Gene deletion of the PACAP/VIP receptor, VPAC2R, alters glycemic responses during metabolic and psychogenic stress in adult female mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the homologous peptide, vasoactive intestinal peptide (VIP), participate in glucose homeostasis using insulinotropic and counterregulatory processes. The role of VIP receptor 2 (VPAC2R) in these opposing actions needs further characterization. In this study, we examined the participation of VPAC2R on basal glycemia, fasted levels of glucoregulatory hormones and on glycemia responses during metabolic and psychogenic stress using gene-deleted (Vipr2-/- ) female mice. The mean basal glycemia was significantly greater in Vipr2-/- in the fed state and after an 8-h overnight fast as compared to wild-type (WT) mice. Insulin tolerance testing following a 5-h fast (morning fast, 0.38 U/kg insulin) indicated no effect of genotype. However, during a more intense metabolic challenge (8 h, ON fast, 0.25 U/kg insulin), Vipr2-/- females displayed significantly impaired insulin hypoglycemia. During immobilization stress, the hyperglycemic response and plasma epinephrine levels were significantly elevated above basal in Vipr2-/- , but not WT mice, in spite of similar stress levels of plasma corticosterone. Together, these results implicate participation of VPAC2R in upregulated counterregulatory processes influenced by enhanced sympathoexcitation. Moreover, the suppression of plasma GLP-1 levels in Vipr2-/- mice may have removed the inhibition on hepatic glucose production and the promotion of glucose disposal by GLP-1. qPCR analysis indicated deregulation of central gene markers of PACAP/VIP signaling in Vipr2-/- , upregulated medulla tyrosine hydroxylase (Th) and downregulated hypothalamic Vip transcripts. These results demonstrate a physiological role for VPAC2R in glucose metabolism, especially during insulin challenge and psychogenic stress, likely involving the participation of sympathoadrenal activity and/or metabolic hormones.

Glucoregulatory disruption in male mice offspring induced by maternal transfer of endocrine disrupting brominated flame retardants in DE-71.

Introduction: Polybrominated diphenyl ethers (PBDEs) are commercially used flame retardants that bioaccumulate in human tissues, including breast milk. PBDEs produce endocrine and metabolic disruption in experimental animals and have been associated with diabetes and metabolic syndrome (MetS) in humans, however, their sex-specific diabetogenic effects are not completely understood. Our past works show glucolipid dysregulation resulting from perinatal exposure to the commercial penta-mixture of PBDEs, DE-71, in C57BL/6 female mice. Methods: As a comparison, in the current study, the effects of DE-71 on glucose homeostasis in male offspring was examined. C57BL/6N dams were exposed to DE-71 at 0.1 mg/kg/d (L-DE-71), 0.4 mg/kg/d (H-DE-71), or received corn oil vehicle (VEH/CON) for a total of 10 wks, including gestation and lactation and their male offspring were examined in adulthood. Results: Compared to VEH/CON, DE-71 exposure produced hypoglycemia after a 11 h fast (H-DE-71). An increased fast duration from 9 to 11 h resulted in lower blood glucose in both DE-71 exposure groups. In vivo glucose challenge showed marked glucose intolerance (H-DE-71) and incomplete clearance (L- and H-DE-71). Moreover, L-DE-71-exposed mice showed altered glucose responses to exogenous insulin, including incomplete glucose clearance and/or utilization. In addition, L-DE-71 produced elevated levels of plasma glucagon and the incretin, active glucagon-like peptide-1 (7-36) amide (GLP-1) but no changes were detected in insulin. These alterations, which represent criteria used clinically to diagnose diabetes in humans, were accompanied with reduced hepatic glutamate dehydrogenase enzymatic activity, elevated adrenal epinephrine and decreased thermogenic brown adipose tissue (BAT) mass, indicating involvement of several organ system targets of PBDEs. Liver levels of several endocannabinoid species were not altered. Discussion: Our findings demonstrate that chronic, low-level exposure to PBDEs in dams can dysregulate glucose homeostasis and glucoregulatory hormones in their male offspring. Previous findings using female siblings show altered glucose homeostasis that aligned with a contrasting diabetogenic phenotype, while their mothers displayed more subtle glucoregulatory alterations, suggesting that developing organisms are more susceptible to DE-71. We summarize the results of the current work, generated in males, considering previous findings in females. Collectively, these findings offer a comprehensive account of differential effects of environmentally relevant PBDEs on glucose homeostasis and glucoregulatory endocrine dysregulation of developmentally exposed male and female mice.

Developmental exposure to indoor flame retardants and hypothalamic molecular signatures: Sex-dependent reprogramming of lipid homeostasis.

Polybrominated diphenyl ethers (PBDEs) are a class of flame-retardant organohalogen pollutants that act as endocrine/neuroendocrine disrupting chemicals (EDCs). In humans, exposure to brominated flame retardants (BFR) or other environmentally persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) and novel organophosphate flame retardants has been associated with increasing trends of diabetes and metabolic disease. However, the effects of PBDEs on metabolic processes and their associated sex-dependent features are poorly understood. The metabolic-disrupting effects of perinatal exposure to industrial penta-PBDE mixture, DE-71, on male and female progeny of C57BL/6N mouse dams were examined in adulthood. Dams were exposed to environmentally relevant doses of PBDEs daily for 10 weeks (p.o.): 0.1 (L-DE-71) and 0.4 mg/kg/d (H-DE-71) and offspring parameters were compared to corn oil vehicle controls (VEH/CON). The following lipid metabolism indices were measured: plasma cholesterol, triglycerides, adiponectin, leptin, and liver lipids. L-DE-71 female offspring were particularly affected, showing hypercholesterolemia, elevated liver lipids and fasting plasma leptin as compared to same-sex VEH/CON, while L- and H-DE-71 male F1 only showed reduced plasma adiponectin. Using the quantitative Folch method, we found that mean liver lipid content was significantly elevated in L-DE-71 female offspring compared to controls. Oil Red O staining revealed fatty liver in female offspring and dams. General measures of adiposity, body weight, white and brown adipose tissue (BAT), and lean and fat mass were weighed or measured using EchoMRI. DE-71 did not produce abnormal adiposity, but decreased BAT depots in L-DE-71 females and males relative to same-sex VEH/CON. To begin to address potential central mechanisms of deregulated lipid metabolism, we used RT-qPCR to quantitate expression of hypothalamic genes in energy-regulating circuits that control lipid homeostasis. Both doses of DE-71 sex-dependently downregulated hypothalamic expression of Lepr, Stat3, Mc4r, Agrp, Gshr in female offspring while H-DE-71 downregulated Npy in exposed females relative to VEH/CON. In contrast, exposed male offspring displayed upregulated Stat3 and Mc4r. Intestinal barrier integrity was measured using FITC-dextran since it can lead to systemic inflammation that leads to liver damage and metabolic disease, but was not affected by DE-71 exposure. These findings indicate that maternal transfer of PBDEs disproportionately endangers female offspring to lipid metabolic reprogramming that may exaggerate risk for adult metabolic disease.

Development and validation of an LC-MS/MS methodology for the quantification of thyroid hormones in dko MCT8/OATP1C1 mouse brain.

The Allan-Herndon Dudley Syndrome (AHDS) is a rare disease caused by the progressive loss of monocarboxylate transporter 8 (MCT8). In patients with AHDS, the absence of MCT8 impairs transport of thyroid hormones (TH) through the blood brain barrier, leading to a central state of TH deficiency. In mice, the AHDS is mimicked by simultaneous deletion of the TH transporters MCT8 and the solute carrier organic anion transporter family member 1c1 (OATP1C1). To support preclinical mouse studies, an analytical methodology was developed and successfully applied for quantifying selected thyroid hormones in mouse whole brain and in specific regions using liquid chromatography tandem mass-spectrometry (LC-MS/MS). An important requirement for the methodology was its high sensitivity since a very low concentration of THs was expected in MCT8/OATP1C1 double-knockout (dko) mouse brain. Seven THs were targeted: L-thyroxine (T4), 3,3´,5-triiodo-L-thyronine-thyronine (T3), 3,3´,5´-triiodo-L-thyronine-thyronine (rT3), 3,3´-diiodo-L-thyronine (3,3´-T2, T2), 3,5-diiodo-L-thyronine (rT2, 3,5-T2), 3-iodo-L-thyronine (T1), 3-iodothyronamine (T1AM). Isotope dilution liquid chromatography triple-quadrupole mass spectrometry methodology was applied for detection and quantification. The method was validated in wild-type animals for mouse whole brain and for five different brain regions (hypothalamus, hippocampus, prefrontal cortex, brainstem and cortex). Instrumental calibration curves ranged from 0.35 to 150 pg/µL with good linearity (r2 >0.996). The limit of quantification was from 0.08 to 0.6 pg/mg, with an intra- and inter-day precision of 4.2-14.02% and 0.4-17.9% respectively, and accuracies between 84.9% and 114.8% when the methodology was validated for the whole brain. In smaller, distinct brain regions, intra- and inter-day precision were 0.6-20.7% and 2.5-15.6% respectively, and accuracies were 80.2-128.6%. The new methodology was highly sensitive and allowed for the following quantification in wild-type mice: (i) for the first time, four distinct thyroid hormones (T4, T3, rT3 and 3,3´-T2) in only approximately 100 mg of mouse brain were detected; (ii) the quantification of T4 and T3 for the first time in distinct mouse brain regions were reported. Further, application of our method to MCT8/OATP1C1 dko mice revealed the expected, relative lack of T3 and T4 uptake into the brain, and confirmed the utility of our analytical method to study TH transport across the blood brain barrier in a preclinical model of central TH deficiency.

Persistent exercise fatigue and associative learning deficits in combination with transient glucose dyshomeostasis in a mouse model of Gulf War Illness.

AIMS: To characterize exercise fatigue, metabolic phenotype and cognitive and mood deficits correlated with brain neuroinflammatory and gut microbiome changes in a chronic Gulf War Illness (GWI) mouse model. The latter have been described in an accompanying paper [1]. MAIN METHODS: Adult male C57Bl/6N mice were exposed for 28 days (5 days/week) to pyridostigmine bromide: 6.5 mg/kg, b.i.d., P.O. (GW1) or 8.7 mg/kg, q.d., P.O. (GW2); topical permethrin (1.3 mg/kg in 100% DMSO) and N,N-diethyl-meta-toluamide (DEET 33% in 70% EtOH) and restraint stress (5 min). Exercise, metabolic and behavioral endpoints were compared to sham stress control (CON/S). KEY FINDINGS: Relative to CON/S, GW2 presented persistent exercise intolerance (through post-treatment (PT) day 161), deficient associative learning/memory, and transient insulin insensitivity. In contrast to GW2, GW1 showed deficient long-term object recognition memory, milder associative learning/memory deficit, and behavioral despair. SIGNIFICANCE: Our findings demonstrate that GW chemicals dose-dependently determine the presentation of exercise fatigue and severity/type of cognitive/mood-deficient phenotypes that show persistence. Our comprehensive mouse model of GWI recapitulates the major multiple symptom domains characterizing GWI, including fatigue and cognitive impairment that can be used to more efficiently develop diagnostic tests and curative treatments for ill Gulf War veterans.

Induction of distinct neuroinflammatory markers and gut dysbiosis by differential pyridostigmine bromide dosing in a chronic mouse model of GWI showing persistent exercise fatigue and cognitive impairment.

AIMS: To characterize neuroinflammatory and gut dysbiosis signatures that accompany exaggerated exercise fatigue and cognitive/mood deficits in a mouse model of Gulf War Illness (GWI). METHODS: Adult male C57Bl/6N mice were exposed for 28 d (5 d/wk) to pyridostigmine bromide (P.O.) at 6.5 mg/kg/d, b.i.d. (GW1) or 8.7 mg/kg/d, q.d. (GW2); topical permethrin (1.3 mg/kg), topical N,N-diethyl-meta-toluamide (33%) and restraint stress (5 min). Animals were phenotypically evaluated as described in an accompanying article [124] and sacrificed at 6.6 months post-treatment (PT) to allow measurement of brain neuroinflammation/neuropathic pain gene expression, hippocampal glial fibrillary acidic protein, brain Interleukin-6, gut dysbiosis and serum endotoxin. KEY FINDINGS: Compared to GW1, GW2 showed a more intense neuroinflammatory transcriptional signature relative to sham stress controls. Interleukin-6 was elevated in GW2 and astrogliosis in hippocampal CA1 was seen in both GW groups. Beta-diversity PCoA using weighted Unifrac revealed that gut microbial communities changed after exposure to GW2 at PT188. Both GW1 and GW2 displayed systemic endotoxemia, suggesting a gut-brain mechanism underlies the neuropathological signatures. Using germ-free mice, probiotic supplementation with Lactobacillus reuteri produced less gut permeability than microbiota transplantation using GW2 feces. SIGNIFICANCE: Our findings demonstrate that GW agents dose-dependently induce differential neuropathology and gut dysbiosis associated with cognitive, exercise fatigue and mood GWI phenotypes. Establishment of a comprehensive animal model that recapitulates multiple GWI symptom domains and neuroinflammation has significant implications for uncovering pathophysiology, improving diagnosis and treatment for GWI.

Persistent autism-relevant behavioral phenotype and social neuropeptide alterations in female mice offspring induced by maternal transfer of PBDE congeners in the commercial mixture DE-71.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous persistent organic pollutants (POPs) that are known neuroendocrine disrupting chemicals with adverse neurodevelopmental effects. PBDEs may act as risk factors for autism spectrum disorders (ASD), characterized by abnormal psychosocial functioning, although direct evidence is currently lacking. Using a translational exposure model, we tested the hypothesis that maternal transfer of a commercial mixture of PBDEs, DE-71, produces ASD-relevant behavioral and neurochemical deficits in female offspring. C57Bl6/N mouse dams (F0) were exposed to DE-71 via oral administration of 0 (VEH/CON), 0.1 (L-DE-71) or 0.4 (H-DE-71) mg/kg bw/d from 3 wk prior to gestation through end of lactation. Mass spectrometry analysis indicated in utero and lactational transfer of PBDEs (in ppb) to F1 female offspring brain tissue at postnatal day (PND) 15 which was reduced by PND 110. Neurobehavioral testing of social novelty preference (SNP) and social recognition memory (SRM) revealed that adult L-DE-71 F1 offspring display deficient short- and long-term SRM, in the absence of reduced sociability, and increased repetitive behavior. These effects were concomitant with reduced olfactory discrimination of social odors. Additionally, L-DE-71 exposure also altered short-term novel object recognition memory but not anxiety or depressive-like behavior. Moreover, F1 L-DE-71 displayed downregulated mRNA transcripts for oxytocin (Oxt) in the bed nucleus of the stria terminalis (BNST) and supraoptic nucleus, and vasopressin (Avp) in the BNST and upregulated Avp1ar in BNST, and Oxtr in the paraventricular nucleus. Our work demonstrates that developmental PBDE exposure produces ASD-relevant neurochemical, olfactory processing and behavioral phenotypes that may result from early neurodevelopmental reprogramming within central social and memory networks.

Maternal transfer of environmentally relevant polybrominated diphenyl ethers (PBDEs) produces a diabetic phenotype and disrupts glucoregulatory hormones and hepatic endocannabinoids in adult mouse female offspring.

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardant chemicals and environmental contaminants with endocrine-disrupting properties that are associated with diabetes and metabolic syndrome in humans. However, their diabetogenic actions are not completely characterized or understood. In this study, we investigated the effects of DE-71, a commercial penta-mixture of PBDEs, on glucoregulatory parameters in a perinatal exposure model using female C57Bl/6 mice. Results from in vivo glucose and insulin tolerance tests and ex vivo analyses revealed fasting hyperglycemia, glucose intolerance, reduced sensitivity and delayed glucose clearance after insulin challenge, decreased thermogenic brown adipose tissue mass, and exaggerated hepatic endocannabinoid tone in F1 offspring exposed to 0.1 mg/kg DE-71 relative to control. DE-71 effects on F0 dams were more limited indicating that indirect exposure to developing offspring is more detrimental. Other ex vivo glycemic correlates occurred more generally in exposed F0 and F1, i.e., reduced plasma insulin and altered glucoregulatory endocrines, exaggerated sympathoadrenal activity and reduced hepatic glutamate dehydrogenase enzymatic activity. Hepatic PBDE congener analysis indicated maternal transfer of BDE-28 and -153 to F1 at a collective level of 200 ng/g lipid, in range with maximum values detected in serum of human females. Given the persistent diabetogenic phenotype, especially pronounced in female offspring after developmental exposure to environmentally relevant levels of DE-71, additional animal studies should be conducted that further characterize PBDE-induced diabetic pathophysiology and identify critical developmental time windows of susceptibility. Longitudinal human studies should also be conducted to determine the risk of long-lasting metabolic consequences after maternal transfer of PBDEs during early-life development.

Interactive Student-Centered Neuroscience Workshops for Sixth Graders Enhance Science Knowledge and Education Attitudes.

UCNeuro, a University of California, Riverside student-run organization, developed, implemented, and tested a school-based supplemental science intervention. The purpose of this intervention was to improve students' neuroscience knowledge and education attitudes and meet, in part, California's new elementary science education standards. The intervention consisted of interactive, hands-on neuroscience workshops on the structure of a neuron, neuron-to-neuron communication, brain structure and function, autonomic nervous system function, and drug effects on the brain. Under the supervision of a faculty neuroscientist, undergraduate students implemented the intervention with 77 sixth-grade students in one school in Riverside County, California. Pre- and post-test results showed increases in students' neuroscience knowledge, confidence in achieving their goals, likeliness to go to college, and desire to attend school. Excitement about learning science material and school learning opportunities did not change after the workshops. We hope that the UCNeuro workshops can be employed and adapted to the existing curriculum to improve knowledge in the life sciences while California's new elementary science standards are being operationalized.

Dysregulation of Hypothalamic Gene Expression and the Oxytocinergic System by Soybean Oil Diets in Male Mice.

Soybean oil consumption has increased greatly in the past half-century and is linked to obesity and diabetes. To test the hypothesis that soybean oil diet alters hypothalamic gene expression in conjunction with metabolic phenotype, we performed RNA sequencing analysis using male mice fed isocaloric, high-fat diets based on conventional soybean oil (high in linoleic acid, LA), a genetically modified, low-LA soybean oil (Plenish), and coconut oil (high in saturated fat, containing no LA). The 2 soybean oil diets had similar but nonidentical effects on the hypothalamic transcriptome, whereas the coconut oil diet had a negligible effect compared to a low-fat control diet. Dysregulated genes were associated with inflammation, neuroendocrine, neurochemical, and insulin signaling. Oxt was the only gene with metabolic, inflammation, and neurological relevance upregulated by both soybean oil diets compared to both control diets. Oxytocin immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus was reduced, whereas plasma oxytocin and hypothalamic Oxt were increased. These central and peripheral effects of soybean oil diets were correlated with glucose intolerance but not body weight. Alterations in hypothalamic Oxt and plasma oxytocin were not observed in the coconut oil diet enriched in stigmasterol, a phytosterol found in soybean oil. We postulate that neither stigmasterol nor LA is responsible for effects of soybean oil diets on oxytocin and that Oxt messenger RNA levels could be associated with the diabetic state. Given the ubiquitous presence of soybean oil in the American diet, its observed effects on hypothalamic gene expression could have important public health ramifications.

Perinatal exposure to organohalogen pollutants decreases vasopressin content and its mRNA expression in magnocellular neuroendocrine cells activated by osmotic stress in adult rats.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are environmental pollutants that produce neurotoxicity and neuroendocrine disruption. They affect the vasopressinergic system but their disruptive mechanisms are not well understood. Our group reported that rats perinatally exposed to Aroclor-1254 (A1254) and DE-71 (commercial mixtures of PCBs and PBDEs) decrease somatodendritic vasopressin (AVP) release while increasing plasma AVP responses to osmotic activation, potentially emptying AVP reserves required for body-water balance. The aim of this research was to evaluate the effects of perinatal exposure to A1254 or DE-71 (30mgkg/day) on AVP transcription and protein content in the paraventricular and supraoptic hypothalamic nuclei, of male and female rats, by in situ hybridization and immunohistochemistry. cFOS mRNA expression was evaluated in order to determine neuroendocrine cells activation due to osmotic stimulation. Animal groups were: vehicle (control); exposed to either A1254 or DE-71; both, control and exposed, subjected to osmotic challenge. The results confirmed a physiological increase in AVP-immunoreactivity (AVP-IR) and gene expression in response to osmotic challenge as reported elsewhere. In contrast, the exposed groups did not show this response to osmotic activation, they showed significant reduction in AVP-IR neurons, and AVP mRNA expression as compared to the hyperosmotic controls. cFOS mRNA expression increased in A1254 dehydrated groups, suggesting that the AVP-IR decrease was not due to a lack of the response to the osmotic activation. Therefore, A1254 may interfere with the activation of AVP mRNA transcript levels and protein, causing a central dysfunction of vasopressinergic system.

A Calibrated Method of Massage Therapy Decreases Systolic Blood Pressure Concomitant With Changes in Heart Rate Variability in Male Rats.

OBJECTIVE: The purpose of this study was to develop a method for applying calibrated manual massage pressures by using commonly available, inexpensive sphygmomanometer parts and validate the use of this approach as a quantitative method of applying massage therapy to rodents. METHODS: Massage pressures were monitored by using a modified neonatal blood pressure (BP) cuff attached to an aneroid gauge. Lightly anesthetized rats were stroked on the ventral abdomen for 5 minutes at pressures of 20 mm Hg and 40 mm Hg. Blood pressure was monitored noninvasively for 20 minutes following massage therapy at 5-minute intervals. Interexaminer reliability was assessed by applying 20 mm Hg and 40 mm Hg pressures to a digital scale in the presence or absence of the pressure gauge. RESULTS: With the use of this method, we observed good interexaminer reliability, with intraclass coefficients of 0.989 versus 0.624 in blinded controls. In Long-Evans rats, systolic BP dropped by an average of 9.86% ± 0.27% following application of 40 mm Hg massage pressure. Similar effects were seen following 20 mm Hg pressure (6.52% ± 1.7%), although latency to effect was greater than at 40 mm Hg. Sprague-Dawley rats behaved similarly to Long-Evans rats. Low-frequency/high-frequency ratio, a widely-used index of autonomic tone in cardiovascular regulation, showed a significant increase within 5 minutes after 40 mm Hg massage pressure was applied. CONCLUSIONS: The calibrated massage method was shown to be a reproducible method for applying massage pressures in rodents and lowering BP.

Permanently compromised NADPH-diaphorase activity within the osmotically activated supraoptic nucleus after in utero but not adult exposure to Aroclor 1254.

Stimulated vasopressin (VP) release from magnocellular neuroendocrine cells in the supraoptic nucleus (SON) of hyperosmotic rats is inhibited by treatment with the industrial polychlorinated biphenyl (PCB) mixture, Aroclor 1254. Because VP responses to hyperosmotic stimulation are regulated by nitric oxide (NO) signaling, we studied NO synthase (NOS) activity in the SON of hyperosmotic rats as potential target of PCB-induced disruption of neuroendocrine processes necessary for osmoregulation. To examine PCB-induced changes in NOS activity under normosmotic and hyperosmotic conditions, male Sprague-Dawley rats were exposed to Aroclor 1254 (30mg/kg/day) in utero and NADPH-diaphorase (NADPH-d) activity was assessed in SON sections at three ages: postnatal day 10, early adult (3-5 months) or late adult (14-16 months). Hyperosmotic treatment increased mean NADPH-d staining density of oil hyperosmotic controls by 19.9% in early adults and 58% in late adulthood vs normosmotic controls. In utero exposure to PCBs reduced hyperosmotic-induced upregulation of NADPH-d activity to control levels in early adults and by 28% in late adults. Basal NADPH-d was reduced in postnatal rats. Rats receiving PCB exposure as early adults orally for 14 days displayed normal responses. Our findings show that developmental but not adult exposure to PCBs significantly reduces NOS responses to hyperosmolality in neuroendocrine cells. Moreover, reduced NADPH-d activity produced by in utero exposure persisted in stimulated late adult rats concomitant with reduced osmoregulatory capacity vs oil controls (375±9 vs 349±5mOsm/L). These findings suggest that developmental PCBs permanently compromise NOS signaling in the activated neuroendocrine hypothalamus with potential osmoregulatory consequences.

Cigarette smoke toxins deposited on surfaces: implications for human health.

Cigarette smoking remains a significant health threat for smokers and nonsmokers alike. Secondhand smoke (SHS) is intrinsically more toxic than directly inhaled smoke. Recently, a new threat has been discovered - Thirdhand smoke (THS) - the accumulation of SHS on surfaces that ages with time, becoming progressively more toxic. THS is a potential health threat to children, spouses of smokers and workers in environments where smoking is or has been allowed. The goal of this study is to investigate the effects of THS on liver, lung, skin healing, and behavior, using an animal model exposed to THS under conditions that mimic exposure of humans. THS-exposed mice show alterations in multiple organ systems and excrete levels of NNAL (a tobacco-specific carcinogen biomarker) similar to those found in children exposed to SHS (and consequently to THS). In liver, THS leads to increased lipid levels and non-alcoholic fatty liver disease, a precursor to cirrhosis and cancer and a potential contributor to cardiovascular disease. In lung, THS stimulates excess collagen production and high levels of inflammatory cytokines, suggesting propensity for fibrosis with implications for inflammation-induced diseases such as chronic obstructive pulmonary disease and asthma. In wounded skin, healing in THS-exposed mice has many characteristics of the poor healing of surgical incisions observed in human smokers. Lastly, behavioral tests show that THS-exposed mice become hyperactive. The latter data, combined with emerging associated behavioral problems in children exposed to SHS/THS, suggest that, with prolonged exposure, they may be at significant risk for developing more severe neurological disorders. These results provide a basis for studies on the toxic effects of THS in humans and inform potential regulatory policies to prevent involuntary exposure to THS.

Altered cardiovascular reactivity and osmoregulation during hyperosmotic stress in adult rats developmentally exposed to polybrominated diphenyl ethers (PBDEs).

Polybrominated diphenyl ethers (PBDEs) and the structurally similar chemicals polychlorinated biphenyls (PCBs) disrupt the function of multiple endocrine systems. PCBs and PBDEs disrupt the secretion of vasopressin (VP) from the hypothalamus during osmotic activation. Since the peripheral and central vasopressinergic axes are critical for osmotic and cardiovascular regulation, we examined whether perinatal PBDE exposure could impact these functions during physiological activation. Rats were perinatally dosed with a commercial PBDE mixture, DE-71. Dams were given 0 (corn oil control), 1.7 (low dose) or 30.6 mg/kg/day (high dose) in corn oil from gestational day (GD) 6 through postnatal day (PND) 21 by oral gavage. In the male offspring exposed to high dose PBDE plasma thyroxine and triiodothyronine levels were reduced at PND 21 and recovered to control levels by PND 60 when thyroid stimulating hormone levels were elevated. At 14-18 months of age, cardiovascular responses were measured in four groups of rats: Normal (Oil, normosmotic condition), Hyper (Oil, hyperosmotic stress), Hyper PBDE low (1.7 mg/kg/day DE-71 perinatally, hyperosmotic stress), and Hyper PBDE high (30.6 mg/kg/day DE-71 perinatally, hyperosmotic stress). Systolic blood pressure (BP), diastolic BP, and heart rate (HR) were determined using tail cuff sphygmomanometry and normalized to pretreatment values (baseline) measured under basal conditions. Hyperosmotic treatment yielded significant changes in systolic BP in PBDE exposed rats only. Hyper PBDE low and high dose rats showed 36.1 and 64.7% greater systolic BP responses at 3h post hyperosmotic injection relative to pretreatment baseline, respectively. No treatment effects were measured for diastolic BP and HR. Hyper and Hyper PBDE rats showed increased mean plasma osmolality values by 45 min after injection relative to normosmotic controls. In contrast to Hyper rats, Hyper PBDE (high) rats showed a further increase in mean plasma osmolality at 3h (358.3±12.4mOsm/L) relative to 45 min post hyperosmotic injection (325.1±11.4mOsm/L). Impaired osmoregulation in PBDE-treated animals could not be attributed to decreased levels of plasma vasopressin. Our findings suggest that developmental exposure to PBDEs may disrupt cardiovascular reactivity and osmoregulatory responses to physiological activation in late adulthood.

Nitric oxide signaling as a common target of organohalogens and other neuroendocrine disruptors.

Organohalogen compounds such as polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE) are global environmental pollutants and highly persistent, bioaccumulative chemicals that produce adverse effects in humans and wildlife. Because of the widespread use of these organohalogens in household items and consumer products, indoor contamination is a significant source of human exposure, especially for children. One significant concern with regard to health effects associated with exposure to organohalogens is endocrine disruption. Toxicological studies on organohalogen pollutants primarily focused on sex steroid and thyroid hormone actions, and findings have largely shaped the way one envisions their disruptive effects occurring. Organohalogens exert additional effects on other systems including other complex endocrine systems that may be disregulated at various levels of organization. Over the last 20 years evidence has mounted in favor of a critical role of nitric oxide (NO) in numerous functions ranging from neuroendocrine functions to learning and memory. With its participation in multiple systems and action at several levels of integration, NO signaling has a pervasive influence on nervous and endocrine functions. Like blockers of NO synthesis, PCBs and PBDEs produce multifaceted effects on physiological systems. Based on this unique set of converging information it is proposed that organohalogen actions occur, in part, by hijacking processes associated with this ubiquitous bioactive molecule. The current review examines the emerging evidence for NO involvement in selected organohalogen actions and includes recent progress from our laboratory that adds to our current understanding of the actions of organohalogens within hypothalamic neuroendocrine circuits. The thyroid, vasopressin, and reproductive systems as well as processes associated with long-term potentiation were selected as sample targets of organohalogens that rely on regulation by NO. Information is provided about other toxicants with demonstrated interference of NO signaling. Our focus on the convergence between NO system and organohalogen toxicity offers a novel approach to understanding endocrine and neuroendocrine disruption that is particularly problematic for developing organisms. This new working model is proposed as a way to encourage future study in elucidating common mechanisms of action that are selected with a better operational understanding of the systems affected.

Neuroendocrine actions of organohalogens: thyroid hormones, arginine vasopressin, and neuroplasticity.

Organohalogen compounds are global environmental pollutants. They are highly persistent, bioaccumulative, and cause adverse effects in humans and wildlife. Because of the widespread use of these organohalogens in household items and consumer products, indoor contamination may be a significant source of human exposure, especially for children. One significant concern with regard to health effects associated with exposure to organohalogens is endocrine disruption. This review focuses on PCBs and PBDEs as old and new organohalogens, respectively, and their effects on two neuroendocrine systems; thyroid hormones and the arginine vasopressin system (AVP). Regarding neuroendocrine effects of organohalogens, there is considerable information on the thyroid system as a target and evidence is now accumulating that the AVP system and associated functions are also susceptible to disruption. AVP-mediated functions such as osmoregulation, cardiovascular function as well as social behavior, sexual function and learning/memory are discussed. For both thyroid and AVP systems, the timing of exposure seems to play a major role in the outcome of adverse effects. The mechanism of organohalogen action is well understood for the thyroid system. In comparison, this aspect is understudied in the AVP system but some similarities in neural processes, shown to be targeted by these pollutants, serve as promising possibilities for study. One challenge in understanding modes of action within neuroendocrine systems is their complexity stemming, in part, from interdependent levels of organization. Further, because of the interplay between neuroendocrine and neural functions and behavior, further investigation into organohalogen-mediated effects is warranted and may yield insights with wider scope. Indeed, the current literature provides scattered evidence regarding the role of organohalogen-induced neuroendocrine disruption in the neuroplasticity related to both learning functions and brain structure but future studies are needed to establish the role of endocrine disruption in nervous system function and development.

Domoic acid induces a long-lasting enhancement of CA1 field responses and impairs tetanus-induced long-term potentiation in rat hippocampal slices.

Domoic acid (DOM) is known to cause hippocampal neuronal damage and produces amnesic effects. We examined synaptic plasticity changes induced by DOM exposure in rat hippocampal CA1 region. Brief bath application of DOM to hippocampal slices produces a chemical form of long-term potentiation (LTP) of CA1 field synaptic potentials. The potentiation cannot be blocked by NMDA receptor antagonist MK-801 but can be blocked by the calcium-calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-62 or cAMP-dependent protein kinase (PKA) inhibitor H-89. DOM-potentiated slices show decreased autophosphorylated CaMKII (p-Thr286), an effect that is also dependent on the activity of CaMKII and PKA. Increased phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR1 (p-Ser831) was seen in DOM-potentiated slices. Therefore, aberrant regulation of CaMKII and GluR1 phosphorylation occurs after DOM application. In addition, tetanus-induced LTP as well as the increase of phosphorylation of CaMKII (p-Thr286) were reduced in DOM-potentiated slices. Compared with brief exposure, slices recovering from prolonged exposure did not show potentiation or altered levels of CaMKII (p-Thr286) or GluR (p-Ser831). However, decreased phosphorylation of GluR1 at Ser845 was seen. These results describe a new chemical form of LTP and uncover novel molecular changes induced by DOM. The observed impairment of tetanus LTP and misregulation of CaMKII and GluR1 phosphorylation may partially account for DOM neurotoxicity and underlie the molecular basis for DOM-induced memory deficit.

Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney.

Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague-Dawley rats were injected intraperitoneal with 3.5M NaCl at a doses ranging from 0.3cm(3)/100g to 0.6cm(3)/100g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p<0.05, r=0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p<0.05, r=0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p<0.05, r=0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats.

In vitro effects of environmentally relevant polybrominated diphenyl ether (PBDE) congeners on calcium buffering mechanisms in rat brain.

Polybrominated diphenyl ethers (PBDEs) are widely used as additive flame-retardants and have been detected in human blood, adipose tissue, and breast milk. Developmental and long-term exposures to these chemicals may pose a human health risk, especially to children. We have previously demonstrated that polychlorinated biphenyls (PCBs), which are structurally similar to PBDEs and cause neurotoxicity, perturb intracellular signaling events including calcium homeostasis and protein kinase C translocation, which are critical for neuronal function and development of the nervous system. The objective of the present study was to test whether environmentally relevant PBDE congeners 47 and 99 are also capable of disrupting Ca(2+) homeostasis. Calcium buffering was determined by measuring (45)Ca(2+)-uptake by microsomes and mitochondria, isolated from adult male rat brain (frontal cortex, cerebellum, hippocampus, and hypothalamus). Results show that PBDEs 47 and 99 inhibit both microsomal and mitochondrial (45)Ca(2+)-uptake in a concentration-dependent manner. The effect of these congeners on (45)Ca(2+)-uptake is similar in all four brain regions though the hypothalamus seems to be slightly more sensitive. Among the two preparations, the congeners inhibited (45)Ca(2+)-uptake in mitochondria to a greater extent than in microsomes. These results indicate that PBDE 47 and PBDE 99 congeners perturb calcium signaling in rat brain in a manner similar to PCB congeners, suggesting a common mode of action of these persistent organic pollutants.