Effects of maternal high-fat diet and sedentary lifestyle on susceptibility of adult offspring to ozone exposure in rats.

Epidemiological and experimental data suggest that obesity exacerbates the health effects of air pollutants such as ozone (O3). Maternal inactivity and calorically rich diets lead to offspring that show signs of obesity. Exacerbated O3 susceptibility of offspring could thus be manifested by maternal obesity. Thirty-day-old female Long-Evans rats were fed a control (CD) or high-fat (HF) (60% calories) diet for 6 wks and then bred. GD1 rats were then housed with a running wheel (RW) or without a wheel (SED) until parturition, creating four groups of offspring: CD-SED, CD-RW, HF-SED and HF-RW. HF diet was terminated at PND 35 and all offspring were placed on CD. Body weight and %fat of dams were greatest in order; HF-SED > HF-RW > CD-SED > CD-RW. Adult offspring were exposed to O3 for two consecutive days (0.8 ppm, 4 h/day). Glucose tolerance tests (GTT), ventilatory parameters (plethysmography), and bronchoalveolar fluid (BALF) cell counts and protein biomarkers were performed to assess response to O3. Exercise and diet altered body weight and %fat of young offspring. GTT, ventilation and BALF cell counts were exacerbated by O3 with responses markedly exacerbated in males. HF diet and O3 led to significant exacerbation of several BALF parameters: total cell count, neutrophils and lymphocytes were increased in male HF-SED versus CD-SED. Males were hyperglycemic after O3 exposure and exhibited exacerbated GTT responses. Ventilatory dysfunction was also exacerbated in males. Maternal exercise had minimal effects on O3 response. The results of this exploratory study suggest a link between maternal obesity and susceptibility to O3 in their adult offspring in a sex-specific manner.

Active vs. sedentary lifestyle from weaning to adulthood and susceptibility to ozone in rats.

The prevalence of a sedentary (SED) life style combined with calorically rich diets has spurred the rise in childhood obesity, which, in turn, translates to adverse health effects in adulthood. Obesity and lack of active (ACT) lifestyle may increase susceptibility to air pollutants. We housed 22-day-old female Long-Evans rats in a cage without (SED) or with a running wheel (ACT). After 10 wk the rats ran 310 ± 16.3 km. Responses of SED and ACT rats to whole-body O3 (0, 0.25, 0.5, or 1.0 ppm; 5 h/day for 2 days) was assessed. Glucose tolerance testing (GTT) was performed following the first day of O3 ACT rats had less body fat and an improved glucose GTT. Ventilatory function (plethysmography) of SED and ACT groups was similarly impaired by O3 Bronchoalveolar lavage fluid (BALF) was collected after the second O3 exposure. SED and ACT rats were hyperglycemic following 1.0 ppm O3 GTT was impaired by O3 in both groups; however, ACT rats exhibited improved recovery to 0.25 and 1.0 ppm O3 BALF cell neutrophils and total cells were similarly increased in ACT and SED groups exposed to 1.0 ppm O3 O3-induced increase in eosinophils was exacerbated in SED rats. Chronic exercise from postweaning to adulthood improved some of the metabolic and pulmonary responses to O3 (GTT and eosinophils) but several other parameters were unaffected. The reduction in O3-induced rise in BALF eosinophils in ACT rats suggests a possible link between a SED lifestyle and incidence of asthma-related symptoms from O3.

Pulmonary sensitivity to ozone exposure in sedentary versus chronically trained, female rats.

Epidemiological data suggest that a sedentary lifestyle may contribute to increased susceptibility for some environmental toxicants. We developed an animal model of active versus sedentary life style by providing female Sprague-Dawley rats with continuous access to running wheels. Sedentary rats were housed in standard cages without wheels. After training for 12 wks, rats were exposed to 0, 0.25, 0.5 or 1.0 ppm ozone [O3 for 5 h/d, 1 d/wk, for 6 wk (N = 10 per group)]. Body composition (%fat, lean and fluid) was monitored noninvasively over the course of the study. Ventilatory parameters [tidal volume, minute ventilation, frequency and enhanced pause (Penh)] were assessed using whole-body plethysmography prior to O3 and 24 h after the 5th O3 exposure. Trained rats lost ∼2% body fat after 12 wk of access to running wheels. Peak wheel activity was reduced by 40% after exposure to 1.0 ppm O3. After the 5th O3 exposure, body weight and %fat were reduced in sedentary but not trained rats. Penh was significantly elevated in sedentary but not trained rats the day after exposure to 1.0 ppm O3. However, lung lavage cell counts and biomarkers of pulmonary inflammation measured 1 day after the final exposure were inconsistently affected by training. Wheel running led to marked physiological responses along with some indication of improved pulmonary recovery from O3 exposure. However, wheel running with O3 exposure may also be a detriment for some pulmonary endpoints. Overall, a sedentary lifestyle may increase susceptibility to O3, but additional studies are needed.

Effect of high-fructose and high-fat diets on pulmonary sensitivity, motor activity, and body composition of brown Norway rats exposed to ozone.

Diet-induced obesity has been suggested to lead to increased susceptibility to air pollutants such as ozone (O3); however, there is little experimental evidence. Thirty day old male and female Brown Norway rats were fed a normal, high-fructose or high-fat diet for 12 weeks and then exposed to O3 (acute - air or 0.8 ppm O3 for 5 h, or subacute - air or 0.8 ppm O3 for 5 h/d 1 d/week for 4 weeks). Body composition was measured non-invasively using NMR. Ventilatory parameters and exploratory behavior were measured after the third week of subacute exposure. Bronchoalveolar lavage fluid (BALF) and blood chemistry data were collected 18 h after acute O3 and 18 h after the fourth week of subacute O3. The diets led to increased body fat in male but not female rats. O3-induced changes in ventilatory function were either unaffected or improved with the fructose and fat diets. O3-induced reduction in exploratory behavior was attenuated with fructose and fat diets in males and partially in females. O3 led to a significant decrease in body fat of males fed control diet but not the fructose or fat diet. O3 led to significant increases in BALF eosinophils, increase in albumin, and reductions in macrophages. Female rats appeared to be more affected than males to O3 regardless of diet. Overall, treatment with high-fructose and high-fat diets attenuated some O3 induced effects on pulmonary function, behavior, and metabolism. Exacerbation of toxicity was observed less frequently.

Episodic ozone exposure in adult and senescent Brown Norway rats: acute and delayed effect on heart rate, core temperature and motor activity.

Setting exposure standards for environmental pollutants may consider the aged as a susceptible population but the few published studies assessing susceptibility of the aged to air pollutants are inconsistent. Episodic ozone (O3) is more reflective of potential exposures occurring in human populations and could be more harmful to the aged. This study used radiotelemetry to monitor heart rate (HR), core temperature (T(c)) and motor activity (MA) in adult (9-12 months) and senescent (20-24 months) male, Brown Norway rats exposed to episodic O3 (6 h/day of 1 ppm O3 for 2 consecutive days/week for 13 weeks). Acute O3 initially led to marked drops in HR and T(c). As exposures progressed each week, there was diminution in the hypothermic and bradycardic effects of O3. Senescent rats were less affected than adults. Acute responses were exacerbated on the second day of O3 exposure with adults exhibiting greater sensitivity. During recovery following 2 d of O3, adult and senescent rats exhibited an elevated T(c) and HR during the day but not at night, an effect that persisted for at least 48 h after O3 exposure. MA was elevated in adults but not senescent rats during recovery from O3. Overall, acute effects of O3, including reductions in HR and T(c), were attenuated in senescent rats. Autonomic responses during recovery, included an elevation in T(c) with a pattern akin to that of a fever and rise in HR that were independent of age. An attenuated inflammatory response to O3 in senescent rats may explain the relatively heightened physiological response to O3 in younger rats.

Ozone induces glucose intolerance and systemic metabolic effects in young and aged Brown Norway rats.

Air pollutants have been associated with increased diabetes in humans. We hypothesized that ozone would impair glucose homeostasis by altering insulin signaling and/or endoplasmic reticular (ER) stress in young and aged rats. One, 4, 12, and 24 month old Brown Norway (BN) rats were exposed to air or ozone, 0.25 or 1.0 ppm, 6 h/day for 2 days (acute) or 2 d/week for 13 weeks (subchronic). Additionally, 4 month old rats were exposed to air or 1.0 ppm ozone, 6 h/day for 1 or 2 days (time-course). Glucose tolerance tests (GTT) were performed immediately after exposure. Serum and tissue biomarkers were analyzed 18 h after final ozone for acute and subchronic studies, and immediately after each day of exposure in the time-course study. Age-related glucose intolerance and increases in metabolic biomarkers were apparent at baseline. Acute ozone caused hyperglycemia and glucose intolerance in rats of all ages. Ozone-induced glucose intolerance was reduced in rats exposed for 13 weeks. Acute, but not subchronic ozone increased α2-macroglobulin, adiponectin and osteopontin. Time-course analysis indicated glucose intolerance at days 1 and 2 (2>1), and a recovery 18 h post ozone. Leptin increased day 1 and epinephrine at all times after ozone. Ozone tended to decrease phosphorylated insulin receptor substrate-1 in liver and adipose tissues. ER stress appeared to be the consequence of ozone induced acute metabolic impairment since transcriptional markers of ER stress increased only after 2 days of ozone. In conclusion, acute ozone exposure induces marked systemic metabolic impairments in BN rats of all ages, likely through sympathetic stimulation.

Susceptibility of adult and senescent Brown Norway rats to repeated ozone exposure: an assessment of behavior, serum biochemistry and cardiopulmonary function.

Ozone (O3) is a pervasive air pollutant that produces pulmonary and cardiovascular dysfunction and possible neurological dysfunction. Young and old individuals are recognized as being susceptible to O3; however, remarkably little is known about susceptibility with senescence. This study explored the pulmonary, cardiovascular and neurological effects of O3 exposure in adult (4 m) and senescent (20 m) Brown Norway rats exposed to 0 or 0.8 ppm O3 for 6 h, 1 d/week, for 17 weeks. Ventilatory function was assessed 1 and 7 d after each exposure (Buxco). Heart rate, blood pressure (tail cuff) and motor activity were measured biweekly. Blood, aorta and bronchoalveolar lavage fluid (BALF) were analyzed 24 h after the last exposure for pulmonary inflammation, serum biomarkers and aorta mRNA markers of vascular disease. Measures of normal ventilatory function declined following each O3 exposure in both adult and senescent rats, however, senescent rats took weeks to exhibit a decline. Evidence for residual respiratory effects of O3 7 d after exposure in both age groups was observed. O3 had no effect on either heart rate or blood pressure, but decreased motor activity in both age groups. BALF indicated mild neutrophilic inflammation and protein leakage in adults. Age affected 17/58 serum analytes, O3 affected 6/58; 2/58 showed an age-O3 interaction. Leptin, adiponectin, lipocalin and insulin were increased in senescent rats. Overall, adult rats exhibited more immediate effects of episodic O3 than senescent rats. Residual effects were, however, obtained in both ages of rat, especially for ventilatory endpoints.

Effects of instilled combustion-derived particles in spontaneously hypertensive rats. Part I: Cardiovascular responses.

Epidemiological studies have reported statistically significant associations between the levels of ambient particulate matter (PM) and the incidence of morbidity and mortality, particularly among persons with cardiopulmonary disease. While similar effects have been demonstrated in animals, the mechanism(s) by which these effects are mediated are unresolved. To further investigate this phenomenon, the cardiovascular and thermoregulatory effects of an oil combustion-derived PM (HP-12) were examined in spontaneously hypertensive (SH) rats. The particle used in this study had considerably fewer water-soluble metals than the residual oil fly ash (ROFA) particles widely used in previous animal toxicity studies, with Zn and Ni constituting the primary water-leachable elements in HP-12. Rats were surgically implanted with radiotelemeters capable of continuously monitoring electrocardiogram (ECG), heart rate (HR), systemic arterial blood pressure (BP), and core temperature (T(co)). Animals were divided into four dose groups and were administered one of four doses of HP-12 suspended in saline vehicle (0.00, 0.83, 3.33, 8.33 mg/kg; control, low, mid, and high dose, respectively) via intratracheal instillation (IT). Telemetered rats were monitored continuously for up to 7 days post-IT, and were sacrificed 4 or 7 days post-IT. Exposures to mid- and high-dose HP-12 induced large decreases in HR (decreasing 30-120 bpm), BP (decreasing 20-30 mmHg), and T(co) (decreasing 1.2-2.6 degrees C). The decreases in HR and BP were most pronounced at night and did not return to pre-IT values until 72 and 48 h after dosing, respectively. ECG abnormalities (rhythm disturbances, bundle branch block) were observed primarily in the high-dose group. This study demonstrates substantial dose-related deficits in cardiac function in SH rats after IT exposure to a low-metal content, combustion-derived particle.

Effects of instilled combustion-derived particles in spontaneously hypertensive rats. Part II: Pulmonary responses.

A consistent association between exposure to high concentrations of ambient particulate matter (PM) and excess cardiopulmonary-related morbidity and mortality has been observed in numerous epidemiological studies, across many different geographical locations. To elicit a similar response in a controlled laboratory setting, spontaneously hypertensive rats were exposed to an oil combustion-derived PM (HP-12) and monitored for changes in pulmonary function and indices of pulmonary injury. Rats were implanted with radiotelemeters to monitor electrocardiogram, heart rate, systemic arterial blood pressure, core temperature, and activity. Animals were divided into four groups and exposed via intratracheal instillation (IT) to suspensions of HP-12 (0.0, 0.83, 3.33, and 8.33 mg/kg; control, low, mid, and high dose, respectively) in saline vehicle. Telemetered rats were monitored continuously for 4-7 days post-IT and pulmonary function was examined using a whole-body plethysmograph system for 6 h/day on post-IT days 1-7. At 24, 96, and 192 h post-IT, bronchoalveolar lavage fluid (BALF) was obtained from subsets of nontelemetered animals in order to assess the impact of HP-12 on biochemical indices of pulmonary inflammation and injury. Immediate dose-related changes in pulmonary function were observed after HP-12 exposure, consisting of decreases in tidal volume (decreasing 12-41%) and increases in breathing frequency (increasing 52-103%), minute ventilation (increasing 12-25%), and enhanced pause (increasing 113-187%). These functional effects were resolved by 7 days post-IT, although some average BALF constituents remained elevated through day 7 for mid- and high-dose groups when compared to those of the saline-treated control group. This study demonstrates significant deficits in pulmonary function, along with significant increases in BALF indices of pulmonary inflammation and injury in SH rats after IT exposure to HP-12.

Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents.

Epidemiologic reports by C.A. Pope III et. al. demonstrated that in the Utah Valley, closure of an open-hearth steel mill over the winter of 1987 was associated with reductions in respiratory disease and related hospital admissions in valley residents. To better examine the relationship between plant-associated changes in ambient particulate matter (PM) and respiratory health effects, we obtained total suspended particulate filters originally collected near the steel mill during the winter of 1986 (before closure), 1987 (during closure), and 1988 (after plant reopening). PM subcomponents were water-extracted from these filters and Sprague-Dawley rats were intratracheally instilled with equivalent masses of extract. Data indicated that 24 hr later, rats exposed to 1986 or 1988 extracts developed significant pulmonary injury and neutrophilic inflammation. Additionally, 50% of rats exposed to 1986 or 1988 extracts had increased airway responsiveness to acetylcholine, compared to 17 and 25% of rats exposed to saline or the 1987 extract, respectively. By 96 hr, these effects were largely resolved except for increases in lung lavage fluid neutrophils and lymphocytes in 1986 extract-exposed rats. Analogous effects were observed with lung histologic assessment. Extract analysis using inductively coupled plasma-mass spectroscopy demonstrated in all three extracts nearly 70% of the mass appeared to be sodium-based salts derived from the glass filter matrix. Interestingly, relative to the 1987 extract, the 1986/1988 extracts contained more sulfate, cationic salts (i.e., calcium, potassium, magnesium), and certain metals (i.e., copper, zinc, iron, lead, strontium, arsenic, manganese, nickel). Although total metal content was (3/4) 1% of the extracts by mass, the greater quantity detected in the 1986 and 1988 extracts suggests metals may be important determinants of the pulmonary toxicity observed. In conclusion, the pulmonary effects induced by exposure of rats to water-based extracts of local ambient PM filters were in good accord with the cross-sectional epidemiologic reports of adverse respiratory health effects in Utah Valley residents.

Residual oil fly ash inhalation in guinea pigs: influence of absorbate and glutathione depletion.

Inhaled urban particulate matter (PM) often contains metals that appear to contribute to its toxicity. These particles first make contact with a thin layer of epithelial lining fluid in the respiratory tract. Antioxidants present in this fluid and in cells might be important susceptibility factors in PM toxicity. We investigated the role of ascorbic acid (C) and glutathione (GSH) as determinants of susceptibility to inhaled residual oil fly ash (ROFA) in guinea pigs (male, Hartley). Guinea pigs were divided into four groups, +C+GSH, +C-GSH, -C+GSH, and -C-GSH, and exposed to clean air or ROFA (< 2.5 micron diameter, 19--25 mg/m(3) nose-only for 2.0 h). C and/or GSH were lowered by either feeding C-depleted diet (1 microg C/kg diet, 2 weeks) and/or by ip injection of a mixture of buthionine-S,R-sulfoximine (2.7 mmol/kg body weight) and diethylmaleate (1.2 mmol/kg, 2 h prior). Nasal lavage (NL) and bronchoalveolar lavage (BAL) fluid and cells were examined at 0 h and 24 h postexposure to ROFA. The C-deficient diet lowered C concentrations in BAL fluid and cells and in NL fluid by 90%, and the GSH-depletion regimen lowered both GSH and C in the BAL fluid and cells by 50%. ROFA deposition was calculated at time 0 from lung Ni levels to be 46 microg/g wet lung. In unexposed animals, the combined deficiency of C and GSH modified the cellular composition of cells recovered in lavage fluid, i.e., the increased number of eosinophils and macrophages in BAL fluid. ROFA inhalation increased lung injury in the -C-GSH group only (evidenced by increased BAL protein, LDH and neutrophils, and decreased BAL macrophages). ROFA exposure decreased C in BAL and NL at 0 h, and increased BAL C and GSH (2- to 4-fold above normal) at 24 h in nondepleted guinea pigs, but had no effect on C and GSH in depleted guinea pigs. Combined deficiency of C and GSH resulted in the highest macrophage and eosinophil counts of any group. GSH depletion was associated with increased BAL protein and LDH, increased numbers of BAL macrophages and eosinophils, and decreased rectal body temperatures. We conclude that combined deficiency of C and GSH increased susceptibility to inhaled ROFA; caused unusual BAL cellular changes; resulted in lower antioxidant concentrations in BAL than were observed with single deficiencies. Antioxidant deficiency may explain increased susceptibility to PM in elderly or diseased populations and may have important implications for extrapolating animal toxicity data to humans.

The spontaneously hypertensive rat as a model of human cardiovascular disease: evidence of exacerbated cardiopulmonary injury and oxidative stress from inhaled emission particulate matter.

Cardiovascular disease is considered a probable risk factor of particulate matter (PM)-related mortality and morbidity. It was hypothesized that rats with hereditary systemic hypertension and underlying cardiac disease would be more susceptible than healthy normotensive rats to pulmonary injury from inhaled residual oil fly ash (ROFA) PM. Eight spontaneously hypertensive (SH) and eight normotensive Wistar-Kyoto (WKY) rats (12-13 weeks old) were implanted with radiotelemetry transmitters on Day -10 for measurement of electrocardiographic (ECG) waveforms. These and other nonimplanted rats were exposed to filtered air or ROFA (containing leachable toxic levels of metals) on Day 0 by nose-only inhalation (ROFA, 15 mg/m(3) x 6 h/day x 3 days). ECGs were monitored during both exposure and nonexposure periods. At 0 or 18 h post-ROFA exposure, rats were assessed for airway hyperreactivity, pulmonary and cardiac histological lesions, bronchoalveolar lavage fluid (BALF) markers of lung injury, oxidative stress, and cytokine gene expression. Comparisons were made in two areas: (1) underlying cardiopulmonary complications of control SH rats in comparison to control WKY rats; and (2) ROFA-induced cardiopulmonary injury/inflammation and oxidative burden. With respect to the first area, control air-exposed SH rats had higher lung and left ventricular weights when compared to age-matched WKY rats. SH rats had hyporeactive airways to acetylcholine challenge. Lung histology revealed the presence of activated macrophages, neutrophils, and hemorrhage in control SHrats. Consistently, levels of BALF protein, macrophages, neutrophils, and red blood cells were also higher in SH rats. Thiobarbituric acid-reactive material in the BALF of air-exposed SH rats was significantly higher than that of WKY rats. Lung inflammation and lesions were mirrored in the higher basal levels of pulmonary cytokine mRNA expression. Cardiomyopathy and monocytic cell infiltration were apparent in the left ventricle of SH rats, along with increased cytokine expression. ECG demonstrated a depressed ST segment area in SH rats. With regard to the second area of comparison (ROFA-exposed rats), pulmonary histology indicated a slightly exacerbated pulmonary lesions including inflammatory response to ROFA in SH rats compared to WKY rats and ROFA-induced increases in BALF protein and albumin were significantly higher in SH rats than in WKY rats. In addition, ROFA caused an increase in BALF red blood cells in SH rats, indicating increased hemorrhage in the alveolar parenchyma. The number of alveolar macrophages increased more dramatically in SH rats following ROFA exposure, whereas neutrophils increased similarly in both strains. Despite greater pulmonary injury in SH rats, ROFA-induced increases in BALF GSH, ascorbate, and uric acid were attenuated when compared to WKY rats. ROFA inhalation exposure was associated with similar increases in pulmonary mRNA expression of IL-6, cellular fibronectin, and glucose-6-phosphate dehydrogenase (relative to that of beta-actin) in both rat strains. The expression of MIP-2 was increased in WKY but attenuated in SH rats. Thus, SH rats have underlying cardiac and pulmonary complications. When exposed to ROFA, SH rats exhibited exacerbated pulmonary injury, an attenuated antioxidant response, and acute depression in ST segment area of ECG, which is consistent with a greater susceptibility to adverse health effects of fugitive combustion PM. This study shows that the SH rat is a potentially useful model of genetically determined susceptibility with pulmonary and cardiovascular complications.

Variable pulmonary responses from exposure to concentrated ambient air particles in a rat model of bronchitis.

Chronic bronchitis may be considered a risk factor in particulate matter (PM)-induced morbidity. We hypothesized that a rat model of human bronchitis would be more susceptible to the pulmonary effects of concentrated ambient particles (CAPs) from Research Triangle Park, NC. Bronchitis was induced in male Sprague-Dawley rats (90-100 days of age) by exposure to 200 ppm sulfur dioxide (SO2), 6 h/day x 5 days/week x 6 weeks. One day following the last SO2 exposure, both healthy (air-exposed) and bronchitic (SO2-exposed) rats were exposed to filtered air (three healthy; four bronchitic) or CAPs (five healthy; four bronchitic) by whole-body inhalation, 6 h/day x 2 or 3 days. Pulmonary injury was determined either immediately (0h) or 18 h following final CAPs exposure. The study protocol involving 0 h time point was repeated four times (study #A, November, 1997; #B, February, 1998; #C and #D, May, 1998), whereas the study protocol involving 18 h time point was done only once (#F). In an additional study (#E), rats were exposed to residual oil fly ash (ROFA), approximately 1 mg/ m(3)x6 h/day x 3 days to mimic the CAPs protocol (February, 1998). The rats allowed 18 h recovery following CAPs exposure (#F) did not depict any CAPs-related differences in bronchoalveolar lavage fluid (BALF) injury markers. Of the four CAPs studies conducted (0 h time point), the first (#A) study (approximately 650 microg/m3 CAPs) revealed significant changes in the lungs of CAPs-exposed bronchitic rats compared to the clean air controls. These rats had increased BALF protein, albumin, N-acetyl glutaminidase (NAG) activity and neutrophils. The second (#B) study (approximately 475 microg/m3 CAPs) did not reveal any significant effects of CAPs on BALF parameters. Study protocols #C (approximately 869 microg/m3 CAPs) and #D (approximately 907 microg/m3 CAPs) revealed only moderate increases in the above mentioned BALF parameters in bronchitic rats exposed to CAPs. Pulmonary histologic evaluation of studies #A, #C, #D, and #F revealed marginally higher congestion and perivascular cellularity in CAPs-exposed bronchitic rats. Healthy and bronchitic rats exposed to ROFA (approximately 1 mg/m3) did not show significant pulmonary injury (#E). Analysis of leachable elemental components of CAPs revealed the presence of sulfur, zinc, manganese, and iron. There was an apparent lack of association between pulmonary injury and CAPs concentration, or its leachable sulfate or elemental content. In summary, real-time atmospheric PM may result in pulmonary injury, particularly in susceptible models. However, the variability observed in pulmonary responses to CAPs emphasizes the need to conduct repeated studies, perhaps in relation to the season, as composition of CAPs may vary. Additionally, potential variability in pathology of induced bronchitis or other lung disease may decrease the ability to distinguish toxic injury due to PM.

Preparation and initial characterization of the compound I, II, and III states of iron methylchlorin-reconstituted horseradish peroxidase and myoglobin: models for key intermediates in iron chlorin enzymes.

To better understand the spectral properties of high valent and oxyferrous states in naturally occurring iron chlorin-containing proteins, we have prepared the oxoferryl compound I derivative of iron methylchlorin-reconstituted horseradish peroxidase (MeChl-HRP) and the compound II and oxyferrous compound III states of iron MeChl-reconstituted myoglobin. Initial spectral characterization has been carried out with UV-visible absorption and magnetic circular dichroism. In addition, the peroxidase activity of iron MeChl-HRP in pyrogallol oxidation has been found to be 40% of the rate for native HRP. Previous studies of oxoferryl chlorins have employed tetraphenylchlorins in organic solvents at low temperatures; stable oxyferrous chlorins have not been previously examined. The present study describes the compound I, II, and III states of histidine-ligated iron chlorins in a protein environment for the first time.

Lung injury from intratracheal and inhalation exposures to residual oil fly ash in a rat model of monocrotaline-induced pulmonary hypertension.

A rat model of monocrotaline (MCT)-induced pulmonary injury/hypertension has been recently used in particulate matter (PM) health effects studies, however, results have been equivocal. Neither the mechanism by which mortality occurs in this model nor the variation in response due to differences in PM exposure protocols (i.e., a bolus dose delivered intratracheally versus a similar cumulative dose inhaled over three days) have been fully investigated. Sprague Dawley rats (SD, 60 d old; 250-300 g) were injected with either saline (healthy) or MCT, 60 mg/kg, i.p. (to induce pulmonary injury/hypertension). Ten days later they were exposed to residual oil fly ash (ROFA), either intratracheally (IT; saline, 0.83 or 3.33 mg/kg) or by nose-only inhalation (15 mg/m3 x 6 h/d x 3 d). Lung histology, pulmonary cytokine gene expression (0 and 18 h postinhalation), and bronchoalveolar lavage fluid (BALF) markers of injury were analyzed (24 and 96 h post-IT; or 18 h post-inhalation). Data comparisons examined three primary aspects, 1) ROFA IT versus inhalation effects in healthy rats; 2) pulmonary injury caused by MCT; and 3) exacerbation of ROFA effects in MCT rats. In the first aspect, pulmonary histological lesions following ROFA inhalation in healthy rats were characterized by edema, inflammatory cell infiltration, and thickening of alveolar walls. Increases in BALF markers of lung injury and inflammation were apparent in ROFA-IT or nose-only exposed healthy rats. Increased IL-6, and MIP-2 expression were also apparent in healthy rats following ROFA inhalation. In regards to the second aspect, MCT rats exposed to saline or air showed perivascular inflammatory cell infiltrates, increased presence of large macrophages, and alveolar thickening. Consistently, BALF protein, and inflammatory markers (macrophage and neutrophil counts) were elevated indicating pulmonary injury. In regards to the third aspect, 58% of MCT rats exposed to ROFA IT died within 96 h regardless of the dose. No mortality was observed using the inhalation protocol. ROFA inhalation in MCT rats caused exacerbation of lung lesions such as increased edema, alveolar wall thickening, and inflammatory cell infiltration. This exacerbation was also evident in terms of additive or more than additive increases in BALF neutrophils, macrophages and eosinophils. IL-6 but not MIP-2 expression was more than additive in MCT rats, and persisted over 18 h following ROFA. IL-10 and cellular fibronectin expression was only increased in MCT rats exposed to ROFA. In summary, only the bolus IT ROFA caused mortality in the rat model of lung injury/hypertension. Exacerbation of histological lesions and cytokine mRNA expression were most reflective of increased ROFA susceptibility in this model.

Low-temperature stabilization and spectroscopic characterization of the dioxygen complex of the ferrous neuronal nitric oxide synthase oxygenase domain.

Nitric oxide (NO), an intercellular messenger and an immuno-cytotoxic agent, is synthesized by the family of nitric oxide synthases (NOS), which are thiolate-ligated, heme-containing monooxygenases that convert L-Arg to L-citrulline and NO in a tetrahydrobiopterin (BH4)-dependent manner, using NADPH as the electron donor. The dioxygen complex of the ferrous enzyme has been proposed to be a key intermediate in the NOS catalytic cycle. In this study, we have generated a stable ferrous-O2 complex of the oxygenase domain of rat neuronal NOS (nNOS) by bubbling O2 through a solution of the dithionite-reduced enzyme at -30 degrees C in a cryogenic solvent containing 50% ethylene glycol. The most stable dioxygen complex is obtained using the oxygenase domain which has been preincubated for an extended length of time at 4 degrees C with BH4/dithiothreitol and NG-methyl-L-arginine, a substrate analogue inhibitor. The O2 complex of the nNOS oxygenase domain thus prepared exhibits UV-visible absorption (maxima at 419 and 553 nm, shoulder at approximately 585 nm) and magnetic circular dichroism spectra that are nearly identical to those of ferrous-O2 cytochrome P450-CAM. Our spectral data are noticeably blue-shifted from those seen at 10 degrees C for a short-lived transient species (lambdamax = 427 nm) for the nNOS oxygenase domain using stopped-flow rapid-scanning spectroscopy [Abu-Soud, H. M., Gachhui, R., Raushel, F. M., and Stuehr, D. J. (1997) J. Biol. Chem. 272, 17349], but somewhat similar to those of a relatively stable O2 adduct of L-Arg-free full-length nNOS (lambdamax = 415-416.5 nm) generated at -30 degrees C [Bec, N., Gorren, A. C. F., Voelder, C., Mayer, B., and Lange, R. (1998) J. Biol. Chem. 273, 13502]. Compared with ferrous-O2 P450-CAM, however, the ferrous-O2 adduct of the nNOS oxygenase domain is considerably more autoxidizable and the O2-CO exchange reaction is noticeably slower. The generation of a stable ferrous-O2 adduct of the nNOS oxygenase domain, as described herein, will facilitate further mechanistic and spectroscopic investigations of this important intermediate.

Essential thiol requirement to restore pterin- or substrate-binding capability and to regenerate native enzyme-type high-spin heme spectra in the Escherichia coli-expressed tetrahydrobiopterin-free oxygenase domain of neuronal nitric oxide synthase.

Nitric oxide (NO) synthases (NOS) are thiolate-ligated heme-, tetrahydrobiopterin (BH(4))-, and flavin-containing monooxygenases which catalyze the NADPH-dependent conversion of L-arginine (L-Arg) to NO AND citrulline. NOS consists of two domains: an N-terminal oxygenase (heme- and BH(4)-bound) domain and a C-terminal reductase (FMN- and FAD-bound) domain. In this study, we have spectroscopically examined the binding of L-Agr and BH(4) to the dimeric, BH(4)-free ferric neuronal NOS (NNOS) oxygenase domain expressed in Escherichia coli separately from the reductase domain. Addition of L-Arg or its analogue inhibitors (N(G)()-methyl-L-Arg, N(G)()-nitro-L-Arg) and BH(4), together with dithiothreitol (DTT), to the pterin-free ferric low-spin oxygenase domain (gamma(MAX): 419, 538, 568 NM) and incubation for 2-3 days at 4 degrees C converted the domain to a native enzyme-type, predominantly high-spin state (gamma(MAX): approximately 395, approximately 512, approximately 650 NM). 7,8-Dihydrobiopterin and other thiols (E.G., beta-mercaptoethanol, cysteine, and glutathione, with less effectiveness) can replace BH(4) and DTT, respectively. the UV-visible absorption spectrum of L-Arg-bound ferric full length NNOS, which exhibits a relatively intense band at approximately 650 NM (epsilon equals 7.5-8 MM(-)(1) CM(-)(1)) due to the presence of a neutral flavin semiquinone, can then be quantitatively reconstructed by combining the spectra of equimolar amounts of the oxygenase and reductase domains. Of particular note, the heme spin-state conversion does not occur in the absence of a thiol even after prolonged (35-48 H) incubation of the oxygenase domain with BH(4) and/or L-Arg under anaerobic conditions. Thus, DTT (or other thiols) plays a significant role(s) beyond keeping BH(4) in its reduced form, In restoring the pterin- and/or substrate-binding capability of the E. coli-expressed, BH(4) free, dimeric NNOS oxygenase domain. Our results in combination with recently available X-ray crystallography and site-directed mutagenesis data suggest that the observed DTT effects arise from the involvement of an intersubunit disulfide bond or its rearrangement in the NOS dimer.

Pulmonary responses to oil fly ash particles in the rat differ by virtue of their specific soluble metals.

Occupational exposure to residual oil fly ash (ROFA) particulate has been associated with adverse respiratory health effects in humans. We hypothesized that ROFA collected at different sites within an oil burning power plant, by virtue of its differing metal and sulfate composition, will induce differential lung injury. Ten ROFA samples collected at various sites within a power plant were analyzed for water- and 1.0 M HCl-leachable arsenic (As), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), vanadium (V), zinc (Zn), and sulfur by inductively coupled plasma-atomic emission spectroscopy. All ROFA samples contained variable amounts of leachable (water-extractable) and 1.0 M HCl-extractable Fe, V, and/or Ni. All other metals, except Zn (ROFA No. 1 contained 3.43 and No. 3, 6.35 micrograms/mg Zn), were present in negligible quantities (< 1.0 microgram/mg) in the water extract. In vivo pulmonary injury from exposure to whole saline suspensions of these ROFA was evaluated. Male, SD rats (60 days old) were intratracheally instilled with either saline or saline suspension of whole ROFA (< 3.0 mass median aerodynamic diameter) at three concentrations (0.833, 3.33, or 8.33 mg/kg). After 24 h, lungs were lavaged and bronchoalveolar lavage fluid (BALF) was analyzed for cellular influx and protein content as well as lactate dehydrogenase (LDH) and N-acetyl glucosaminidase (NAG) activity and total hemoglobin as indicators of lung injury. ROFA-induced increases in BALF protein and LDH, but not neutrophilic inflammation, were associated with its water-leachable total metal, Ni, Fe, and sulfate content. However, the neutrophilic response following ROFA exposure was positively correlated with its water-leachable V content. Modest lung injury was observed with the ROFA samples which contained the smallest amounts of water-leachable metals. The ability of ROFA to induce oxidative burst in alveolar macrophage (AM) was determined in vitro using a chemiluminescence (CL) assay. AM CL signals in vitro were greatest with ROFA containing primarily soluble V and were less with ROFA containing Ni plus V. In summary, ROFA-induced in vivo acute pulmonary inflammation appears to be associated with its water-leachable V content; however, protein leakage appears to be associated with its water-leachable Ni content. ROFA-induced in vitro activation of AM was highest with ROFA containing leachable V but not with Ni plus V, suggesting that the potency and the mechanism of pulmonary injury will differ between emissions containing V and Ni.