1. My Presentation

3. 1 A NTHONY W ATKINS, 2 A NNIE J ARABEK, 1 J ACK H ARKEMA 1 Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824. 2 U.S. Environmental Protection Agency, Research Triangle Park, NC.

4. Chlorine: The Oxidizing Halogen  High Reactivity with Other Elements & Versatility in Reactions  Toxic Effects in its Gaseous State  Morphometric Assessment of Toxicity in Nasal Airway

5. High Reactivity & Reaction Versatility High solubility in water: Production of Chloride Acids. Reactions with numerous elements: Hydrogen, oxygen, organic compounds, alkali and transition metals, etc. Participates in an array of reactions: chlorination, hydrochlorination, etc., to create chemical intermediates. Intermediates used to create end-products: disinfectants, aerosols, pesticides, textiles, paint removers, and bleaches. Used as an effective chemical warfare agent in War World I and the Iraqi War due to its high solubility property. Gori, 1994.

6. Toxic Effects in its Gaseous State Cl 2 (g) + H 2 0(l) HOCl(aq) + HCl(aq) Reaction Mimicked in Nasal Airway (Chlorine and Moist Lining) Cl 2 (g) + H 2 0(l) HOCl(aq) + HCl(aq) Mucosal Water *Subsequent Ionization follows after this reaction.* Winder, 2001.

7. Kinetics of Nasal Epithelial Tissue Responses to Inhaled Chlorine Cl 2 Necrosis – Inflammation – Hyperplasia – Mucous Cell Metaplasia

8. Assessing the Degree of Toxicity Chlorine toxicity in the nasal airways is measured by morphometry: examining the amount of mucous-cell metaplasia that has occurred in the proximal airway (accumulation of mucosubstances). Haber’s Law is used as the primary relationship to determine the degree of toxicity present in the body. C x T = Total Dose Concentration (ppm)Time [Duration] (days) x Haber’s Law Zwart and Wouterson, 1988; Hoyle et. al., 2010

9. Purpose and Hypothesis Purpose: To determine the severity of nasal injury in rats exposed to various exposure regimens to evaluate the contribution of concentration (c) and time (t; duration) of exposure. Hypothesis: The exposure regimen, rather than the total dose, determines the manifestation and magnitude of chlorine- induced nasal pathology.

10. Rat Nasal Anatomy and Histology Sagittal view of the rat nose (without septum). S = squamous, I = incisor, T/R = transitional / respiratory epithelium, HP = hard palate, O = olfactory epithelium, OB = olfactory bulb, and NPD = nasopharyngeal duct. Dashed blue line demarcates region of T/R epithelium.

11. Female F344 Rats 0.5 ppm x 10 Days; 6h/day 1.0 ppm x 5 Days; 6h/day Exposure Regimen (c x t) Morphometric Analysis of Mucous Cell Metaplasia (Volume Density) MT = Maxilloturbinate The Experimental Design

12. Results of Study

13. Nasal Histopathology: Concentration- and Time-Dependent Responses to Cl 2

14. Nasal Histopathology: Persistence of Cl 2 - Induced Nasal Toxicity

15. Dose-Dependent Responses to 5-Day Cl 2 Exposure: Intraepithelial Mucus in Maxilloturbinates

16. Dose-Dependent Responses to 10-Day Cl 2 Exposure: Intraepithelial Mucus in Maxilloturbinates

17. Time-Dependent Responses to Cl 2 Exposure: Intraepithelial Mucus

18. SummarySummary 5- and 10-day Cl 2 exposure caused mucous cell metaplasia in nasal epithelium. Amount of mucous cell metaplasia was both time (t)- and concentration (c)-dependent. Rats exposed to the higher c for the shorter t had significantly less intraepithelial mucus compared to rats exposed to the lower c for the longer t.

19. The exposure regimen, rather than total dose (c x t), should be used to estimate chlorine-induced mucous cell metaplasia. Future studies are needed to determine how other Cl 2 -induced nasal lesions are dependent on (c x t). Conclusions & Need of Future Studies (Summer 2012)

20. Current Study (Fall 2012) Continuation of investigating different parameters to support hypothesis (the exposure regimen versus the total dose). First inflammatory response and parameters (neutrophils)

21. Final Study (Fall 2012 / Spring 2013) Final study of investigating different parameters to support hypothesis (the exposure regimen versus the total dose). Second inflammatory response and parameters (eosinophils)

22. AcknowledgementsAcknowledgements Dr. Jack Harkema (PI) Annie Jarabek (EPA) Experimental Pathology & Toxicology Lab U.S. Environmental Protection Agency CVM (College of Veterinary Medicine) Summer Research Program NIH Grant R25 HL103156

23. ReferencesReferences  Gori, G. B. (1994). Chapter 2: Chlorine. Regulatory Toxicology and Pharmacology 20: S69-S125.  Hoyle, G. W., W. Chang, J. Chen, C. F. Schlueter, and R. J. Rando. (2010). Deviations from Haber’s Law for Multiple Measures of Acute Lung Injury in Chlorine-Exposed Mice. Toxicological Sciences 118: 696-703.  Winder, C. (2001). The Toxicology of Chlorine. Environmental Research Section A 85: 105-114.  Zwart, A. and R. A. Woutersen. (1988). Acute inhalation toxicity of chlorine in rats and mice: time-concentration- mortality relationships and effects on respiration. Journal of Hazardous Materials 19: 195-208.