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Environmental chemicals are a persistent and pervasive part of everyday life. A subset of environmental chemicals are xenoestrogens, compounds that bind to the estrogen receptor (ER) and drive estrogen-related processes. One such chemical, benzophenone-3 (BP3), is a common chemical in sunscreen. It is a potent UV protectant but also is quickly absorbed through the skin. While it has been approved by the FDA, there is a renewed interest in the safety of BP3, particularly in relation to breast cancer. The focus of this study was to examine the impact that BP3 has on triple negative breast cancer (TNBC) through alterations to cells in the immune microenvironment. In this study, we exposed female mice to one of two doses of BP3 before injecting them with a TNBC cell line. Several immune endpoints were examined both in the primary tissues and from in vitro studies of T cell behavior. Our studies revealed that in the lung tumor microenvironment, exposure to BP3 not only increased the number of metastases, but also the total area of tumor coverage. We also found that BP3 caused alterations in immune populations in a tissue-dependent manner, particularly in T cells. Taken together, our data suggest that while BP3 may not directly affect the proliferation of TNBC, growth and metastasis of TNBC-derived tumors can be altered by BP3 exposures via the alterations in the immune populations of the tumor microenvironment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PCB 126 is a pervasive, dioxin-like chemical pollutant which can activate the aryl hydrocarbon receptor (AhR). Despite being banned from the market, PCB 126 can be detected in breast milk to this day. The extent to which interindividual variation impacts the adverse responses to this chemical in the breast tissue remains unclear. This study aimed to investigate the impact of 3 nM PCB 126 on gene expression in a panel of genetically diverse benign human breast epithelial cell (HBEC) cultures and patient derived breast tissues. Six patient derived HBEC cultures were treated with 3 nM PCB 126. RNAseq was used to interrogate the impact of exposure on differential gene expression. Gene expression changes from the top critical pathways were confirmed via qRT-PCR in a larger panel of benign patient derived HBEC cultures, as well as in patient-derived breast tissue explant cultures. RNAseq analysis of HBEC cultures revealed a signature of 144 genes significantly altered by 3 nM PCB 126 treatment. Confirmation of 8 targets using a panel of 12 HBEC cultures and commercially available breast cell lines demonstrated that while the induction of canonical downstream target gene, CYP1A1, was consistent across our primary HBECs, other genes including AREG, S100A8, IL1A, IL1B, MMP7, and CCL28 exhibited significant variability across individuals. The dependence on the activity of the aryl hydrocarbon receptor was confirmed using inhibitors. PCB 126 can induce significant and consistent changes in gene expression associated with xenobiotic metabolism in benign breast epithelial cells. Although the induction of most genes was reliant on the AhR, significant variability was noted between genes and individuals. These data suggest that there is a bifurcation of the pathway following AhR activation that contributes to the variation in interindividual responses., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ex vivo mammary explant systems are an excellent model to study interactions between epithelium and stromal cell types because they contain physiologically relevant heterotypic interactions in the background of genetically diverse patients. The intact human mammary tissue, termed patient-derived explant (PDE), can be used to investigate cellular responses to a wide variety of external stimuli in situ. For this study, we examined the impact of cytokines or environmental chemicals on macrophage phenotypes. We demonstrate that we can polarize macrophages within human breast tissue PDEs toward M1 or M2 through the addition of interferon-γ (IFNγ) + lipopolysaccharide (LPS) or interleukin (IL)-4 + IL-13, respectively. Elevated expression levels of M(IFNγ + LPS) markers (HLADRA and CXCL10) or M(IL-4 + IL-13) markers (CD209 and CCL18) were observed in cytokine-treated tissues. We also examined the impact of the endocrine-disrupting chemical, benzophenone-3, on PDEs and measured significant, yet varying effects on macrophage polarization. Furthermore, a subset of the PDEs respond to IL-4 + IL-13 through downregulation of E-cadherin and upregulation of vimentin which is reminiscent of epithelial-to-mesenchymal transition (EMT) changes. Finally, we were able to show immortalized nonmalignant breast epithelial cells can exhibit EMT characteristics when exposed to growth factors secreted by M(IL-4 + IL-13) macrophages. Taken together, the PDE model system is an outstanding preclinical model to study early tissue-resident immune responses and effects on epithelial and stromal responses to stimuli found both endogenously in the breast and exogenously as a result of exposures., (© 2020 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology Inc.)

We aimed to develop effective radioligands for quantifying brain O -linked-β- N -acetyl-glucosamine ( O -GlcNAc) hydrolase (OGA) using positron emission tomography in living subjects as tools for evaluating drug target engagement. Posttranslational modifications of tau, a biomarker of Alzheimer's disease, by O -GlcNAc through the enzyme pair OGA and O -GlcNAc transferase (OGT) are inversely related to the amounts of its insoluble hyperphosphorylated form. Increase in tau O -GlcNAcylation by OGA inhibition is believed to reduce tau aggregation. LSN3316612, a highly selective and potent OGA ligand [half-maximal inhibitory concentration (IC 50 ) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [ 3 H]LSN3316612 imaged and quantified OGA in postmortem brains of rat, monkey, and human. The presence of fluorine and carbonyl functionality in LSN3316612 enabled labeling with positron-emitting fluorine-18 or carbon-11. Both [ 18 F]LSN3316612 and [ 11 C]LSN3316612 bound reversibly to OGA in vivo, and such binding was blocked by pharmacological doses of thiamet G, an OGA inhibitor of different chemotype, in monkeys. [ 18 F]LSN3316612 entered healthy human brain avidly (~4 SUV) without radiodefluorination or adverse effect from other radiometabolites, as evidenced by stable brain total volume of distribution (V T ) values by 110 min of scanning. Overall, [ 18 F]LSN3316612 is preferred over [ 11 C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: The objective of this investigation was to examine the distribution of galcanezumab and a control immunoglobulin 4 antibody containing the same constant regions as galcanezumab, into peripheral and central tissues., Methods: Galcanezumab and a control immunoglobulin 4 antibody were radioiodinated with Iodine-125 to specific activities of 0.11 mCi/mg and 0.16 mCi/mg, respectively. At 24, 72, and 168 hours following subcutaneous injection of either antibody (4 mg/kg), cerebrospinal fluid and plasma were obtained followed by saline perfusion to remove residual blood and collection of selected tissues for determination of Iodine-125 content by gamma counting., Results: The peak plasma levels of Iodine-125 galcanezumab and Iodine-125 control immunoglobulin 4 were observed at 72 hours and remained high at 168 hours post-dose. The rank order of tissue levels was dura mater = spleen > trigeminal ganglia ≫hypothalamus = spinal cord = prefrontal cortex = cerebellum. Iodine-125 galcanezumab levels in peripheral tissue (dura mater, spleen, and trigeminal ganglia) averaged 5% to 11% of plasma, whereas all of the central nervous system (CNS) tissue levels and the cerebrospinal fluid levels were < 0.4% of plasma. Distribution of the antibodies into the dura mater and the trigeminal ganglia was similar to that observed in the spleen and significantly greater than exposure in the brain or spinal cord., Conclusions: The central levels of galcanezumab were relatively low, which would favor the dura mater and trigeminal ganglia as sites of action for its observed clinical efficacy. However, a central site of action cannot be excluded.

Background Many patients with migraines suffer from allergies and vice versa, suggesting a relationship between biological mechanisms of allergy and migraine. It was proposed many years ago that mast cells may be involved in the pathophysiology of migraines. We set out to investigate the relationship between mast cell activation and known neurogenic peptides related to migraine. Methods Cultured human mast cells were assayed for the presence of neuropeptides and their receptors at the RNA and protein level. Immunohistochemistry analyses were performed on tissue resident and cultured mast cells. Mast cell degranulation assays were performed and pituitary adenylate cyclase-activating polypeptide (PACAP) activity was measured with a bioassay. Results We found that cultured and tissue resident human mast cells contain PACAP in cytoplasmic granules. No other neurogenic peptide known to be involved in migraine was detected, nor did mast cells express the receptors for PACAP or other neurogenic peptides. Furthermore, mast cell degranulation through classic IgE-mediated allergic mechanisms led to the release of PACAP. The PACAP released from mast cells was biologically active, as demonstrated using PACAP receptor reporter cell lines. We confirmed existing literature that mast cell degranulation can also be induced by several neurogenic peptides, which also resulted in PACAP release. Conclusion Our data provides a potential biological explanation for the association between allergy and migraine by demonstrating the release of biologically active PACAP from mast cells.

Metabolic reprograming is a hallmark of cancer cells. However, the roles of pre-existing differences in normal cells metabolism toward cancer risk is not known. In order to assess pre-existing variations in normal cell metabolism, we have quantified the inter-individual variation in oxidative metabolism of normal primary human mammary epithelial cells (HMECs). We then assessed their response to selected cytokines such as insulin growth factor 1 (IGF1) and tumor necrosis factor alpha (TNFα), which are associated with breast cancer risk. Specifically, we compared the oxidative metabolism of HMECs obtained from women with breast cancer and without cancer. Our data show considerable inter-individual variation in respiratory activities of HMECs from different women. A bioenergetic parameter called pyruvate-stimulated respiration (PySR) was identified as a key distinguishing feature of HMECs from women with breast cancer and without cancer. Samples showing PySR over 20% of basal respiration rate were considered PySR +ve and the rest as PySR -ve . By this criterion, HMECs from tumor-affected breasts (AB) and non-tumor affected breasts (NAB) of cancer patients were mostly PySR -ve (88% and 89%, respectively), while HMECs from non-cancer patients were mostly PySR +ve (57%). This suggests that PySR -ve/+ve phenotypes are individual-specific and are not caused by field effects due to the presence of tumor. The effects of IGF1 and TNFα treatments on HMECs revealed that both suppressed respiration and extracellular acidification. In addition, IGF1 altered PySR -ve/+ve phenotypes. These results reveal individual-specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk., (© 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

Background: Secreted frizzled-related protein 1 (SFRP1) expression is down-regulated in a multitude of cancers, including breast cancer. Loss of Sfrp1 also exacerbates weight gain as well as inflammation. Additionally, loss of SFRP1 enhances TGF-β signaling and the downstream MAPK pathway. TGF-β has been shown to increase the expression of Early Growth Response 2 (EGR2), a transcription factor implicated in immune function in a wide variety of cell types. The work described here was initiated to determine whether SFRP1 modulation affects TGF-β mediated EGR2 expression in mammary tissues as well as macrophage polarization., Methods: Real-time PCR analysis was performed to examine EGR2 expression in human and murine mammary epithelial cells and tissues in response to SFRP1 modulation. Chemical inhibition was employed to investigate the roles TGF-β and MAPK signaling play in the control of EGR2 expression in response to SFRP1 loss. Primary murine macrophages were isolated from Sfrp1 -/- mice and stimulated to become either M1 or M2 macrophages, treated with recombinant SFRP1, and real-time PCR was used to measure the expression of murine specific M1/M2 markers [Egr2 (M2) and Gpr18 (M1)]. Immunohistochemical analysis was used to measure the expression of human specific M1/M2 markers [CD163 (M2) and HLA-DRA (M2)] in response to rSFRP1 treatment in human mammary explant tissue., Results: Knockdown of SFRP1 expression increases the expression of EGR2 mRNA in human mammary epithelial cells and addition of rSFRP1 decreases the expression of EGR2 when added to explant mammary gland tissues. Chemical inhibition of both TGF-β and MAPK signaling in Sfrp1 -/- or knockdown mammary epithelial cells results in decreased expression of EGR2. Stimulated murine macrophages obtained from Sfrp1 -/- mice and treated with rSFRP1 exhibit a reduction in Egr2 expression and an increase in Gpr18 mRNA expression. Human mammary explant tissue treated with rSFRP1 decreases CD163 protein expression whereas there was no effect on the expression of HLA-DRA., Conclusions: Loss of SFRP1 likely contributes to tumor progression by altering the expression of a critical transcription factor in both the epithelium and the immune system.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a K b of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists., (Copyright © 2016 The Author(s).)