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Markers of Pulmonary Toxicity

Markers of Pulmonary Toxicity. Gary E. Hatch, Ph.D. Pulmonary Toxicology Branch Experimental Toxicology Division Email: Hatch.Gary@epa.gov Phone: 919-541-2658 October 3, 2006. IMPORTANT TERMS. Biological marker or Biomarker : 'Indicator signaling events in a biological system'

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Markers of Pulmonary Toxicity

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  1. Markers of Pulmonary Toxicity Gary E. Hatch, Ph.D. Pulmonary Toxicology Branch Experimental Toxicology Division Email: Hatch.Gary@epa.gov Phone: 919-541-2658 October 3, 2006

  2. IMPORTANT TERMS Biological marker or Biomarker: 'Indicator signaling events in a biological system' Exposure markers Effect markers Susceptibility markers Non-invasive: Measurable in blood or urine Homologous: Applicable across species Harmonized: Markers predictive of both cancer and non-cancer effects Mechanism of action: The complete sequence of biological events --> toxic effect Mode of action: Less-detailed than 'mechanism' Source: Human Health Research Strategy, U.S. E.P.A., ORD, 2003

  3. DESIRABLE QUALITIES in a Marker of Toxicity - Rapid, simple, easy - Predictive of human pathology - Non-invasive - Sensitive - Validated scientifically - Accepted by scientists and public

  4. POSSIBLE TEST QUESTIONS - Definitions - What are the desirable qualities of biomarkers of toxicity? - Choose one marker and evaluate its strengths and weaknesses. - Outline a protocol for an experiment that could either 1) remove a weakness or reduce an uncertainty in an existing marker or 2) find a new marker.

  5. READING MATERIAL Examples of experiments on markers: Finding a new marker: intratracheal instillation with bacterial infectivity Linking rat to human: ozone dose Linking acute to chronic: phosgene toxicity

  6. EXAMPLES: MARKERS OF PULMONARY TOXICITY Gross appearance of the lung Clinical signs: whole body Microscopic changes Lung physiology changes Lung and lung lavage fluid Cellular Biochemical Genomic Markers of Dose Markers of Susceptibility Bacterial infectivity marker Non-invasive markers Blood and urine

  7. GROSS APPEARANCE of the LUNG HUMAN LUNG Normal Lung Emphysema Fibrosis, cut away Fibrosis

  8. GROSS APPEARANCE of the LUNG ASBESTOSIS in the HUMAN LUNG

  9. THE NEED FOR MARKERS To predict and prevent bad experiences, such as: Crystalline silica: miners silicosis Tobacco smoke: cancer, emphysema, COPD Asbestos: cancer Beryllium metal: fibrosis Paraquat (herbicide) : edema, fibrosis

  10. How were these agents first established as respiratory toxicants? Human Epidemiology - Polycyclic aromatic hydrocarbons (roofing tar) - Airborne particulate matter - Tobacco smoke - Asbestos Animal studies - Ozone - Aldehydes - Carbon tetrachloride - Nitrogen oxides

  11. NITROGEN DIOXIDE OZONE PHOSGENE CARBON CARBON TETRACHLORIDE TETRACHLORIDE CHLORINE DIOXIDE CHLORINE Prototype Oxidant Pollutant Molecules NITROGEN DIOXIDE NITROGEN DIOXIDE OZONE OZONE PHOSGENE PHOSGENE CHLORINE DIOXIDE CHLORINE DIOXIDE CHLORINE CHLORINE

  12. CLINICAL SIGNS: Whole Body INFARED THERMAL CAMERA PHOTOS OF A MOUSE EXPOSED TO PHOSGENE Control air exposed Same mouse ~3 minutes in subclinical level of phosgene

  13. CLINICAL SIGNS: Whole Body INVASIVE CORE TEMPERATURE USING PERITONEAL RADIO-TELIMETER

  14. LUNG PHYSIOLOGY CHANGES: Human FEV1 - "FORCED EXPIRATORY VOLUME IN ONE SECOND" Measures "the inability to take a deep breath." Affected by asthma, aging, irritant exposure, COPD Might sometimes be interpreted as a protective response.

  15. LUNG PHYSIOLOGY CHANGES: HUMAN FEV1, AGING, SMOKING

  16. LUNG PHYSIOLOGY CHANGES: Animal: "PenH" Assessment of Airway Obstruction in Unanesthetized Unrestrained Rodents (Buxco) analysis software aerosol chamber control unit nebulizer plethysmograph preamplifier flow regulator Courtesy: Steve Gavett, EPA, 541-2555

  17. Wheel Running Activity of Mice Exposed to Ozone Night-time wheel running Decreased as ozone concentrations increased Black = ozone exposure Tepper et al, T.A.P. 64: 317, 1982

  18. CLINICAL SIGNS: Whole Body PERITONEAL IRRITANCY ASSAY Control CdSO4 150 ug /kg i.p. Mice injected I.P. with and irritating test material develop visible indentations above the hind legs. Sensory irritation induced by metal containing fly ash (Hatch et al, 1982 Fundam. Appl. Toxicol. 2:77)

  19. MICROSCOPIC CHANGES: Light Microscopy of rat lung CONTROL LUNG TISSUE SECTION, 10 X, trichrome stain for collagen

  20. MICROSCOPIC CHANGES: Light Microscopy 12 WEEKS EXPOSURE of RATS to 0.2 ppm phosgene (20x) green = collagen Trichrome Stain

  21. LUNG BIOCHEMICAL MARKERS: Chronic effect: FIBROSIS Biochemical Determination of Hydroxyproline Phosgene Dose 12 weeks 12 weeks + recovery 4 weeks Kodavanti et al, 1992

  22. MICROSCOPIC CHANGES: Scanning Electron Microscopy Rat terminal airway epithelium 1 day post exposure to 1 ppm ozone for 8 hr. Normal rat terminal broncheolar epithelium Paige and Plopper, Air Pollution and Health, 1999 p.539

  23. MICROSCOPIC CHANGES "Comet Assay" for detection of single cell DNA strand breaks Normal cell Severe injury Mild injury Cells are suspended in agar and electrophoresed in the presence of NaOH then stained for DNA. Broken DNA strands migrate into the gel. [[Ref.

  24. MICROSCOPIC CHANGES HISTOCHEMICAL STAINING FOR APOPTOSIS 17 day pregnant mouse mammary glands of the p53Arg-Leu transgenic (left) and a control (right) mouse stained using the TUNEL assay indicate apoptotic cells. Only one apoptotic cell is present in the upper right hand corner of the control, but approximately 20% of the cells stained positively in the line expressing the p53Arg-Leu transgene. From: B Li, FS Kittrell, D Medina, JM Rosen (1995). Delay of dimethylbenz(a)anthracene-induced mammary tumorigenesis in transgenic mice by apoptosis induced by an unusual mutant p53 protein. Mol. Carcinogenesis 14:75-83.

  25. MICROSCOPIC CHANGES: Dividing cells -- Repair VISUALIZING PROLIFERATING CELLS: BRDU labeling Light micrographs of the centriacinar regions of the lung with immunohistochemical identification of proliferating cells. (A) filtered air-exposed mouse, (D) ozone-exposed mouse. Proliferating cells are recognized as densely BrdU labeling-positive (seen as dots centering around the bronchiolar-alveolar duct junctions). Scale bar = 100 µm. From: M Yu, X Zheng, H Witschi, K E Pinkerton. The Role of Interleukin-6 in Pulmonary Inflammation and Injury Induced by Exposure to Environmental Air Pollutants. Toxicological Sciences 68, 488-497 (2002)

  26. GENOMIC MARKERS: Whole lung homogenate Results from a 2 hr ozone exposure (5 ppm, 2 hr) Clontech array of 588 genes. Lung homogenate (~40 cell types). 67 increased > 2x 76 decreased Many oncogenes activated OF NOTE: - Insulin like growth factor - VEGF receptor - c-fos, c-met, c-jun, ras Nadadur, Hatch., 2005

  27. 1.0 ppm 0.5 ppm 0.25 ppm Air Exposure: 4 hr TOXIC EFFECT MARKER: Lung lavage fluid protein concentration PHOSGENE DOSE RESPONSE in Rats 2000 * 1800 * 1600 * * 1400 * 1200 BAL Protein, mg/ml * 1000 * * * 800 600 400 200 72 0 24 48 Hours Post Exposure Hatch et al, 2001. Toxicol Indust. Health 17: 285-293

  28. LUNG LAVAGE MARKERS: Inflammatory cell changes Guinea Pig Alveolar Macrophages eosinophils macrophages

  29. 100 Time Course Dose Response: Rat lung lavage neutrophils following phosgene inhalation 80 60 1.0 ppm % NEUTROPHILS 40 0.5 ppm 20 0.25 ppm 0 72 24 48 Hours post Exposure Air Exposure: 4 hr LUNG LAVAGE FLUID TOXICITY MARKER: Inflammatory cell influx Hatch et al, 2001. Toxicol Indust. Health 17: 285-293

  30. MARKERS of INTERNAL DOSE: Labeled phosgene AUTORADIOGRAPHY OF PHOSGENE REACTION PRODUCT AT THE NASAL EPITHELIAL SURFACE Visualization of the reaction product of inhaled labeled phosgene on the surface of the rat nasal cavity. A transverse section of the nasal turbinate is shown. The dark line on most of the airway surface (except in the pocket) is carbon-14 containing reaction product of carbon-14 labeled phosgene (Cl214C=O). Rats were exposed for 3 minutes to 1 ppm of the labeled phosgene and killed immediately after exposure. (Hatch, G.E. and Morgan, K.).

  31. Ozone Generator BIOMARKERS of INTERNAL DOSE: Oxygen-18 labeled ozone GENERATION OF 18O3 FOR INHALATION EXPOSURES Air 5% 18O2 in argon Inhalation Chamber 18O3

  32. Resting Exercising Resting F344 Rat Human F344 Rat (0.4 ppm, (0.4 ppm, (2.0 ppm, 2 hours) 2 hours) 2 hours) BIOMARKERS OF INTERNAL DOSE: Linking animal to human Rats underestimate human ozone dose: 18O incorporation into lung following 18o3 50 Bronchoalveolar lavage cells BAL High Speed Pellet BAL High Speed Supernatant Lavaged Lung Excess 18O, ug / g dry 25 0 Hatch et al, 1994 Am J. Respir. Crit Care Med 150: 676

  33. MARKERS OF SUSCEPTIBILITY ANTIOXIDANT SUBSTANCES in LUNG and LINING FLUID Ascorbate Urate Glutathione Alpha-tocopherol

  34. MARKERS OF SUSCEPTIBILITY: Species comparisons

  35. FEATURES Defense Occurs at Air-Liquid Interface Phagocyte mediators are important Extracellular defenses are important BACTERIAL INFECTIVITY ANIMAL MODEL Pollutants increase susceptibility to bacterial infection Clearance Effect spectrum Lung Bacteria Morbidity viruses pollutants “stress” diet, age, genetics Mortality

  36. IMMUNE BIOMARKERS OF PULMONARY TOXICITY Gardner, et al 1982

  37. NO 2 COMPARISON OF TWO MARKERS: Sensitivity of dose response Streptococcus Infectivity vs. Lung Permeability 100 2.5 Phosgene 90 Phosgene 80 2.0 70 NO2 60 1.5 BAL Fluid Protein, mg/ml Ozone % Excess Mortality Due to Infection 50 40 1.0 Ozone 30 20 0.5 10 0 0.0 0.01 0.10 1.00 10.00 100.00 Approximate Amount in Lungs, m g / mouse

  38. NON-INVASIVE MARKER OF OXIDATIVE STRESS: ISOPROSTANE Fatty acid: Arachidonate radical OXIDATION Type III Isoprostane, measured by GCMS or ELISA

  39. 18O in urine of rats exposed to 5.0 ppm 18O3 (2 hr) 25 20 Urine from18O3exposed 15 EXCESS 18O ug / g dry 10 5 Urine from untreated rats 0 Post-exposure time, hr -5 10 20 30 40 50 60 70 80 90 100 NON-INVASIVE MARKER: Urinary products of lung repair from labeled ozone

  40. SUMMARY: MARKERS OF PULMONARY TOXICITY Gross appearance of the lung Clinical signs: whole body Microscopic changes Lung physiology changes Lung and lung lavage fluid Cellular Biochemical Genomic Markers of Dose Markers of Susceptibility Bacterial infectivity marker Non-invasive markers Blood and urine

  41. SUMMARY DESIRABLE QUALITIES in a Marker of Toxicity - Rapid, simple, easy - Predictive of human pathology - Non-invasive - Sensitive - Validated scientifically - Accepted by scientists and public

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