Acute exposure of early-life stage zebrafish (Danio rerio) to Deepwater Horizon crude oil impairs glomerular filtration and renal fluid clearance capacity.

The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any defects in kidney development and function are likely associated with a decreased ability to osmoregulate. Previous work has shown that early-life stage (ELS) zebrafish (Danio rerio) acutely exposed to Deepwater Horizon (DWH) crude oil exhibit transcriptional changes in key genes involved in pronephros development and function, as well as pronephric morphological defects and whole-animal osmoregulatory impairment. The objective of this study was to examine the acute effects of crude oil exposure during zebrafish ELS on pronephros function by assessing its fluid clearance capacity and glomerular filtration integrity. Following a 72-h exposure to control conditions, 20% or 40% dilutions of high-energy water-accommodated fractions (HEWAF) of DWH crude oil, zebrafish were injected into the common cardinal vein either with fluorescein-labeled (FITC) 70-kDa dextran to assess glomerular filtration integrity or with FITC-inulin to assess pronephric clearance capacity. Fluorescence was quantified after the injections at predetermined time intervals by fluorescence microscopy. The results demonstrated a diminished pronephric fluid clearance capacity and failed glomerular perfusion when larvae were exposed to 40% HEWAF dilutions, whereas only a reduced glomerular filtration selectivity was observed in zebrafish previously exposed to the 20% HEWAF dilution.

Probiotics Function as Immunomodulators in the Intestine in C57Bl/6 Male Mice Exposed to Inhaled Diesel Exhaust Particles on a High-Fat Diet.

Epidemiological studies reveal a correlation between air pollution exposure and gastrointestinal (GI) diseases, yet few studies have investigated the role of inhaled particulate matter on intestinal integrity in conjunction with a high-fat (HF) diet. Additionally, there is currently limited information on probiotics in mitigating air-pollutant responses in the intestines. Thus, we investigated the hypothesis that exposure to inhaled diesel exhaust particles (DEP) and a HF diet can alter intestinal integrity and inflammation, which can be attenuated with probiotics. 4-6-w-old male C57Bl/6 mice on a HF diet (45% kcal fat) were randomly assigned to be exposed via oropharyngeal aspiration to 35 µg of DEP suspended in 35 µL of 0.9% sterile saline or sterile saline (CON) only twice a week for 4 w. A subset of mice was treated with 0.3 g/day of Winclove Ecologic® barrier probiotics (PRO) in drinking water throughout the duration of the study. Our results show that DEP exposure ± probiotics resulted in increased goblet cells and mucin (MUC)-2 expression, as determined by AB/PAS staining. Immunofluorescent quantification and/or RT-qPCR showed that DEP exposure increases claudin-3, occludin, zona occludens (ZO)-1, matrix metalloproteinase (MMP)-9, and toll-like receptor (TLR)-4, and decreases tumor necrosis factor (TNF)-α and interleukin (IL)-10 expression compared to CON. DEP exposure + probiotics increases expression of claudin-3, occludin, ZO-1, TNF-α, and IL-10 and decreases MMP-9 and TLR-4 compared to CON + PRO in the small intestine. Collectively, these results show that DEP exposure alters intestinal integrity and inflammation in conjunction with a HF diet. Probiotics proved fundamental in understanding the role of the microbiome in protecting and altering inflammatory responses in the intestines following exposure to inhaled DEP.

Transcriptomic responses and apoptosis in larval red drum (Sciaenops ocellatus) co-exposed to crude oil and ultraviolet (UV) radiation.

Ultraviolet (UV) radiation can significantly increase the toxicity of polycyclic aromatic hydrocarbons (PAHs) in crude oil to early life stage (ELS) fishes through photo-induced /photo-enhanced toxicity. However, little is known about the sub-lethal effects and mechanisms of photo-induced PAH toxicity in ELS fishes. The present study investigated apoptosis and global transcriptomic effects in larval red drum (Sciaenops ocellatus) (24-72 h post-fertilization) following co-exposure to oil (0.29-0.30 µg/L ∑PAH50) and UV. Apoptosis was quantified using the TUNEL assay, and transcriptomic effects were assessed using RNA sequencing analysis. Apoptotic fluorescence was greatest in the eyes and skin following 24 and 48 h co-exposure to oil and UV, indicating photo-induced toxicity. Consistent with these phenotypic responses, pathways associated with phototransduction, eye development, and dermatological disease were among the top predicted pathways impacted. The present study is the first to provide global transcriptomic analysis of UV and oil co-exposure in an ELS fish.

Traffic-generated air pollution - Exposure mediated expression of factors associated with demyelination in a female apolipoprotein E-/- mouse model.

Epidemiology studies suggest that exposure to ambient air pollution is associated with demyelinating diseases in the central nervous system (CNS), including multiple sclerosis (MS). The pathophysiology of MS results from an autoimmune response involving increased inflammation and demyelination in the CNS, which is higher in young (adult) females. Exposure to traffic-generated air pollution is associated with neuroinflammation and other detrimental outcomes in the CNS; however, its role in the progression of pathologies associated with demyelinating diseases has not yet been fully characterized in a female model. Thus, we investigated the effects of inhalation exposure to mixed vehicle emissions (MVE) in the brains of both ovary-intact (ov+) and ovariectomized (ov-) female Apolipoprotein (ApoE-/-) mice. Ov + and ov- ApoE-/- mice were exposed via whole-body inhalation to either filtered air (FA, controls) or mixed gasoline and diesel vehicle emissions (MVE: 200 PM µg/m3) for 6 h/d, 7 d/wk., for 30 d. We then analyzed MVE-exposure mediated alterations in myelination, the presence of CD4+ and CD8+ T cells, reactive oxygen species (ROS), myelin oligodendrocyte protein (MOG), and expression of estrogen (ERα and ERß) and progesterone (PROA/B) receptors in the CNS. MVE-exposure mediated significant alterations in myelination across multiple regions in the cerebrum, as well as increased CD4+ and CD8+ staining. There was also an increase in ROS production in the CNS of MVE-exposed ov- and ov + ApoE-/- mice. Ov- mice displayed a reduction in cerebral ERα mRNA expression, compared to ov + mice; however, MVE exposure resulted in an even further decrease in ERα expression, while ERß and PRO A/B were unchanged across groups. These findings collectively suggest that inhaled MVE-exposure may mediate estrogen receptor expression alterations associated with increased CD4+/CD8+ infiltration, regional demyelination, and ROS production in the CNS of female ApoE-/- mice.

Inhalation exposure to silver nanoparticles induces hepatic inflammation and oxidative stress, associated with altered renin-angiotensin system signaling, in Wistar rats.

Silver nanoparticles (AgNPs) have become increasingly popular in the biomedical field over the last few decades due to its proven antibacterial property. Previous scientific studies have reported that one of the major organs responsible for detoxification of AgNPs is the liver. The liver is also the primary organ responsible for secretion of angiotensinogen (AGT), a key signaling molecule involved in the renin-angiotensin system (RAS), which plays an important role in maintaining cardiac output and vascular pressure. The aim of this study was to assess any potential changes in the RAS-associated gene signaling, inflammatory response, and hepatocellular toxicity resulting from AgNP exposure. To do this, 6-week-old, male Wistar rats were exposed to a subacute inhalation exposure of AgNP (200 ppb/days over 4 h/days exposure, for 5 d) and their livers were analyzed for alterations in RAS components, inflammation, and oxidative stress. Real time qPCR analysis showed that AgNP-exposure resulted in a significant increase in hepatic AGT, angiotensin converting enzyme (ACE)-1, and ACE-2 mRNA expression. Expression of inflammatory markers interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were also upregulated with AgNP-exposure, compared to controls. Furthermore AgNP-exposure mediated a significant increase in hepatic expression of catalase, and superoxide dismutase, and oxidative stress, as assessed via 8-Oxo-2'-deoxyguanosine staining. Increased oxidative stress was associated with increased monocyte/macrophage-2 staining in the liver of AgNP-exposed rats. Such findings indicate that subacute inhalation exposure to AgNPs mediate increased hepatic RAS signaling, associated with inflammation, macrophage infiltration, and oxidative stress.

Vehicle emissions-exposure alters expression of systemic and tissue-specific components of the renin-angiotensin system and promotes outcomes associated with cardiovascular disease and obesity in wild-type C57BL/6 male mice.

Exposure to air pollution from traffic-generated sources is known to contribute to the etiology of inflammatory diseases, including cardiovascular disease (CVD) and obesity; however, the signaling pathways involved are still under investigation. Dysregulation of the renin-angiotensin system (RAS) can contribute to CVD and alter lipid storage and inflammation in adipose tissue. Our previous exposure studies revealed that traffic-generated emissions increase RAS signaling, further exacerbated by a high-fat diet. Thus, we investigated the hypothesis that exposure to engine emissions increases systemic and local adipocyte RAS signaling, promoting the expression of factors involved in CVD and obesity. Male C57BL/6 mice (6-8 wk old) were fed either a high-fat (HF, n = 16) or low-fat (LF, n = 16) diet, beginning 30d prior to exposures, and then exposed via inhalation to either filtered air (FA, controls) or a mixture of diesel engine + gasoline engine vehicle emissions (MVE: 100 µg PM/m3) via whole-body inhalation for 6 h/d, 7 d/wk, 30d. Endpoints were assessed via immunofluorescence and RT-qPCR. MVE-exposure promoted vascular adhesion factors (VCAM-1, ICAM-1) expression, monocyte/macrophage sequestration, and oxidative stress in the vasculature, associated with increased angiotensin II receptor type 1 (AT1) expression. In the kidney, MVE-exposure promoted the expression of renin, AT1, and AT2 receptors. In adipose tissue, both HF-diet and MVE-exposure mediated increased epididymal fat pad weight and adipocyte hypertrophy, associated with increased angiotensinogen and AT1 receptor expression; however, these outcomes were further exacerbated in the MVE + HF group. MVE-exposure also induced inflammation, monocyte chemoattractant protein (MCP)-1, and leptin, while reducing insulin receptor and glucose transporter, GLUT4, expression in adipose tissue. Our results indicate that MVE-exposure promotes systemic and local adipose RAS signaling, associated with increased expression of factors contributing to CVD and obesity, further exacerbated by HF diet consumption.

Exposure to diesel exhaust particles results in altered lung microbial profiles, associated with increased reactive oxygen species/reactive nitrogen species and inflammation, in C57Bl/6 wildtype mice on a high-fat diet.

BACKGROUND: Exposure to traffic-generated emissions is associated with the development and exacerbation of inflammatory lung disorders such as chronic obstructive pulmonary disorder (COPD) and idiopathic pulmonary fibrosis (IPF). Although many lung diseases show an expansion of Proteobacteria, the role of traffic-generated particulate matter pollutants on the lung microbiota has not been well-characterized. Thus, we investigated the hypothesis that exposure to diesel exhaust particles (DEP) can alter commensal lung microbiota, thereby promoting alterations in the lung's immune and inflammatory responses. We aimed to understand whether diet might also contribute to the alteration of the commensal lung microbiome, either alone or related to exposure. To do this, we used male C57Bl/6 mice (4-6-week-old) on either regular chow (LF) or high-fat (HF) diet (45% kcal fat), randomly assigned to be exposed via oropharyngeal aspiration to 35 µg DEP, suspended in 35 µl 0.9% sterile saline or sterile saline only (control) twice a week for 30 days. A separate group of study animals on the HF diet was concurrently treated with 0.3 g/day of Winclove Ecologic® Barrier probiotics in their drinking water throughout the study. RESULTS: Our results show that DEP-exposure increases lung tumor necrosis factor (TNF)-α, interleukin (IL)-10, Toll-like receptor (TLR)-2, TLR-4, and the nuclear factor kappa B (NF-κB) histologically and by RT-qPCR, as well as Immunoglobulin A (IgA) and Immunoglobulin G (IgG) in the bronchoalveolar lavage fluid (BALF), as quantified by ELISA. We also observed an increase in macrophage infiltration and peroxynitrite, a marker of reactive oxygen species (ROS) + reactive nitrogen species (RNS), immunofluorescence staining in the lungs of DEP-exposed and HF-diet animals, which was further exacerbated by concurrent DEP-exposure and HF-diet consumption. Histological examinations revealed enhanced inflammation and collagen deposition in the lungs DEP-exposed mice, regardless of diet. We observed an expansion of Proteobacteria, by qPCR of bacterial 16S rRNA, in the BALF of DEP-exposed mice on the HF diet, which was diminished with probiotic-treatment. CONCLUSIONS: Our findings suggest that exposure to DEP causes persistent and sustained inflammation and bacterial alterations in a ROS-RNS mediated fashion, which is exacerbated by concurrent consumption of an HF diet.

Exposure to traffic-generated air pollution promotes alterations in the integrity of the brain microvasculature and inflammation in female ApoE-/- mice.

Traffic-generated air pollutants have been correlated with alterations in blood-brain barrier (BBB) integrity, which is associated with pathologies in the central nervous system (CNS). Much of the existing literature investigating the effects of air pollution in the CNS has predominately been reported in males, with little known regarding the effects in females. As such, this study characterized the effects of inhalation exposure to mixed vehicle emissions (MVE), as well as the presence of female sex hormones, in the CNS of female ApoE-/- mice, which included cohorts of both ovariectomized (ov-) and ovary-intact (ov+) mice. Ov + and ov- were placed on a high-fat diet and randomly grouped to be exposed to either filtered-air (FA) or MVE (200 PM/m3: 50 µg PM/m3 gasoline engine + 150 µg PM/m3 from diesel engine emissions) for 6 h/d, 7d/wk, for 30d. MVE-exposure resulted in altered cerebral microvascular integrity and permeability, as determined by the decreased immunofluorescent expression of tight junction (TJ) proteins, occludin, and claudin-5, and increased IgG extravasation into the cerebral parenchyma, compared to FA controls, regardless of ovary status. Associated with the altered cerebral microvascular integrity, we also observed an increase in matrix metalloproteinases (MMPs) -2/9 activity in the MVE ov+, MVE ov-, and FA ov- groups, compared to FA ov+. There was also elevated expression of intracellular adhesion molecule (ICAM)-1, inflammatory interleukins (IL-1, IL-1ß), and tumor necrosis factor (TNF-α) mRNA in the cerebrum of MVE ov + and MVE ov- animals. IκB kinase (IKK) subunits IKKα and IKKß mRNA expressions were upregulated in the cerebrum of MVE ov- and FA ov- mice. Our findings indicate that MVE exposure mediates altered integrity of the cerebral microvasculature correlated with increased MMP-2/9 activity and inflammatory signaling, regardless of female hormones present.

Exposure to Traffic-Generated Pollutants Exacerbates the Expression of Factors Associated with the Pathophysiology of Alzheimer's Disease in Aged C57BL/6 Wild-Type Mice.

BACKGROUND: Multiple studies report a strong correlation between traffic-generated air pollution-exposure and detrimental outcomes in the central nervous system (CNS), including Alzheimer's disease (AD). Incidence of AD is rapidly increasing and, worldwide, many live in regions where pollutants exceed regulatory standards. Thus, it is imperative to identify environmental pollutants that contribute to AD, and the mechanisms involved. OBJECTIVE: We investigated the effects of mixed gasoline and diesel engine emissions (MVE) on the expression of factors involved in progression of AD in the hippocampus and cerebrum in a young versus aged mouse model. METHODS: Young (2 months old) and aged (18 months old) male C57BL/6 mice were exposed to either MVE (300µg/m3 PM) or filtered air (FA) for 6 h/d, 7 d/wk, for 50 d. Immunofluorescence and RT-qPCR were used to quantify oxidative stress (8-OHdG) and expression of amyloid-ß protein precursor (AßPP), ß secretase (BACE1), amyloid-ß (Aß), aryl hydrocarbon receptor (AhR), cytochrome P450 (CYP) 1B1, angiotensin-converting enzyme (ACE1), and angiotensin II type 1 (AT1) receptor in the cerebrum and hippocampus, in addition to cerebral microvascular tight junction (TJ) protein expression. RESULTS: We observed age-related increases in oxidative stress, AhR, CYP1B1, Aß, BACE1, and AT1 receptor in the CA1 region of the hippocampus, and elevation of cerebral AßPP, AhR, and CYP1B1 mRNA, associated with decreased cerebral microvascular TJ protein claudin-5. MVE-exposure resulted in further promotion of oxidative stress, and significant increases in AhR, CYP1B1, BACE1, ACE1, and Aß, compared to the young and aged FA-exposed mice. CONCLUSION: Such findings suggest that MVE-exposure exacerbates the expression of factors in the CNS associated with AD pathogenesis in aged populations.

Exposure to Crude Oil Induces Retinal Apoptosis and Impairs Visual Function in Fish.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil are known to impair visual development in fish. However, the underlying mechanism of PAH-induced toxicity to the visual system of fish is not understood. Embryonic zebrafish (Danio rerio) at 4 h post fertilization were exposed to weathered crude oil and assessed for visual function using an optokinetic response, with subsequent samples taken for immunohistochemistry and gene expression analysis. Cardiotoxicity was also assessed by measuring the heart rate, stroke volume, and cardiac output, as cardiac performance has been proposed to be a contributing factor to eye-associated malformations following oil exposure. Larvae exposed to the highest concentrations of crude oil (89.8 µg/L) exhibited an increased occurrence of bradycardia, though no changes in stroke volume or cardiac output were observed. However, genes important in eye development and phototransduction were downregulated in oil-exposed larvae, with an increased occurrence of cellular apoptosis, reduced neuronal connection, and reduced optokinetic behavioral response in zebrafish larvae.

Effects of inhaled air pollution on markers of integrity, inflammation, and microbiota profiles of the intestines in Apolipoprotein E knockout mice.

Air pollution exposure is known to contribute to the progression of cardiovascular disease (CVD) and there is increasing evidence that dysbiosis of the gut microbiome may also play a role in the pathogenesis of CVD, including atherosclerosis. To date, the effects of inhaled air pollution mixtures on the intestinal epithelial barrier (IEB), and microbiota profiles are not well characterized, especially in susceptible individuals with comorbidity. Thus, we investigated the effects of inhaled ubiquitous air-pollutants, wood-smoke (WS) and mixed diesel and gasoline vehicle exhaust (MVE) on alterations in the expression of markers of integrity, inflammation, and microbiota profiles in the intestine of atherosclerotic Apolipoprotein E knockout (ApoE-/-) mice. To do this, male 8 wk-old ApoE-/- mice, on a high-fat diet, were exposed to either MVE (300 µg/m3 PM), WS; (∼450 µg/m3 PM), or filtered air (FA) for 6 h/d, 7 d/wk, for 50 d. Immunofluorescence and RT-PCR were used to quantify the expression of IEB components and inflammatory factors, including mucin (Muc)-2, tight junction (TJ) proteins, matrix metalloproteinase (MMP)-9, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß, as well as Toll-like receptor (TLR)-4. Microbial profiling of the intestine was done using Illumina 16S sequencing of V4 16S rRNA PCR amplicons. We observed a decrease in intestinal Muc2 and TJ proteins in both MVE and WS exposures, compared to FA controls, associated with a significant increase in MMP-9, TLR-4, and inflammatory marker expression. Both WS and MVE-exposure resulted in decreased intestinal bacterial diversity, as well as alterations in microbiota profiles, including the Firmicutes: Bacteroidetes ratio at the phylum level. Our findings suggest inhalation exposure to either MVE or WS result in alterations in components involved in mucosal integrity, and also microbiota profiles and diversity, which are associated with increased markers of an inflammatory response.

The effects of subacute inhaled multi-walled carbon nanotube exposure on signaling pathways associated with cholesterol transport and inflammatory markers in the vasculature of wild-type mice.

Exposure to multi-walled carbon nanotubes (MWCNTs) has been associated with detrimental cardiovascular outcomes; however, underlying mechanisms have not yet been fully elucidated. Thus, we investigated alterations in proatherogenic and proinflammatory signaling pathways in C57Bl6/ mice exposed to MWCNTs (1 mg/m3) or filtered air (FA-Controls), via inhalation, for 6 h/day, 14d. Expression of mediators of cholesterol transport, namely the lectin-like oxidized low-density lipoprotein receptor (LOX)-1 and ATP-binding cassette transporter (ABCA)-1, inflammatory markers tumor necrosis factor (TNF)-α and interleukin (IL)-1ß/IL-6, nuclear-factor kappa-light-chain-enhancer of activated B cells (NF-κB), intracellular/vascular adhesion molecule(s) (VCAM-1, ICAM-1), and miRNAs (miR-221/-21/-1), associated with cardiovascular disease (CVD), were analyzed in cardiac tissue and coronary vasculature. Cardiac fibrotic deposition, matrix-metalloproteinases (MMP)-2/9, and reactive oxygen species (ROS) were also assessed. MWCNT-exposure resulted in increased coronary ROS production with concurrent increases in expression of LOX-1, VCAM-1, TNF-α, and MMP-2/9 activity; while ABCA-1 expression was downregulated, compared to FA-Controls. Additionally, trends in fibrotic deposition and induction of cardiac TNF-α, MMP-9, IκB Kinase (IKK)-α/ß, and miR-221 mRNA expression were observed. Analysis using inhibitors for nitric oxide synthase or NADPH oxidase resulted in attenuated coronary ROS production. These findings suggest that subacute inhalation MWCNT-exposure alters expression of cholesterol transporter/receptors, and induces signaling pathways associated with inflammation, oxidative stress, and CVD in wild-type mice.

Identification of chemical components of combustion emissions that affect pro-atherosclerotic vascular responses in mice.

Combustion emissions cause pro-atherosclerotic responses in apolipoprotein E-deficient (ApoE/⁻) mice, but the causal components of these complex mixtures are unresolved. In studies previously reported, ApoE⁻/⁻ mice were exposed by inhalation 6 h/day for 50 consecutive days to multiple dilutions of diesel or gasoline exhaust, wood smoke, or simulated "downwind" coal emissions. In this study, the analysis of the combined four-study database using the Multiple Additive Regression Trees (MART) data mining approach to determine putative causal exposure components regardless of combustion source is reported. Over 700 physical-chemical components were grouped into 45 predictor variables. Response variables measured in aorta included endothelin-1, vascular endothelin growth factor, three matrix metalloproteinases (3, 7, 9), metalloproteinase inhibitor 2, heme-oxygenase-1, and thiobarbituric acid reactive substances. Two or three predictors typically explained most of the variation in response among the experimental groups. Overall, sulfur dioxide, ammonia, nitrogen oxides, and carbon monoxide were most highly predictive of responses, although their rankings differed among the responses. Consistent with the earlier finding that filtration of particles had little effect on responses, particulate components ranked third to seventh in predictive importance for the eight response variables. MART proved useful for identifying putative causal components, although the small number of pollution mixtures (4) can provide only suggestive evidence of causality. The potential independent causal contributions of these gases to the vascular responses, as well as possible interactions among them and other components of complex pollutant mixtures, warrant further evaluation.

Inhaled diesel emissions alter atherosclerotic plaque composition in ApoE(-/-) mice.

Recent epidemiological studies suggest that traffic-related air pollution may have detrimental effects on cardiovascular health. Previous studies reveal that gasoline emissions can induce several enzyme pathways involved in the formation and development of atherosclerotic plaques. As a direct comparison, the present study examined the impact of diesel engine emissions on these pathways, and further examined the effects on vascular lesion pathology. Apolipoprotein E-null mice were simultaneously placed on a high-fat chow diet and exposed to four concentrations, plus a high concentration exposure with particulates (PM) removed by filtration, of diesel emissions for 6 h/day for 50 days. Aortas were subsequently assayed for alterations in matrix metalloproteinase-9, endothelin-1, and several other biomarkers. Diesel induced dose-related alterations in gene markers of vascular remodeling and aortic lipid peroxidation; filtration of PM did not significantly alter these vascular responses, indicating that the gaseous portion of the exhaust was a principal driver. Immunohistochemical analysis of aortic leaflet sections revealed no net increase in lesion area, but a significant decrease in lipid-rich regions and increasing trends in macrophage accumulation and collagen content, suggesting that plaques were advanced to a more fragile, potentially more vulnerable state by diesel exhaust exposure. Combined with previous studies, these results indicate that whole emissions from mobile sources may have a significant role in promoting chronic vascular disease.

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.