Multicenter Ozone Study in oldEr Subjects (MOSES): Part 1. Effects of Exposure to Low Concentrations of Ozone on Respiratory and Cardiovascular Outcomes.

INTRODUCTION: Exposure to air pollution is a well-established risk factor for cardiovascular morbidity and mortality. Most of the evidence supporting an association between air pollution and adverse cardiovascular effects involves exposure to particulate matter (PM). To date, little attention has been paid to acute cardiovascular responses to ozone, in part due to the notion that ozone causes primarily local effects on lung function, which are the basis for the current ozone National Ambient Air Quality Standards (NAAQS). There is evidence from a few epidemiological studies of adverse health effects of chronic exposure to ambient ozone, including increased risk of mortality from cardiovascular disease. However, in contrast to the well-established association between ambient ozone and various nonfatal adverse respiratory effects, the observational evidence for impacts of acute (previous few days) increases in ambient ozone levels on total cardiovascular mortality and morbidity is mixed.Ozone is a prototypic oxidant gas that reacts with constituents of the respiratory tract lining fluid to generate reactive oxygen species (ROS) that can overwhelm antioxidant defenses and cause local oxidative stress. Pathways by which ozone could cause cardiovascular dysfunction include alterations in autonomic balance, systemic inflammation, and oxidative stress. These initial responses could lead ultimately to arrhythmias, endothelial dysfunction, acute arterial vasoconstriction, and procoagulant activity. Individuals with impaired antioxidant defenses, such as those with the null variant of glutathione S-transferase mu 1 (GSTM1), may be at increased risk for acute health effects.The Multicenter Ozone Study in oldEr Subjects (MOSES) was a controlled human exposure study designed to evaluate whether short-term exposure of older, healthy individuals to ambient levels of ozone induces acute cardiovascular responses. The study was designed to test the a priori hypothesis that short-term exposure to ambient levels of ozone would induce acute cardiovascular responses through the following mechanisms: autonomic imbalance, systemic inflammation, and development of a prothrombotic vascular state. We also postulated a priori the confirmatory hypothesis that exposure to ozone would induce airway inflammation, lung injury, and lung function decrements. Finally, we postulated the secondary hypotheses that ozone-induced acute cardiovascular responses would be associated with: (a) increased systemic oxidative stress and lung effects, and (b) the GSTM1-null genotype. METHODS: The study was conducted at three clinical centers with a separate Data Coordinating and Analysis Center (DCAC) using a common protocol. All procedures were approved by the institutional review boards (IRBs) of the participating centers. Healthy volunteers 55 to 70 years of age were recruited. Consented participants who successfully completed the screening and training sessions were enrolled in the study. All three clinical centers adhered to common standard operating procedures (SOPs) and used common tracking and data forms. Each subject was scheduled to participate in a total of 11 visits: screening visit, training visit, and three sets of exposure visits, each consisting of the pre-exposure day, the exposure day, and the post-exposure day. The subjects spent the night in a nearby hotel the night of the pre-exposure day.On exposure days, the subjects were exposed for three hours in random order to 0 ppb ozone (clean air), 70 ppb ozone, and 120 ppm ozone, alternating 15 minutes of moderate exercise with 15 minutes of rest. A suite of cardiovascular and pulmonary endpoints was measured on the day before, the day of, and up to 22 hours after, each exposure. The endpoints included: (1) electrocardiographic changes (continuous Holter monitoring: heart rate variability [HRV], repolarization, and arrhythmia); (2) markers of inflammation and oxidative stress (C-reactive protein [CRP], interleukin-6 [IL-6], 8-isoprostane, nitrotyrosine, and P-selectin); (3) vascular function measures (blood pressure [BP], flow-mediated dilatation [FMD] of the brachial artery, and endothelin-1 [ET-1]; (4) venous blood markers of platelet activation, thrombosis, and microparticle-associated tissue factor activity (MP-TFA); (5) pulmonary function (spirometry); (6) markers of airway epithelial cell injury (increases in plasma club cell protein 16 [CC16] and sputum total protein); and (7) markers of lung inflammation in sputum (polymorphonuclear leukocytes [PMN], IL-6, interleukin-8 [IL-8], and tumor necrosis factor-alpha [TNF-α]). Sputum was collected only at 22 hours after exposure.The analyses of the continuous electrocardiographic monitoring, the brachial artery ultrasound (BAU) images, and the blood and sputum samples were carried out by core laboratories. The results of all analyses were submitted directly to the DCAC.The variables analyzed in the statistical models were represented as changes from pre-exposure to post-exposure (post-exposure minus pre-exposure). Mixed-effect linear models were used to evaluate the impact of exposure to ozone on the prespecified primary and secondary continuous outcomes. Site and time (when multiple measurements were taken) were controlled for in the models. Three separate interaction models were constructed for each outcome: ozone concentration by subject sex; ozone concentration by subject age; and ozone concentration by subject GSTM1 status (null or sufficient). Because of the issue of multiple comparisons, the statistical significance threshold was set a priori at P < 0.01. RESULTS: Subject recruitment started in June 2012, and the first subject was randomized on July 25, 2012. Subject recruitment ended on December 31, 2014, and testing of all subjects was completed by April 30, 2015. A total of 87 subjects completed all three exposures. The mean age was 59.9 ± 4.5 years, 60% of the subjects were female, 88% were white, and 57% were GSTM1 null. Mean baseline body mass index (BMI), BP, cholesterol (total and low-density lipoprotein), and lung function were all within the normal range.We found no significant effects of ozone exposure on any of the primary or secondary endpoints for autonomic function, repolarization, ST segment change, or arrhythmia. Ozone exposure also did not cause significant changes in the primary endpoints for systemic inflammation (CRP) and vascular function (systolic blood pressure [SBP] and FMD) or secondary endpoints for systemic inflammation and oxidative stress (IL-6, P-selectin, and 8-isoprostane). Ozone did cause changes in two secondary endpoints: a significant increase in plasma ET-1 (P = 0.008) and a marginally significant decrease in nitrotyrosine (P = 0.017). Lastly, ozone exposure did not affect the primary prothrombotic endpoints (MP-TFA and monocyte-platelet conjugate count) or any secondary markers of prothrombotic vascular status (platelet activation, circulating microparticles [MPs], von Willebrand factor [vWF], or fibrinogen.).Although our hypothesis focused on possible acute cardiovascular effects of exposure to low levels of ozone, we recognized that the initial effects of inhaled ozone involve the lower airways. Therefore, we looked for: (a) changes in lung function, which are known to occur during exposure to ozone and are maximal at the end of exposure; and (b) markers of airway injury and inflammation. We found an increase in forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1) after exposure to 0 ppb ozone, likely due to the effects of exercise. The FEV1 increased significantly 15 minutes after 0 ppb exposure (85 mL; 95% confidence interval [CI], 64 to 106; P < 0.001), and remained significantly increased from pre-exposure at 22 hours (45 mL; 95% CI, 26 to 64; P < 0.001). The increase in FVC followed a similar pattern. The increase in FEV1 and FVC were attenuated in a dose-response manner by exposure to 70 and 120 ppb ozone. We also observed a significant ozone-induced increase in the percentage of sputum PMN 22 hours after exposure at 120 ppb compared to 0 ppb exposure (P = 0.003). Plasma CC16 also increased significantly after exposure to 120 ppb (P < 0.001). Sputum IL-6, IL-8, and TNF-α concentrations were not significantly different after ozone exposure. We found no significant interactions with sex, age, or GSTM1 status regarding the effect of ozone on lung function, percentage of sputum PMN, or plasma CC16. CONCLUSIONS: In this multicenter clinical study of older healthy subjects, ozone exposure caused concentration-related reductions in lung function and presented evidence for airway inflammation and injury. However, there was no convincing evidence for effects on cardiovascular function. Blood levels of the potent vasoconstrictor, ET-1, increased with ozone exposure (with marginal statistical significance), but there were no effects on BP, FMD, or other markers of vascular function. Blood levels of nitrotyrosine decreased with ozone exposure, the opposite of our hypothesis. Our study does not support acute cardiovascular effects of low-level ozone exposure in healthy older subjects. Inclusion of only healthy older individuals is a major limitation, which may affect the generalizability of our findings. We cannot exclude the possibility of effects with higher ozone exposure concentrations or more prolonged exposure, or the possibility that subjects with underlying vascular disease, such as hypertension or diabetes, would show effects under these conditions.

Health effects of oxygenated fuels.

The use of oxygenated fuels is anticipated to increase over the next decades. This paper reviews the toxicological and exposure information for methyl tertiary-butyl ether (MTBE), a fuel additive, and methanol, a replacement fuel, and discusses the possible health consequences of exposure of the general public to these compounds. For MTBE, the health effects information available is derived almost exclusively from rodent studies, and the exposure data are limited to a few measurements at some service stations. Based on these data, it appears unlikely that the normal population is at high risk of exposure to MTBE vapor. However, in the absence of health and pharmacokinetic data in humans or in nonhuman primates, this conclusion is not strongly supported. Similarly, there are a number of uncertainties to take into consideration in estimating human risk from the use of methanol as a fuel. Although methanol may be toxic to humans at concentrations that overwhelm certain enzymes involved in methanol metabolism, the data available provide little evidence to indicate that exposure to methanol vapors from the use of methanol as a motor vehicle fuel will result in adverse health effects. The uncertainties in this conclusion are based on the lack of information on dose-response relationship at reasonable, projected exposure levels and of studies examining end points of concern in sensitive species. In developing a quantitative risk assessment, more needs to be known about health effects in primates or humans and the range of exposure expected for the general public for both compounds.

The 4S polycyclic aromatic hydrocarbon-binding protein: immunohistochemical localization in mice.

The 4S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a cytoplasmic protein that binds PAHs with specificity and high affinity. We have used antisera for the PBP and unlabeled peroxidase anti-peroxidase immunohistochemistry to demonstrate its possible localization in cell types known to have xenobiotic metabolizing capabilities. Cellular sites of the PBP in liver, lung and kidney of C57BL/6 and DBA/2 mice were probed. The PBP was visualized in hepatocytes throughout the liver lobule and was not preferentially located in either centrilobular or periportal areas. However, cellular heterogeneity with respect to PBP content was clearly evident in the hepatocyte population. The positive reactivity correlated with substantial levels of benzo[a]pyrene (B[a]P) binding in liver cytosol. In the lung, the PBP was found in the bronchiolar epithelium and the alveolar septa, and was localized in ciliated and non-ciliated Clara and alveolar type II cells as well as in alveolar macrophages. In the kidney, the glomeruli and epithelia of proximal and distal convoluted tubules and collecting ducts were labeled. Staining for the PBP was greatest in the apical region of the pyramid and was localized in the epithelial lining of the collecting ducts. Relatively lower levels of the PBP were detected in the lung and kidney than in the liver. Staining was localized in the cytoplasmic compartment of cells in all tissues examined. Similar immunoreactivities were exhibited in the tissues of both C57BL/6 and DBA/2 mice. Treatment with beta-naphthoflavone (beta NF) altered neither the intensity nor pattern of immunostaining. Furthermore, treatment with beta NF or isosafrole has no effect on the Kd and Bmax of B[a]P binding to liver cytosolic PBP. The results of our experiments demonstrate localization of the PBP to sites of active physiological response to PAH exposure.

Properties of the specific binding site for arginine vasopressin in rat hippocampal synaptic membranes.

In a previous paper (Pearlmutter, A. F., Constantini, M. G., and Loeser, B. (1983) Peptides 4, 335-341), we have shown that saturable, high-affinity binding sites for [3H]arginine vasopressin (AVP) are located in rat brain membrane preparations. Binding was dependent upon the presence of Ni2+ and could be dissociated by EDTA. In the hippocampus, [3H]AVP binding could be localized to synaptic membranes. In this paper, we characterize in more depth the specificity of [3H]AVP binding to a crude hippocampal synaptic preparation and the metabolism of [3H]AVP in our synaptic preparation. By means of HPLC analysis we demonstrate that the radioactive material specifically bound to hippocampal synaptic membranes is intact [3H]AVP. The ability of analogues of AVP to displace the high-affinity, specific binding of [3H]AVP parallels closely the potency of these analogues to inhibit the extinction of avoidance behavior. In the presence of membrane and Ni2+, [3H]AVP has a half-life of 7 h. In the absence of Ni2+, the half-life of [3H]AVP is 1.2 h. Fractionation by high-pressure liquid chromatography of the supernatant from the incubation media not containing Ni2+ yields three peaks of radioactivity. Analysis of the biological activity of the [3H]AVP peak and the two non-AVP peaks which represent breakdown products show the following: (a) the [3H]AVP peak (52%) and peak III (8%) bind to fresh membranes and (b) peak II (40%) has no binding activity. Although Ni2+, Co2+, benzamidine, and phenanthroline can prevent [3H]AVP degradation, only Ni2+ and, to a much lesser extent, Co2+, can potentiate specific [3H]AVP binding. The results show that AVP-specific binding has properties which parallel its biological activity in behavioral assays; that, ultimately, proteolysis by membrane-bound peptidases inactivates AVP; and that Ni2+ acts both by preventing AVP breakdown and by potentiating specific binding.

Kinetics of DNA repair synthesis induced by bleomycin and N-methyl-N-nitrosourea in isolated rat liver nuclei.

Purified rat liver nuclei were incubated in the presence of a labeled deoxyribonucleoside triphosphate and bleomycin (an antitumor agent) or N-methyl-N-nitrosourea (MNU, a direct-acting carcinogen) to compare their abilities to induce DNA repair synthesis. It was found that bleomycin induced the incorporation of [3H]dTMP and, to a lesser extent, [3H]dCMP and [3H]dAMP, whereas MNU induced incorporation of [3H]dAMP exclusively. The bleomycin-induced DNA repair was linear with time, whereas the MNU-induced repair was more complex, requiring an induction period and lasting only 90 minutes. The extent of incorporation measured after a 15- or 30-minute preexposure to bleomycin was proportional to the time of preexposure and the repair reaction was completed within 30 minutes. The extent of incorporation measured after preexposure to MNU increased less than proportionally with the time of preexposure and the repair reaction lasted a total of approximately 90 minutes. The interactions between bleomycin and MNU damage and repair were also examined. It was found that, following preexposure to MNU and subsequent repair in the presence of bleomycin, the repair activities induced by the two compounds were not additive. Our data are in agreement with previous studies on the base excision induced by bleomycin and MNU and suggest that the in vitro nuclear system reflects the physiological changes induced by the interaction of a compound with DNA.

Characterization of 3H-AVP binding sites in particulate preparations of rat brain.

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni greater than Co greater than Mg greater than Mn greater than no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Level and turnover of polyadenylate-containing ribonucleic acid in Neurospora crassa in different steady states of growth.

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of polyadenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5--5.2%). Using a kinetic approach, an equation was derived which allowed determination of the average half-lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10-min pulse with [5-3H]uridine when they were in the exponential phase. It was found that the determined half-lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady-state level of mRNA.

Levels of the ribonucleoside triphosphates and rate of RNA synthesis in Neurospora crassa.

The levels of the four ribonucleoside triphosphate (ATP, GTP, UTP and CTP) have been determined in Neurospora crassa in three conditions of exponential growth (on glucose, acetate and glycerol) as well as in the course of a shift-up and a shift-down transition of growth between two of them. Although in some cases the pools appear proportional to the rate of synthesis of ribosomal RNA, this seems not to be strictly dependent on the level of the nucleotides.