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1ANTHONY
1Department
WATKINS, 2ANNIE JARABEK, 1JACK HARKEMA
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
Cl2
Cl2 Cl2
Cl2
Cl2
Cl2
Cl2
Necrosis – Inflammation – Hyperplasia – Mucous Cell Metaplasia
Cl2
Assessing the Degree of Toxicity
Haber’s Law
C x T = Total Dose
Concentration (ppm) x Time [Duration] (days)
• 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.
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 chlorineinduced 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.
The Experimental Design
Female
F344 Rats
1.0 ppm x 5
Days; 6h/day
Exposure
Regimen (c x t)
Morphometric Analysis of Mucous Cell
Metaplasia (Volume Density)
MT = Maxilloturbinate
0.5 ppm x 10
Days; 6h/day
Results of Study
Nasal Histopathology: Concentrationand Time-Dependent Responses to Cl2
Nasal Histopathology: Persistence of Cl2Induced Nasal Toxicity
Dose-Dependent Responses to 5-Day Cl2 Exposure:
Intraepithelial Mucus in Maxilloturbinates
0 ppm (Filtered Air)
0.5 ppm Cl2 Gas
1.0 ppm Cl2 Gas
2
Volume Density (nl/mm of basal lamina)
0.6
0.5
**
*
0.4
0.3
*
0.2
0.1
0.0
1-Day Post Exposure
*
**
5-Day Post Exposure
Significantly different from the respective 0 ppm Filtered Air treatment group (p<0.05)
Significantly different from the same treatment in different exposure groups (p<0.05)
2
Volume Density (nl/mm basal lamina)
Dose-Dependent Responses to 10-Day Cl2 Exposure:
Intraepithelial Mucus in Maxilloturbinates
2.5
0 ppm (Filtered Air)
0.5 ppm Cl2 Gas
1.0 ppm Cl2 Gas
**
*
2.0
1.5
1.0
*
0.5
0.0
1-Day Post Exposure
*
**
*
10-Day Post Exposure
Significantly different from the respective 0 ppm Filtered Air treatment group (p<0.05)
Significantly different from the same treatment in different exposure groups (p<0.05)
2
Volume Density (nl/mm basal lamina)
Time-Dependent Responses to Cl2 Exposure:
Intraepithelial Mucus
2.5
**
*
0 ppm (Filtered Air)
0.5ppm Cl2 Gas
1.0ppm Cl2 Gas
2.0
1.5
1.0
*
0.5
*
0.0
5-Day Exposure
*
**
10-Day Exposure
Significantly different from the respective 0ppm Filtered air treatment group (p<0.05)
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.
Conclusions & Need of Future Studies
(Summer 2012)
• 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).
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-concentrationmortality relationships and effects on respiration. Journal of
Hazardous Materials 19: 195-208.