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Morphometric Assessment of Concentration- and Time-Dependent Injury in the Nasal Airways

of Rats Exposed to Chlorine Gas

1ANTHONY WATKINS, 2ANNIE JARABEK, 1JACK HARKEMA

1Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI 48824.

2U.S. Environmental Protection Agency, Research Triangle Park, NC.

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

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.

Toxic Effects in its Gaseous State

Cl2(g) + H20(l)

HOCl(aq) + HCl(aq)

Reaction Mimicked in Nasal Airway (Chlorine and Moist Lining)

Cl2(g) + H20(l) HOCl(aq) + HCl(aq) Mucosal Water

*Subsequent Ionization follows after this reaction.*

Winder, 2001.

Kinetics of Nasal Epithelial Tissue Responses to Inhaled Chlorine

Cl2

Cl2

Cl2 Cl2Cl2Cl2

Cl2

Cl2Cl2 Cl2

Cl2

Necrosis – Inflammation – Hyperplasia – Mucous Cell Metaplasia

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

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.

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.

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

Results of Study

Nasal Histopathology: Concentration- and Time-Dependent Responses to Cl2

Nasal Histopathology: Persistence of Cl2-Induced Nasal Toxicity

Dose-Dependent Responses to 5-Day Cl2 Exposure: Intraepithelial Mucus in Maxilloturbinates

Vo

lum

e D

en

sit

y (n

l/mm

2 o

f b

as

al l

am

ina

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6 0 ppm (Filtered Air) 0.5 ppm Cl2 Gas

*Significantly different from the respective 0 ppm Filtered Air treatment group (p<0.05)

1-Day Post Exposure 5-Day Post Exposure

1.0 ppm Cl2 Gas

Significantly different from the same treatment in different exposure groups (p<0.05)**

*

***

Dose-Dependent Responses to 10-Day Cl2 Exposure: Intraepithelial Mucus in Maxilloturbinates

Vo

lum

e D

en

sit

y (n

l/m

m2 b

as

al

lam

ina)

0.0

0.5

1.0

1.5

2.0

2.5 0 ppm (Filtered Air)

0.5 ppm Cl2 Gas

Significantly different from the respective 0 ppm Filtered Air treatment group (p<0.05)

1-Day Post Exposure 10-Day Post Exposure

1.0 ppm Cl2 Gas

Significantly different from the same treatment in different exposure groups (p<0.05)***

*

*

*

**

Time-Dependent Responses to Cl2 Exposure: Intraepithelial Mucus

Vo

lum

e D

en

sit

y (n

l/m

m2 b

as

al

lam

ina

)

0.0

0.5

1.0

1.5

2.0

2.5 0 ppm (Filtered Air)

0.5ppm Cl2 Gas

Significantly different from the respective 0ppm Filtered air treatment group (p<0.05)

1.0ppm Cl2 Gas

5-Day Exposure 10-Day Exposure

Significantly different from the same treatment in different exposure groups (p<0.05)***

**

***

Summary

• 5- and 10-day Cl2 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.

• 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 Cl2-induced nasal lesions are dependent on (c x t).

Conclusions & Need of Future Studies (Summer 2012)

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)

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)

Acknowledgements

• 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

References 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.