regions (DMRs) defined as minimum number of CpN =5, absolute change in percent methylation >10%, and FDR of <0.05. Enrichment analysis of transcription factor binding motifs within DMRs and single CpG sites (± 200 bp) was performed using HOMER.
Results: PFOA treatment resulted in decreased expression of the transcription factors early growth response protein 1 (EGR1), nuclear receptor Nur77 (NR4A1) , early growth response protein 2 (EGR2), krueppel-like factor 10 (KLF10) and fos-related antigen 1 (FOSL1), which are key genes linked to impaired hepatic insulin signaling, lipid metabolism, steatosis and fibrosis (q<0.05) in undifferentiated hepatocytes. Differentiated hepatocytes showed decreased expression of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α), peroxisome proliferator-activated receptor γ (PPARγ), forkhead box protein O1 (FOXO1) and increased expression of pyruvate dehydrogenase kinase isozyme 4 (PDK4), genes linked to regulation of lipid metabolism and insulin signaling (q <0.05). MethylSeq analysis identified changes in DMRs located in exons, introns, and intergenic regions, with 57 DMRs in undifferentiated hepatocytes (46 with gains; 11 with losses; q <0.05) and 75 DMRs in differentiated hepatocytes (32 with gains; 41 with losses; q<0.05). HOMER identified 29 known transcription factor binding motifs with changes in methylation in undifferentiated hepatocytes (p<0.05) and 19 in differentiated hepatocytes after PFOA treatment (p<0.05), with significant changes in the EGR1 consensus sequence identified in both comparisons.
Conclusions: We conclude hepatocyte progenitor cells exposed to low dose PFOA results in changes in DNA methylation and expression of key metabolic genes linked to NAFLD, notably EGR1, a gene previously linked to NAFLD, suggesting PFOA exposure in utero has lasting effects.

Hepatocytes Exposed to PFOA Prior to Differentiation Leads to Epigenetic Changes in Genes Linked With NAFLD

BPA, a widespread endocrine-disrupting compound (EDC), plays a significant role in the decline of female fertility. Granulosa cells are susceptible to EDCs and are important for oocyte competency. This research focuses on epigenetic mechanisms in granulosa cells that are disrupted by BPA, with a specific focus on microRNA-21(miR-21), which is crucial for oocyte maturation and primarily recognized as an antiapoptotic regulator. miR-21 targets the apoptotic genes PDCD4 and PTEN and limits their expression, resulting in an antiapoptotic effect. Previous studies in our laboratory demonstrate that BPA induces apoptosis and simultaneously increases miR-21 expression, leading to our hypothesis that BPA induces apoptosis in a miR-21 independent manner in bovine granulosa cells.To test this hypothesis, in vitro cultured granulosa cells were transfected with miR-21 inhibitor or scramble with or without BPA, followed by knockdown validation and apoptotic measurements. Using flow cytometry, transfection efficiencies were measured in time- and dose-dependent experiments, showing optimal knock-down greater than 95% at 0.5µM for 12 hrs. miR-21 expression measured by qPCR showed a significant decrease within inhibited groups (p=0.0008, n=3). Although PDCD4 and PTEN mRNA expression remained unaffected, PDCD4 protein expression is inhibited cells was significantly increased (p=0.0233, n=4), suggesting a miR-21 role in PDCD4 translation. PTEN protein was unaffected by miR-21 inhibition.In further experiments, transfected cells were exposed to BPA, and apoptosis was examined. Using Annexin V/PI staining, a significant decrease in healthy cells and an increase in early apoptotic cells within inhibited untreated groups was shown (p=0.0395 and 0.0193, n=3). This finding is likely due to antiapoptotic gene inhibition. BPA exposure resulted in significantly decreased healthy cells, increased late apoptotic cells (p=0.0047 and 0.0145, n=3), with no changes in necrotic cells. Furthermore, no differences were found between inhibited and non-inhibited groups, suggesting that BPA-induced apoptosis is miR-21 independent.Additionally, three proapoptotic (BAX, BAD, Casp-9) and two antiapoptotic (Bcl-2 and HSP70) genes were quantified by qPCR. Unexpectedly, BPA increased the expression of all genes at the mRNA level (p= 0.0021, 0.0283, 0.0143, 0.025, 0.0145, n=4). Quantification of protein levels will reveal whether this change is functional. Moreover, no differences were found between inhibited and non-inhibited groups, suggesting these changes are likely miR-21 independent. Lastly, initiator caspase 9 activity was measured after 6, 12, 24, 48, 72 hrs of BPA exposure, as caspases are primary effectors of apoptosis. 6 and 12hrs timepoints showed no changes; however, at 24 and 48 hrs significantly increased caspase activity was detected (p=0.0087 and 0.0014, n=4). At 72hrs, BPA exposure was lethal, resulting in insufficient cell numbers for measurements.Overall, this research focuses on effects of EDCs on apoptotic regulation within granulosa cells and aims to uncover epigenetic consequences of BPA's mechanism of action, ultimately affecting oocyte competency and fertility.

The effects of Bisphenol A on miR-21 expression and miR-21 mediated apoptosis in bovine granulosa cells.

Endocrine-disrupting compounds (EDCs) elicit adverse responses in the body, including the disruption of female reproductive functions. A common EDC is Bisphenol A (BPA), which is a frequently used plasticizer. Due to the negative effects of BPA on human health, production of BPA-free products using analogs, such as BPS and BPF, has increased; yet their effects are still largely unknown. Adequate oocyte maturation requires proper functioning of the surrounding environment, consisting of granulosa cells that impact oocyte competency. Granulosa cells are also susceptible to BPA disruption, affecting oocyte maturation and, ultimately, fertility.
In our lab, we showed that BPA and BPS significantly increased apoptosis in bovine embryos through increased DNA fragmentation (Saleh et al., 2021). However, the apoptotic pathway used is not fully characterized. Thus, the aim of this study is to investigate how bisphenols affect granulosa cell viability and through which apoptotic pathway. In vitro cultured granulosa cells were exposed for 12 or 48 hrs to six treatment groups: control, vehicle, estradiol, BPA, BPS and BPF at a low dose (0.005 mg/mL) and at the BPA Lowest Observed Adverse Effect Level (LOAEL-0.05 mg/mL). Live cells were counted using trypan exclusion. A significant decrease in the number of live cells after BPA treatment at the LOAEL dose for 12 hrs was observed (P=0.02, n=9).
 mRNA expression of three proapoptotic (BAX, BAD, Casp9) and two antiapoptotic (HSP70 and BCl-2) genes was quantified by qPCR. A significant increase in BAX transcript levels after 12 hrs of exposure to BPA and BPF at the LOAEL dose (P=0.046, P= 0.033, n=6) as well as in BAD (P= 0.049, n=3) and Casp9 (P=0.049, n=4) levels after BPA exposure at the LOAEL dose was detected. Interestingly, at the 48 hr timepoint, a significant increase in BAD (P= 0.018, n= 9), BAX (P=0.002, n=10), and Casp9 (P=0.015, n=5) transcript levels was observed only following BPF treatment at the LOAEL dose. Despite expecting a decrease, both anti-apoptotic transcripts, at 12 and 48 hrs, showed increased levels following exposure to BPA and/or BPF at the LOAEL dose. This suggests an early first line of defense or shock response. Future protein analysis will help in further understanding these unexpected findings.
To quantify apoptosis levels, flow cytometry together with Annexin V/PI staining was used. Results indicate that after 48 hrs of treatment, BPA at the LOAEL dose significantly increases late apoptosis (P=0.004, n=5) and necrosis (P=0.0014, n=5), while showing a significant decrease in the number of healthy cells (P=0.0007, n=5). Preliminary data also show a similar pattern after BPA treatment for 12 hrs. This research aims to further our knowledge of the mechanisms by which bisphenols affect oocyte competency and suggests that BPF might have equivalent potency and effects to BPA.

BPA and BPF, but not BPS, alter pro- and anti-apoptotic transcript levels in granulosa cells, affecting cell viability.

Humans are exposed to a mixture of endocrine disrupting chemicals (EDCs) in everyday life, believed to contribute to a range of non-communicable diseases. While the adverse health impacts of exposure to individual EDCs have been extensively investigated, studies on effects of real-world exposures characterized by chronic low doses of a mixture of chemicals, are only recently emerging. Biosolids derived from human wastes, exemplify both the array and dosage of chemicals humans are exposed to in real life and represents a novel model to investigate the risk posed by the EDCs. Previous studies in sheep have demonstrated aberrant reproductive and metabolic phenotypic effects in offsprings, as a result of maternal biosolid exposure. We hypothesize that EDC-mediated disruption in the maternal metabolic milieu is a potential mediator of the aberrant phenotypic outcomes in offsprings in the biosolid-exposure model. Recent untargeted metabolomic studies provide evidence of the technique's ability to unravel biomarkers of both exposure and effect by identifying the disrupted endogenous metabolite profile and xenobiotic mixture, respectively. In this study, we sought to identify biosolid exposure-induced maternal metabolic fingerprints that could influence fetal programming.
Ewes were grazed on either organic fertilizer or biosolid-treated pastures from 1 month before mating until close to parturition. Plasma from blood samples collected on day 90 of the 147-day gestation period, from ewes carrying singleton pregnancies (Control n=15, Treatment n=15) were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry. Metabolites were identified by matching the retention time, mass and isotope profile to authentic standards or metabolome databases using Agilent's MassHunter Find by Molecular Feature workflow (v7.0).
Untargeted metabolomics identified 1006 and 1295 annotated features from the negative and positive mode respectively. Principal component analysis and orthogonal signal correction partial least-squares discriminant analysis discriminated the metabolomes of control sheep and sheep grazed in biosolid-treated pasture. The key differential metabolites identified by variable importance in projection score primarily belonged to the super-class organic acids, fatty acids, benzenoids, glycerophospholipids and organoheterocyclic compounds. These differentially expressed metabolites are implicated in metabolic pathways including carnitine synthesis, alanine metabolism, glucose alanine cycle, phosphatidyl choline biosynthesis and glutathione metabolism. Some of the top differential metabolites- oleamide, pyroglutamic acid and 15-HETrE have been implicated in fetal development. Oleamide, an endocannabinoid, has growth inhibitory properties. 15-HETrE modulates arachidonic acid metabolism and upregulate PPARγ expression, which plays a pivotal role in fetal development and metabolic outcomes.
These findings indicate that biosolids exposure results in alterations in the maternal metabolome which likely contributes to the aberrant phenotypic outcomes reported in offspring of biosolid-exposed mothers. This is the first study to apply metabolomics to validate the effect of real-life EDC exposure in sheep, a translationally relevant precocial model with a developmental trajectory similar to humans.

Exposure to a Real Life Environmental Chemical Mixture Alters the Metabolite Profile in the Plasma of Sheep: An Untargeted Metabolomics Study.

Prenatal exposure to bisphenol-A (BPA), an ubiquitously present endocrine-disrupting chemicals, induces peripheral insulin resistance as well as lipotoxicity and oxidative stress in the liver and muscle along with tissue-specific transcriptional changes in female sheep. To gain a mechanistic understanding of contributors to these liver and muscle-specific transcriptional changes, we hypothesized that prenatal BPA exposure impacts non-coding RNA (ncRNA), regulators of transcriptional and post-transcriptional control of coding RNAs. We sequenced ncRNA (including lncRNA, microRNA, snoRNA, and snRNA) in the liver and muscle obtained during the late follicular phase from 21-month-old control and prenatal BPA-treated (0.5mg/kg/day from days 30 to 90 of gestation; Term: 147days) female sheep (n=4 control and 4 prenatal BPA treated). The sequence data were subjected to differential expression analysis and multivariate modeling to identify specific ncRNA signatures. In the liver, 77 lncRNA (49 down, 28 up), 14 microRNAs (6 down, 8 up), 127 snoRNAs (63 down, 64 up) and 55 snRNAs (15 down, 40 up) were dysregulated (FDR<0.1, absolute log2FC>0.5) with BPA exposure. In the muscle 6 lncRNA were upregulated and 65 snoRNAs (47 down, 18 up) were dysregulated (FDR<0.1, absolute log2FC>0.5). Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) plots identified microRNAs MIR200B, MIR409, MIR30B, and MIR154A in the liver and miRNAs MIR26B, MIR29A, MIR125B, and MIR127 in muscle and several uncharacterized ncRNAs as signatures of prenatal BPA exposure. Multiple ncRNA signatures correlated with coding genes including LCORL, MED17, and ZNF41 in the liver, but none were correlated in the muscle. Among the coding genes which correlated with ncRNA, LCORL is reported to be related to steatosis of the liver in geese, a feature also observed in livers from prenatal BPA-treated female sheep. Since lipid accumulation in the liver may result from increased fatty acid biosynthesis, MED17 which is involved in transcriptional activation of lipogenic genes that correlated with multiple ncRNA in the liver may be a contributor to the steatosis observed in this model. In addition, MIR200B and MIR30B in the liver and MIR26B and MIR29A in the muscle are linked to lipid dysfunction, endoplasmic reticulum stress, and fibrosis. These findings provide mechanistic clues into the role of ncRNA in the prenatal BPA-induced lipid accumulation and oxidative stress in the liver and muscle, contributors to metabolic dysfunction observed in this female sheep model of gestational BPA exposure. In addition, findings from this precocial model of human translational relevance is a step towards gaining mechanistic insights in the programming of metabolic dysfunctions from environmental exposure to BPA, an endocrine disruptor of public health relevance.

Non-coding RNA Signatures of Prenatal BPA Exposure in Liver and Muscle of Female Sheep

Introduction: Many patients in the US with hypothyroidism receive suboptimal treatment with thyroid hormone. Sustained off-target TSH levels have been shown to cause serious harm to patients, including increased risk of cardiovascular disease and excess mortality over time. Suboptimal treatment may also impact hospital outcomes. The primary objective of the study was to identify differences in hospital outcomes between patients with and without hypothyroidism.Methods: This is a retrospective cohort study that utilized the propensity score-based fine stratification method to balance covariates. Patient data was extracted from a large, US-based, IBM MarketScan® Commercial Claims and Encounters Database from January 1, 2008 to December 31, 2015. Participants included patients 64 years and younger who had commercial insurance and a thyrotropin (TSH) level collected prior to a medical or surgical hospital admission. Covariates included age, sex, US region, type of admission (medical vs surgical), year of admission, and co-morbidities. The primary exposure was defined as clinical hypothyroidism, which was divided into 4 subgroups based on pre-hospitalization TSH level: low (TSH <0.40 mIU/L), normal (TSH 0.40 – 4.50 mIU/L), intermediate (TSH 4.51 – 10.00 mIU/L), and high (TSH >10.00 mIU/L). Non-hypothyroidism was the non-exposure (control). The primary outcomes were length of stay (LOS), in-hospital mortality, and readmission outcomes following medical and surgical admission.Results: A total of 46,235 patients were included in the final study population, of whom 9,394 had a diagnosis of hypothyroidism. Those with a high pre-hospitalization TSH level had a LOS that was 1.2 days longer (95% CI [1.1 – 1.3]; p = 0.001), a 41% higher risk of 30-day readmission (RR 1.41, 95% CI [1.13 – 1.75]; p = 0.002), and 33% higher rate of 90-day readmission (RR 1.33, 95% CI [1.13 – 1.56]; p <0.001) compared to balanced controls. Patients with intermediate TSH levels exhibited increased likelihood of 30-day readmission (RR 1.20, 95% CI [1.01 – 1.43]; p = 0.04). Conversely, those with a normal pre-hospitalization TSH level had a decreased risk of in-hospital mortality (RR 0.55, 95% CI [0.34 – 0.89]; p = 0.02) and 90-day readmission (RR 0.91, 95% CI [0.85 – 0.99]; p = 0.02).Conclusions: Suboptimal treatment of hypothyroidism appears to be associated with worse hospital outcomes, including longer LOS and higher rate of readmission. Public health strategies designed to deliver appropriate, consistent outpatient treatment of hypothyroidism are needed to improve hospital outcomes in those patients with off-target TSH levels.

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