Stress Drivers of Glucose Dynamics during Ozone Exposure Measured Using Radiotelemetry in Rats

Abstract

Background: Inhaled irritant air pollutants may trigger stress-related metabolic dysfunction associated with altered circulating adrenal-derived hormones. Objectives: We used implantable telemetry in rats to assess real-time changes in circulating glucose during and after exposure to ozone and mechanistically linked responses to neuroendocrine stress hormones. Methods: First, using a cross-over design, we monitored glucose during ozone exposures (0.0, 0.2, 0.4, and 0.8 ppm) and nonexposure periods in male Wistar Kyoto rats implanted with glucose telemeters. A second cohort of unimplanted rats was exposed to ozone (0.0, 0.4 or 0.8 ppm) for 30 min, 1 h, 2 h, or 4 h with hormones measured immediately post exposure. We assessed glucose metabolism in sham and adrenalectomized rats, with or without supplementation of adrenergic/glucocorticoid receptor agonists, and in a separate cohort, antagonists. Results: Ozone (0.8 ppm) was associated with significantly higher blood glucose and lower core body temperature beginning 90 min into exposure, with reversal of effects 4-6 h post exposure. Glucose monitoring during four daily 4-h ozone exposures revealed duration of glucose increases, adaptation, and diurnal variations. Ozone-induced glucose changes were preceded by higher levels of adrenocorticotropic hormone, corticosterone, and epinephrine but lower levels of thyroid-stimulating hormone, prolactin, and luteinizing hormones. Higher glucose and glucose intolerance were inhibited in rats that were adrenalectomized or treated with adrenergic plus glucocorticoid receptor antagonists but exacerbated by agonists. Discussion: We demonstrated the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced glucose metabolic dysfunction and mechanism in a rodent model. Stress hormones assessment with real-time glucose monitoring may be useful in identifying interactions among irritant pollutants and stress-related illnesses.

Headnote

Background: Inhaled irritant air pollutants may trigger stress-related metabolic dysfunction associated with altered circulating adrenal-derived hormones.

Objectives: We used implantable telemetry in rats to assess real-time changes in circulating glucose during and after exposure to ozone and mechanistically linked responses to neuroendocrine stress hormones.

Methods: First, using a cross-over design, we monitored glucose during ozone exposures (0.0, 0.2, 0.4, and 0.8 ppm) and nonexposure periods in male Wistar Kyoto rats implanted with glucose telemeters. A second cohort of unimplanted rats was exposed to ozone (0.0, 0.4 or 0.8 ppm) for 30 min, 1 h, 2 h, or 4 h with hormones measured immediately post exposure. We assessed glucose metabolism in sham and adrenalectomized rats, with or without supplementation of adrenergic/glucocorticoid receptor agonists, and in a separate cohort, antagonists.

Results: Ozone (0.8 ppm) was associated with significantly higher blood glucose and lower core body temperature beginning 90 min into exposure, with reversal of effects 4-6 h post exposure. Glucose monitoring during four daily 4-h ozone exposures revealed duration of glucose increases, adaptation, and diurnal variations. Ozone-induced glucose changes were preceded by higher levels of adrenocorticotropic hormone, corticosterone, and epinephrine but lower levels of thyroid-stimulating hormone, prolactin, and luteinizing hormones. Higher glucose and glucose intolerance were inhibited in rats that were adrenalectomized or treated with adrenergic plus glucocorticoid receptor antagonists but exacerbated by agonists.

Discussion: We demonstrated the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced glucose metabolic dysfunction and mechanism in a rodent model. Stress hormones assessment with real-time glucose monitoring may be useful in identifying interactions among irritant pollutants and stress-related illnesses. https://doi.org/10.1289/EHP11088

Introduction

Among many environmental risk factors, air pollution accounted for over 70% of all environmental causes of human mortality in 20191 and has been associated with altered neurobehavioral performance in children2 and metabolic diseases in a panel study.3 Increased incidence of Alzheimer disease,4 late-life cognitive decline,5 anxiety,6 and risk of violent behavior7 were associated with exposure to air pollutants. Moreover, associations were found between air pollution and concurrent exacerbation of diabetes and Alzheimer disease.5 Those with Alzheimer8 and Parkinson disease9 also often suffer from diabetes, suggesting potential neural contribution to systemic diseases and mechanistic linkages. Recently, it has become apparent that inhaled irritant air pollutants might be perceived as stressors by the autonomic nervous system, resulting in stimulation of neuroendocrine axes that mediate acute effects in the lung and periphery.10 Because stress responses are dynamic and central nervous system (CNS)-regulated to induce reversible peripheral changes,11'12 real-time assessment is necessary for in-depth understanding of the impact of stress response on health and resiliency.

Neural mechanisms of integrated peripheral stress response and the regulation of its temporality involve highly complex interactive communication between multiple brain regions and the neuroendocrine system.13 With the emerging link between air pollution, stress,14 and neurocognition,15 the role of the neuroendocrine system deserves further exploration. Psychosocial and environmental stressors are proposed to be the primary contributors to chronic disease susceptibility.16'17 Chronic alterations in the levels of circulating stress hormones, especially glucocorticoids, were linked to psychological disorders and cardiometabolic diseases.18 Centrally mediated release of stress hormones and peripheral responses to stress are plastic, and these hormones are temporally and spatially regulated such that adverse effects following acute stressor exposure are often reversible on discontinuation of stress.19 However, when the neuroendocrine system is impaired or overactive, disease may ensue.20 Thus, the temporal assessment of the stress dynamics is critical to understanding an individual's susceptibility to environmental insults.

Monitoring of the stress response through real-time assessment of circulating Cortisol and glucose in humans is not common. However, in children recovering from surgery, blood glucose and Cortisol were measured to assess stress.21 Individuals with type 2 diabetes, when subjected to acute moderate psychological stress (Trier Social Stress Test), had spikes in blood glucose as determined using real-time glucose monitors.22 Very recently, a wireless graphene-based sweat stress-sensing mHealth system was developed for dynamic and noninvasive assessment of Cortisol in sweat.23 The use of new techniques for dynamic stress assessment will be valuable for determining the health impact of stressors on the body. This approach in air pollution studies could promote the evaluation of short- and long-term effects on metabolic health and resiliency and provide diagnostic and mechanistic insights.

We showed that an acute single exposure to ozone induced a classic stress response associated with increases in circulating catecholamines, glucocorticoids, lipid metabolites, and glucose in rats,24 consistent with previously reported higher Cortisol and lipid metabolites in humans after ozone exposure relative to air.25 However, the neuroendocrine stress response is temporal and might be reversible in healthy individuals.11'12 To link ozone-induced alterations in metabolic processes with neuroendocrine stress, it is critical to determine the dynamicity of peripheral metabolic effects and its relation to neuroendocrine changes. The purpose of the current study was to determine the temporality of glucose changes during and after ozone exposure in a rat model using implantable radiotelemetry and to assess the linkages between glucose and neurohormone response. We hypothesized that higher blood glucose levels during ozone exposure in rats when compared with those of air-exposed rats would be due to ozone-induced alterations of neuroendocrine and stress hormones. Real-time glucose measures were coupled with separate temporal measures of stress hormones, as well as mechanistic studies to assess the role of stress responses in observed differences in glucose levels between ozone-exposed and nonexposed groups.

Materials and Methods

Animals

Male Wistar Kyoto (WKY) rats for all studies were purchased from Charles River Laboratories, Inc., at 10-12 wk of age and maintained in our Association for Assessment and Accreditation of Laboratory Animal Care-approved U.S. Environmental Protection Agency (U.S. EPA) animal facility. Animals were pair-housed in polycarbonate cages with hard wood chips or pine-shavings bedding and EnviroDry enrichment material (Lab Supply), except when stated. Animal rooms were maintained on a 12-h light/dark cycle [0600-1800 hours (6 A.M.-6 P.M.)] at ~ 22°C and 50% relative humidity. They were provided free access to Purina (5001) pellet rat chow and water ad libitum, unless stated during experimental procedures. All animal protocols were approved by the U.S. EPA's Institutional Animal Care and Use Committee prior to starting studies, and we followed National Institutes of Health guide for the care and use of rats (National Institutes of Health Publication No. 8023; ncbi.nlm. nih.gov/books/NBK54050/).

Experimental Protocols

Data from four distinct cohorts of male rats are designated as: a) Telemetry, b) Time Course, c) adrenalectomy (AD) agonists [(32-adrenergic receptor (AR) + glucocorticoid receptor (GR)], and d) antagonists ((3-AR and GR). A detailed breakdown of these studies is provided in Table 1. The goal of the glucose telemetry study was to describe real-time glucose and body temperature data over 7 wk of episodic ozone exposures and glucose tolerance tests (GTTs) in rats; no data from this study have previously been published. The goal of the hormonal time course study was to describe the temporal response to acute ozone exposure on circulating pituitary, adrenal, and metabolic hormones as well as free fatty acids related to glucose changes; corticosterone, epinephrine, and leukocyte trafficking data from the same cohort of rats were previously published.26 The goal of the AD antagonists study was to describe the effects of surgical elimination of adrenal hormones and demonstrate whether epinephrine and glucocorticoid receptor agonists ((32-AR plus GR agonists) treatment was sufficient to restore ozone-induced hyperglycemia (significant difference in blood glucose after ozone exposure) and glucose intolerance in AD rats; ventilatory parameters, pulmonary protein leakage, and inflammation data from the same cohort of rats were previously published.27 The goal of the (3-AR± GR Antagonists study was to determine whether pharmaceutical antagonism of (3-AR and/or GR caused the same effects as surgical removal of adrenal hormones on ozone-induced responses and to characterize the differential role of glucocorticoids and catecholamines; ventilatory parameters and pulmonary protein leakage and inflammation data from same cohort of rats have been previously published.28

Telemetry Study: Glucose Telemetry Surgeries

Eight male WKY rats (at 13 wk of age) were surgically implanted with Data Sciences International, Inc., (DSI) glucose telemeters (HD-XG) using aseptic techniques (Figure SI; Table SI). Anesthesia was induced by vaporized isoflurane inhalation [4%, 1-2 liters per minute (LPM) of O2] and maintained during the surgery (2%-3%, 1-2 LPM of O2). Once anesthetized, analgesic meloxicam (2 mg/kg, in saline, subcutaneous; s.c.) and artificial tear ointment were provided before surgery. Anesthesia was continuously checked by toe pinch. A trained surgeon implanted the sensor in the descending abdominal aorta and the transmitter was anchored to muscle in the abdominal cavity. The blood glucose (Nova Biomedical) sensor uses glucose oxidase to convert glucose and oxygen into gluconic acid and hydrogen peroxide. The amount of hydrogen peroxide, which is proportional to the amount of glucose, reacts with a noble metal electrode to transfer electrons and create a current.29 The glucose telemetry system included a reference electrode with an electronics/battery (Ag/ AgCl) with a separate lead. This apparatus was sutured to the inner abdominal wall module into the midline abdominal muscle (Figure SI). During abdominal wall closure, the abdominal cavity was washed with saline (15 mL/kg), and rats were given bupiva-caine (1 mg/kg, in saline s.c). Recovery took place on heating pads under close observation of distress signals; once awake, the rats were placed into their home cages and administered with meloxicam (1 mg/kg, in saline s.c.) 24, 48, and 72 h after the surgery.

Blood glucose concentration data acquisition prior to exposure. After surgery, rats were individually housed in cages with pine-shavings bedding. Rats were provided with water and powdered as well as pelleted standard Purina (5001) rat chow ad libitum. Blood glucose level and core body temperature measurements were sent via radio signals and collected using receivers placed under each cage. The recording for each rat began soon after the surgery and continued until the end of the experiment (~ 9 wk). The cages were placed over receivers (RPC-1; DSI) and data were simultaneously collected in a computer placed in an adjacent isolated room (Figure SI). For verification and calibration purposes, single point calibrations were carried out. As recommended, these calibrations were done twice weekly throughout the study based on the protocol explained by Brockway et al. at Data Sciences International, Inc.29 Data used for calibration are excluded from analysis and graphs. Interpolation of glucose levels and electric current detected by the sensor was corrected for differences in body temperature vs. room temperature, merged and analyzed for each animal with a resolution of 1 min using Dataquest software acquisition system (DSI). The Dataquest acquisition system (DSI) included a telemetry signal receiver for each animal, a data matrix for analyzing receiver signals, and a computer. These telemetry devices allowed continuous sampling of eight animals that are individually housed in cages with receivers directly underneath. To avoid signal mixing between two animals, the animal racks were equipped with stainless-steel dividers. The software allowed device configuration and the protocol for continuous data collection as described previously.29

Timeline of telemetered rat experiments. Two weeks after surgery, rats were exposed in pairs whole body to ozone (0.2, 0.4, or 0.8 ppm) or filtered air (0.0 ppm) in Rochester style "Hinners" chambers, using a crossover exposure design such that each rat was exposed to each concentration across the first 4 wk of testing (Table SI). We previously reported that hyperglycemia and glucose intolerance noted after an acute ozone exposure were reversible next day24 and that hyperglycemia, glucose intolerance, and lung injury/inflammation noted after weekly ozone exposure (3 d/wk) for 12 wk were also reversible on 1 wk of recovery in home cages.30 Thus, crossover design was appropriate for real-time glucose telemetry because the weekly washout was sufficient to clear effects. Briefly, rats for the first exposure were randomized in pairs for 0.0 (clean air), 0.2, 0.4, or 0.8 ppm of ozone exposure for 4 h. A different set of receivers were placed in each ozone exposure chamber to acquire data while animals were being exposed. After 1 wk of washout period, these exposures were repeated, but the targeted ozone exposure concentration for each pair of rats was changed. This experiment was repeated a total of four times over 4 wk to cover all the targeted ozone concentrations for all rats (n = l independent recordings). Rat #6 was discarded from the experiment due to malfunction of the sensor and/or transmitter. During week 5 and 6, these seven telemetered rats were randomized into two groups, air and 0.8 ppm ozone, and again using crossover design, they were exposed for 4 h, once per week for 2 wk (Table SI), and glucose levels were continuously monitored (n = 7-8 per concentration). Immediately after each exposure, a GTT was performed using a glucometer as described in the section titled "GTT for All Rat Experiments" in addition to continuous recording of glucose through telemetry. Finally, during week 7, animals were divided into air (n = 3) and ozone (n = 4), and at that time they were exposed to air or 0.8 ppm ozone 4 h/d for four consecutive days to determine adaptation on repeated exposure (Table SI). Because animals were exposed for four consecutive days during a single week, a crossover design was not possible.

Glucose/temperature telemetry data acquisition and analysis during exposures. During the day of exposure, animals were transported to the nearby exposure room, where a control and three ozone exposure chambers were preprepared with a different set of receivers connected to an independent but identical data acquisition system to acquire data during exposure for each rat. Within ~ 5 minutes of removal from their home cages, animals were immediately placed at designated locations (with receivers placed underneath) in the exposure chambers with wire-mesh bottom and walls, and data were collected with the data acquisition system during exposure. Rats were placed at two opposite corners of the chamber to avoid signal mixing. All exposures were aligned to minute 0 when 80% of target ozone concentration was reached. For each minute, glucose level and temperature were averaged from exposed rats (n = 7/concentration). Immediately following the exposure, animals were likewise transported to their home cages for continued data acquisition. The data collected during nonexposure periods when telemetered rats are in their home cages and during air or ozone exposure periods were temporally aligned to tally continuous recording each day and during the entire experimental period. Glucose and temperature data with a resolution of 1 min was averaged to prepare graphs. As mentioned above, for the adaptation study (seventh week), because of the daily exposure for 4 d, crossover design could not be used, and the sample size was thus n = 3 for 0.0 ppm and n = 4 for 0.8 ppm ozone. Multiple hours of data after the exposures stopped were included to follow days of recovery during nonexposure periods.

Ozone generation and exposure for all rat experiments.

Ozone exposure methods have been described in detail in previous publications.24'28'31 Briefly, ozone was generated from oxygen using a silent arc discharge generator (OREC) and metered into the chambers using mass flow controllers (Coastal Instruments Inc.). The chamber ozone concentrations were recorded by photometric analyzers (API Model 400; Teledyne). Mean chamber temperature, relative humidity, and air flow were recorded hourly. For each study, the target and actual concentrations of ozone achieved in the chamber and the chamber atmospheric data are shown in Table S1. Because some data from were previously published for time course,26 adrenalectomy agonists,27 and (3-AR ± GR antagonists studies,28 the chamber data for these studies have been previously published.

GTTsfor all rat experiments. For glucose telemetry, adrenalectomy agonist, and (3-AR ± GR antagonists studies, GTTs were performed in rats after air or ozone exposure as previously described.24 Because rats underwent air or ozone exposure for 4 h prior to GTT when no food was provided, this served as fasting for GTTs. Immediately after or within 30 min of air or ozone exposure, baseline blood glucose concentrations (0 min) were determined by tail prick using a sterile 23-gauge needle with a Bayer Contour Glucometer. For glucose telemetry animals, a different glucometer and strips were used (Nova Biomedical StatStrip Xpress; DSI). Rats were then injected with 20% D-glucose [20% pharmaceutical grade D-glucose; Covetrus; diluted to 2 g/kg/10 mL, intraperitoneal (i.p.)]. Glucose levels were measured by tail prick at 30,60,90, and 120 min.

Time Course Study: Temporal Assessment of Hormones and Free Fatty Acids in Rats

The goals of this study were to determine the time course of neuroendocrine, adrenal, and metabolic hormone changes and to ascertain how these changes are linked to real-time glucose dynamics. Data collected from these rats on leukocyte trafficking, corticosterone, and epinephrine have been previously published (Table l).26 This study included a cohort of healthy male 12- to 13-wk-old WKY rats that were exposed to clean air or ozone at two concentrations (0.4 and 0.8 ppm) for 30 min, 1 h, 2 h, or 4 h (n = 6-8 per group) and necropsied within 15 min after each time point to collect blood samples for assessment of various pituitary-derived, adrenal and metabolic hormones as well as free fatty acids.

Necropsy and blood samples collection for Time Course study. Rats were necropsied within 15 min after each exposure period. Necropsies were performed in a staggered manner. Rats were euthanized with Fatal Plus (sodium pentobarbital, Virbac AH, Inc; >200 mg/kg, i.p.). Blood samples were collected from the abdominal aorta directly in vacutainer serum separator tubes and EDTA tubes for serum and plasma separation, respectively. Blood samples were spun at 3,500 Xg for 15 min at 4°C; serum and plasma samples were aliquoted and stored at - 80 °C until analysis.

Plasma and serum analysis of hormones and free fatty acids in the Time Course study. Serum levels of pituitary, adrenal and metabolic hormones, as well as free fatty acids were analyzed in the time course study to compare temporality of hormone changes with that of real-time glucose dynamics after ozone exposure. Plasma levels of epinephrine (adrenaline) and corticosterone were quantified using ELISA kits from Rocky Mountain Diagnostics (Catalog No. BA E-5100R) and Arbor Assays (Catalog No. K014), respectively. Samples were processed according to kit protocol and read on a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices). The data for these two hormones were recently published in table form along with immune effects of ozone.26 Serum pituitary hormone levels for adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), prolactin (PRL), luteinizing hormone (LH), growth hormone (GH), and follicle stimulating hormone (FSH) were determined using MILLIPLEX MAP Rat Pituitary Magnetic Bead Panel following manufacturer's protocol (Catalog No. RPTMAG-86K; Merck-Millipore), and plates were read on a Luminex 200 System (MilliporeSigma). Serum insulin and leptin were quantified using Meso Scale kits (Catalog No. K151BZC and K151BYC, respectively; Meso Scale Discovery) and MESO QuickPlex SQ 120 platform to detect electrochemiluminescence signals (Mesoscale Discovery Inc.). Serum free fatty acids were measured using kits from Cell Biolabs, Inc. (Catalog No. STA-618). This assay was adapted for use on a Konelab Arena 30 clinical analyzer (Thermo Chemical Lab Systems).

AD Agonists Study: AD and Sham (SH) Surgeries, Drug Treatments, and Ozone Exposure

To determine the roles of adrenal-derived stress hormones in ozone-induced changes in glucose, we adrenalectomized rats concomitantly with restoration of adrenal hormone function using pharmacological agonists. Data from rats in the AD Agonist study on pulmonary protein leakage and inflammation have been previously published (Table SI).27 Male WKY rats (12-13 wk old) underwent total bilateral AD or SH surgeries.27 Briefly, rats were anesthetized with ketamine (25-50 mg/kg in saline, i.p.), and once anesthetized, injected with the analgesic buprenorphine (0.02 mg/kg/mL in saline, s.c). On achievement of anesthesia, rats were subcutaneously injected with transponders with temperature sensors (BMDS, IPTT-300 model; Avidity Science LLC) on the dorsal shoulder to determine subcutaneous temperature.27 During the aseptic surgery, anesthesia was maintained by inhalation of vaporized isoflurane through nose cone (~ 3%, 1-2 LPM of O2). Animal surgeons from Charles River Laboratories, Inc., performed the surgeries. Animals were placed in sternal recumbency, and dorsal incision was made. Adrenals from both sides were removed. Surgical wound clips were used to clip the skin and close the incision. SH surgeries were performed using the same anesthesia and surgical approaches as AD except for the removal of adrenal glands. Rats were recovered on heating pads and assessed for signs of distress and pain. Once the rats were awake, meloxicam (0.2 mg/kg in saline, s.c.) was given followed by buprenorphine (0.02 mg/mL/kg in saline, s.c, every 8-12 h two times) for analgesia. After the surgery, AD rats received water with 0.9% NaCl to maintain adequate salt:water balance in the absence of mineralocorticoids eliminated due to AD along with other adrenal-derived stress hormones. All animals were provided with powdered as well as pelleted food ad libitum. The rats were pair-housed with EnviroDry enrichment/nesting material and allowed to recover for 4-6 d prior to drug treatment.

Because AD, in addition to reducing circulating catecholamines and glucocorticoids, could also eliminate mineralocorticoids, we used pharmacological intervention that mimics catecholamines' plus glucocorticoids' effects on AR and GR and examined whether this treatment was sufficient to restore ozone-induced hyperglycemia and glucose intolerance in AD rats. The SH and AD rats each were randomized by body weight into four groups [vehicle:air, vehi-cle:ozone, clenbuterol (CLEN; (32-AR agonist that mimics effect of epinephrine) plus dexamethasone (DEX; GR agonist that mimics the effect of glucocorticoids):air, and CLEN+DEX:ozone], resulting in eight total groups (n = 8/group). In brief, after 4-6 d of recovery from surgeries, SH and AD rats were treated with vehicles (saline 1 mL/kg as control for CLEN, i.p.) and corn oil (1 mL/kg as control for DEX, s.c.) or CLEN hydrochloride, a long acting (32-AR agonist (0.2 mg/kg in saline, i.p.) and GR agonist, DEX (2 mg/mL corn oil/kg, s.c). CLEN injections were followed by DEX. The drug treatment began 1 d prior to start of ozone exposure and continued the day of air or ozone exposure in the morning at ~ 0600hours (6 A.M.). CLEN and DEX doses are comparable to those used in other controlled experiments using rodents and are sufficient to induce bronchodilation and immunosuppression, respectively.32'33 On the second day after drug treatment, these rats were exposed to air or 0.8 ppm ozone for 4 h. Immediately after exposure, whole body plethysmography was performed, and these data were previously published27; within 30 min of the end of the exposure period, GTTs were performed as described under section titled, "GTTs for all rat experiments."

Antagonists Study: Treatment of Rats with /S-AR and GR Antagonists and Ozone Exposure

To determine the influence of pharmacological blockade of adrenal stress hormone receptors on ozone-induced hyperglycemia and glucose intolerance, rats were injected with (3-AR and/or GR antagonists and assessed for effects on ozone-induced hyperglycemia and glucose intolerance. Healthy male WKY rats 12-13 wk old were treated with a) (3-AR antagonist propranolol (PROP), b) GR antagonist mifepristone (MIFE), or c) both in combination to determine their influence on ozone-induced hyperglycemia and glucose intolerance. For each assessment, rats were randomized by body weight into four groups (vehicle/air, drug/air, vehicle/ozone, drug/ozone, n = 8/group). In the first assessment, rats were injected with either sterile saline (vehicle; 1 mL/kg, i.p.) or PROP hydrochloride (Sigma-Aldrich; 10 mg/kg in saline, i.p.). In the second assessment, rats were injected with pharmaceutical grade corn oil (vehicle; 1 mL/kg, s.c.) or MIFE (Cayman Chemical Co.; 30 mg/kg in corn oil, s.c). In the third assessment, rats were injected with vehicles, saline (1 mL/kg, i.p.) followed by corn oil (1 mL/kg, s.c.) or drugs, PROP (10 mg/kg, i.p.) followed by MIFE (30 mg/kg, s.c.) (PROP+MIFE). These three assessments allowed for determining the effects of blocking (3-AR or GR individually or in combination. To assure complete inhibition of (3-AR and GR receptors, the daily morning treatment began 7 d prior to the air or ozone exposure and was continued the day of exposure. The concentration and treatment duration for PROP was based on prior publication showing its effectiveness in reducing leukocyte migration in a rat model (Table SI).34 MIFE treatment at this concentration has been shown to reduce ethanol withdrawal severity in rats.35 After the final drug treatment, rats were exposed to filtered air or 0.8 ppm ozone for 4 h as described in the section titled "Ozone generation and exposure for all rat experiments." Immediately after the exposure period, whole body plethysmography was performed, and within 30 min GTTs were performed as described under the section titled "GTTs for all rat experiments."

Statistics

For all analyzed end points, a threshold of p < .05 was used to determine significant effects. Outliers were identified using the box plot method, defined as above Q3 + 1.5 interquartile range (IQR) or below Ql - 1.5 IQR and discarded. Analysis of glucose telemetry data was done by assessing peak glucose levels and area under the curve (AUC) for set time points (i.e., during exposure, during GTT, etc.) (Excel Table SI). AUC was calculated using the trapezoidal method as previously described.36 Briefly, the area was constrained to the X values provided, using a baseline of 0, and dividing the total area into smaller trapezoids created by connecting the points to approximate the area under a function over the given interval. Significant effects of exposure were determined using one-way analysis of variance (ANOVA) with a Tukey's post hoc comparison to assess differences between ozone concentrations or a Student's f-test when only one ozone concentration was used. To determine the relationship between glucose and animal body temperature, a linear regression was performed. To compare the similarities of glucose telemetry and handheld glucometer, significant effects were examined using a two-way ANOVA (measurement technique, exposure) with a Tukey's post hoc comparison used for pair-wise comparisons. To determine adaptation over time, a two-way repeated measures ANOVA was performed (exposure, day) with Tukey's post hoc comparison used for pair-wise comparisons (Excel Table SI). For analysis of the effects of ozone on serum pituitary and adrenal hormones and serum metabolic hormones and free fatty acids, a two-way repeated measures ANOVA (exposure, time) was performed and followed by Tukey's post hoc comparisons for each ozone concentration (Excel Table S2). To explore the effects of adrenalectomy and/or pharmaceutical interventions on ozone's effects on glucose and glucose tolerance, a three-way (exposure, drug, surgery) or two-way (exposure, drug) ANOVA was performed with Tukey's post hoc test used for pair-wise comparisons (Excel Table S3). GraphPad Prism 9 (version 9.1.2; GraphPad Software) was used for statistical analysis and graph generation. Statistical summaries pertaining to all data presented in figures is provided in Excel Table SI (Telemetry), Excel Table S2 (Time Course), and Excel Table S3 (AD Agonists and (3-AR ± GR Antagonist) with a detailed breakdown of all statistical analyses, effect size calculations, and notes on any outliers removed. Group means and standard deviations for all analyses are also provided in these tables. Raw data can be found in Excel Table S4 (Telemetry), Excel Table S5 (Time Course), and Excel Table S6 (AD Agonists and (3-AR ± GR Antagonist).

Results

Telemetry Study

Real-time in vivo glucose monitoring during and after a single ozone exposure. We used a novel real-time blood glucose telemetry system (Figure 1A; Figure SI)29 to determine temporality and longevity of ozone-induced glucose metabolic alterations in rats. Using a crossover design with the seven telemetered rats, we obtained independent readings at each concentration for a weekly 4-h exposure to air or ozone with 1-wk washout between exposures (Table S2). Blood glucose levels began to increase ~ 90 min into the 4-h ozone exposure relative to air controls, but only in the 0.8-ppm group (Figure IB; Excel Table S4). In the 0.8-ppm ozone exposure group, blood glucose levels peaked between 2.5 and 3 h (Figure IB), followed by a second peak between 4-5 h relative to levels at the start of exposure (Figure 1C; Excel Table S4). A third peak of blood glucose levels in the 0.8 ppm ozone group was noted roughly 1 h after the beginning of dark cycle (and 4 h post cessation of ozone exposure) when rodents were active and feeding (Figure 1C; Excel Table S4). The core body temperature began to drop ~ 90 min into exposure in 0.8-ppm ozone group relative to air controls and continued this trend until the end of exposure (Figure ID). After the termination of 4-h exposure, the core body temperature in the 0.8-ppm ozone group begun to return to air control levels, and 6-8 h post exposure the core body temperature in the 0.8-ppm ozone group returned to preexposure levels (Figure ID). Glucose levels at 90 min were negatively associated with core body temperature in the 0.8 ppm ozone group, but without the multiple peaks observed in glucose levels (Figure IE and IF). Outside of the exposure period (during the dark cycle), when glucose levels were high, core body temperature was also high for rodents (Figure 2A and 2B). However, changes in in blood glucose and body temperature during and immediately after the exposure period were in the opposite direction.

After completing 4-wk exposure using crossover design, animals were assigned to the air or the 0.8-ppm ozone group for two subsequent weeks. On the fifth and sixth week, rats were exposed to air or 0.8 ppm ozone for 4 h using crossover design (alternating exposure assignment to air or ozone each week) (Table S2), and GTTs were performed immediately following exposure in telemetered rats (Figure S2A). Rat exposed to 0.8 ppm ozone had higher levels of circulating glucose, as well as higher peak glucose levels and greater AUC, suggesting glucose intolerance (Figure S2); findings were consistent with our previous studies involving postexposure assessment.24 The telemetry data for blood glucose after bolus glucose injection was consistent with the data obtained through a handheld glucometer (Figure S2A), with both finding significantly higher levels of basal glucose (Figure S2B) and greater AUC (Figure S2C) during the 0.8-ppm exposure in comparison with the control. Further, when data from 0, 30, 60, 90, and 120 min were compared using the handheld technique and telemetry, there were no statistically significant differences in peak glucose or glucose AUC (Excel Table SI).

Real-time glucose monitoring during repeated ozone exposures. After a single 4-h 0.8 ppm ozone exposure, differences in circulating glucose were not noted during subsequent days without exposure (Figure 2A). The diurnal changes were apparent with all animals showing higher levels of glucose at nighttime compared to daytime.37 On week 7, animals were exposed to air or 0.8 ppm ozone (4 h each day) for 4 consecutive days to determine whether adaptation occurred with regard to glucose and body temperature during repeated daily exposure. Because of the repeated exposures during week 7 (daily for 4 d), crossover design was not possible, and sample sizes were therefore smaller than for all other experiments (air, " = 3; ozone, n = 4). Rats exposed to ozone at 0.8 ppm 4 h/day for 4 consecutive days had significantly higher peak glucose and greater glucose AUC only on days 1 and 2, with a notable lack of a significant difference in blood glucose during and right after the exposure on the third and fourth day, despite continued daily exposure (Figure 2C; Excel Table SI). Near complete adaptation to ozone exposure was evident by the third day, despite continued exposure on third and fourth day, with no significant differences between rats exposed to ozone at 0.8 ppm and control rats observed in peak glucose or glucose AUC on day 3 or day 4 (Figure 2C; Excel Table SI). The adaptation was also noted in hypothermia on the third day (Figure 2D).

Time Course Study

Temporality of ozone-induced hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes activation.

Because the method for continuous monitoring of corticosterone in animals is still not available, we exposed a separate cohort of naive rats to air or ozone for variable durations spanning a 4-h time frame to assess temporal changes in adrenal-derived and other neuroendocrine hormones (Figure 3A). This study followed a paradigm similar to that of real-time glucose monitoring but used a distinct cohort of rats at each time point during 4-h ozone exposure to assess serum samples for key pituitary, adrenal-derived, metabolic hormones, and free fatty acids (Figure 3). ACTH level was higher than levels in control or 0.4 ppm ozone-exposed rats 30 min into the 0.8-ppm ozone exposure and peaked at 1 h, likely reflecting the activation of the HPA axis and concomitant ACTH release from the anterior pituitary (Figure 3B). Consistent with this, we noted that the levels of circulating corticosterone in rats were similarly higher in rats exposed to 0.8 ppm ozone starting at 1 h into ozone exposure prior to the increase in glucose and remained significantly higher until 4 h of exposure (Figure 3B), despite the restoration of ACTH to control group levels at this time point. The data on corticosterone and epinephrine were previously published in a table form are reproduced here as a line graph.26 It is important to note that no significant differences in plasma corticosterone were measured during the 0.4-ppm ozone exposure until the 4 h time point, at which point levels were significantly higher than in both control rats and those exposed to 0.8 ppm ozone, suggesting that the stress response was concentration dependent.

After 30 min of 0.8 ppm ozone exposure, the levels of epinephrine were higher in comparison with the air group, as recently reported.26 The levels of epinephrine remained high in ozone-exposed rats as determined during 4-h exposure. Rats exposed to the lower concentration of ozone (0.4 ppm) also showed a higher level of epinephrine, although the time required for this increase was longer, because it was evident only after 4 h of exposure (Figure 3B).38

Temporal changes in hypothalamic-pituitary-thyroid (HPT) and hypothalamic-pituitary-gonadal (HPG) hormones during ozone exposure. Because ozone-mediated stimulation of the neuroendocrine system may also influence other hypothalamic stress pathways such as HPT and HPG axes,39 we next assessed temporal effects of ozone exposure on relevant hormones. Ozone exposure was associated with lower TSH levels relative to air controls in a time- and concentration-dependent manner, which occurred sooner (1 h) at 0.8 ppm and was temporally linked to higher levels of corticosterone and preceded the glucose response with 0.8 ppm ozone (Figure 3B). We also assessed pituitary hormones involved in gonadal axis including follicle stimulating hormone

(FSH), prolactin (PRL), and luteinizing hormone (LH) (Figure 3B). LH and PRL levels were lower in rats exposed to 0.8ppm ozone in comparison with air-exposed animals. The temporal assessment showed depletion of circulating PRL as early as 1 h into ozone exposure concomitant with peak ACTH levels. However, lower LH levels were not measured until 2 h of exposure (Figure 3B), and the levels of FSH were not significantly different after ozone exposure (Figure 3B).

Because pituitary-derived GH was involved in cell growth and regeneration processes37 and tied to metabolic changes,40 we assessed the kinetics of growth hormone changes and noted that a delayed but concentration-dependent increase in GH occurred at 4 h of ozone exposure, suggesting that the anabolic processes are being activated (Figure 3B). A similar temporal pattern was noted with higher levels of leptin during ozone exposure (Figure 3C). Relative to air-exposed rats, the higher levels of circulating free fatty acids were noted as early as 1 h after ozone exposure, suggesting the early activation of lipolytic activity in adipose tissue coincided with changes in circulating adrenal-derived hormones, but a delayed difference in GH (higher levels at 4 h, although not statistically significant; Figure 3B) coincided with leptin levels (Figure 3C).

AD Agonists Study: The Role of Epinephrine and Corticosterone in Mediating Hyperglycemia

Adrenal-derived epinephrine and glucocorticoids are the major regulators of liver metabolic processes during stress,41 and adrenalectomy diminished ozone-induced hyperglycemia and glucose intolerance in rats.31 Therefore, we further assessed the roles of adrenal-derived stress hormones in mediating differences in circulating glucose after ozone exposure (Figure 4). Differences in blood glucose during the glucose tolerance test were not evident in vehicle-treated AD rats exposed to ozone in comparison with those exposed to filtered air, which confirmed our earlier findings.31 Moreover, all animals treated with CLEN+DEX had markedly higher glucose levels during the GTT, in both SH and AD rats exposed to air. Further, this response was exacerbated in rats exposed to ozone (Figure 4B).

Antagonists Study: The Role of Epinephrine and Corticosterone in Mediating Hyperglycemia

Pharmacological interventions involving inhibition of AR and GR allowed examination of the role of each hormone receptor individually or in combination. In all rats exposed to ozone blood glucose levels at baseline were higher than those exposed to filtered air. In rats exposed to filtered air and those exposed to 0.8 ppm ozone, no differences were observed in baseline blood glucose and in blood glucose 30 min after the glucose bolus in those given PROP or MIFE individually and those given the vehicle. A noticeably lower glucose level was observed at 60-120 min post glucose bolus in rats that received MIFE in comparison with those that received the vehicle in the ozone-exposed groups; a similar trend was observed for PROP alone, but this decrease at 60-120 min post glucose bolus was not significant (Excel Table S3). Rats given PROP+MIFE in combination had significantly lower baseline glucose levels in comparison with those given the vehicle in the ozone-exposed group. Similarly, rats given the PROP+MIFE combination demonstrated a greater difference in glucose levels from vehicle at 60-120 min after glucose bolus in the ozone-exposed group. These results suggest the involvement of both epinephrine and glucocorticoids in mediating glucose increases during ozone exposure (Figure 4C; Excel Table S3).

Discussion

We used real-time glucose telemetry in rats, combined with temporal assessment of neuroendocrine hormones to demonstrate in rodents a contextual relationship between ozone-induced stress response and changes in glucose homeostasis. We further demonstrated a mechanistic link between glucose metabolic alterations and adrenal-derived stress hormones using AD and pharmacological interventions of (3-AR and GR. The real-time monitoring of glucose and stress hormones may serve as immediate biomarkers of ozone-induced pulmonary stress and the interactive impacts of other reactive environmental contaminants and nonchemical stressors. Impairment in the neuroendocrine stress response and/ or the activities of adrenal hormones following irritant air pollutant or stressor exposures could impair metabolic response and increase susceptibility for chronic diseases.

Hyperglycemia is one of the earliest markers of stress-induced homeostatic changes that was consistently noted after a single ozone exposure in rats24'31'42 and other reactive gas exposures, such as acrolein.43 Here, we showed that glucose levels began to rise in the circulation ~ 90 min into 0.8 ppm ozone exposure and coincided with a decrease in core body temperature and that reversal of these effects occurred 4-6 h after termination of 4-h ozone exposure (Figure 2B). However, on daily 4-h ozone exposures for 3 or more consecutive days, the ozone-associated differences in blood glucose (Figure 2C) and core body temperature (Figure 2D) were no longer evident, reflective of stress adaptation. We showed that ozone-induced differences in blood glucose during exposure were preceded by measured differences in levels of circulating anterior pituitary-derived hormones and adrenal-derived epinephrine and corticosterone, linking the activation of SAM and HPA axes and the inhibition of HPT and HPG axes to hyperglycemia. Further, eliminating adrenal-derived stress hormones from circulation through AD or pharmacologically inhibiting stress hormone receptors, the data suggest that (3-AR and GR lowered ozone-induced hyperglycemia and that treatment with agonists restored ozone-induced hyperglycemia in AD rats. Combined, these results demonstrate the mechanistic role of adrenal-derived stress hormones in mediating glucose metabolic alterations and highlight the utility of real-time glucose measurement as a sensitive marker that reflects the impacts of irritant air-pollutant exposure, which may be useful in linking source to health outcomes.

Acute ozone exposure was shown to activate stress-responsive regions in the rat brain.44 The release of stress hormones by activation of SAM and HPA axes is integral in mediating key metabolic processes that channel energy resources where needed by acting on AR and GR.45 Glucocorticoid feedback regulation on HPA activity and the roles of mineralocorticoids and catecholamines are implicated in adaptation and the plasticity of organismal neural stress responses.19 However, the full understanding of molecular mechanisms linked to impaired stress adaptation and neuropsychiatric disorders is still lacking. The adaptation of rats to ozone effects on blood glucose and core body temperature on the third consecutive day of exposure suggested a neuroendocrine contribution to adaptation.46 This adaptation response was not evident on the second day of ozone exposure. Moreover, one week of no exposure washout period was associated with the loss of adaptation here and even in rats exposed to ozone 3 d/wk for 12 consecutive weeks,30 indicating remarkable plasticity that might be influenced by stressor type, potency, and longevity of exposure. Given the contribution of glucocorticoids in stress adaptation18 and the link between changes in circulating Cortisol25 and chronic neurobehavioral and metabolic diseases,47'48 the temporal assessment of changes in glucose and Cortisol could delineate health status, resiliency, and longevity of stressor effects. The loss of dynamicity or oscillatory rhythms in glucocorticoids was an important indicator of chronic health issues,49 suggesting that in some individuals the stress mechanisms might be impaired, and thus, the temporal monitoring of stress may provide additional insights on disease severity.

Real-time glucose monitoring is now more frequently employed for individuals with diabetes who require repeated assessment of blood glucose.50 In this study, we were able to assess changes in blood glucose during GTTs and from food intake during nighttime in rats. Thus, real-time glucose assessment offers the opportunity to study interactive effects of diet, metabolic disease, and stress induced by irritant air-pollutant exposures. Ozone-induced stress exacerbated preex-istent hyperglycemia as reported in diabetic Goto Kakizaki rats51 and exacerbated other stressors in patients with diabetes.52 Thus, the temporal assessment of glucose and its mechanistic linkage to adrenal- derived stress hormones could provide insights on stress, overall physiological status, and underlying metabolic health condition.

Combined, evidence of temporal, directionally different, and concentration-dependent changes in hormones associated with

SAM, HPA, HPT, and HPG axes during acute ozone exposure indicated that the stress response likely involved a complex interplay of multiple neuroendocrine pathways. The selective activation of HPA and SAM axes was evident first by higher levels of ACTH within 30 min of ozone exposure followed by higher levels of circulating corticosterone in rats. The higher epinephrine and corticosterone levels in response to ozone relative to air group were associated with concurrently lower levels of hormones involved in the HPT and HPG axes. These responses were consistent with previously reported acute psychosocial stress-induced increased ACTH and Cortisol, which were associated with depletion of gonadal hormones in men and women.53 This response was in contrast, however, with higher thyroid hormone and corticosterone after exercise in rats in comparison with nonexercising controls.54 With regard to HPG hormones, PRL was decreased as early as 30 min, and this effect was not observed in AD rats39; these findings suggested the possible involvement of glucocorticoids in regulation of the stress axis and complex interplay of multiple neurohormonal axes in orchestrating a stress response to ozone. Although circulating glucocorticoids might also inhibit LH secretion after ozone exposure, a role of gonadotropin-releasing hormone and gonadotropin inhibitory hormone was likely in the ozone-induced inhibition of LH. In rats, stress exposure was associated with lower LH levels.55 These findings indicate that stress responses might not be uniform between stressor types and that ozone might impact specific neuroendocrine pathways that involve input from multiple interactive signaling processes in the brain to develop an integrated and temporally regulated host response. Together, these data suggest that there are dynamic relationships between ozone-induced changes in neuroendocrine stress pathways, HPA activation, and glucose homeostasis.

Circulating corticosterone, cytokines, exhausting exercise, caloric deprivation, and even sepsis were linked to depletion of TSH, T4, and T3.56 Acute exposure to ozone depleted circulating thyroxine and TSH in rats.57 We have shown that AD reversed ozone-induced inhibition of TSH release, suggesting a role for circulating adrenal-derived stress hormones.39 Pituitary TSH release was regulated by hypothalamic thyrotropin releasing hormone (TRH) with feedback controls on HPT at different levels.58 Thus, stressor-specific differences in activation vs. inhibition of given hormonal systems may impact downstream physiological responses. The mechanisms by which pituitary hormones such as ACTH and GH increased, whereas TSH, LH, and PRL decreased after ozone exposure, might involve selective activation or inhibition of upstream regulators of hormonal responses. Those include likely activation of corticotropin-releasing hormone neurons, up-regulation of FK506, a scaffolding protein involved in glucocorticoid feedback, and other neurotropic factors within the stress-responsive regions impacted by ozone.19'44

We have previously reported that ozone exposure was associated with pulmonary59 and liver60 transcriptional changes in rats reflective of processes that regulate metabolism, cell cycle and regeneration.61'62 Because pituitary-derived GH involved in these processes in humans37 was shown to regulate insulin and lipid metabolic process in rats,60 we assessed kinetics of GH changes and noted a delayed response, where GH levels were significantly higher following 4-h ozone exposure in comparison with those of vehicle rats, suggesting that anabolic processes might be activated. Similar patterns were noted for leptin, which regulates satiety at the level of the hypothalamus.59 However, rats exhibited higher circulating free fatty acids as soon as 1 h into the ozone exposure, suggesting an early activation of lipolytic activity in adipose tissue coinciding with changes in circulating adrenal-derived hormones but a delayed increase in GH coinciding with leptin release from adipose tissue. These regulatory mechanisms could be altered by repeated stressor exposures or underlying psychiatric or systemic disease conditions.

Our approach focused on establishing a causal role of SAM and HPA axes activation on ozone-induced glucose metabolic response using AD and stress hormone receptor agonists/antagonists. Circulating epinephrine and glucocorticoids regulate glucose metabolic processes, in addition to regulating other physiological and immunological processes through their action on receptors in multiple tissues, including the liver and pancreas.63 Our results here suggest that AD or treatment with pharmacological blockers of (3-AR plus GR inhibited ozone-induced hyperglycemia and glucose intolerance, whereas the combination of (3-AR and GR agonists amplified ozone-induced hyperglycemia and glucose intolerance even in AD rats (Figure 4). We have previously shown that ozone exposure was associated with increased gluconeogenesis and inhibition of glucose-mediated insulin secretion in rats.30'64 Each AR and GR subtype might be selectively influencing different processes of glucose metabolism, such as gluconeogenesis, and (3-cell insulin secretion in the liver and pancreas, respectively. These differences in rats exposed to acute ozone suggest that adrenal-derived hormones regulated glucose homeostatic changes induced by ozone.

Stress response is proportional to stressor severity and duration, and is directed to the affected organ system through coordinated involvement of multiple organs.1213 Furthermore, in a rat study, this response was reversible upon stress discontinuation and in some cases, even after continued stressor application (habituation).46 However, the reversibility of these physiological stressor effects in healthy individuals could be impaired in susceptible individuals both at the CNS and peripheral organ levels, contributing to health burdens from environmental exposure.49 Based on evidence presented in this paper on the lack of glucose and core body temperature differences on the third consecutive day of ozone exposure, we surmise that evaluation of the dynamicity of this response through real-time glucose and Cortisol monitoring could unravel critical information on the magnitude and persistence of stress from environmental exposures and its impairment in individuals with preexisting diseases, including psychosocial and metabolic. Using ozone inhalation as an example, we here demonstrated the utility of such an approach to monitor the dynamics of stressor effects on health that is amenable in humans with currently available technologies.65

This study assessed responses after exposure to only ozone. Whereas we have shown similar changes in stress hormones and glucose after exposure to another gaseous irritant, acrolein,43 this response may be linked to irritancy and should not be generalized to all pollutants and pollution mixtures. Similarly, the nature and timing of stress responses to other pollutants may vary, leading to differences in the organ being affected and the pathology. The ozone concentration that showed major effects was over an order of magnitude higher than what might be encountered in U.S. cities with nonattainment for National Ambient Air Quality Standard66; however, ambient ozone levels have been associated with health effects.67 Rodent studies showed that the effective ozone dose at the airway lining could be critical in initiating the biological response, and given higher levels of antioxidants in airway lining of rodents relative to humans,68 the ozone concentration that produce effects in rodents could be higher than humans. The airway lining antioxidants were considered a first line of defense68 and thus might have increased the threshold for ozone effects in rats, leading to lack of clear dose response at 0.4 ppm ozone in this study. Moreover, this study assessed only acute health outcomes in healthy animals after inhaled ozone exposure; however, the mechanisms by which repeated exposures and underlying health conditions increase disease susceptibility can be better studied using longer exposures in models of compromised health status. One of the important limitations of the study is the lack of consideration of female rats in the study design. The sex differences are likely tightly linked to neuroendocrine system, making it important to consider sex differences. We acknowledge this limitation, but our justification for using males for this study came from our previous research, which showed that WKY females were less sensitive than males to secondhand smoke-induced lung inflammation and pathology.69

In conclusion, we monitored the dynamicity of metabolic effects of a prototypic reactive air pollutant, ozone, in real-time by assessment of blood glucose using telemetry in rats. During ozone exposure, differences from control animals in blood glucose and core body temperature were preceded by higher levels of circulating adrenal-derived stress hormones in rats. Further, we showed that exposure to ozone resulted in rapid differences in hormones associated with anabolic (ACTH, GH) neuroendocrine pathways (higher hormone levels in ozone-exposed rats), suggesting that sympathetically mediated adrenal medullary release of epinephrine and HPA-mediated release of glucocorticoids might be influencing hormones linked to HPT and HPG axes (lower hormone levels in ozone-exposed rats). These dynamic neuroendocrine changes were followed by higher glucose levels. Our data support the idea that adrenal-derived stress hormones mediated glucose increases based on the evidence that AD and pharmacological inhibitors of stress hormones reduced and agonists enhanced ozone-induced glucose increases and glucose intolerance. Stress processes affected by ozone exposure depicted how stress pathways and pollution might interact to alter resilience to pollution. Because dynamic changes in circulating stress hormones likely mediate interactive metabolic effects of reactive air pollutants such as ozone, this approach of continuous monitoring of glucose and stress hormones may be useful in assessing health effects and susceptibility variations in epidemiological studies.

Acknowledgments

The authors thank M. I. Gilmour of the U.S. EPA, D. L. Costa of the University of North Carolina (formerly of the U.S. EPA) and A. Egorov of the U.S. EPA for their critical review of the manuscript. The authors also acknowledge the help of M. Higuchi and A. Malek Khan of the U.S. EPA for ozone inhalation exposures.

A.R.H, S.J.S., and U.P.K. designed experiments, data collection, and interpretation, manuscript preparation; T.W.J, conducted data analysis, statistical evaluation, and manuscript preparation; J.S.H., A.A.M.-R. conducted data analysis and manuscript review; C.K.W.-C. conducted data interpretation and manuscript review; M.C.S., D.I.A., C.N.M., A.K.F., M.S.H., and R.G. conducted data collection and manuscript review; A.J.G., and D.D.-S. provided guidance and assisted in manuscript review.

The research described in this article has been reviewed by the Center for Public Health and Environmental Assessment, U.S. EPA, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does the mention of trade names of commercial products constitute endorsement or recommendation for use. All opinions expressed in this paper are the authors' and do not necessarily reflect the policies and views of U.S. Department of Energy (U.S. DOE) or Oak Ridge Associated Universities (ORAU)/Oak Ridge Institute for Science and Education (ORISE).

This research was supported by the intramural research program of the U.S. EPA. Partial support also came through an appointment of A.R.H. to the U.S. EPA Research Participation Program administered through an interagency agreement between the U.S. DOE and the U.S. EPA. ORISE is managed by ORAU under U.S. DOE contract number DE-SC0014664.

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