Ovarian status modulates endocrine and neuroinflammatory responses to a murine mammary tumor.

Patients with breast cancer have increased circulating inflammatory markers and mammary tumors increase neuroinflammation in rodent models. Menopausal status is not only important in the context of breast cancer as circulating estrogen influences tumor progression, but also because estrogen is anti-inflammatory and an essential modulator of endocrine function in the brain and body. Here, we manipulated "menopause" status (ovary-intact and ovariectomized) in an estrogen receptor (ER)+ mouse mammary tumor model to determine the extent to which ovarian status modulates: 1) tumor effects on estrogen concentrations and signaling in the brain, 2) tumor effects on estrogen-associated neurobiology and inflammation, and 3) the ability for tumor resection to resolve the effects of a tumor. We hypothesized that reduced circulating estradiol (E2) after an ovariectomy exacerbates tumor-induced peripheral and central inflammation. Notably, we observed ovarian-dependent modulation on tumor-induced peripheral outcomes, including E2-dependent processes and, to a lesser degree, circulating inflammatory markers. In the brain, ovariectomy exacerbated neuroinflammatory markers in select brain regions and modulated E2-related neurobiology due to a tumor and/or resection. Overall, our data suggest that ovarian status has moderate implications for tumor-induced alterations in neuroendocrinology and neuroinflammation and mild effects on peripheral inflammatory outcomes in this murine mammary tumor model.

Paclitaxel Chemotherapy Elicits Widespread Brain Anisotropy Changes in a Comprehensive Mouse Model of Breast Cancer Survivorship: Evidence From In Vivo Diffusion Weighted Imaging.

Breast cancer is one of the most common diseases in the United States with 1 in 8 women developing the disease in her lifetime. Women who develop breast cancer are often post-menopausal and undergo a complex sequence of treatments including surgery, chemotherapy, and aromatase inhibitor therapy. Both independently and through potential interactions, these factors and treatments are associated with behavioral comorbidities reported in patients (e.g., fatigue), although the underlying neurobiological mechanisms are poorly understood. Currently, brain imaging is the most feasible way to assess neurobiology in patients. Indeed, breast cancer patients display alterations in white matter connections and chemotherapy is associated with decreased white and gray matter in the corpus callosum and cortex as well as decreased hippocampal volume. However, imaging in breast cancer rodent models is lacking, impeding translation of the mechanistic neurobiological findings made possible through modeling. Furthermore, current rodent models of breast cancer often lack the complexity of typical multimodal breast cancer treatments, thereby limiting translational value. The present study aimed to develop a comprehensive model of post-menopausal breast cancer survival using immunocompetent ovariectomized mice, including an orthotopic syngeneic tumor, surgical tumor removal, chemotherapy, and aromatase inhibitor therapy. Using this model, we systematically investigated the cumulative effects of chemotherapy and hormone replacement therapy on neurostructure and behavior using diffusion weighted imaging, open field test, and spontaneous alternation test. Our previous findings, in a simplified chemotherapy-only model, indicate that this regimen of chemotherapy causes circulating and central inflammation concurrent with reduced locomotor activity. The current study, in the more comprehensive model, has recapitulated the peripheral inflammation coincident with reduced locomotor activity as well as demonstrated that chemotherapy also drives widespread changes in brain anisotropy. Validating the clinical relevance of this comprehensive rodent breast cancer model will allow for additional neurobiological investigations of the interactions among various cancer components associated with behavioral comorbidities, as well as the relationship between these mechanisms and neurostructural imaging changes that can be measured in cancer patients.

Mammary tumors alter the fecal bacteriome and permit enteric bacterial translocation.

BACKGROUND: Cancer patients experience gastrointestinal and behavioral symptoms, and are at increased risk of systemic infection and inflammation. These conditions are a major source of morbidity and decreased quality of life prior to cancer treatment, but poorly defined etiologies impede successful treatment. The gastrointestinal microbiota shape inflammation, influence cancer progression and treatment, and colonize tumors. However, research has not directly determined if peripheral tumors influence the microbiome and intestinal physiology, thus influencing gastrointestinal and behavioral symptoms. Therefore, the purpose of this study was to examine consequences of orthotopic, syngeneic mammary tumor implantation, growth, and resection on fecal bacteriome composition and intestinal barrier function in relation to systemic inflammation and enteric bacterial translocation in mice. METHODS: Female mice were randomized to 3 experimental groups: sham surgical control, tumor recipients, and tumor recipients later receiving tumor-resection. Mice were sacrificed three weeks after tumor implantation or resection for collection of stool, colon, spleen, and brain tissue and analysis. RESULTS: Tumor-bearing mice exhibited several markers of colonic barrier disruption, including dampened expression of tight junction proteins (Cldn1 and Ocln) and elevated circulating lipopolysaccharide binding protein (LBP). Compromised colonic barrier integrity was associated with altered fecal bacterial profiles in tumor-mice, including lower relative abundance of Lactobacillus, but higher Bacteroides. Consistent with colonic barrier disruption and altered microbiomes, tumor-mice displayed markers of systemic inflammation including splenomegaly, higher splenic bacterial load, and elevated splenic and brain pro-inflammatory cytokines. Several  bacteria cultured from spleens had 16S rRNA gene amplicons matching those in fecal samples, suggesting they were of intestinal origin. Fecal Lactobacillus was highly-interrelated to physiological parameters disrupted by tumors via correlation network analysis. Tumor resection ameliorated circulating LBP, splenomegaly, and splenic cytokines, but not other parameters associated with loss of colonic barrier integrity and bacterial translocation. CONCLUSIONS: Orthotopic mammary tumors alter the microbiome, reduce intestinal barrier function, increase translocation of enteric bacteria, and alter systemic inflammation. This provides insight into how tumors commence gastrointestinal and behavioral symptoms prior to treatment, and identify targets for future therapeutics, such as probiotic Lactobacillus supplementation.

A novel targeted approach to delineate a role for estrogen receptor-ß in ameliorating murine mammary tumor-associated neuroinflammation.

PURPOSE: Circulating estrogens in breast cancer patients and survivors are often extremely low due to menopause and estrogen-reducing cancer treatments. Simultaneously, circulating inflammatory markers, and inflammatory proteins in brains of rodent tumor models, can be elevated and correlate with debilitating neurological and psychological comorbidities. Because estrogen has anti-inflammatory properties in the brain, we hypothesized that mammary tumor-induced neuroinflammation is driven, in part, by reduced brain estrogen signaling. METHODS: An ovariectomized mouse model of postmenopausal breast cancer utilizing the ERα-positive 67NR mammary tumor cell line was used for these experiments. A novel, orally bioavailable, and brain penetrant ERß agonist was administered daily via oral gavage. Following treatment, estrogen-responsive genes were measured in brain regions. Central and circulating inflammatory markers were measured via RT-qPCR and a multiplex cytokine array, respectively. RESULTS: We present novel findings that peripheral mammary tumors alter estrogen signaling genes including receptors and aromatase in the hypothalamus, hippocampus, and frontal cortex. Mammary tumors induced peripheral and central inflammation, however, pharmacological ERß activation was not sufficient to reduce this inflammation. CONCLUSIONS: Data presented here suggest that compensating for low circulating estrogen with ERß brain activation is not sufficient to attenuate mammary tumor-induced neuroinflammation, and is therefore not a likely candidate for the treatment of behavioral symptoms in patients. The novel finding that mammary tumors alter estrogen signaling-related genes is a clinically relevant advancement to the understanding of how peripheral tumor biology modulates neurobiology. This is necessary to predict and prevent behavioral comorbidities (e.g., cognitive impairment) prevalent in cancer patients and survivors.

Tumor-Induced Cardiac Dysfunction: A Potential Role of ROS.

Cancer and heart diseases are the two leading causes of mortality and morbidity worldwide. Many cancer patients undergo heart-related complications resulting in high incidences of mortality. It is generally hypothesized that cardiac dysfunction in cancer patients occurs due to cardiotoxicity induced by therapeutic agents, used to treat cancers and/or cancer-induced cachexia. However, it is not known if localized tumors or unregulated cell growth systemically affect heart function before treatment, and/or prior to the onset of cachexia, hence, making the heart vulnerable to structural or functional abnormalities in later stages of the disease. We incorporated complementary mouse and Drosophila models to establish if tumor induction indeed causes cardiac defects even before intervention with chemotherapy or onset of cachexia. We focused on one of the key pathways involved in irregular cell growth, the Hippo-Yorkie (Yki), pathway. We used overexpression of the transcriptional co-activator of the Yki signaling pathway to induce cellular overgrowth, and show that Yki overexpression in the eye tissue of Drosophila results in compromised cardiac function. We rescue these cardiac phenotypes using antioxidant treatment, with which we conclude that the Yki induced tumorigenesis causes a systemic increase in ROS affecting cardiac function. Our results show that systemic cardiac dysfunction occurs due to abnormal cellular overgrowth or cancer elsewhere in the body; identification of specific cardiac defects associated with oncogenic pathways can facilitate the possible early diagnosis of cardiac dysfunction.

Light at night exacerbates metabolic dysfunction in a polygenic mouse model of type 2 diabetes mellitus.

AIMS: Electric lighting is beneficial to modern society; however, it is becoming apparent that light at night (LAN) is not without biological consequences. Several studies have reported negative effects of LAN on health and behavior in humans and nonhuman animals. Exposure of non-diabetic mice to dim LAN impairs glucose tolerance, whereas a return to dark nights (LD) reverses this impairment. We predicted that exposure to LAN would exacerbate the metabolic abnormalities in TALLYHO/JngJ (TH) mice, a polygenic model of type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: We exposed 7-week old male TH mice to either LD or LAN for 8-10 weeks in two separate experiments. After 8 weeks of light treatment, we conducted intraperitoneal glucose tolerance testing (ipGTT) followed by intraperitoneal insulin tolerance testing (ipITT). In Experiment 1, all mice were returned to LD for 4 weeks, and ipITT was repeated. KEY FINDINGS: The major results of this study are i) LAN exposure for 8 weeks exacerbates glucose intolerance and insulin resistance ii) the effects of LAN on insulin resistance are reversed upon return to LD, iii) LAN exposure results in a greater increase in body weight compared to LD exposure, iv) LAN increases the incidence of mice developing overt T2DM, and v) LAN exposure decreases survival of mice with T2DM. SIGNIFICANCE: In conclusion, LAN exacerbated metabolic abnormalities in a polygenic mouse model of T2DM, and these effects were reversed upon return to dark nights. The applicability of these findings to humans with T2DM needs to be determined.

miR-155 deletion modulates lipopolysaccharide-induced sleep in female mice.

Immune signaling is known to regulate sleep. miR-155 is a microRNA that regulates immune responses. We hypothesized that miR-155 would alter sleep regulation. Thus, we investigated the potential effects of miR-155 deletion on sleep-wake behavior in adult female homozygous miR-155 knockout (miR-155KO) mice and littermate controls (WT). Mice were implanted with biotelemetry units and EEG/EMG biopotentials were recorded continuously for three baseline days. miR-155KO mice had decreased bouts of NREM and REM sleep compared with WT mice, but no differences were observed in the length of sleep bouts or total time spent in sleep-wake states. Locomotor activity and subcutaneous temperature did not differ between WT and miR-155KO mice. Following baseline recordings, mice were sleep-deprived during the first six hours of the rest phase (light phase; ZT 0-6) followed by an 18 h recovery period. There were no differences between groups in sleep rebound (% sleep and NREM δ power) after sleep deprivation. Following recovery from sleep deprivation, mice were challenged with a somnogen (viz., lipopolysaccharide (LPS)) one hour prior to the initiation of the dark (active) phase. Biopotentials were continuously recorded for the following 24 h, and miR-155KO mice displayed increased wakefulness and decreased NREM sleep during the dark phase following LPS injection. Additionally, miR-155KO mice had reduced EEG slow-wave responses (0.5-4 Hz) compared to WT mice. Together, our findings indicate that miR-155 deletion attenuates the somnogenic and EEG delta-enhancing effects of LPS. Abbreviations: ANOVA: analysis of variance; EEG: electroencephalogram; EMG: electromyogram; h: hour; IL-1: interleukin-1; IL-6: interleukin-6; IP: intra-peritoneal; LPS: lipopolysaccharide; miR/miRNA: microRNA; miR-155KO: miR-155 knockout; NREM: non-rapid eye movement; REM: rapid eye movement; TNF: tumor necrosis factor; SWS: slow-wave sleep; WT: wild-type.

Effects of light at night on laboratory animals and research outcomes.

Light has substantial influences on the physiology and behavior of most laboratory animals. As such, lighting conditions within animal rooms are potentially significant and often underappreciated variables within experiments. Disruption of the light/dark cycle, primarily by exposing animals to light at night (LAN), disturbs biological rhythms and has widespread physiological consequences because of mechanisms such as melatonin suppression, sympathetic stimulation, and altered circadian clock gene expression. Thus, attention to the lighting environment of laboratory animals and maintaining consistency of a light/dark cycle is imperative for study reproducibility. Light intensity, as well as wavelength, photoperiod, and timing, are all important variables. Although modern rodent facilities are designed to facilitate appropriate light cycling, there are simple ways to modify rooms to prevent extraneous light exposure during the dark period. Attention to lighting conditions of laboratory animals by both researchers and research care staff ensures best practices for maintaining animal welfare, as well as reproducibility of research results. (PsycINFO Database Record

Artificial light at night alters behavior in laboratory and wild animals.

Life has evolved to internalize and depend upon the daily and seasonal light cycles to synchronize physiology and behavior with environmental conditions. The nightscape has been vastly changed in response to the use of artificial lighting. Wildlife is now often exposed to direct lighting via streetlights or indirect lighting via sky glow at night. Because many activities rely on daily and seasonal light cues, the effects of artificial light at night could be extensive, but remain largely unknown. Laboratory studies suggest exposure to light at night can alter typical timing of daily locomotor activity and shift the timing of foraging/food intake to the daytime in nocturnal rodents. Additionally, nocturnal rodents decrease anxiety-like behaviors (i.e., spend more time in the open and increase rearing up) in response to even dim light at night. These are all likely maladaptive responses in the wild. Photoperiodic animals rely on seasonal changes in day length as a cue to evoke physiological and behavioral modifications to anticipate favorable and unfavorable conditions for survival and reproduction. Light at night can mask detection of short days, inappropriately signal long days, and thus desynchronize seasonal reproductive activities. We review laboratory and the sparse field studies that address the effects of exposure to artificial light at night to propose that exposure to light at night disrupts circadian and seasonal behavior in wildlife, which potentially decreases individual fitness and modifies ecosystems.

Elevated aggressive behavior in male mice with thyroid-specific Prkar1a and global Epac1 gene deletion.

Alterations in circulating thyroid hormone concentrations are associated with several psychological and behavioral disorders. In humans, behavioral disorders such as anxiety, depression, and attention-deficit hyperactivity disorder can be associated with thyroid disease. The Tpo-Cre;Prkar1aflox/flox;Epac1-/- (R1A-Epac1KO) mice, originally bred to investigate the role of exchange protein directly activated by cAMP (Epac1) in follicular thyroid cancer, displayed self-mutilating and aggressive behaviors during casual observation. To assess these atypical responses, behavioral testing was conducted with the R1A-Epac1KO mice, as well as their single knockout counterparts, the thyroid-specific Prkar1a-/- and global Epac1-/- mice. Mice of all three genotypes demonstrated increased aggressive behavior against an intruder mouse. In addition, Epac1-/- mice increased response to an auditory stimulus, and the Prkar1a-/- and R1A-Epac1KO mice increased swimming behavior in the Porsolt forced swim test. Both Prkar1a-/- mice and R1A-Epac1KO mice have increased circulating thyroxine and corticosterone concentrations. Although hyperthyroidism has not been previously associated with aggression, increased thyroid hormone signaling might contribute to the increased aggressive response to the intruder mouse, as well as the increased swimming response. Mice with a genetic background of Tpo-Cre;Prkar1aflox/flox;Epac1-/- are aggressive, and both the thyroid-specific knockout of Prkar1a and global knockout of Epac1 likely contribute to this aggressive behavior. This study supports the hypothesis that altered thyroid signaling and aggressive behavior are linked.

Light at night as an environmental endocrine disruptor.

Environmental endocrine disruptors (EEDs) are often consequences of human activity; however, the effects of EEDs are not limited to humans. A primary focus over the past ∼30years has been on chemical EEDs, but the repercussions of non-chemical EEDs, such as artificial light at night (LAN), are of increasing interest. The sensitivity of the circadian system to light and the influence of circadian organization on overall physiology and behavior make the system a target for disruption with widespread effects. Indeed, there is increasing evidence for a role of LAN in human health, including disruption of circadian regulation and melatonin signaling, metabolic dysregulation, cancer risk, and disruption of other hormonally-driven systems. These effects are not limited to humans; domesticated animals as well as wildlife are also exposed to LAN, and at risk for disrupted circadian rhythms. Here, we review data that support the role of LAN as an endocrine disruptor in humans to be considered in treatments and lifestyle suggestions. We also present the effects of LAN in other animals, and discuss the potential for ecosystem-wide effects of artificial LAN. This can inform decisions in agricultural practices and urban lighting decisions to avoid unintended outcomes.

Depressive-like behavior is elevated among offspring of parents exposed to dim light at night prior to mating.

Rates of major depressive disorder (MDD) have steadily increased over the past 50 years. Many factors have been implicated in the etiology of depressive disorders and environmental influences are being increasingly recognized. The increase in depression rates has coincided with increased artificial nighttime lighting. Exposure to light at night (LAN) has been associated with increased depressive-like behavior in rodents and decreased mood in humans. However, relatively little is known on the multigenerational effects of dLAN on affect. In this study, we exposed adult male and female Siberian hamsters (Phodopus sungorus) to either DARK (0lx) or dim LAN (5lx) for 9 weeks, then paired animals in a full factorial design; all animals were thereafter housed in dark nights. Offspring were gestated and reared in dark nights, then tested in adulthood for depressive-like behaviors and hippocampal expression of glucocorticoid (GR) and melatonin (MT1) receptor expression. Maternal exposure to dLAN decreased sucrose preference, time to first float bout in the Porsolt swim test, and GR expression in the hippocampus. Paternal exposure to dLAN increased time spent floating, and increased hippocampal GR expression. Overall, our results suggest that chronic exposure of parents to light at night has multigenerational effects on offspring depressive-like behavior. If these results pertain to humans, then our data suggest that LAN may contribute to the rapidly rising rates of major depressive disorder in industrialized and developing countries.

Parental Exposure to Dim Light at Night Prior to Mating Alters Offspring Adaptive Immunity.

Exposure to dim light at night (dLAN) disrupts natural light/dark cycles and impairs endogenous circadian rhythms necessary to maintain optimal biological function, including the endocrine and immune systems. We have previously demonstrated that white dLAN compromises innate and cell mediated immune responses in adult Siberian hamsters (Phodopus sungorus). We hypothesized that dLAN has transgenerational influences on immune function. Adult male and female Siberian hamsters were exposed to either dark nights (DARK) or dLAN (~5 lux) for 9 weeks, then paired in full factorial design, mated, and thereafter housed under dark nights. Offspring were gestated and reared in dark nights, then tested as adults for cell-mediated and humoral immunity. Maternal exposure to dLAN dampened delayed type hypersensitivity (DTH) responses in male offspring. Maternal and paternal exposure to dLAN reduced DTH responses in female offspring. IgG antibodies to a novel antigen were elevated in offspring of dams exposed to dLAN. Paternal exposure to dLAN decreased splenic endocrine receptor expression and global methylation in a parental sex-specific manner. Together, these data suggest that exposure to dLAN has transgenerational effects on endocrine-immune function that may be mediated by global alterations in the epigenetic landscape of immune tissues.

Atrazine alters expression of reproductive and stress genes in the developing hypothalamus of the snapping turtle, Chelydra serpentina.

Atrazine is an herbicide used to control broadleaf grasses and a suspected endocrine disrupting chemical. Snapping turtles lay eggs between late May and early June, which could lead to atrazine exposure via field runoff. Our goal was to determine whether a single exposure to 2ppb or 40ppb atrazine during embryogenesis could induce short- and long-term changes in gene expression within the hypothalamus of snapping turtles. We treated eggs with atrazine following sex determination and measured gene expression within the hypothalamus. We selected genes a priori for their role in the hypothalamus-pituitary-gonad or the hypothalamus-pituitary-adrenal axes of the endocrine system. We did not identify any changes in gene expression 24-h after treatment. However, at hatching AR, Kiss1R, and POMC expression was upregulated in both sexes, while expression of CYP19A1 and PDYN was increased in females. Six months after hatching, CYP19A1 and PRLH expression was increased in animals treated with 2ppb atrazine. Our study shows persistent changes in hypothalamic gene expression due to low-dose embryonic exposure to the herbicide atrazine with significant effects in both the HPG and HPA axes. Effects reported here appear to be conserved among vertebrates.