Industrial Toxicology and Pathology Short Course 2012 Respiratory Response to Toxic Injury: Upper Respiratory Tract (Nasal Toxicology) Jack R. Harkema July 26, 2012 Nose and Society 40 million cases of chronic sinusitis Cases of allergic rhinitis also increasing Increase in the identification of nasal toxicants New understanding of the molecular makeup of olfaction: 2004 Nobel Prize (Drs. Axel and Buck) utline Nasal Airway Anatomy and Histology Nasal Toxicity of Inhaled zone Nasal Toxicity of Inhaled Nanoparticles lfactory Toxicity of Inhaled Satratoxin G (Mycotoxin in Stachybotris chatarum) Future Studies, Summary and Conclusions 1
Comparative Anatomy of the Respiratory Tract Rat Human Nasal Airway Nasopharynx Terminal Bronchiole Respiratory Bronchiole Alveolar Duct Alveolus Bronchus Larynx Trachea Pulmonary Artery Pulmonary Vein Alveolar Capillary Bed Comparative Nasal Airway Structure and Function Human Monkey Rat Volume (cm 3 ) 16 19 8 0.4 Turbinate Anatomy Simple Simple Complex lfactory Epithelial Surface Area Small <10% Moderate 20 30% Large 50% Breathing ronasal ronasal Nasal Computer Assisted Reconstruction of Rat Nasal MRI Proximal T2 Nasal Section Distal T4 Nasal Section 2
Comparative Anatomy of Nasal Airways Harkema JR et al. Chapter 6 Nose, Sinus, Pharynx and Larynx. In Comparative Anatomy and Histology: A Mouse and Human Atlas, Eds. Treuting PM and Dintz SM, Academic Press, 2012 Nasal Airway of the Monkey Nose Nasal Mucosa of the Monkey Nose Harkema JR et al. Chapter 6 Nose, Sinus, Pharynx and Larynx. In Comparative Anatomy and Histology: A Mouse and Human Atlas, Eds. Treuting PM and Dintz SM, Academic Press, 2012 3
Proximal Nasal Airway of the Mouse Harkema JR et al. Chapter 6 Nose, Sinus, Pharynx and Larynx. In Comparative Anatomy and Histology: A Mouse and Human Atlas, Eds. Treuting PM and Dintz SM, Academic Press, 2012 Proximal Nasal Airway of the Mouse Harkema JR et al. Chapter 6 Nose, Sinus, Pharynx and Larynx. In Comparative Anatomy and Histology: A Mouse and Human Atlas, Eds. Treuting PM and Dintz SM, Academic Press, 2012 Distal Nasal Airways of the Mouse Harkema JR et al. Chapter 6 Nose, Sinus, Pharynx and Larynx. In Comparative Anatomy and 4
lfactory System 1) Main lfactory rgan (ordors in general) 2) Vomeronasal rgan (chemicals in social and sexual activities) 3) Septal lfactory rgan (same odors as main olfactory system and an alerting role) lfactory Epithelium lfactory sensory neurons (SN) Axons are unmyelinated (0.1 0.7 mm in diameter) Sustentacular cells Basal cells Microvillous cells Lamina Propria lfactory nerve fascicles (bundles of SN axons) Schwann cells surrounding bundles of SN axons Bowman s Glands Blood vessels Connective tissue lfactory Mucosa Farbman AI. Cell Biology of lfaction, Cambridge University Press,1992 Unique Characteristics of the lfactory Chemosensory Neurons Dendrites exposed to the external environment Axons project directly to the forebrain without synapsing in the thalamus (conduit to the CNS) Capacity for continued postnatal neurogenesis 5
7/20/2012 lfactory Marker Protein Dendritic Cilia SC SN BC N lfactory Sensory Neuron (Receptor Cells) Dendrite: Knob and Cilia 0.1 m 1-2 m 50 mm 9(2)+2 No dynein arms not motile 0.3 m Farbman AI. Cell Biology of lfaction, Cambridge University Press,1992 dorant Receptors and rganization of the lfactory System 2004 Nobel Award Drs. Axel and Buck 6
lfactory Genes & Receptors 3% of human genome devoted to identifying odors (1000 odorant receptor genes RG) Humans and mice have 5 million olfactory sensory neurons (SN) Bloodhound has 220 million SN 1 RG per SN 1,800 olfactory axon convergence centers in the lfactory Bulb = Glomeruli Each RG corresponds to 2 glomeruli zone ( 3 ) ne of the most reactive chemicals xidant gas in photochemical smog 131 million people (50% of the U.S. population) live in communities where average ambient concentrations exceed the NAAQS Respiratory toxicant causing airway inflammation and remodeling Long-term exposure causes an increase in mortality Comparative Nasal Toxicity of zone Calderon Garciduenas et al. Am. J. Pathol. 140: 225 32, 1992 Harkema et al. Am. J. Pathol. 127:90 96, 1987 Harkema et al. Am. J. Pathol. 128:29 44, 1987 Harkema et al. Toxicol. Pathol. 17: 525 535, 1989 7
3 Induced Remodeling of Maxilloturbinate in Rat Kinetics of Nasal Tissue Responses to Inhaled zone Kinetics of zone Induced Mucous Cell Metaplasia in Rat Transitional Epithelium Cho HY et al., Toxicol Sci 51:135 145, 1999 8
Schematic of zone Exposure Protocol for Infant Monkeys Carey SA et al. Am J Physiol Lung Cell Mol Physiol 2011;300:L242 L254 Imaging, 3D modeling, & Histopathologic Methods Carey SA et al. Toxicol Pathol 2007;35:27 40 Site and Exposure Specific Nasal Mucosal Injury Carey SA et al. Am J Physiol Lung Cell Mol Physiol 2011;300:L242-L254 9
Nasal Morphometry of zone Induced Nasal Epithelial Injury in Infant Rhesus Monkeys Carey SA et al. Am J Physiol Lung Cell Mol Physiol 2011;300:L242 L254 Effect of 1 and 11 cycle 3 exposure on the intracellular concentrations of GSH (A), GSSG (B), ascorbate (AH2; C), and uric acid (UA; D) in the nasal mucosa from the anterior MT, posterior MT, and anterior ET of infant monkeys. Carey SA et al. Am J Physiol Lung Cell Mol Physiol 2011;300:L242 L254 Correlations Among Sites of zone Induced Nasal Airway Injury, Predicted zone Flux, and Epithelial Type Carey SA et al. Toxicol Pathol 2007;35:27 40 10
Inhaled Particle Deposition in Airways Fractional Deposition of Inhaled Particles in the Human Respiratory Tract (ICRP Model, 1994; Nose breathing) Is the nose a potential target for toxic injury caused by inhaled nanoparticles? Carbon Black Nanoparticles Combustion derived nanoparticles Industrially produced for colouring enamels, acrylics, and plastics, as well as inks and paints Low toxicity, low solubility without organics or metals Pulmonary carcinogen in rats with long term exposures and under overload conditions Donaldson et al. Particle and Fibre Toxicology 2:1 14, 2005 11
Effects of Inhaled Carbon Black in Three Rodent Species: Study Design and Methods Female F344 rats, B6C3F1 mice, and Syrian Golden Hamsters Rodents exposed by inhalation to 0, 1, 7, or 50 mg/m 3 of high surface area carbon black (HSCB; 17nm primary particle diameter) for 6 h/day, 5 days/wk for 13 wk Some rats exposed to 50 mg/m 3 of low surface area CB (LSCB; 70nm primary diameter) for 6 h/day, 5 days/wk for 13 wk Rodents were sacrificed 1 day, 13 wk, or 11 mo post exposure Nasal and pulmonary airways were processed for microscopic and morphometric examination Elder et al., Toxicol Sci. 2005; 88(2), 614 629 Pulmonary Effects of Inhaled Carbon Black in Laboratory Rats Initial lung burdens were similar in high dose HSCB and LSCB Surface area burdens were equivalent for mid dose HSCB and high dose LSCB LSCB cleared faster from the lung (less retention) than HSCB More pulmonary pathology in HSCB than in LSCB Particle surface area is an important factor in target tissue dose and, therefore, toxic effects Toxicity in Rats>Mice>Hamsters Elder et al., Toxicol Sci. 2005; 88(2), 614 629 Nasal Histopathology in Rodents Exposed to Inhaled Carbon Black Particles in nasal mucosa Epithelial hyperplasia Mucous cell metaplasia Inflammation (rhinitis) Atrophy of turbinate bone Hyalinosis (chitinase) Dose dependent severity No LSCB related lesions 12
HSCB Induced Histopathology in Maxilloturbinates of Rats Air Control HSCB 50mg/m 3 H&E AB/PAS HSCB in Nasal Mucosa In Nasal Transitional and Respiratory Mucosa In Mucosal Macrophage 100 m tb E bg In lfactory Mucosa HSCB in lfactory Epithelium, Nerves, and Bulb CP lfactory Nerves lfactory Bulb lfactory Epithelium CP CB Dorsal Meatus 50 m 100 m 13
11 Months After HSCB Exposure: Maxilloturbinate e lp tb HSCB Induced Mucous Cell Metaplasia in Transitional Epithelium Lining the Rat Maxilloturbinates Volume Density (nl/mm 2 ) 5 4 3 2 1 HSCB LSCB 0 0 1 7 50 50 Airborne Carbon Black Concentration (mg/m 3 ) Air (0 mg/m 3 ) HSCB (50 mg/m 3 ) LSCB (50 mg/m 3 ) Mucous Cell Metaplasia in Rats and Mice, but not Hamsters, Exposed to HSCB 0 mg/m 3 Air 50 mg/m 3 HSCB R e tb bv e 0 mg/m 3 tb bv M H bv tb e e tb e tb bv tb e Volume Density (nl/mm 2 ) 5 4 3 2 1 0 Rats Mice Hamsters a 0 1 7 50 Airborne Carbon Black Concentration (mg/m 3 ) a b 14
Interspecies Differences in the Severity of HSCB Induced Mucous Cell Metaplasia at ne Day after Exposure Proximal Nasal Tissue Sections Distal Species T1 T2 T3 T4 Rat Mouse - - Hamster - - - HSCB Induced Rhinitis in Rats, but not in Mice or Hamsters Air control (0 mg/m 3 ) HSCB (50 mg/m 3 ) Neutrophils in Nasal Mucosa Lining the Midseptum (T2) of Rats Exposed to Carbon Black Neutrophils in Nasal Mucosa of the Midseptum (T2) of Rodents Exposed to Carbon Black Particles Numeric Cell Density (Neutrophils/mm Basal Lamina) 120 100 80 60 40 20 0 1 day PE 13 weeks PE 0 Air 50 HSCB 50 LSCB Numeric Cell Density (neutrophils/mm BL) 120 100 80 60 40 20 0 Rats Mice Hamsters ND 0 50 HSCB Airborne Carbon Black Concentration (mg/m3) ND Airborne Carbon Black Concentration (mg/m3) Time Post Exposure: 1 day Summary of Results Rats exposed to 7 or 50 mg/m 3 HSCb had chronic rhinitis, epithelial hyperplasia, and mucous cell metaplasia at one day post exposure Magnitude of HSCb induced nasal lesions was siteand dose dependent, and attenuated with time post exposure Aggregates of HSCB found in nasal mucosa, olfactory nerves and olfactory bulb Rats exposed to 1 mg/m 3 HSCb had no nasal lesions Rats exposed to 50 mg/m 3 LSCb had minimal nasal lesions HSCB induced nasal toxicity was species dependent: Rat > Mice > Hamsters 15
Conclusions The nose is a potential target tissue for nanoparticle induced toxicity and a possible route of exposure to the brain As in the lung, particle surface area is an important determinant in the nasal toxicity caused by inhaled carbon black Future studies are needed to determine the underlying mechanisms responsible for nasal toxicity caused by nanoparticles The effects of nanoparticles on the human nasal airways is yet unknown. Black Mold, Trichothecenes and Satratoxins Black Mold Stachybotrys chatarum saprophytic fungus that grows on cellulosic materials Trichothecenes low molecular weight (200 500D) mycotoxins produced by Stachybotrys, Fusarium, and other molds Bind to ribosomes, inhibit protein synthesis, and induce ribotoxic stress Satratoxins macrocyclic trichothecenes produced by Stachybotrys Experimental Design and Methods 7 8 wk old, C57Bl/6 Mice Single intranasal instillation of Satratoxin G (SG; 500 µg/kg BW) in saline or saline alone (controls) Necropsy at 6h, 1 day, 3 days, 7days or 28 days after instillation Nasal and olfactory bulb tissues were processed for microscopic, morphometric, and molecular analysis Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099 1107 16
Summary of SG Induced Histopathology in Nose and Brain Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099 1107 Kinetics of SG Induced Atrophy of E Epithelial Thickness ( m) 70 60 50 40 30 20 10 0 Control Satratoxin-G Multiple Single Instillation Instillations A A A A B B C D E F 1 3 7 28 6 Days Post Instillation Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099 1107 SG Induced Apoptosis of E at 24h Post Instillation C Relative mrna Expression (Fold Increase) 20 15 10 5 0 Fas FasL Control Satratoxin G p75 p53 Bax Bcl-2 Apaf-1 Pro-/Anti-Apoptotic Genes Cas-3 Cad Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099 1107 17
SG induced Loss of MP Positive lfactory Sensory Neurons Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099 1107 MP Positive Cells/mm 700 600 500 400 300 200 100 0 A Single Instillation B A D A 1 3 7 28 6 Days Post Instillation C A Multiple Instillations B A C SG Induced Atrophy of lfactory Marker Protein (MP) Positive lfactory Sensory Neurons A B A) Normal MP expression in olfactory nerve and glomerular layers of olfactory bulb (B) B) Expression of MP in B 7 days after SG instillation C) Site of SG induced atrophy of B () C Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099 1107 Effects of Trichothecenes on E A B H 1 2 16 13 H 12 3 4 9 10 11 8 7 H 15 4 5 6 14 H H H H 2C 15 Deoxynivalenol H CH 3 H Isosatratoxin F 5 4 H 15 4 H 15 Verrucarin A H 1 2 H 16 13 H 12 CH 3 9 10 3 11 H T 2 toxin 8 7 H 4 5 6 Satratoxin G C CH 2 15 C CH 2 H H 3C CH C CH 3 CH 3 CH 3 18
STG Induced Inflammatory Responses in Nose and Brain A C D Relative mrna Expression (Fold Increase) B Relative mrna Expression (Fold Increase) 1000 Control Satratoxin-G lfactory 100 Epithelium 10 2 1 0 1 5 Control Satratoxin-G 4 lfactory 3 Bulb IL-1 IL-1 IL-6 TNF- MIP-2 IL-1 IL-1 IL-6 TNF- MIP-2 E G Inflammatory Cytokines Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099 1107 F H Carey SA et al. Toxicol Pathol 2012 online, in press Satratoxin G Induced lfactory Injury in Rhesus Monkeys Carey SA et al. Toxicol Pathol. 2012; online, in press 19
Satratoxin G Induced Loss of SN in Rhesus Monkeys Carey SA et al. Toxicol Pathol. 2012; online, in press. Satratoxin G Induced Apoptosis of SN in Rhesus Monkeys Carey SA et al. Toxicol Pathol. 2012; online, in press Satratoxin G Induced Rhinitis in Rhesus Monkeys Carey SA et al. Toxicol Pathol. 2012; online, in press 20
Conclusions Nasal airways and brain are potential target sites for toxicity caused by inhalation exposure to macrocylic trichothecene mycotoxins that may be found at high concentrations in the indoor air of moldcontaminated, water damaged buildings. Quantitative assessments of human exposures and epidemiological studies are needed to determine the potential risks to human health. Future Studies: Multi Scale Modeling for Airway Dosimetry Rat vs. Human Nasal Breathing 0.6 ppm Acrolein 434 ml/min flow 0.6 ppm Acrolein 13.8 L/min flow Slide courtesy of Dr. Rick Corley Summary and Conclusions Nasal Airway Anatomy and Histology Nasal Toxicity of Inhaled zone Nasal Toxicity of Inhaled Nanoparticles lfactory Toxicity of Inhaled Satratoxin G (Mycotoxin in Stachybotris chatarum) The nose is a target organ for many inhaled toxicants. Nasal responses to toxicants are dependant on many factors (e.g., physicochemical characteristics, dosimetry, tissue sensitivity, host, species). Results from future laboratory animal studies will be able to better predict human risk to toxic injury. 21