A songbird strategically modifies its blinking behavior when viewing human faces.

Even though blinking is necessary to maintain clear vision in many species, blinking is likely costly because it temporarily impairs vision. Given this cost, individuals can strategically modify their blinking behavior to minimize information loss. We tested whether a songbird species modifies its blinking behavior when viewing potential threats (human faces). We recorded the blinking behavior of captive great-tailed grackles (Quiscalus mexicanus) before, during, and after they viewed human face stimuli or control stimuli (tree bark as well as scrambled versions of human faces and tree bark). We found that the birds inhibited their blinking behavior the most when viewing human faces versus controls. In addition, they inhibited their blinking behavior more when viewing human faces that were directed rather than averted. Furthermore, when viewing the human faces, their blinking behavior was modified based on reactivity. These results suggest that a songbird can strategically modify its blinking behavior based on its perceived level of risk.

Omega-3 polyunsaturated fatty acids improve endothelial function in humans at risk for atherosclerosis: A review.

Epidemiology studies and clinical trials show that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can prevent atherosclerotic morbidity and evidence suggests this may be mediated by improving endothelial dysfunction. Endothelial dysfunction is characterized by reduced vasodilation and a pro-inflammatory, pro-thrombotic state, and is an early pathological event in the development of atherosclerosis. Flow-mediated dilation (FMD), a gold standard for assessing endothelial dysfunction, is a predictor of future cardiovascular events and coronary heart disease risk. Notably, risk factors for endothelial dysfunction include classic risk factors for atherosclerosis: Elevated lipids, diabetes, hypertension, elevated BMI, cigarette smoking, and metabolic syndrome. In this paper, we review the ability of n-3 PUFAs to improve endothelial dysfunction in individuals with classic risk factors for atherosclerosis, but lacking diagnosed atherosclerotic disease, with the goal of identifying those individuals that might gain the most vasoprotection from n-3 PUFA supplements. We include trials using eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or alpha-linolenic acid (ALA) alone, or EPA+DHA; and assessing endothelial function by FMD, forearm blood flow, or peripheral arterial tonometry. We found that n-3 PUFAs improved endothelial dysfunction in 16 of 17 studies in individuals with hyperlipidemia, elevated BMI, metabolic syndrome, or that smoked cigarettes, but only in 2 of 5 studies in diabetics. Further, these trials showed that use of EPA+DHA consistently improve endothelial dysfunction; ALA-enriched diets appear promising; but use of EPA or DHA alone requires further study. We conclude that individuals with hyperlipidemia, elevated BMI, metabolic syndrome, or that smoke could derive vaosprotective benefits from EPA+DHA supplementation.

Role of CYP1A1 in modulating the vascular and blood pressure benefits of omega-3 polyunsaturated fatty acids.

The mechanisms that mediate the cardiovascular protective effects of omega 3 (n-3) polyunsaturated fatty acids (PUFAs) have not been fully elucidated. Cytochrome P450 1A1 efficiently metabolizes n-3 PUFAs to potent vasodilators. Thus, we hypothesized that dietary n-3 PUFAs increase nitric oxide (NO)-dependent blood pressure regulation and vasodilation in a CYP1A1-dependent manner. CYP1A1 wild-type (WT) and knockout (KO) mice were fed an n-3 or n-6 PUFA-enriched diet for 8 weeks and were analyzed for tissue fatty acids and metabolites, NO-dependent blood pressure regulation, NO-dependent vasodilation of acetylcholine (ACh) in mesenteric resistance arterioles, and endothelial NO synthase (eNOS) and phospho-Ser1177-eNOS expression in the aorta. All mice fed the n-3 PUFA diet showed significantly higher levels of n-3 PUFAs and their metabolites, and significantly lower levels of n-6 PUFAs and their metabolites. In addition, KO mice on the n-3 PUFA diet accumulated significantly higher levels of n-3 PUFAs in the aorta and kidney without a parallel increase in the levels of their metabolites. Moreover, KO mice exhibited significantly less NO-dependent regulation of blood pressure on the n-3 PUFA diet and significantly less NO-dependent, ACh-mediated vasodilation in mesenteric arterioles on both diets. Finally, the n-3 PUFA diet significantly increased aortic phospho-Ser1177-eNOS/eNOS ratio in the WT compared with KO mice. These data demonstrate that CYP1A1 contributes to eNOS activation, NO bioavailability, and NO-dependent blood pressure regulation mediated by dietary n-3 PUFAs.

Dibenzo[def,p]chrysene (DBC) suppresses antibody formation in spleen cells following oral exposures of mice.

Dibenzo[def,p]chrysene (DBC) is a potent environmental carcinogen in rodents, fish, and human cells examined in culture. There are numerous similarities between the patterns of cytochrome P-450 (P450) activation of DBC and its covalent binding to DNA and proteins with another polycyclic aromatic hydrocarbon (PAH), 7,12-dimethylbenz[a]anthracene (DMBA). Our lab has previously shown that DMBA produces immunosuppression in rodents and human cell systems. Therefore, the purpose of these studies was to examine the immunotoxicity of DBC in a rodent model that was found to be sensitive to the immunosuppressive effects of DMBA. Data showed that DBC had similar potency to DMBA in producing suppression of a T-dependent antibody response (TDAR) and altered spleen cell subsets in a similar manner as DMBA when DMBA was given by gavage for 5 d in corn oil to mice at doses of 1-100 mg/kg total cumulative doses. T-cell-independent antigen (TNP-Ficoll) responses were quantitatively less sensitive to DBC suppression. It was also found that as with DMBA, DBC produced a persistent immunosuppression, which lasted for at least 4 wk following dosing with a novel pill method for self-administration of DBC. In conclusion, DBC appears to possess many of the same characteristics of DMBA in terms of its immunotoxicity.

Elevated blood pressure in cytochrome P4501A1 knockout mice is associated with reduced vasodilation to omega-3 polyunsaturated fatty acids.

In vitro cytochrome P4501A1 (CYP1A1) metabolizes omega-3 polyunsaturated fatty acids (n-3 PUFAs); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), primarily to 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP), respectively. These metabolites have been shown to mediate vasodilation via increases in nitric oxide (NO) and activation of potassium channels. We hypothesized that genetic deletion of CYP1A1 would reduce vasodilatory responses to n-3 PUFAs, but not the metabolites, and increase blood pressure (BP) due to decreases in NO. We assessed BP by radiotelemetry in CYP1A1 wildtype (WT) and knockout (KO) mice±NO synthase (NOS) inhibitor. We also assessed vasodilation to acetylcholine (ACh), EPA, DHA, 17,18-EEQ and 19,20-EDP in aorta and mesenteric arterioles. Further, we assessed vasodilation to an NO donor and to DHA±inhibitors of potassium channels. CYP1A1 KO mice were hypertensive, compared to WT, (mean BP in mmHg, WT 103±1, KO 116±1, n=5/genotype, p<0.05), and exhibited a reduced heart rate (beats per minute, WT 575±5; KO 530±7; p<0.05). However, BP responses to NOS inhibition and vasorelaxation responses to ACh and an NO donor were normal in CYP1A1 KO mice, suggesting that NO bioavailability was not reduced. In contrast, CYP1A1 KO mice exhibited significantly attenuated vasorelaxation responses to EPA and DHA in both the aorta and mesenteric arterioles, but normal vasorelaxation responses to the CYP1A1 metabolites, 17,18-EEQ and 19,20-EDP, and normal responses to potassium channel inhibition. Taken together these data suggest that CYP1A1 metabolizes n-3 PUFAs to vasodilators in vivo and the loss of these vasodilators may lead to increases in BP.

A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice.

Oral gavage dosing can induce stress and potentially confound experimental measurements, particularly when blood pressure and heart rate are endpoints of interest. Thus, we developed a pill formulation that mice would voluntarily consume and tested the hypothesis that pill dosing would be significantly less stressful than oral gavage. C57Bl/6 male mice were singly housed and on four consecutive days were exposed to an individual walking into the room (week 1, control), a pill being placed into the cage (week 2), and a dose of water via oral gavage (week 3). Blood pressure and heart rate were recorded by radiotelemetry continuously for 5h after treatment, and feces collected 6-10h after treatment for analysis of corticosterone metabolites. Both pill and gavage dosing significantly increased mean arterial pressure (MAP) during the first hour, compared to control. However, the increase in MAP was significantly greater after gavage and remained elevated up to 5h, while MAP returned to normal within 2h after a pill. Neither pill nor gavage dosing significantly increased heart rate during the first hour, compared to control; however, pill dosing significantly reduced heart rate while gavage significantly increased heart rate 2-5h post dosing. MAP and heart rate did not differ 24h after dosing. Lastly, only gavage dosing significantly increased fecal corticosterone metabolites, indicating a systemic stress response via activation of the hypothalamic-pituitary-adrenal axis. These data demonstrated that this pill dosing method of mice is significantly less stressful than oral gavage.

Interactive Effects of Omega-3 Polyunsaturated Fatty Acids and Secondhand Smoke in Mice and Human Subjects.

Active smoking and secondhand smoke (SHS) exposure increase the risk of cardiovascular morbidity and mortality. Active smoking is associated with reduced levels of omega-3 polyunsaturated fatty acids (n-3 PUFA) and studies show that n-3 PUFA supplementation can improve smoking-induced vascular dysfunction. However, the relationship between n-3 PUFA and SHS exposure has not been studied. Fat-1 transgenic mice, which convert n-6 to n-3 PUFA, were fed diets with n-3 PUFA or without (n-6 PUFA diet), exposed to air or SHS for 4 weeks, and vasoreactivity, antioxidant indices, and omega-3 index (percent eicosapentaenoic + docosahexaenoic acids in RBC) measured. Compared to air-exposed mice, SHS-enhanced aortic constriction in mice fed the n-6 PUFA diet (omega-3 index, 5.9 ± 0.2%; mean ± SE), but not in mice fed the n-3 PUFA diet (omega-3 index, 7.8 ± 0.6%). SHS also significantly induced mRNA expression of cytochrome P4501A1, NADPH:quinone oxidoreductase, heme oxygenase-1, and angiotensinogen in adipose tissue, and increased antioxidant capacity only in mice on the n-6 PUFA diet. Notably, SHS reduced the omega-3 index by 1.0 percentage point (p = 0.003), compared to air-exposed mice irrespective of diet. Additionally, we recruited human nonsmokers (NS) with and without SHS exposure (n = 40) 19-40 years old and measured the omega-3 index and antioxidant capacity. In human subjects SHS exposure was associated with a significantly lower omega-3 index (NS, 4.4 ± 1.1%; NS + SHS, 3.2 ± 1.0%; mean ± SD, p = 0.002) and higher antioxidant capacity (p < 0.001) than unexposed NS. Thus, SHS exposure is associated with lower levels of n-3 PUFA in mice and humans; however, an omega-3 index of ~ 8% in mice has vasoprotective and antioxidant properties.

NFATc3 contributes to intermittent hypoxia-induced arterial remodeling in mice.

Sleep apnea (SA) is defined as intermittent respiratory arrest during sleep and affects up to 20% of the adult population. SA is also associated with an increased incidence of hypertension and peripheral vascular disease. Exposing rodents to intermittent hypoxia during sleep mimics the cyclical hypoxia/normoxia of SA. We have previously shown that in mice and rats intermittent hypoxia induces ET-1 upregulation and systemic hypertension. Furthermore, intermittent hypoxia (IH) in mice increases nuclear factor of activated T cells isoform 3 (NFATc3) transcriptional activity in aorta and mesenteric arteries, whereas the calcineurin/NFAT inhibitor cyclosporin A prevents IH-induced hypertension. More importantly, NFATc3 knockout (KO) mice do not develop IH-induced hypertension. The goals of this study were to determine the role of NFATc3 in IH-induced arterial remodeling and whether IH-induced NFATc3 activation is mediated by ET-1. Oral administration of both a dual (bosentan) and a selective endothelin receptor type A antagonist (PD155080) during 2 days of IH exposure attenuated NFAT activation in aorta and mesenteric arteries. Rho kinase inhibition with fasudil also prevented IH-induced NFAT activation. Mesenteric artery cross-sectional wall thickness was increased by IH in wild-type (WT) and vehicle-treated mice but not in bosentan-treated and NFATc3 KO mice. The arterial remodeling in mesenteric arteries after IH was characterized by increased expression of the hypertrophic NFATc3 target smooth muscle-alpha-actin in WT but not in KO mice. These results indicate that ET-1 is an upstream activator of NFATc3 during intermittent hypoxia, contributing to the resultant hypertension and increased wall thickness.

Overview of developmental heart defects by dioxins, PCBs, and pesticides.

The developing cardiovascular system is a sensitive target of many environmental pollutants, including dioxins, dioxin-like polychlorinated biphenyls (PCBs), and some pesticides such as methyl parathion. Laboratory research has utilized a variety of vertebrate models to elucidate potential mechanisms that mediate this cardioteratogenicity and to establish the sensitivity of different species for predicting potential risk to environmental and human health. Studies of dioxin and dioxin-like PCBs have illustrated that piscine, avian, and mammalian embryos exhibit cardiovascular structural changes and functional deficits, although the specific characteristics vary among the individual models. Piscine models typically exhibit reduced blood flow, altered heart looping, and reduced heart size and contraction rate. The chick embryo exhibits extensive cardiac dilation, thinner ventricle walls, and reduced responsiveness to chronotropic stimuli, while the murine embryo exhibits reduced heart size. It is notable that in all models the dioxin-associated cardioteratogenicity is associated with increases in cardiovascular apoptosis and decreases in cardiocyte proliferation. While the cardiotertogenicity in piscine and avian species is associated with overt morbidity and mortality, that is not the case for the murine embryo. However, murine offspring exposed during development to dioxin exhibit cardiac hypertrophy and an increased sensitivity to a second cardiovascular insult in adulthood. Thus, although the mammalian embryo is less sensitive to cardiovascular defects by dioxin and dioxin-like compounds, developmental exposure increases the risk of cardiovascular disease later in life. The impact of developmental exposure to dioxin-like chemicals on human cardiovascular disease susceptibility is not known. However, recent animal research has confirmed human epidemiology studies that dioxin exposure in adulthood is associated with hypertension and cardiovascular disease.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Vascular Dysfunction That is Dependent on Perivascular Adipose and Cytochrome P4501A1 Expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is associated with hypertension in humans and animals, and studies suggest that cytochrome P4501A1 (Cyp1a1) induction and vascular dysfunction may contribute. We investigated the role of perivascular adipose tissue (PVAT) and Cyp1a1 in TCDD-induced vascular dysfunction. Cyp1a1 wild-type (WT) and knockout (KO) male mice were fed a dough pill containing 1,4-p-dioxane (TCDD vehicle control) on days 0 and 7, or 1000 ng/kg TCDD on day 0 and 250 ng/kg TCDD on day 7. mRNA expression of Cyp1a1 was assessed on days 3, 7, and 14, and of Cyp1b1, 1a2, angiotensinogen, and phosphodiesterase 5a on day 14. Dose-dependent vasoconstriction to a thromboxane A2 mimetic (U46619), and vasorelaxation to acetylcholine and a nitric oxide donor (S-nitroso-N-acetyl-DL-penicillamine, SNAP), were investigated in the aorta with and without PVAT. Cyp1a1 and 1a2 mRNA was induced in aorta of WT mice only with PVAT, and Cyp1a1 induction was sustained through day 14. TCDD significantly enhanced constriction to U46619 in WT mice and inhibited relaxation to both acetylcholine and SNAP, but only in the presence of PVAT. The effects of TCDD on U46619 constriction and SNAP relaxation were not observed in Cyp1a1 KO mice. Finally, in aorta + PVAT of WT mice TCDD significantly induced expression of angiotensinogen and phosphodiesterase 5a both of which could contribute to the TCDD-induced vascular dysfunction. These data establish PVAT as a TCDD target which is critically involved in mediating vascular dysfunction. TCDD enhances vasoconstriction via the thromboxane/prostanoid (TP) receptor and inhibits vasorelaxation via nitric oxide (NO) signaling. This TCDD-induced vascular dysfunction requires perivascular adipose (PVAT) and cytochrome P4501a1 (CYP1a1) induction.

Associations of medium and long chain omega-3 polyunsaturated fatty acids with blood pressure in Hispanic and non-Hispanic smokers and nonsmokers.

Epidemiology studies and clinical trials have shown that omega-3 polyunsaturated fatty acids (n-3 PUFAs) are inversely associated with blood pressure. We sought to determine the influence of cigarette smoking and Hispanic ethnicity on this association. Age- and sex-matched smokers and nonsmokers (n = 98) 19-50 years old lacking cardiovascular disease were recruited. Systolic and diastolic blood pressure (SBP, DBP), heart rate, HbA1c, lipids, BMI, and RBC fatty acids were measured. The omega-3 index (percent eicosapentaenoic and docosahexaenoic acid, EPA+DHA, in RBCs) was significantly lower in smokers (Smokers: 3.19 ± 0.86%; Nonsmokers, 3.88 ± 1.05%, p = 0.001) and Hispanics (Hispanic 3.32 ± 0.93%; Non-Hispanic, 3.82 ± 1.03%, p = 0.006). DHA exhibited a significant inverse association with BP in both smokers and nonsmokers, while alpha-linolenic acid (ALA) exhibited a significant positive association with BP only in smokers. Multiple regression analyses showed that BMI, DHA, smoking status, and smoking status*ALA interaction significantly predicted SBP (p < 0.0001, R2 = 0.44) and DBP (p < 0.0001, R2 = 0.33), while ethnicity had no effect. The observed lower BP when DHA levels are high suggests a possible protective role of DHA on BP in normotensive smokers and nonsmokers. Additionally, the observed higher BP when ALA levels are high only in smokers suggests that ALA may influence the BP-lowering effects of chronic smoking.

In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: effects on fetal and adult cardiac gene expression and adult cardiac and renal morphology.

The mouse heart is a target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during fetal development, and microarray analysis demonstrates significant changes in expression of cardiac genes involved in extracellular matrix (ECM) remodeling. We tested the hypothesis that developmental TCDD exposure would disrupt cardiac ECM expression and be associated with changes in cardiac morphology in adulthood. In one study, time-pregnant C57BL/6 mice were dosed with corn oil or 1.5, 3.0, or 6.0 microg TCDD/kg on gestation day (GD) 14.5 and sacrificed on GD 17.5, when changes in fetal cardiac mRNA expression were analyzed using quantitative PCR. TCDD induced mRNA expression of genes associated with ECM remodeling (matrix metalloproteinase 9 and 13, preproendothelin-1 [preproET-1]), cardiac hypertrophy (atrial natriuretic peptide, beta-myosin heavy chain, osteopontin), and aryl hydrocarbon receptor (AHR) activation (cytochrome P4501A1, AHR repressor). Further, all TCDD-induced changes required the AHR since gene expression was not altered in AHR knockout fetuses. In a second study, time-pregnant mice were treated with corn oil or 6.0 microg TCDD/kg on GD 14.5, and male offspring were assessed for changes in cardiac gene expression and cardiac and renal morphology at 3 months. All TCDD-induced changes in cardiac gene expression observed fetally, except for preproET-1, remained induced in the hearts of adult male offspring. Adult male offspring of TCDD-exposed dams also displayed cardiac hypertrophy, decreased plasma volume, and mild hydronephrosis. These results demonstrate that in utero and lactational TCDD exposures alter cardiac gene expression and cardiac and renal morphology in adulthood, which may increase the susceptibility to cardiovascular dysfunction.

Perinatal 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure sensitizes offspring to angiotensin II-induced hypertension.

In utero and lactational exposure of mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to cardiac hypertrophy and hydronephrosis in adulthood. We tested the hypothesis that perinatal TCDD exposure increases the susceptibility to cardiovascular disease when offspring are exposed to a common cardiovascular disease risk factor, angiotensin II (Ang II). Pregnant C57BL/6N mice were exposed to corn oil (control) or 6.0 microg/kg TCDD on gestation day 14.5. Male offspring were then exposed to a subpressor (0.1 mg/kg/day) or pressor (0.7 mg/kg/day) dose of Ang II at 3.5 months and cardiac morphology and blood pressure analyzed, respectively. Perinatal TCDD exposure increased left ventricular cavity dilation during diastole, and wall thickness during diastole and systole. While Ang II stimulated an increase in wall thickness, the degree of increase was equivalent between control and TCDD offspring. In contrast, perinatal TCDD exposure did not alter basal blood pressure. However, Ang II increased systolic blood pressure more rapidly and to a greater degree in TCDD offspring. Further, Ang II stimulated renal myofibroblast differentiation and collagen deposition to a greater degree, and tended to increase procollagen I mRNA in TCDD offspring, compared to controls. These data suggest that perinatal TCDD exposure increases the susceptibility of offspring to renal fibrosis and hypertension in adulthood.

Effects of in ovo exposure to 3,3',4,4'-tetrachlorobiphenyl (PCB 77) on heart development in tree swallow (Tachycineta bicolor).

Tree swallow (Tachycineta bicolor) eggs from 2 uncontaminated sites, the Patuxent Research Refuge (Laurel, MD, USA) and the Cobleskill Reservoir (Cobleskill, NY, USA) were dosed with polychlorinated biphenyl (PCB) 77 to evaluate effects on the developing cardiovascular system. To ensure embryonic viability, treatments were administered into the air cell at embryonic day 2.5 including: untreated (control), vehicle (filtered sterilized fatty acid mixture), 100 ng/g and 1000 ng/g egg. Eggs were dosed in the field with 0.2 µL/egg, returned to the nest, collected at embryonic day 13, hatched in the laboratory, and necropsied. The PCB 77-treated hatchlings were compared with uninjected, vehicle-injected, and environmentally exposed hatchlings collected from a PCB-contaminated Upper Hudson River (NY, USA) site. The PCB 77-treated embryos showed no effects on hatching success or hatchling mortality, heart index, or morphological measures of 4 distinct heart layers (heart width, length, septal thickness, total and ventricular cavity area) compared with controls. Hatchlings that had received PCB 77 exhibited increased incidence of a cardiomyopathy and absence of the ventricular heart wall compact layer (Chi square test; p < 0.001); environmentally exposed embryos showed no apparent effects. The compact layer is essential in development and overall heart function for ventricular cardiomyocyte proliferation and normal heart contraction. The finding that in ovo exposure to PCB 77 resulted in distinct cardiomyopathy has implications for long-term individual fitness. Environ Toxicol Chem 2018;37:116-125. © 2017 SETAC.

Crosstalk between the aryl hydrocarbon receptor and hypoxia on the constitutive expression of cytochrome P4501A1 mRNA.

Endothelial cells are a target of halogenated aromatic hydrocarbon toxicity following aryl hydrocarbon receptor (AHR) activation. Further, evidence suggests that AHR has a physiological function in endothelial cells in the absence of exogenous ligands. Understanding these "normal" functions of AHR may help to reveal the mechanisms that contribute to the toxicity of xenobiotic ligands. Thus, this study focused on the crosstalk between hypoxia and AHR in the absence of exogenous ligands. Constitutive CYP1A1 mRNA was measured by real time PCR in human pulmonary microvascular endothelial cells exposed to hypoxia (1 or 2.5% O2), 25 nM AHR siRNA, 25 nM hypoxia-inducible factor (HIF)-2alpha siRNA, or their combinations. Hypoxia significantly induced known hypoxia-regulated genes, and this induction was highly attenuated by HIF-2alpha siRNA, suggesting that HIF-2alpha is a primary mediator of hypoxic responses in these cells. Hypoxia also significantly reduced CYP1A1 mRNA and this reduction was also attenuated by HIF-2alpha siRNA. As expected, AHR siRNA significantly reduced constitutive CYP1A1 mRNA. While the combination of hypoxia plus AHR siRNA reduced CYP1A1 mRNA more than either treatment alone, the reduction was less than additive, suggesting that hypoxia and AHR deficiency may share a common pathway in reducing CYP1A1 expression. Finally, hypoxia significantly reduced AHR mRNA and this reduction was completely prevented by HIF-2alpha siRNA. In conclusion, constitutive CYP1A1 mRNA expression is dependent on AHR and is reduced by hypoxia via a HIF-2alpha-dependent mechanism, which may be mediated by a HIF-2alpha-dependent reduction of AHR expression.

Omega-3 Polyunsaturated Fatty Acids Protect Against Cigarette Smoke-Induced Oxidative Stress and Vascular Dysfunction.

In cigarette smokers endothelial dysfunction, measured by flow-mediated dilation (FMD), precedes cardiovascular disease (CVD) and can be improved by supplementation with n - 3 polyunsaturated fatty acids (PUFAs). We developed a mouse model of cigarette smoke (CS)-induced endothelial dysfunction that resembles impaired FMD observed in human cigarette smokers and investigated the mechanism by which n - 3 PUFAs mediate vasoprotection. We hypothesized that loss of nitric oxide (NO)-dependent vasodilation in CS-exposed mice would be prevented by dietary n - 3 PUFAs via a decrease in oxidative stress. C57BL/6 mice were fed a chow or n - 3 PUFA diet for 8 weeks and then exposed to mainstream CS or filtered air for 5 days, 2 h/day. Mesenteric arterioles were preconstricted with U46619 and dilated by stepwise increases in pressure (0-40 mmHg), resulting in increases in flow, ± inhibitor of NO production or antioxidant, Tempol. Markers of oxidative stress were measured in lung and heart. CS-exposed mice on a chow diet had impaired FMD, resulting from loss of NO-dependent dilation, compared with air exposed mice. Tempol restored FMD by normalizing NO-dependent dilation and increasing NO-independent dilation. CS-exposed mice on the n - 3 PUFA diet had normal FMD, resulting from a significant increase in NO-independent dilation, compared with CS-exposed mice on a chow diet. Furthermore, n - 3 PUFAs decreased two CS-induced markers of oxidative stress, 8-epiprostaglandin-F2α levels and heme oxygenase-1 mRNA, and significantly attenuated CS-induced cytochrome P4501A1 mRNA expression. These data demonstrate that dietary n - 3 PUFAs can protect against CS-induced vascular dysfunction via multiple mechanisms, including increasing NO-independent vasodilation and decreasing oxidative stress.

Dietary Omega-3 Polyunsaturated Fatty Acids Prevent Vascular Dysfunction and Attenuate Cytochrome P4501A1 Expression by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) found in fish protect against cardiovascular morbidity and mortality; however, many individuals avoid fish consumption due to concerns about pollutants. We tested the hypothesis that n-3 PUFAs would prevent vascular dysfunction induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). C57Bl/6 male mice were fed a chow or n-3 PUFA diet for 10 weeks and were exposed to vehicle or 300 ng/kg/d TCDD during the final 2 weeks on each diet. Aortic vasoconstriction mediated by arachidonic acid (AA) ± SKF525 (P450 inhibitor) or SQ29548 (thromboxane/prostanoid [TP] receptor antagonist) was assessed. RBC fatty acids and expression of n-3 and n-6 PUFA metabolites were analyzed. Cytochrome P4501A1 (CYP1A1), CYP1B1, and aryl hydrocarbon receptor (AHR) expression was measured. TCDD significantly increased AA-mediated vasoconstriction on a chow diet by increasing the contribution of P450s and TP receptor to the constriction response. In contrast, the n-3 PUFA diet prevented the TCDD-induced increase in AA vasoconstriction and normalized the contribution of P450s and TP receptor. Although TCDD increased the levels of AA vasoconstrictors on the chow diet, this increase was prevent by the n-3 PUFA diet. Additionally, the n-3 PUFA diet significantly increased the levels of n-3 PUFA-derived vasodilators and TCDD increased these levels further. Interestingly, the n-3 PUFA diet significantly attenuated CYP1A1 induction by TCDD without a significant effect on AHR expression. These data suggest that n-3 PUFAs can prevent TCDD-induced vascular dysfunction by decreasing vasoconstrictors, increasing vasodilators, and attenuating CYP1A1 induction, which has been shown previously to contribute to TCDD-induced vascular dysfunction.

Early developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure decreases chick embryo heart chronotropic response to isoproterenol but not to agents affecting signals downstream of the beta-adrenergic receptor.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes cardiovascular toxicity in laboratory animals, including alteration in several processes in which beta-adrenergic receptor (beta-AR) signaling plays important roles. Thus, our laboratory investigated the effects of TCDD on beta-AR expression and signal transduction. Fertile chicken eggs were injected with vehicle (corn oil), 0.24 or 0.3 pmol TCDD/g egg on incubation day 0 (D0) or D5. On D10, heart function was assessed by ECG in ovo. Exposure to TCDD increased the incidence of arrhythmias and decreased the positive chronotropic responsiveness of the heart to isoproterenol. The reduced beta-AR responsiveness was, in part, independent of any overt morphological changes in the heart as chick embryos exposed to TCDD on D5 displayed an intermediate responsiveness to beta-AR agonist in the absence of the dilated cardiomyopathy observed in chick embryos exposed to TCDD on D0. TCDD did not decrease the chronotropic response of the heart to agents that stimulate signals downstream of the beta-AR. In fact, TCDD-exposed embryos were more sensitive than controls to forskolin, increasing heart rates (HR) 21.8 +/- 3.5 beats per min (bpm) above baseline versus control values at 6.3 +/- 2.7 bpm above baseline. TCDD exposure also augmented the negative chronotropic response of the heart to verapamil, decreasing HR -23.2 +/- 7.4 bpm relative to baseline versus control embryos at -12.7 +/- 5.9 bpm below baseline. Finally, the mean cardiac beta1-AR mRNA expression in D10 embryos was not significantly altered by exposure to TCDD on D0. These findings establish that a functional end point of the developing chick heart is sensitive to TCDD exposure and that the TCDD-induced reduction in beta-AR responsiveness may result from alterations in signal transduction upstream of adenylyl cyclase.

Endothelin-1-mediated increase in reactive oxygen species and NADPH Oxidase activity in hearts of aryl hydrocarbon receptor (AhR) null mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor characterized to play a role in detection and adaptation to environmental stimuli. Genetic deletion of the AhR results in cardiac hypertrophy that is mediated primarily by endothelin-1 (ET-1); ET-1 has been implicated in the elevation of reactive oxygen species (ROS) in the heart, which are thought to contribute to several cardiovascular disorders, including cardiac hypertrophy. Thus, we tested the novel hypothesis that ET-1 induces ROS in AhR null mice via ET(A) receptor activation. We first confirmed the presence of ROS in the hearts of AhR null mice by measuring superoxide (O2*-)-dependent oxidation of dihydroethidium. Ethidium fluorescence was increased 10-fold in the hearts of AhR null mice, compared to the wild type. Then, to elucidate whether ET-1 mediated the increase in ROS, mice were chronically treated with 100 ng/kg/day of the ET(A) receptor antagonist BQ-123. In AhR null mice, BQ-123 significantly reduced elevated plasma 8-isoprostane, a systemic end product of phospholipid oxidation by ROS, and cardiac thiobarbituric acid reactive substances (TBARS), a nonspecific assessment of ROS production. Furthermore, BQ-123 reduced both cardiac lucigenin chemiluminescence and cardiac mRNA expression of NAD(P)H oxidase subunits gp91phox, p47phox, and p67phox in AhR null mice below the levels observed in wild-type mice. These findings demonstrate that ET-1 activation of ET(A) receptors mediates an increase in ROS that is associated with cardiac hypertrophy in AhR null mice. In addition, the ET-1-mediated increase in ROS appears to be initiated via increased NAD(P)H oxidase activity.

Inhibition of neovascularization by environmental agents.

The formation of new blood vessels, neovascularization, occurs by two unique processes: vasculogenesis, the de novo assembly of blood vessels from angioblast precursors, and angiogenesis, the formation of new capillary sprouts from preexisting vessels. There are many potential targets by which environmental pollutants may inhibit neovascularization and thus there are many possible phenotypic outcomes. Two examples of environmental pollutants that have been demonstrated to inhibit neovascularization include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical halogenated aromatic hydrocarbon, and constituents found in environmental tobacco smoke. Studies have shown that TCDD disrupts neoangiogenesis by inhibiting the expression of angiogenic stimuli as well as by reducing the responsiveness of endothelial cells to those stimuli. Additionally, studies have shown that constituents of environmental tobacco smoke, including pyradine and pyrazine derivatives, can potently inhibit the angiogenic process of branching as well as the vasculogenic process involved in capillary plexus formation. Further, the inhibition of neovascularization by either TCDD or environmental tobacco smoke constituents is associated with reduced endothelial cell proliferation and altered expression of extracellular matrix proteins. Future research that identifies the specific angiogenic signaling pathways that are disrupted by these pollutants will improve our ability to assess their risk to human health. Finally, it is likely that many other environmental pollutants impact neovascularization; however, very few have been studied in sufficient detail. Thus, additional research also is needed to identify those environmental agents that mediate their toxicity by disrupting neovascularization.