General Research Strategy
	Status of the Research Program

General Research Protocol for Studies of Complex Atmospheres

The Center's current research strategy is to create a new detailed database on pollutant mixture composition vs. health responses to serve as a platform for statistical analyses determining the species and combinations of air contaminants most closely associated with the different types of health responses associated with air pollution in population studies. To create the database, a consistent set of atmospheric analyses and health assays will be used to determine the dose-response relationships of the effects of several complex atmospheres having different, but overlapping, compositions. Both the composition of the inhaled atmospheres and the health responses will be measured more comprehensively than in previous laboratory or population studies. This new database will allow relationships between composition and health responses to be examined across a continuum of concentrations and combinations of pollutants in a manner that existing data sets do not allow. The use of "real world" atmospheres will also provide direct comparisons of the health effects of emissions from several sources whose health impacts are currently debated. The program's general research matrix is outlined in the following table.

	Irritation & Inflammation	Allergies & Asthma	Defenses against Infection	Heart &  Lung Function	Cancer
Diesel exhaust      (contemporary, outdated)	+	+	+	+	+
Gasoline exhaust      (on-road, off-road)	+	+	+	+	+
Road dust      (paved, unpaved)	+	+	+	+	+
Wood smoke      (hardwood, softwood)	+	+	+	+	+
Tobacco smoke	+	+	+	+	+
Cooking fumes       (vegetable, meat)	+	+	+	+	+
Coal combustion emissions	+	+	+	+	+

1. Body weight and clinical signs throughout 6 months of exposure
   
   
2. After 1 week or 6 months of exposure:
   
   

   1. Blood: cell counts and differentials, hemoglobin, clinical chemistry (19 parameters), coagulation parameters (fibrinogen, thrombin-antithrombin, factor VII)
      
      

   2. Bronchoalveolar lavage: cell counts and differentials, lactate dehydrogenase, protein, alkaline phosphatase, oxidized and reduced glutathione, beta glucuronidase, TNFa, MIP-2, IL-1, macrophage production of superoxide and hydrogen peroxide.
      
      

   3. Necropsy/Histopathology: complete necropsy, fix all major organs, fix left lung at standard pressure, freeze right lobes for DNA/RNA analyses and archive, light microscopy of respiratory tract, heart, liver, spleen, kidney, and bronchial lymph nodes.

   B.   Carcinogenic Potential

   Cancer incidence is associated statistically with air pollution. Because it is impractical to conduct lifetime cancer bioassays of all the atmospheres, shorter-term responses known to be related to the potential for carcinogenesis will be used. These assays will not directly indicate human cancer risk, but will serve to characterize and compare the relative potential cancer hazards of the atmospheres and their components. Responses will be evaluated in both F344 rats and Strain A/J mice. Methylation of DNA is a common mechanism of chemical carcinogenesis; thus, both the total level of DNA methylation in lung tissue and methylation of selected cancer-related genes will be measured. Oxidative damage to DNA can be caused by inhaled materials; thus, the level of oxidative DNA adducts in lung tissue will be measured. The frequency of micronuclei in polychromatic erythrocytes from bone marrow will be determined as an assay for clastogenicity. As a short-term screen for tumorigenesis, the induction of lung adenomas in Strain A mice exposed for 6 months, then held without exposure for an additional 6 months will be determined. In addition to the animal assays, samples of particles and semi-volatile organic compounds taken from the exposure atmosphere will be assayed for mutagenic potential using the Ames bacterial reverse mutation assay.

   Subjects: Male and female young adult F344/CrlBR rats and Strain A/J mice

   Protocol:

   1. After 1 week or 6 months of exposure:
      
      

      1. Oxidative damage to lung DNA: 8-hydroxy-deoxyguanine adduct level, M1G adduct level, abasic (apurinic and apyrimidinic) sites
         
         

      2. Methylation of lung DNA: global hypermethylation, methylation of CpG islands in p16^INK4a and MGMT genes
         
         

      3. Clastogenesis: micronuclei in immature bone marrow reticulocytes from femur (mice only)
         
         

      4. Histopathology of neoplastic and pre-neoplastic lesions
         
         

   2. Lung tumorigenesis in A/J mice through 6 months following the end of the 6-month exposure

   C.   Pulmonary Immune Responses and Airway Reactivity

   Air pollution is associated with exacerbation of asthma and allergic rhinitis. The potential role of pollutants in causing these conditions is uncertain. Some components found in air pollution have been shown to amplify allergic responses of animals and humans under high-dose laboratory conditions. Two issues regarding interactions between pollution and respiratory allergic responses will be addressed in the NERC studies. In one assay, the potential for concurrent exposure to affect the development of an airway immune response will be examined by sensitizing mice to antigen during exposure, followed by both specific (antigen) and nonspecific (bronchoconstrictor) challenge and measurement of airway constrictive responses, antibody production, and inflammatory responses. In another assay, the potential for exposure to increase responses in mice having pre-existing immune responses will be examined by establishing an allergic airway response before pollutant exposure, then measuring the response again after a few days of exposure to the atmospheres. BALB/c mice will be used because the primary concern for asthma arises from morbidity in children, and young mice of this strain are the only model for which the specific assays to be used have been adequately demonstrated.

   Subjects: Male BALB/c mice exposed to ovalbumin (OVA) antigen

   Protocol:

   1. Effect on development of allergic airway response: expose for 2 months while immunizing and challenging, then measure responses. Responses include specific and nonspecific airway reactivity [Penh] during inhalation challenge with OVA and methacholine, levels of lung mRNA for IL-2, IL-4, IL-5, IL-9, IL-13 IFNg, GADPH, and ribosomal protein L-32 antibody, bronchoalveolar lavage [see parameters in A.2.b. above], total and OVA-specific IgG₁, IgG_2a, and IGE in lavage fluid and serum, and histopathology of lung and associated lymph nodes.
      
      

   2. Exacerbation of pre-existing allergic airway response: immunize and measure specific and nonspecific airway responses before exposure, expose 3 days, then measure airway and other responses (same as above).
      
      

   D.   Resistance to Respiratory Infection

   The occurrence of pulmonary infections, particularly bacterial pneumonia in the elderly and viral infections in infants, is associated statistically with air pollution. Air contaminants are thought to affect the host defense mechanisms important in resistance to infectious agents. Rather than studying each defense mechanism separately, we will examine the effect of exposure to the pollutant atmospheres on the integrated defenses of the respiratory tract by challenging C57/B6 mice with Pseudomonas aeruginosa bacteria. The colonization of the lung (amount of viable agent remaining at a given time) will be the key outcome parameter. The C57/B6 mouse is the best-characterized model for this assay.

   Subjects: Young adult male C57/B6 mice challenged with Pseudomonas aeruginosa

   Protocol: After 1 week or 6 months of exposure:

   1. Instill bacteria intratracheally, allow 18 hours for lung colonization/clearance.
      
      

   2. Homogenize lungs, incubate cultures of serial dilutions of lung homogenate, and count colonies after incubation
      
      

   3. Lung histopathology, retain frozen lung and spleen for potential assays
      
      

   E.   Cardiac Effects

   Epidemiological data indicate an association between short-term increases in airborne pollutants, cardiovascular mortality and morbidity, and subclinical changes in the electrocardiogram (ECG). These changes have primarily been observed in elderly subjects with pre-existing cardiac disease. Laboratory studies using high concentrations of various air contaminants have produced ECG changes in old rats, rats treated to induce pulmonary hypertension, normal dogs, and dogs with experimentally induced coronary occlusion. Changes have been noted both during exposure and during days following exposure. The numbers of animals required to evaluate cardiac effects with robust statistics and the numbers of exposure groups preclude the use of dogs for the NERC studies. We will examine the impact of a 1-week exposure on the cardiac function of mid-aged rats having genetic hypertension. This strain of rats has been shown to have increased sensitivity to inhaled ambient particles.

   Subjects: Male and female SHR/crib (spontaneously hypertensive) rats

   Protocol:

   1. Monitor ECG intermittently via telemetry from implanted transducers 24 hours/day during and for 4 days after 7 consecutive days of exposure
      
      

   2. Analyze ECG for heart rate, heart rate variability, and waveform abnormalities
      
      

   3. Histopathology of lung, heart, and major vessels; archive frozen tissues
      
      

   
   

   For more information, view the Center’s web site at www.nercenter.org, or contact:
   
   

   Matthew D. Reed, PhD, DABT, NERC Study Director
   Lovelace Respiratory Research Institute
   Albuquerque, NM 87108 USA
   Phone: 505-348-9451
   Fax: 505-348-4980
   E-mail: mreed@lrri.org
   

         Or:

   Joe L. Mauderly, DVM, NERC Director
   Lovelace Respiratory Research Institute
   Albuquerque, NM 87108 USA
   Phone: 505-348-9432
   Fax: 505-348-4983
   E-mail: jmauderl@lrri.org